Prescott's Microbiology 9th Edition

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The author team of Prescott’s Microbiology continues the tradition of past editions by providing a balanced, comprehensive introduction to all major areas of microbiology. This balance makes Microbiology appropriate for microbiology majors and mixed majors courses. The authors have introduced a number of pedagogical elements designed to facilitate student learning. They also remain focused on readability, artwork, and the integration of several key themes (including evolution, ecology and diversity) throughout the text, making an already superior text even bett

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ninth edition Prescotts Microbiology Joanne M. Willey HOFSTRA UNIVERSITY Linda M. Sherwood MONTANA STATE UNIVERSITY Christopher J. Woolverton KENT STATE UNIVERSITY ��onnect Lear R_ Succeed·

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The McGraw·Hi Companies 1 � �onnect Lear B.. Succeed" PRESCOTTS MICROBIOLOGY NINTH EDITION Published by McGraw-Hill a business unit of Te McGraw-Hill Companies Inc. 1221 Avenue of the Americas New York NY 10020 . Copyright© 2014 by Te McGraw-Hill Companies Inc. All rights reserved. Printed in the United States of America. Previous editions© 20112008 and 2005. No part of this publication may be reproduced or distributed in any form or by any means or stored in a database or retrieval system without the prior written consent of Te McGraw-Hill Companies Inc. including but not limited to in any network or other electronic storage or transmission or broadcast for distance learning. Some ancillaries including electronic and print components may not be available to customers outside the United States. Tis book is printed on acid-free paper. 12 3 4 56 7 8 9 0 DOW/DOW 1 0 9 8 7 6 54 3 ISBN 978-0-07-340240-6 MHID 0-07-340240-0 Senior Vice President Products Markets: Kurt L. Strand Vice President General Manager Products Markets: Marty Lange Vice President Content Production Technology Services: Kimberly Meriwether David Managing Director: Michael S. Hackett Director Biology: Lynn M. Breithaupt Brand Manager: AmyL. Reed Director of Development: Rose Koos Development Editor: Kathleen Timp/Angela FitzPatrick Director of Digital Content Development: Barbekka Hurtt Ph.D. Digital Product Manager: Amber Bettcher Content Project Manager: Sandy Wille Senior Buyer: Sandy Ludovissy Senior Designer: David W Hash Cover/Interior Designer: Christopher Reese Cover Image: Sebastian KaulitzkiAlamy Lead Content Licensing Specialist: Carrie K. Burger Photo Research: Mary Reeg Compositor: AptaraC Inc. Typeface: 10/12 Minion Pro Printer: R. R. Donnelley All credits appearing on page or at the end of the book are considered to be an extension of the copyright page. Library of Congress Cataloging-in-Publication Data Cataloging-in-Publication Data has been requested from the Library of Congress. Te Internet addresses listed in the text were accurate at the time of publication. Te inclusion of a website does not indicate an endorsement by the authors or McGraw-Hill and McGraw-Hill does not guarantee the accuracy of the information presented at these sites. ww w.mhhe.com

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Joanne M. Willey has been a professor at Hofstra University on Long Island New York since 1993 where she is Professor of Microbiology she holds a joint appointment with the Hofstra University School of Medicine. Dr. Willey received her B. A. in Biology from the University of Pennsylvania where her interest in microbiology began with work on cyanobacterial growth in eutrophic streams. She earned her Ph.D. in biological oceanography specializing in marine microbiology from the Massachusetts Institute of Technology­ Woods Hole Oceanographic Institution Joint Program in 1987. She then went to Harvard University where she spent her postdoctoral fellowship study ing the flamentous soil bacterium Streptomyces coelicolor. Dr. Willey continues to investigate this fascinating microbe and has coauthored a number of publications that focus on its complex developmental cycle. She is an active member of the American Society for Microbiology ASM and served on the editorial board of the journal Applied and Environmental Microbiology for nine years and as Chair of the Division of General Microbiology. Dr. Willey regularly teaches microbiology to biology majors as well as medical students. She also teaches courses in cell biology marine microbiology and laboratory techniques in molecular genetics. Dr. Willey lives on the north shore of Long Island with her husband and two sons. She is an avid runner and enjoy s skiing hiking sailing and reading. She can be reached at joanne.m.willeyhofstra.edu. About the Authors Linda M. Sherwood is a member of the Department of Microbiology at Montana State University. Her interest in microbiology was sparked by the last course she took to complete a B.S. degree in Psychology at Western Illinois University. She went on to complete an M.S. degree in Microbiology at the University of Alabama where she studied histidine utilization by Pseudomonas acidovorns. She subsequently earned a Ph.D. in Genetics at Michigan State University where she studied sporulation in Saccharomyces cerevisiae. She briefly lef the microbial world to study the molecular biology of dunce fruit fies at Michigan State University before moving to Montana State University. Dr. Sherwood has always had a keen interest in teaching and her psychology training has helped her to understand current models of cognition and learning and their implications for teaching. Over the years she has taught courses in general microbiology genetics biology microbial genetics and microbial physiology. She has served as the editor for ASMs Focus on Microbiology Education and has participated in and contributed to numerous ASM Conferences for Undergraduate Educators ASMCUE. She also has worked with K-12 teachers to develop a kit-based unit to introduce microbiology into the elementary school curriculum and has coauthored with Barbara Hudson a general microbiology laboratory manual Explortions in Microbiology: A Discovery Approach published by Prentice-Hall. Her association with McGraw-Hill began when she prepared the study guides for the ffh and sixth editions of Microbiology. Her non­ academic interests focus primarily on her family. She also enjoys reading hiking gardening and traveling. She can be reached at lsherwoodmontana.edu. Christopher J. Woolverton is founding professor of Environmental Health Science College of Public Health at Kent State University Kent OH and is the Director of the Kent State University KSU Center for Public Health Preparedness overseeing its BSL-3 Training Facility. Dr. Woolverton serves on the KSU graduate faculty of the College of Public Health the School of Biomedical Sciences and the Department of Biological Sciences. He holds a joint appointment at Akron Childrens Hospital Akron OH. He earned his B.S. in Biology from Wilkes College PA and his M.S. and Ph.D. in Medical Microbiology from West Virginia University School of Medicine. He spent two years as a postdoctoral fellow at UNC-Chapel-Hill. Dr. Woolvertons current research is focused on real-time detection and identifcation of pathogens using a liquid crystal LC biosensor that he patented in 2001. Dr. Woolverton has published and lectured widely on the mechanisms by which LCs act as biosensors and on the LC characteristics of microbial proteins. Professor Woolverton teaches microbiology communicable diseases immunology prevention and control of disease and microbial physiology. He is on the faculty of the National Institutes of Health National Biosafety and Biocontainment Training Program teaching laboratory safety risk assessment decontamination strategies and bioterrorism readiness. An active member of the American Society for Microbiology Woolverton serves on its Board of Education and as the editor-in-chief of its Journal of Microbiology and Biology Education. Woolverton and his wife Nancy have three daughters a son-in-law and a grandson. He enjoys time with his family ultra-light hiking and camping and is an avid cyclist. His e-mail address is cwoolverkent.edu. iii

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Digital Tools for Your Success 8 I LOG Save time with auto-graded assessmen Gather powerful performance data. McGraw-Hill ConnectPius® Microbiology provides online presentation assignment and assessment solutions connecting your students with the tools and resources theyll need to achieve success. Homework and Assessment With ConnectPlus Microbiology you can deliver auto-graded assignments quizzes and tests online. A robust set of interactive questions and activities using high-quality art from the text­ book and animations is presented. Assignable content is avail­ able for every Learning Outcome in the book and is categorized according to the ASM Curriculum Guidelines. As an instruc­ tor you can edit existing questions and author entirely new tTI" hlonC librr JIILI U\LUI noUUI \ Customize your lecture with tools such as PowerPoin� presentations animations and editable art from the textbook. An instruc­ tors manual for the text and lab manual as well as answer keys to in-text questions save you time in developing your course. eDOOK ConnectPlus Microbiology pro­ vides students 24/7 online access to a media-rich version of the book allowing seamless integra­ tion of text media and assess­ ments. Learn more at www.mcgrawhillconnect.com. iv Detailed Reports Track individual student performance­ by question assignment or in relation to the class overall-with detailed grade reports. Integrate grade reports easily with Learning Management Systems LMS such as WebCT and Black­ board-and much more. Lecture Capture McGraw-Hill Tegrit� records and dis­ tributes your class lecture with just a click of a button. Students can view any­ time anywhere via computer or mobile device. Indexed as you record students can use keywords to fnd exactly what they want to study.

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Digital Tools for Your Success I LearnSmart� A diagnostic adaptive learning system to increase preparedness. Now Available for the Ninth Edition McGraw-Hill LearnSmartTM is an adaptive learning system de­ signed to help students learn faster study more efciently and retain more knowledge for greater success. LearnSmart assesses a students knowledge of course content through a series of adap­ tive questions. It pinpoints concepts the student does not under­ stand and maps out a personalized study plan for success. Tis innovative study tool also has features that allow instructors to see exactly what students have accomplished and a built-in assessment tool for graded assignments. Visit www.mhlearnsmart.com for a demonstration. l I .. "- " - � 21 Wt tt.r G1 � Clttd f 01 from Uc CCII membrneo a Icl bra cl Laboratory Exercses in Microbiology Ninth Edition John P. Harley has revised this labora­ tory manual to accompany the ninth edition of Prescotts Microbiology. Te class-tested exercises are modular to allow instructors to easily incorporate them into their course. Tis balanced introduction to each area of microbiol­ ogy now also has accompanying Con- •crete With McGraw-Hill CreateM you can easily rearrange chapters combine material nect content for additional homework and assessment opportunities. In addition all artwork from the lab manual is now available through the Instructor Resources in Connect for incorporation into lectures. from other content sources and quickly upload content you have written such as your course syllabus or teaching notes. Find the content you need in Create by searching through thousands of leading McGraw-Hill textbooks. Arrange your book to ft your teaching style. Create even allows you to personalize your books appearance by selecting the cover and adding your name school and course information. Order a Create book and youll receive a complimentary print review copy in three to fve business days or a complimentary electronic review copy eComp via e-mail in minutes. Go to ww .mcgrawhillcreate.com today and register to experience how McGraw-Hill Create empowers you to teach your students your way. v

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A Modern Approach to Microbiology Evolution as a Framework Introduced immediately in chapter 1 and used as an overarching theme throughout evolution helps unite microbiological con­ cepts and provides a framework upon which students can build their knowledge. Separate Chapters on Bacteria and Archaea In recognition of the importance and prevalence of archaea the structure genetics and taxonomic and physiologic diversity of these microbes are now covered in chapters that are separate from those about bacteria. An Introduction to the Entire Microbial World Now covered in chapters 3-6 the separate chapters on the structure and function of bacteria and archaea are followed by the discussion of eukaryotic cells preceding viruses. vi Secondary Lymphoid Organs and Tissues The 8plecn is the most highly organized secondary lymphoid organ.lt is a largeorganlocated inthe abdominal cavitythat functions to flter the blood and trap blood-borne particles to be ass�ssed for foreignness by phagocytes fgure 33.14. Mac­ rophages and dendritic cells are present in abundance and once trapped by splenic macrophages or dendritic cells a pathogen is phagocytosed killed and digested. 1he resulting antigens are presented to lymphocytes activating a specifc im­ mune response. Lymph nodes lk at the junctions oflymphaticvessds where macro phages md dendritic cells trap particles that enter the lym­ phaticsyslemfgure33.14c.Ifaparlicle isfolndlobe foreignil is then phagocyosed and degraded and the resulting antigens arcprcscntcdto lymphocytcs. Lymphoid tissues are found througholl the body as highly organi7.d or loosely associated cellular complexes figure 33.14. Some lymphoid cells are closely associated with specifc tissues such as skin skin-associated lymphoid tissue or SALT and mu­ cous membranes mucosal-associated lymphoid tissue or MALT. SALT and MALT arc good examples ofhighlyorgani7d lymphoidtissues that featuremacrophagessurrounded by spe­ cifc areas ofB and T lymphocytes and sometimes dendritic cells. Loosely associated lymphoid tissue is best represented by the bronchial-associated lymphoid tissue BALT because it lack cellular partitioning. Te primary role of these lymphoid tissues is to efiently organi7eleukocytes to increase intcraction be­ tween the innate and the adaptive arms of the immune response. lhus the lymphoid tissues serve as the interface between the in­ nate resistance mechanisms and adaptive immunity of a host. We now discuss these tissues in more detail Despite the skins defenses at times pathogenic microorgan­ isms gain access to the tissue under the skn surface. Here they encounter a spedalized set of cells called the kin-associated lmphoid tisue SALT fgure 33.15. The major fnction of SALT is to confne microbial invaders to the area immediately underlying the epidermisand to preventthemfomgaining ac­ cess to the bloodstream. One type of SALT cell is the Langcr­ hans cl a dendritic ccll that phagocytoses microorganisms that penetrate th� skin. Once the Langerhans cell has int�mal­ i7ed a foreign particle or microorganism it migrates from the epidermis t nearby lymph nodes where it presents antigen to activate nearby lymphocytes inducing a specifc immnne re­ sponse to that antigen. This dendritic cell-lymphocyte interac­ tion illustrates another bridge between innate resistance and adaptive immunity. The epidermis also contains another type of SALT cell called the intraepidfrmal lymphocytf figure 33.15 a spe­ cialized T cellhavingpotentcytolyticand immunoregulatory responses to antigen. These cells are strategically located in the skin so that they can intercept any antigens that breach the frst line of defense. Most of these specialized SALT cells have limiLed rcceptordiversityandhavelikelyevolvedto recogni7ecommon skin pathogen patterns. Te specialized lymphoid tissue in mllcous membranes is called mucosa-associated lymphoid tissue MALT. There are sneral types of MALT. Te system mol studied is the gut­ associated lymphoid tis�ue GALT. GALT includes the ton­ sils adenoids difllse lymphoid areas along the gut and specialized regionsinthe intestine calledPeyerspatches. Less well-organized MALT also occurs in the respiratory system and Aboutthe Authors 111 Preface iv Part One Introduction to Microbiology --- 1 The Evolution ofMicroorganrm� and Microbiology 1 2 Micro5topy 22 3 BacteriaiCeiiStructure 42 4 ArchaeaiCeiiStructure 82 5 EukaryoticCeiiStructure 92 6 Viruses and Other Acellular lnfedklus Agents Part Tw Microbial Nutrition Growth and Control 7 MicrobiaiGrowth 133 8 ControlofMicroorganismsin theEnvironlent 9 AntimicrobiaiChemotherapy 189 Part Tree Microbial Metabolism 10 lntroductklntoMetaboWsm 210 11 Catabolism:EnergyReleaseandConservation 12 Anabolism:TheUseofEnergyin8iosynthesis 266 Part Four Microbial Molecular Biology and Genetics 13 BacteriaiGenomeReplicationandExpression 287 14 RegulationofBicteriaiCellularProcesses 325 15 Eukaryotic and Archaeal Genome Replication andExpression 353 16 MechanismsofGeneticVariation 372 17 Recombinant DNA Technology 18 Microbial Genomics 424 Part Five The Diversity of the Microbial World 19 Microbial Taxonomy and the Evolution of Diversity 20 TheArhaea 469 21 TheDeinococciMollicutesandNonproteobacterial Gram-Negative Bacteria 489 22 TheProteobacteria 509 24 Actinobacteria: The High G + C Gram-Positive Bacteria 555 2STheProtists 568 21 TheFungiEumycta 588 Part Six Ecology and Symbiosis 28 BiogeochemicaiCyclingandGiobal ClimateChange 632 29 Methods In Microbial Ecology 141 30 Microorgani5ms in Marine and Fre5hwater Ecosystems 660 31 Microorgani5m5inTerre5triaiEcostems 679 32 Microbiallnteractions 699 Part Sevn Pathogenicity and Host Response 33 lnnateHostResistance 723 34 Adaptivelmmunity 753 35 Pathogenicity andlnfection 789 Part Eight Microbial Diseases Detection and Their Control 36 ClinicaiMicrobiolo gyandlmmunology 808 37 Epidemiology and Public Health Microbiology 38 HumanDiseasesCausedbyVirusesandPrions 854 39 HumanDiseasesCausedbyBacteria 888 40 HumanDiseasesCausedb FungiandProtists Part Nine Applied Microbiology 41 MicrobiologyofFood 958 42 Biotechnologyand lndustriaiMicrobiology 43 Applied Environmental Microbiology 996 Appendix 1 A Review of the Chemistr ofBiologicaiMolecules A -1 Appendix2 Common Metabolic Pathways Glossary G-1 Credits C-1 23 Firmicutes:ThelowG+CGram-PositiveBicteria 542 Index 1-1 Molecular Microbiology and Immunology Te ninth edition includes updates on genetics biotechnology genomics and immunology. Te discussion of eukaryotic and archaeal genetics has been expanded and makes up a separate chapter to reflect the relatedness of genetic information flow. A streamlined discussion of immunity with enhanced detail be­ tween innate and adaptive linkages helps students grasp the complexity and specifcity of immune responses.

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A Modern Approach to Microbiology figurelll .Amrfleacei Ito lahsando fl causing eutophication-an increase in nutrient lthat stmula�thegof alim­ itednumberof oml therebydb­ ingthe ecooftheseaero�m�. Bycontraslmic nt cre­ lultlnthe od ofmunonium tomore nlrattth�n eanbeinodby pbnt andmlcrobe. asoneedapedc ruofC:N:PT heprofd tc tionoomt ihleKtra nitte toNand lhe rnctt g:nllotue nlto�nodes. This cycle ofnic.oo/ di on feledbyNH+intoducedasftiliris respoosiblefrtehighestN00leve ls in 650.000U. What arethen�u.nus of di•­ rupting lhccarbon andnitrogencycles Globllclimate change itthemost obvi­ ous example. k is important to keep in mind that weaber is oot the same a1 cli­ mate. While North America has 1ufered lomtoftheholl«l tll1llmtnon recordln thepurdecad eaingle dayr week in Julytlt is panioubrlyhot 8 not by it· sel£evicknce olgloN\clima te ebange. Globfl clim•techange is mmuredovtr decad eswdindudnn1•nypenmete11 lucha.urf�tempenolureonlandand Flgure28 .12 .tur• llndHumm·Mid tlnfutnoonlht"ll rOCJ t. on. and In tb . atmmpbere and trope· MICRO INQUIIV "ombrniri optMrerates ofprecipitationandfre- quency of extreme Wather. Based on these analyr� the average global temperature has incr 0.74"Ca00th is rise is di rectly corrdatedwithfuilfuel com- bustion toCO fpre2S.l3 .Dtpending onthe rte ofconlln· nedincrease ingreenhouse gases.the average global surface temperatu reis predict edtorise betweenl.land6.4"Cl2100. Mo imporunt queston is how will mic re�poOO 1 a cgwluoe forthevast majority of ls history miCrganimu h bun the d of elememzl Cllng. cin rl activitesw illhavea major ic ontherae andr ae ofgreenbouse gas�umutionadgl obal elimae ee.Ihe rolemierobes pltnbalaneingcvbon andlitn fuxethunpentdne warouesofmearchltricroblal e. Retie�. tf ¥i 1. listlhnetemlouHgoml.DiSth tiroriQim 2.D1 theroleDfreot• in the controiDCO. l. Howdo�"9"inthe nitrogencyd"caused bylrtilization lnflu.ncetheurbon� 4. GIVen hit "idl mkroblal �roup ha• an optimum temperall re rmJ 1or l"- miglt you predict cl�nges to a soli mlcrollal com ll nity1ng inyourgeog�plica�7 Flgure2 8.13 GlobaiAnnuai-MeanSAirTm.per1ture Change. DanidefrvedfmmthPmrtromlogic.l•laOOnnetwOO:Goddard lnstituteforSpaceSiero:.hnpta.Ji".n"".JIislem¢rapW Special Interest Essays Organized into four themes-Microbial Diversity Ecology Techniques Applications Historical High­ lights and Disease-these focused and interesting essays provide additional insight to relevant topics. � Microbial Diversity E 3.1 Gram Positive and Gram Negative or Monoderms and Diderms The importance of the Gram stain in the history of microbi­ ology cannot be overstated. The Gram stain reaction was fr many years one of the critical pieces of information used by bacterial taxonomists to construct taxa and it is still useful in identifying bacteria in clinical settings. The initial studies done to diferentiate bacteria that stained Gram positive from those that stain Gram negative were done using modd organisms such as Bacillus subtilis Gram positive and Esch­ erichia coli Gram negative. At the time it was thought that all other bacteria would have similar cell wall structures. However as the cell walls of more bacteria hav been charac­ terized it has become apparent that it may be misleading to refer to bacteria as Gram positive or Gram negative. In other words the long-held models of Gram-positive and Gram­ negative cell walls do not hold true for aU bacteria. Recently Iain Sutclife has proposed that microbiologists stop refer­ ring to bacteria as either Gram positive or Gram negative. He suggests that instead we should more precisely describe bac­ terial cell envelope architectures by fcusing on the observa­ tion that some bacteria have envelopes with a single membrane-the plasma membrane as seen in typical Gram­ positive bacteria-while others have envelopes with two membranes-the plasma membrane and an outer membrane as seen in typical Gram-negative bacteria. He proposed call­ ing the former monoderms and the latter diderms. But why make this change Sutclife begins by pointing out that some bacteria staining Gram positive are actually diderms and some staining Gram negative are actually moooderms. By referring to Gram-positive-staining diderms as Gram-positive bacteria It is too easy to mislead scientists and many a budding microbiologist into thinkil bacterium has a typical Gram-positive envelope. gues that by relating cell envelope architecture tot enies of various bacterial taxa we may gain insig evolution of these architectures. He notes that th• micutes and Actinobacterta are composed almost of monoderm bacteria whereas almost all othe phyla consist of diderms. There are interesting exceptions to the rela phylogeny and cl envelope structure. For instanet of the genus Mycobacterium e.g. M. tubercula to the predominantly monoderm phylum Acti1 Mycobacteria have cell walls that consist of pep and an outer membrane. The outer membrane is of mycolic adds rather than the phospholipid� polysaccharides LPSs f�und in the typical Gnu cells outer membrane. t Suborder Corynet section 24.1 Members of the genus Deinococcus are anotb ing exception. Tese bacteria stain Gram positive derms. Their cell envelopes consist of the plasma · what appears to be a typical Gram-negative cdl � outer S-layer. Their outer membrane is distinctivt lacks LPS. Deinococd are not unique in this respe I is now known that there are several t with c branes that substitute other molecules for LPS. Soune:SutclifI.C.lIO.A phylum level perpectiveonbl�teticenMvelope �t ch i c wr • . lrendsU ie ro blol . fBI0/ .. 64-70. . 21st-Century Microbiology Prescotts Microbiology leads the way with updated text devoted to global climate change biofuels and microbial fuel cells. For more see chapters 28 30 42 and 43. Metagenomics and the Human Microbiome Te updated genomics chapter covers the technical aspects of metagenomics and the human microbiome is discussed in the context of microbial interactions in chapters 18 and 32. Laboratory Safety Refecting forthcoming recommendations from the American Society for Microbiology chapter 37 provides specifc guidance for laboratory best practices to help instructors provide safe con­ ditions during the teaching of laboratory exercises. - _ Disease 26.1 White-Nose Syndrome Is Decimating North American Bat Populations Bats evoke all kinds of images. Some people immediately think of vampire bats and are repulsed. Others think of the large fruit bats ofen called flying foxes. If you have spent a summer evening outdoors on the east coast ofNorth America mosquitoes and the small bats that eat them may come to mind. A new scene can now be added to these: bats with white fungal hyphae growing around their muzzles box fg­ ure . This is the hallmark of white-nose syndrome WNS and if its rate of infection continues unchecked it is projected t eliminate the most common bat species In eastern North America Myotis lucifgus by 2026. WNS was first spotted in 2006 among bats hibernating in a cave near Albany NY. Scientists qukkly became alarmed for two reasons. First it spreads rapidly-its known to occur in at least six bat species and is now fund fom the mid-Atlantic United States northward into Canada Ontario Quebec and New Brunswick and as fr west as Oklahoma. Second it is deadly. A population of bats declines from 30 to 99 in any given infected hibernacula the place where bats hibernate which unfortunatdy rhymes with Dracula. WNS is caused by the ascomycete Geomyces destructans. It colonizes a bats wings muzzle and ears where it first Geomyces destructans causes WNS. A little brown bat yotis lucifgus with the white fungal hyphae for which WNS is named. erodes the epidermis and then invades the underlying skin and connective tissue. Despite the name WNS the primary site of infection and the anatomical site harmed most is the wing. Wings provide a large surfce area fr colonization and once infected the thin layer of skin is easily damaged leading to adverse physiological changes during hibernation. These in turn result in premature awakening loss of essential fat reserves and strange behavior. Where did this pathogen come from and why does it infect bats The best hypothesis regarding Its origin Is that humans inadvertently brought it from Europe where it causes mild infection in at least one hibernating bat species. This makes G. destructans an apparent case of pathogen pollution-the human introduction of invasive pathogens of wildlife and domestic animal populations that threaten bio­ diversity and ecosystem function. Te capacity of G. destructans to sweep through bat populations results from a "perfect storm" of host- and pathogen-associated factors. G. destructans is psychrophilic with a growth optimum around trC it does not grow above 20°C. All infected bat species hibernate in cold and humid environments such as caves and mines. Because their meta­ bolic rate is drastically reduced during hibernation their body temperature reaches that of their surroundings be­ tween 2 and 7°C. Tus WNS is only seen in hibernating bats or those that have just emerged from hibernation. When metabolically active the bats body temperature is too V-tm to support pathogen growth. While it is too late to save the estimated 6 million bats that have already succumbed to WNS microbiologists con­ servationists and government agencies are trying to limit the continued decline in bat populations. Caves have been clo�ed to human trafc and protocols for decontamination afer visiting hibernacula have been developed to limit the spread from cave to cave. Although we cannot cure sick bats it is our responsibility to stop the continued spread of this pathogen. Re•dmor:FrctWFera/.2UIU.Aneme r tucausuregioMipop ul•tlon col/1ps• of• common NartAm•rc•n Nt 1p1cir. S�itnca 319:679-682.

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Student-Friendly Organization Viruses and Other Acellular Infectious Agents Mustard Catsup and Viruses D�:� �� e e n�:g : ::�:: �:� g: 1 1:: 111 d : �: r : � n1i�ed States.Hot dogsardluochmeats arepopularatoutingssuch asblsebill mesandin lunchescarried towarkorschooi.Y eteachyearintfe Urlited Stat . apprmimately1600peopleare sickenedby•bacterium that can wntominate the meat and. even worse. survive ari grow when the"" atis properlyrefrigerated. Thediseasecul. . itisLis terimonocytogenesaGram-positive rOO fourtinsoil arimanyotherenvironrental sites.lt isnot orlycoldtolerant butsaltandaddtolerantas weii.AithoLgh itisinthe minorleagoeswhen compared to someofthe big hitters offo OObome disease e.g. So/monel/a ffltericoit isofcoocernfortwo reasons:who itkillsanchowrTInyitk�ls. L. /O ytvgfesUrgets theyOlllgand oldpregrntwomenand immunocompromised individuals about 15ofthose infted die. ltseffectonpregnantwo "" nis partkularlyheortbreaking.The cantaloupe sedan outbreokoflisteriosisin20state sin theUnited Stile ich infected over l and k�led over 20 Viruses as agents of good will come as a surprise to many. Typically we thinkofthemasmajorcausesofdi sease.Howeverviruses areignilicamfor otherreaens.Theyarevitalmembersofaquatic ecosys tems.Ttlerethe interact with cellulor mkrobe sand contribute to the mo\ement ganic Bica/wntrolafmkroorganisms tian8.7 Readiness Check: fased onwhatyouhivelearnedpreviousfy youshoul dbe ableto I Defnetheterm acellular I Compareand contrast ingeneralterms virusesviroidssatelitesaOO prionssectionU woman usually only sufers mild flu like symptoms however. these 6.1 Viruses innocuoussymptomsbelie thefactthatthechildshe carries isin serious danger. Herpregnaocyoftenendsin miscarriageor stillbirth.Newborns After reading this section you should be �ble to infected with the bacterium are likely to develop meningitis. Many will die • Defne the terms virology bacterioptloges and ptloges as a result.Thme whosurviveoftenhaveneurologicaldisorders. • Litorganism thatarehoststo viruses Currentlypregnant wamenarecoonseledagainst eatingrBldy-to-eat foodunlesstheyhavebeencookedpriorto consumption.fowever L. monacyrogene� is koown to contaminate many fods other than tot dogs andthesecant alway sbeheated.ln2006th�U.S.FoodandDrug AdministrationFD Aappro•ed anewapproachto preventlisteriosis: spraying •irusesthatattackanddestroythebacteriuman reody- to-eatcold cutsalldlllncheonmeats.lnother words.the viruseoareafood additive The"" thodissafetecausethe viruses onlyattackL.mooone.not New Newsworthy Stories-Each chapter begins with a real-life story illustrating the relevance of the content cov­ ered in the upcoming text. New Readiness Check-Te introduction to each chapter includes a skills checklist that defnes the prior knowledge a student needs to understand the material that follows. New Learning Outcomes-E very section in each chapter begins with a list of content-based activities students should be able to perform afer reading. Sinceapproval.the uoeofvirusesto controlthetransmissionof listeriosisbyo therfoodshasbeenstudied . Unfununatelytho sestudiesdid-"-"""".""---------- ootindudefodssuchas freshfruit.ln2011L f enes-contaminated Micro Inqu iry- Select fgures throughout every chapter contain probing questions adding another assessment opportunity for the student. viii MICRO INQUIRY ldotheemptywirem1iocfcchrtothf cei/f�rtM·tiro.irlOiMffitmrhelcotct//1 Animation Icon-T is sym­ bol indicates material pre­ sented in the text is also accompanied by an anima­ tion on the text website at www.mhhe.com/willey9. Cross-Referenced Notes­ In-text references refer stu­ dents to other parts of the book to review. Re trieve Infer Apply­ Questions within the nar­ rative of each chapter assist students in mastering sec­ tion concepts before mov­ ing on to other topics.

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Student-Friendly Organization Vivid Instructional Art Program- Three­ dimensional renditions and bright attractive colors enhance learning. r�ugll i tiMl. H:c�· th.� \� -c aiie th c micmo•glnN:l5 within a "ki\1 - l. LJI d1�m�u" wUd LUii.i will l� so:�ts _ idliiL .f �· rv l"��· l i l i�gr•til r tlf mifQ� - Rr _ o ll th t � iioruo�i Qjll.Y r��­ n:n itln in linn 333 fiKuo.in� nn " Pl.T�n t p nliL1T N md r.t J.o�iu:. \� tY .� . u:.SOI�I�ontriUt\IUiUIt� lS �tl �LT"df morc .iuil rt g l rd i tfl h e rr.ult pmcrn rfOjnitin bv plm � .. •vo:�- k� Recognition of Foreignness I he lonin-irndnt mcchlL\i5mN an g .· rm - l i L\ C �rwd:d. ret·tor llfd Sl·stt:W . ·J :� .rtil wu�ulu Illtms VUJ:ou lo m �n •. itfrt flll :hr�r"1 art n:��n i 1� fQ afi�lf lh:rrx - 1f• f•�un• 33.11 . 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Polym�tas� Chin R�action • ��� pymno.. duin ru�hnn ICRJ �11.w ""311 �m4lnl\ uf •J"d lr D::A iu�n""" lu b.· inor�·•d in JJl.tlliOO ltll.ld� OJtillt� JiUt� J.8 • l:Rna.� mJromu1app� i.37ifln�. rtllcni�uoit� . irl ���..� ur du n in g actd in iliiptv�lk md ftll:u:i· �icoc� 17.3 Cloning Vctor ilnd Creating Rfcombin.nt DNA • I h tr � �rc tur l-.. ot c.kmi l18\Ctr: ��k\�mk5 vii".J-.. - �mk . 5 itx. ulilkial �1:moM•m-�. Cbni:tl �-�·w n �e�rtlly """ I \JS th ref.o m p"f "i � 1n oli�in of rtt•linlK a d·ota :c mar k. - and a mul tk ot in b t� lr fl��huk.. WW� 17. Ji�l 17.10 1.U. 7.1lj • 11rnM.nll1ll_PI03:hto�:lnllfi.ITOIig..\lbtl: ICI.T ux. 0.\A tu lx iCIT\l.. w iLn .t:.· ut:x· K�LricLitm �J��·.w.e .x �Jt�·wes �o tlu.l co.w.all-l� li.\� �mls �r� �tncrad. I�� . .. tTnr an- IJN.•\ l: . dnn�d �n Th�n lnr�ud in th�· flC« l oflNA lil"" · -.h i·h �o l �·L �� til blll �tn of l hv\hotiest� · .rhl� oIX thf. n�-� fnFmCTitlilT5inTilthoI.TilT. • Ouct llx cell u�illim. lluJuid b d� t . . 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List of Content Changes Each chapter has been thoroughly reviewed and many have un­ dergone signifcant revision. All now feature pedagogical ele­ ments including a Readiness Check for the chapter and Learning Outcomes for each section therein. Part I Chapter 1- Evolution is the driving force of all biological sys­ tems this is made clear by introducing essential concepts of mi­ crobial evolution frst. Chapter 3-Coverage of bacterial cellular structure and function. Te chapter now includes a discussion of nutrient uptake in the section on bacterial plasma membranes. Chapter 4-G rowing understanding of the distinctive character­ istics of archaea has warranted the creation of a new chapter that focuses on their cell structure and function. Comparisons to bac­ teria are made throughout the chapter. Chapter 5- An introduction to eukaryotic cell structure and function with emphasis on eukaryotic microbes. More de­ tailed information on protist and fungal cells is presented in chapters 25 Te Protists and 26 Te Fungi which also focus on the diversity of these microbes. Comparisons between bac­ teria archaea and eukaryotes are included throughout the chapter. Chapter 6- Tis chapter entitled Viruses and Other Acellular In­ fectious Agents surveys the essential morphological physiologi­ cal and genetic elements of viruses as well as viroids satellites and prions. Tis chapter completes our four-chapter introduction of microbial life. Part II Chapter 7-Reorganized to initially focus on the growth of mi­ crobes outside the laboratory including growth in oligotrophic environments and the environmental factors that infuence microbial reproduction. Topics related to laboratory culture of microbes follow. Chapter 8- Reorganized to refect emphasis on interruption of nor­ mal growth and reproduction fnctions to control microorganisms. Chapter 9-Content focuses on the mechanism of action of each antimicrobial agent and stresses usage to limit drug resistance. Part III Chapter 10- Tis introduction to metabolism includes a new section that outlines the nature of biochemical pathways and X introduces the concept of metabolic fux through the intercon­ nected biochemical pathways used by cells. Chapter 11- Te chapter now begins with an introduction to metabolic diversity and nutritional types. Chapter 12-Updated coverage ofCOrfxation pathways. PartlY Chapter 13-Now focuses on bacterial genetic information fow with improved coverage of bacterial promoters sigma factors termination of DNA replication transcription cycle and protein folding and secretion. Chapter 14-Now focuses on the regulation of bacterial cellular processes. Te coverage of regulation of complex cellular behav­ iors has been signifcantly updated and expanded including new material on cyclic dimeric GMP. Chapter 15-A new chapter that considers eukaryal and archaeal genome replication and expression together. In both cases the discussion has been updated and expanded and refects the simi­ larity of information fow as carried out by members of Archaea and Eukarya. Chapter 16-Covers mutation repair and recombination in the context of processes that introduce genetic variation into popula­ tions. Tis is now related to the evolution of antibiotic-resistant bacteria. Chapter 17- Te use of recombinant DNA approaches to con­ struct a synthetic genome is highlighted. Chapter 18-New principles and applications of genomic tech­ niques including massively parallel genome sequencing and single cell genome sequencing are now reviewed. Te growing importance of metagenomics to environmental microbiology and its use in exploring the human microbiome are introduced here. PartY Chapter 19-Microbial evolution introduced in chapter 1 is ex­ panded with a complete discussion of the endosymbiotic theory and the concept and defnition of a microbial species. Chapter 20- Expanded coverage of archaeal physiology includes new fgures presenting archaeal-specifc anabolic and catabolic pathways. Te evolutionary advantage of each pathway is dis­ cussed in the context of archaeal ecology. Chapter 21-Now includes mycoplasmas in keeping with Bergey Manual new fgures illustrating the life cycle of Chlamydia are included.

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List of Content Changes Chapter 22-Expanded coverage of proteobacterial physiology with content on Cl metabolism including several fgures. Chapter 24- Increased coverage of streptomycetes with new graphics illustrating their life cycle and their importance in anti­ biotic production. Chapter 27-Updated discussion of virus taxonomy and phylog­ eny including increased coverage of archaeal viruses and the CRISPR/CAS system. Part VI Chapter 28- The description of each nutrient cycle is accom­ panied by a new "student-friendly" figure that distinguishes between reductive and oxidative reactions. Expanded cover­ age of the interaction between nutrient cycles is also newly illustrated. Chapter 29-Tis chapter continues to emphasize culture-based techniques as the "gold standard" and reviews some new innova­ tive approaches. Te chapter also discusses a variety of culture­ independent techniques used to assess populations and communities. Chapter 30-Updated and expanded discussion of freshwater microbiology is complemented by discussion of carbon cycling in the open ocean and its implications for global climate change. Chapter 31-New and updated coverage of mycorrhizae with an emphasis on host-microbe communication and evolutionary similarities to rhizobia. Chapter 32-Microbial relationships are presented along with human-microbe interactions helping to convey the concept that the human body is an ecosystem. New and increased coverage of the human microbiome. Part VII Chapter 33-Reorganized and updated this chapter on innate host resistance provides in-depth coverage of physical and chemical components of the nonspecifc host response fol­ lowed by an overview of cells tissues and organs of the im­ mune system. Tis includes a step-by-step discussion of how microorganisms and damaged tissues are identifed by the host using pattern recognition to remove them. Discussions of phagocytosis and inflammation are updated and reflect mo­ lecular mechanisms. Te groundwork is laid for a full apprecia­ tion of the connections between the adaptive and innate arms of the immune system. Chapter 34-Reorganized and updated to enhance linkages be­ tween innate and adaptive immune activities. Discussions inte­ grate cell biology physiology and genetics concepts to present the immune system as a unifed response having various compo­ nents. Implications of dysfunctional immune actions are also discussed. Chapter 35-Tis chapter has been re-titled Pathogenicity and Infection refecting its emphasis on microbial strategies for survival that can lead to human disease. Te essential elements required for a pathogen to establish infection are introduced and virulence mechanisms highlighted. It follows the immu­ nology chapters to stress that the host-parasite relationship is dynamic with adaptations and responses ofered by both host and parasite. Part VIII Chapter 36-Tis chapter has been updated to reflect the work­ flow and practice of a modern clinical laboratory. Emphasis is on modern diagnostic testing to identif infectious disease. Chapter 37-Expanded focus on the important role of labora­ tory safety especially in the teaching laboratory. Discussion em­ phasizes modern epidemiology as an investigative science and its role in preventative medicine. Disease prevention strategies are highlighted. Chapter 38-Updated and expanded coverage includes viral pathogenesis and common viral infections. Chapter 39-Expanded coverage of bacterial organisms and their common methods leading to human disease. Chapter 40-Refocused to reflect disease transmission routes as well as expanded coverage of fungal and protozoal diseases. Part IX Chapter 41-Expanded discussion of probiotics in the context of the human microbiome. Chapter 42-Tis chapter has been reorganized to illustrate the importance of industrial microbiology by presenting common microbial products-including biofuels-frst. Tis is followed by an updated discussion of strain development including in vivo and in vitro directed evolution. Chapter 43-Updated discussion of water purifcation wastewater treatment and bioremediation. Tis includes the development and use of microbial fel cells. xi

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Acknowledgments We would like to thank the Reviewers who provided constructive reviews of every chapter. Teir specialized knowledge helped us assimilate more reliable sources of information and fnd more efective ways of expressing an idea for the student reader. Reviewers Tamarah Adair Baylor University Richard Adler University of Michigan-Dearborn Fernando Agudelo-Silva College of Marin Shivanthi Anandan Drexel University Penny Antley University of Louisiana at Lafayette Suzanne Barth Te University of Texas at Austin Larry Barton University of New Mexico Nancy Boury Iowa State University Ginger Brininstool Louisiana State University-Baton Rouge Linda Bruslind Oregon State University Alison Buchan University of Tennessee Jim Buritt University of Wisconsin-Stout Martha Smith Caldas Kansas State University Joseph Caruso Florida Atlantic University-Boca Raton Andrei Chistoserdov University of Louisiana at Lafayette Carlton Cooper University of Delaware Susan Deines Colorado State University John Dennehy Queens College James Dickson Iowa State University Ronald Dubreuil University of Illinois at Chicago Paul Dunlap University of Michigan-Ann Arbor Mary Farone Middle Tennessee State University Babu Fathepure Oklahoma State University-Stillwater Kathy Feldman University of Connecticut Storrs Bernard Frye University of Texas Arlington Sandra Gibbons University of Illinois at Chicago Elizabeth Good University of Illinois at Urbana-Champaign Melanie Grifn Kennesaw State University Janet Haynes Long Island University Brooklyn Michael Ibba Te Ohio State University David Jenkins Ie University of Alabama Birmingham Dennis Kitz Southern Illinois University Edwardsville James Koukl Ie University of Texas at Tyler Shashi Kumar Saint Mary Mercy Hospital Jefrey Leblond Middle Tennessee State University Richard Long University of South Carolina Jean Lu Kennesaw State University xii Mark McBride University of Wisconsin-Milwaukee Vance McCracken Southern Illinois University Edwardsville Donald Mcgarey Kennesaw State University Robert McLean Texas State University Tamara Mcnealy Clemson University Rita Moyes Texas AM University Karen Nakaoka Weber State University Comer Patterson Texas AM University College Station Ed Perry Faulkner State Community College Tomas Pistole University of New Hampshire Ronald Porter Penn State University-University Park Jackie Reynolds Richland College Margaret Richey Centre College Veronica Riha Madonna University Timberley Roane University of Colorado Denver Jerry Sanders University of Michigan-Flint Pratibha Saxena Te University of Texas at Austin Mark Schneegurt Wichita State University Sasha A. Showsh University ofWisconsin-Eau Claire Khalifah Sidik University of Illinois College of Medicine at Rockford Deborah Siegele Texas AM University Jack Steiert Missouri State University Raji Subramanian NOVA Community College Annandale Karen Sullivan Louisiana State University-Baton Rouge Cristina Takacs-Vesbach University of New Mexico Monica Tischler Benedictine University Virginia Young Mercer University Jianmin Zhong Humboldt State University Te authors wish to extend their gratitude to our editors Kathy Lowenberg Kathleen Timp Angela FitzPatrick Sandy Wille and Lynn Breithaupt. We would also like to thank our photo editor Mary Reeg and the tremendous talent and patience displayed by the artists. We are also very grateful to the many reviewers who provided helpful criticism and analysis. Finally we thank our spouses and children who provided support and tolerated our absences mental if not physical while we completed this demanding project.

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Contents About the Authors iii 5.3 Cytoplasm of Eukaryotes 96 5.4 Organelles of the Secretory Preface iv and Endocytic Pathways 97 5.5 Organelles Involved in Genetic Control Part One Introduction to Microbiology of the Cell 101 0 5.6 Organelles Involved in Energy Conservation 103 The Evolution of Microorganisms 5.7 External Structures 104 and Microbiology 1 Microbial Diversity Ecology 5.1 1.1 Members of the Microbial World 1 There Was an Old Woman Who Swallowed a Fly 1 06 1.2 Microbial Evolution 4 5.8 Comparison of Bacterial Archaeal 1.3 Microbiology and Its Origins 11 and Eukaryotic Cells 108 1.4 Microbiology Today 17 G Viruses and Other Acellular Infectious Agents 112 Microscopy 22 6.1 Viruses 112 2.1 Lenses and the Bending of Light 22 6.2 Virion Structure 113 2.2 Light Microscopes 23 Microbial Diversity Ecology 6.1 2.3 Preparation and Staining of Specimens 31 Host-Independent Growth of an Archaeal Virus 114 2.4 Electron Microscopy 34 6.3 Viral Multiplication 119 2.5 Scanning Probe Microscopy 39 6.4 Types of Viral Infections 124 0 6.5 Cultivation and Enumeration of Viruses 127 Bacterial Cell Structure 42 6.6 Viroids and Satellites 129 3.1 The "Prokaryote" Controversy 42 6.7 Prions 130 3.2 A Typical Bacterial Cell 43 3.3 Bacterial Plasma Membranes 47 3.4 Bacterial Cell Walls 53 Part Two Microbial Nutrition Growth and Control Microbial Diversity Ecology 3.1 0 Microbial Growth Gram Positive and Gram Negative or 133 Monoderms and Diderms 54 7.1 Reproductive Strategies 133 3.5 Cell Envelope Layers Outside the Cell Wall 61 7.2 Bacterial Cell Cycle 134 3.6 Bacterial Cytoplasm 62 Microbial Diversity Ecology 7.1 3.7 External Structures 69 Cytokinesis Without FtsZ 137 3.8 Bacterial Motility and Chemotaxis 72 7.3 Influences of Environmental Factors 3.9 Bacterial Endospores 76 on Growth 141 0 7.4 Microbial Growth in Natural Environments 149 Archaeal Cell Structure 82 7.5 Laboratory Culture of Cellular Microbes 154 4.1 A Typical Archaeal Cell 82 7.6 Growth Curve: When One Becomes 4.2 Archaeal Cell Envelopes 84 Two and Two Become Four ... 160 4.3 Archaeal Cytoplasm 87 7.7 Measurement of Microbial Population Size 164 4.4 External Structures 88 7.8 Continuous Culture of Microorganisms 168 4.5 Comparison of Bacteria and Archaea 90 s s Control of Microorganisms in the Environment 172 Eukaryotic Cell Structure 92 8.1 Principles of Microbial Control 172 5.1 A Typical Eukaryotic Cell 92 8.2 The Pattern of Microbial Death 174 5.2 Eukaryotic Cell Envelopes 94 8.3 Mechanical Removal Methods 175 xiii

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8.4 Physical Control Methods 177 11.7 Anaerobic Respiration 247 8.5 Chemical Control Agents 180 11.8 Fermentation 248 8.6 Evaluation of Antimicrobial 11.9 Catabolism of Organic Molecules Other Agent Effectiveness 184 Than Glucose 251 8.7 Biological Control of Microorganisms 186 11.10 Chemolithotrophy 253 c Antimicrobial Chemotherapy Microbial Diversity Ecology 11.1 189 Acid Mine Drainage 255 9.1 The Development of Chemotherapy 189 11.11 Phototrophy 256 9.2 General Characteristics G 2 of Antimicrobial Drugs 190 Anabolism: The Use of Energy 9.3 Determining the Level in Biosynthesis 266 of Antimicrobial Activity 193 12.1 Principles Governing Biosynthesis 266 9.4 Antibacterial Drugs 195 12.2 Precursor Metabolites 268 9.5 Antifungal Drugs 201 12.3 C0 2 Fixation 269 9.6 Antiviral Drugs 203 12.4 Synthesis of Carbohydrates 272 9.7 Antiprotozoan Drugs 205 12.5 Synthesis of Amino Acids 274 9.8 Factors Influencing Antimicrobial 12.6 Synthesis of Purines Pyrimidines Drug Effectiveness 206 and Nucleotides 281 12.7 Lipid Synthesis 283 Part Three Microbial Metabolism 0 Introduction to Metabolism 210 10.1 Metabolism: Important Principles Part Four Microbial Molecular Biology and Genetics and Concepts 211 3 10.2 ATP: The Major Energy Currency of Cells 213 Bacterial Genome Replication 10.3 Redox Reactions: Reactions of Central and Expression 287 Importance in Metabolism 215 13.1 DNA as Genetic Material 288 10.4 Electron Transport Chains: Sets 13.2 Nucleic Acid and Protein Structure 288 of Sequential Redox Reactions 216 13.3 DNA Replication in Bacteria 293 10.5 Biochemical Pathways 219 13.4 Bacterial Gene Structure 301 10.6 Enzymes and Ribozymes 220 13.5 Transcription in Bacteria 304 10.7 Regulation of Metabolism 224 13.6 The Genetic Code 309 13.7 Translation in Bacteria 311 Catabolism: Energy Release and Conservation 230 13.8 Protein Maturation and Secretion 319 11.1 Metabolic Diversity 4 and Nutritional Types 230 Regulation of Bacterial Cellular Processes 325 11.2 Chemoorganotrophic Fueling Processes 232 14.1 Levels of Regulation 326 11.3 Aerobic Respiration 235 14.2 Regulation ofTranscription Initiation 326 11.4 From Glucose to Pyruvate 235 14.3 Regulation ofTranscription 11.5 Tricarboxylic Acid Cycle 239 Elongation 333 11 .6 Electron Transport and Oxidative 14.4 Regulation of Translation 336 Phosphorylation 239 14.5 Regulating Complex Cellular Processes 338 xiv

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Contents 5 Eukaryotic and Archaeal Genome Replication 18.5 Proteomics 437 and Expression 353 18.6 Systems Biology 440 15.1 Why Consider Eukaryotic and Archaeal 18.7 Comparative Genomics 440 Genetics Together 354 18.8 Metagenomics 443 15.2 DNA Replication 354 15.3 Transcription 358 Part Five The Diversity of the Microbial World 15.4 Translation and Protein Maturation and 9 Localization 363 Microbial Taxonomy and the Evolution 15.5 Regulation of Cellular Processes 367 of Diversity 447 6 19.1 Introduction to Microbial Taxonomy 448 Mechanisms of Genetic Variation 372 19.2 Taxonomic Ranks 449 16.1 Mutations 372 19.3 Exploring Microbial Taxonomy and Phylogeny 450 16.2 Detection and Isolation of Mutants 378 19.4 Phylogenetic Trees 456 16.3 DNA Repair 380 19.5 Evolutionary Processes and the Concept 16.4 Creating Additional Genetic Variability 383 of a Microbial Species 459 16.5 Transposable Elements 385 19.6 Bergeys Manual of Systematic Bacteriology 464 16.6 Bacterial Conjugation 387 Microbial Diversity Ecology 19.1 16.7 Bacterial Transformation 393 "Official" Nomenclature Lists- A Letter from Bergeys 465 16.8 Transduction 396 co TheArchaea 469 16.9 Evolution in Action: The Development of 20.1 Overview of the Archaea 470 Antibiotic Resistance in Bacteria 398 20.2 Phylum Crenarchaeota 476 7 Recombinant DNA Technology 404 20.3 Phylum Euryarchaeota 480 1 17 .1 Key Developments in Recombinant The Deinococci Mollicutes and DNA Technology 405 Nonproteobacterial Gram-Negative Bacteria 489 Techniques Applications 17.1 21.1 Aquificae and Thermotogae 490 Streptavidin-Biotin Binding and Biotechnology 410 21.2 Deinococcus-Thermus 490 17.2 Polymerase Chain Reaction 411 21.3 Class Mollicutes Phylum Tenericutes 491 17.3 Cloning Vectors and Creating 21.4 Photosynthetic Bacteria 494 Recombinant DNA 412 21.5 Phylum Planctomycetes 501 Techniques Applications 17.2 21.6 Phylum Chlamydiae 501 How to Build a Microorganism 416 21.7 Phylum Spirochaetes 504 17.4 Construction of Genomic Libraries 417 21.8 Phylum Bacteroidetes 506 17.5 Introducing Recombinant DNA 21.9 Phylum Verrucomicrobia 507 into Host Cells 418 17.6 Expressing Foreign Genes i2 The Proteobacteria 509 in Host Cells 419 22.1 Class Alphaproteobacteria 510 cs 22.2 Class Betaproteobacteria 518 Microbial Genomics 424 22.3 Class Gammaproteobacteria 522 18.1 Determining DNA Sequences 424 Microbial Diversity Ecology 22.1 18.2 Genome Sequencing 429 Bacterial Bioluminescence 530 18.3 Bioinformatics 431 22.4 Class Deltaproteobacteria 533 18.4 Functional Genomics 433 22.5 Class Epsilonproteobacteria 538 XV

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G 3 Firmicutes: The Low G + C Gram-Positive Bacteria 542 Part Six Ecology and Symbiosis 23.1 Class Clostridia 543 0 Biogeochemical Cycling and Global 23.2 Class Bacilli 547 Climate Change 632 4 28.1 Biogeochemical Cycling 633 Actinobacteria: The High G + C 28.2 Global Climate Change 642 Gram-Positive Bacteria 555 G 9 24.1 Order Actinomycetales 557 Methods in Microbial Ecology 646 24.2 Order Bifidobacteriales 566 29.1 Culturing Techniques 647 29.2 Assessing Microbial Diversity 651 65 The Protists 29.3 Assessing Microbial Community Activity 655 568 io 25.1 Overview of Protists 569 Microorganisms in Marine and Freshwater 25.2 Supergroup Excavata 571 Ecosystems 660 25.3 Supergroup Amoebozoa 573 30.1 Water as a Microbial Habitat 661 25.4 Supergroup Rhizaria 574 30.2 Microorganisms in Marine Ecosystems 662 25.5 Supergroup Chromalveolata 577 30.3 Microorganisms in Freshwater Ecosystems 672 25.6 Supergroup Archaeplastida 584 Microorganisms in Terrestrial Ecosystems 679 c6 31.1 Soils as a Microbial Habitat 680 The Fungi Eumycota 588 31.2 Microorganisms in the Soil Environment 683 26.1 Overview of Fungal Biology 590 31.3 Microbe-Plant Interactions 684 26.2 Chytridiomycota 593 31.4 The Subsurface Biosphere 696 26.3 Zygomycota 593 0 26.4 Glomeromycota 594 Microbial Interactions 699 26.5 Ascomycota 595 32.1 Microbial Interactions 700 26.6 Basidiomycota 598 Microbial Diversity Ecology 32.1 Disease 26.1 Wolbachia pipienris: The Worlds Most Infectious Microbe 701 White-Nose Syndrome Is Decimating North American Bat Populations 599 32.2 Human-Microbe Interactions 713 26.7 Microsporidia 601 Microbial Diversity Ecology 32.2 Do Bacteria Make People F at 714 c 7 Viruses 32.3 Normal Microbiota of the Human Body 715 604 27.1 Virus Taxonomy and Phylogeny 604 27.2 Double-Stranded DNA Viruses 606 Part Seven Pathogenicity and Host Response Microbial Diversity Ecology 27.1 3 Innate Host Resistance What Is a Virus 617 723 27.3 Single-Stranded DNA Viruses 617 33.1 Innate Resistance Overview 724 27.4 RNA Viruses: Unity Amidst Diversity 619 33.2 Physical and Mechanical Barrier 27.5 Double-Stranded RNA Viruses 620 Defenses of Innate Resistance 725 27.6 Plus-Strand RNA Viruses 622 33.3 Chemical Mediators in Innate Resistance 728 27.7 Minus-Strand RNA Viruses 624 33.4 Cells Tissues and Organs 27.8 Retroviruses 626 of the Immune System 735 27.9 Reverse Transcribing DNA Viruses 33.5 Phagocytosis 743 628 33.6 Inflammation 748 xvi

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Contents G 4 37.3 Measuring Infectious Disease Frequency 835 Adaptive Immunity 753 37.4 Patterns of Infectious Disease 34.1 Overview of Adaptive Immunity 753 in a Population 836 34.2 Antigens 755 Historical Highlights 37.4 34.3 Types of Adaptive Immunity 756 "Typhoid Mary" 837 34.4 Recognition of Foreignness 757 37.5 Emerging and Reemerging Infectious 34.5 T-Cell Biology 760 Diseases and Pathogens 839 34.6 B-Cell Biology 764 37.6 Health-Care-Associated Infections 841 34.7 Antibodies 767 37.7 Prevention and Control of Epidemics 843 Techniques Applications 34.1 Historical Highlights 37.5 Monoclonal Antibody Therapy 776 The First Immunizations 846 34.8 Action of Antibodies 777 37.8 Bioterrorism Preparedness 848 34.9 Acquired Immune Tolerance 778 Historical Highlights 37.6 34.10 Immune Disorders 779 1346- The First R ecorded Biological Warfare Attack 849 G 5 Pathogenicity and Infection 789 8 35.1 Pathogenicity and Infectious Disease 790 Human Diseases Caused by Viruses 35.2 Viru lence 793 and Prions 854 35.3 Exposure and Transmission 802 38.1 Airborne Diseases 855 Historical Highlights 35.1 38.2 Arthropod-Borne Diseases 865 The First Indications of P erson-to-Person 38.3 Direct Contact Diseases 865 Spread of an Infectious Disease 803 38.4 Food-Borne and Waterborne Diseases 878 Historical Highlights 38.1 A Brief History of Polio 881 Part Eight Microbial Diseases Detection 38.5 Zoonotic Diseases 881 and Their Control Disease 38.2 6 Clinical Microbiology and Immunology 808 Viral Hemorrhagic F evers: A Microbial History Lesson 882 36.1 Overview of the Clinical Microbiology 38.6 Prion Diseases 885 Laboratory 808 36.2 Biosafet y 809 9 Human Diseases Caused by Bacteria 888 36.3 Identification of Microorganisms from Specimens 812 39.1 Airborne Diseases 888 39.2 Arthropod-Borne Diseases 898 36.4 Clinical Immunology 820 39.3 Direct Contact Diseases 901 f 7 Epidemiology and Public Health Microbiology 830 Disease 39.1 37.1 Epidemiology 830 A Brief History of S yphilis 909 Historical Highlights 37.1 Disease 39.2 The Birth of Public Health in the Biofilms 910 United S tates 831 39.4 Food-Borne and Waterborne Diseases 915 Historical Highlights 37.2 Techniques Applications 39.3 John Snow the First Epidemiologist 832 Clostridial Toxins as Therapeutic Agents: 37.2 Epidemiological Methods 832 B enefits of Natures Most T oxic Proteins 91 9 Historical Highlights 37.3 39.5 Zoonotic Diseases 924 SAR S : E volution of a Virus 833 39.6 Opportunistic Diseases 926 xvii

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0 Human Diseases Caused by Fungi and Protists 932 2 Biotechnology and Industrial Microbiology 979 40.1 Pathogenic Fungi and Protists 932 42.1 Major Products of Industrial Microbiology 980 40.2 Airborne Diseases 934 42.2 Biofuel Production 982 40.3 Arthropod-Borne Diseases 937 42.3 Growing Microbes in Industrial Settings 983 Disease 40.1 42.4 Microorganisms Used in A Brief History of Malaria 938 Industrial Microbiology 985 40.4 Direct Contact Diseases 944 42.5 Agricultural Biotechnology 990 40.5 Food-Borne and 42.6 Microbes as Products 992 Waterborne Diseases 948 3 40.6 Opportunistic Diseases 952 Applied Environmental Microbiology 996 43.1 Water Purification and Sanitary Analysis 996 Techniques Applications 43.1 Part Nine Applied Microbiology Waterborne Diseases Water Supplies 1 and Slow Sand Filtration 999 Microbiology of Food 958 43.2 Wastewater Treatment 1001 41.1 Microbial Growth and Food Spoilage 959 43.3 Microbial Fuel Cells 1008 41.2 Controlling Food Spoilage 961 43.4 Biodegradation and Bioremediation 1009 41.3 Food-Borne Disease Outbreaks 964 41.4 Detection of Food-Borne Pathogens 967 Appendix 1 A Review of the Chemistry 41.5 Microbiology of Fermented Foods 969 of Biological Molecules A-1 Techniques Applications 41.1 Appendix 2 Common Metabolic Pathways A-9 Chocolate: The Sweet Side of Fermentation 970 41.6 Probiotics 976 Glossary G-1 Credits C-1 Index 1-1 xviii

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About the Authors iii Preface iv Part One Introduction to Microbiology 1 The Evolution of Microorganisms and Microbiology 2 Microscopy 22 3 Bacterial Cell Structure 42 4 Archaeal Cell Structure 82 5 Eukaryotic Cell Structure 92 6 Viruses and Other Acellular Infectious Agents 112 Part Two Microbial Nutrition Growth and Control 7 Microbial Growth 133 8 Control of Microorganisms in the Environment 172 9 Antimicrobial Chemotherapy 189 Part Three Microbial Metabolism 10 Introduction to Metabolism 210 11 Catabolism: Energy Release and Conservation 230 12 Anabolism: The Use of Energy in Biosynthesis 266 Part Four Microbial Molecular Biology and Genetics 13 Bacterial Genome Replication and Expression 287 14 Regulation of Bacterial Cellular Processes 325 15 Eukaryotic and Archaeal Genome Replication and Expression 353 16 Mechanisms of Genetic Variation 372 17 Recombinant DNA Technology 404 18 Microbial Genomics 424 Part Five The Diversity of the Microbial World 19 Microbial Taxonomy and the Evolution of Diversity 447 20 The Archaea 469 21 The Deinococci Mollicutes and Nonproteobacterial Gram-Negative Bacteria 489 22 The Proteobacteria 509 23 Firmicutes: The Low G + C Gram-Positive Bacteria 542 Brief Contents 24 Actinobacteria: The High G + C Gram-Positive Bacteria 555 25 The Protists 568 26 The Fungi Eumycota 588 27 Viruses 604 Part Six Ecology and Symbiosis 28 Biogeochemical Cycling and Global Climate Change 632 29 Methods in Microbial Ecology 646 30 Microorganisms in Marine and Freshwater Ecosystems 660 31 Microorganisms in Terrestrial Ecosystems 679 32 Microbiallnteractions 699 Part Seven Pathogenicity and Host Response 33 Innate Host Resistance 723 34 Adaptive Immunity 753 35 Pathogenicity and Infection 789 Part Eight Microbial Diseases Detection and Their Control 36 Clinical Microbiology and Immunology 808 37 Epidemiology and Public Health Microbiology 38 Human Diseases Caused by Viruses and Prions 39 Human Diseases Caused by Bacteria 888 40 Human Diseases Caused by Fungi and Protists Part Nine Applied Microbiology 41 Microbiology of Food 958 830 854 932 42 Biotechnology and Industrial Microbiology 979 43 Applied Environmental Microbiology 996 Appendix 1 A Review of the Chemistry of Biological Molecules A-1 Appendix 2 Common Metabolic Pathways A-9 Glossary G-1 Credits C -1 Index 1-1

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1 The Evolution of Microorganisms and Microbiology Over 2000 Potential Planets Discovered I n February 2012 the National Aeronautics and Space Administration NASA reported that over 2000 potential planets had been discovered by the 2009 Kepler mission. Using a telescope in space the light emanating from stars as far as 3000 light-years away had been monitored every half-hour. The Keplertelescope identifed planets as they circulated their star and caused a brief decrease in emitted light just as an object is detected as a blip by radar a blip of "darkness" indicates a planet. Unless you are a science fction fan you might wonder why NASA is interested in fnding planets. By fnding other planets scientists can gather evidence to support or refute current models of planet formation. These models predict a process that is chaotic and violent. Planets are thought to begin as dust particles circling around newly formed stars. As these particles collide they grow in size forming larger chunks. Eventually a series of such collisions results in planet-sized bodies. Astrobiologists are interested in identifying characteristics of a planet that may allow it to support life. Using Earth as a model they hypothesize that life-supporting planets will share many features with Earth. But how will life be recog­ nized Again scientists look to life on Earth to answer this question and increasingly they are turning to microbiologists for help. Earth formed 4.5 billion years ago. Within the next billion years the frst cellular life forms-microbes-appeared. Since that time microorgan­ isms have evolved and diversifed to occupy virtually every habitat on Earth: from oceanic geothermal vents to the coldest Arctic ice. The diversity of cellular microorganisms is best exemplifed by their metabolic capabilities. Some carry out respiration just as animals do. Others perform photosynthe­ sis rivaling plants in the amount of carbon dioxide they capture forming organic matter and releasing oxygen into the atmosphere. Indeed Prochlorococcus a cyanobacterium formerly called a blue-green alga is thought to be the most abundant photosynthetic organism on Earth and Artists rendition of the six planets orbiting a star called Kepler-11. The drawing is based on observations made of the system by the Kepler spacecraft on August 262010. Some are Earth-sized and may be habitable by lfe. thus a major contributor to the functioning of the biosphere. In addition to these familiar types of metabolism other microbes are able to use inorganic molecules as sources of energy in both oxic oxygen available and anoxic no oxygen conditions. It is these microbes that are of particular interest to NASA scientists as it is thought that the organisms on other planets may have similar unusual metabolisms. Our goal in this chapter is to introduce you to this amazing group of organisms and to outline the history of their evolution and discovery. Microbiology is a biological science and as such much of what you will learn in this text is similar to what you have learned in high school and college biology classes that focus on large organisms. But microbes have unique properties so microbiology has unique approaches to understanding them. These too will be introduced. But before you delve into this chapter check to see if you have the background needed to get the most from it. Readiness Check: Based on what you have learned previously you should be able to: t List the features of eukaryotic cells that distinguish them from other cell types t List the attributes that scientists use to determine if an object is alive 1.1 Members of the Microbial World After reading this section you should be able to: • Diferentiate the biological entities studied by microbiologists from those studied by other biologists • Explain Carl Woeses contributions in establishing the three domain system for classifying cellular life • Provide an example of the importance to humans of each of the major types of microbes • Determine the type of microbe e.g. bacterium fungus etc. when given a description of a newly discovered microbe

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2 CHAPTER 1 I The Evolution of Microorganisms and Microbiology Organisms and biological entities studied by microbiologists includes e.g. e.g. e.g. • • • Yeasts Algae Escherichia Molds Protozoa col Slime molds e.g. • Methanogens I can be composed of includes composed of composed of composed of ckJ Figure 1.1 Concept Map Showing the Types of Biological Entities Studied by Microbiologists. M 1 C RO IN Q u 1 RY How would you alter this concept map so that it also distinguishes the cellular organisms from each other Microorganisms are defned as those organisms and acellular biological entities too small to be seen clearly by the unaided eye fgure 1.1 . Tey are generally 1 millimeter or less in diam­ eter. Although small size is an important characteristic of mi­ crobes it alone is not sufcient to defne them. Some cellular microbes such as bread molds and flamentous photosynthetic microbes are actually visible without microscopes. Tese mac­ roscopic microbes are ofen colonial consisting of small aggre­ gations of cells. Some macroscopic microorganisms are multicellular. Tey are disting uished from other multicellular life forms such as plants and animals by their lack of highly dif­ ferentiated tissues. Most unicellular microbes are microscopic. However there are interesting exceptions as we describe in chapter 3. In summary cellular microbes are usually smaller than 1 millimeter in diameter ofen unicellular and if multi­ cellular lack diferentiated tissues. Te diversity of microorganisms has always presented a challenge to microbial taxonomists. Te early descriptions of cellular microbes as either plants or animals were too simple. For instance some microbes are motile like animals but also have cell walls and are photosynthetic like plants. Such mi­ crobes cannot be placed easily into either kingdom. An im­ portant breakthrough in microbial taxonomy arose from studies of their cellular architecture when it was discovered that cells exhibited one of two possible "foor plans." Cells that came to be called prokaryotic cells Greek pro before and karyon nut or kernel organisms with a primordial nucleus have an open foor plan. Tat is their contents are not divided into compartments "rooms" by membranes "walls". Te most obvious characteristic of these cells is that they lack the membrane-delimited nucleus observed in eukaryotic cells Greek eu true and karyon nut or kernel. Eukaryotic cells not only have a nucleus but also many other membrane-bound organelles that separate some cellular materials and processes from others. Tese observations eventually led to the development of a classifcation scheme that divided organisms into fve kingdoms: Monera Protista Fungi Animalia and Plantae. Microorganisms except for viruses and other acellular infectious agents which have their own classifcation system were placed in the frst three kingdoms. In this scheme all organisms with prokaryotic cell structure were placed in Monera. Te fve-kingdom system was an important development in microbial taxonomy but it is no longer accepted by microbiologists. Tis is because not all "prokaryotes" are the same and therefore should not be grouped together in a single kingdom. Furthermore it is currently argued that the term prokaryote is not meaningful and should be abandoned. As we describe next this discovery required several advances in the tools used to study microbes. �I Te rokaryote" controversy section 3.1 Great progress has been made in three areas that profoundly afect microbial classifcation. First much has been learned about the detailed structure of microbial cells from the use of electron microscopy. Second microbiologists have determined the biochemical and physiological characteristics of many dif­ ferent microorganisms. Tird the sequences of nucleic acids and

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proteins from a wide variety of organisms have been compared. Te comparison of ribosomal RNA rRNA begun by Carl Woese in the 1970s was instrumental in demonstrating that there are two very diferent groups of organisms with prokary­ otic cell architecture: Bacteria and Archaea. Later studies based on rRNA comparisons showed that Protista is not a cohesive taxonomic unit i.e. taxon and that it should be divided into three or more kingdoms. Tese studies and others have led many taxonomists to reject the fve-kingdom system in favor of one that divides cellular organisms into three domains: Bacteria sometimes referred to as true bacteria or eubacteria Archaea sometimes called archaeobacteria or archaebacteria and Eukar y a all eukaryotic organisms fgure 1.2 . We use this system throughout the text. A brief description of the three domains and of the microorganisms placed in them follows. �I Nucleic acids appendix I Proteins appendix I Members of domain Bacteria are usually single-celled or­ ganisms.1 Most have cell walls that contain the structural mol­ ecule peptidoglycan. Although most bacteria exhibit typical prokaryotic cell structure i.e. they lack a membrane-bound nucleus a few members of the unusual phylum Planctom y cetes have their genetic material surrounded by a membrane. Tis inconsistency is another argument made for abandoning the term "prokaryote." Bacteria are abundant in soil water and air including sites that have extreme temperatures pH or sa­ linity. Bacteria are also major inhabitants of our skin mouth and intestines. Indeed more microbial cells are found in and on the human body than there are human cells. Tese microbes begin to colonize humans shortly afer birth. As the microbes establish themselves they contribute to the development of the bodys immune system. Tose microbes that inhabit the large intestine help the body digest food and produce vitamins. In these and other ways microbes help maintain the health and well-being of their human hosts. �I Ph y lum Planctom y cetes section 21.5 Unfortunately some bacteria cause disease and some of these diseases have had a huge impact on human history. In 1347 the plague Black Death an arthropod-borne disease struck Europe with brutal force killing one-third of the population about 25 million people within four years. Over the next 80 years the disease struck repeatedly eventually wiping out 75 of the European population. Te plagues efect was so great that some historians believe it changed European culture and prepared the way for the Renaissance. Because of such plagues it is easy for people to think that all bacteria are patho­ gens but in fact relatively few are. Most play benefcial roles from global impact to maintaining human health. Tey break down dead plant and animal material and in doing so cycle elements in the biosphere. Furthermore they are used exten­ sively in industry to make bread cheese antibiotics vitamins enzymes and other products. 1.1 Members of the Microbial World 3 1- rRNA sequence change � Unrsolved brnching orer Figure 1.2 Universal Phylogenetic Tree. These evolutionary relationships are based on rRNA sequence comparisons. To save space many lineages have not been identifed. MICRO 1 N Q u 1 RY How many of the taxa listed in the fgure include microbes Members of domain Archaea are distinguished from bacte­ ria by many features most notably their distinctive rRNA sequences lack of peptidoglycan in their cell walls and unique membrane lipids. Some have unusual metabolic characteristics such as the methanogens which generate methane natural gas. Many archaea are found in extreme environments including those with high temperatures thermophiles and high concen­ trations of salt extreme halophiles. Although some archaea are members of a community of microbes involved in gum disease in humans their role in causing disease has not been clearly established. Domain Eukar y a includes microorganisms classifed as protists or fungi. Animals and plants are also placed in this domain. Protists are generally unicellular but larger than most bacteria and archaea. Tey have traditionally been di­ vided into protozoa and algae. Despite their use none of these terms has taxonomic value as protists algae and protozoa do 1 In this text the term bacteria s. bacterium is used to refer to those microbes belonging to domain Bacteria and the term archaea s. archaean is used to refer to those that belong to domain Archaea. In some publications the term bacteria is used to refer to all cells having prokaryotic cell structure. That is not the case in this text.

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4 CHAPTER 1 I The Evolution of Microorganisms and Microbiology not form cohesive taxa. However for convenience we use them here. Te major types of protists are algae protozoa slime molds and water molds. Algae are photosynthetic. Tey together with cyanobacteria produce about 75 of the planets oxygen and are the foundation of aquatic food chains. Protozoa are unicellular animal-like protists that are usually motile. Many free-living protozoa function as the principal hunters and grazers of the microbial world. Tey obtain nutrients by ingesting organic matter and other microbes. Tey can be found in many diferent environments and some are normal inhabitants of the intestinal tracts of animals where they aid in digestion of complex materi­ als such as cellulose. A few cause disease in humans and other animals. Slime molds are protists that behave like protozoa in one stage of their life cycle but like fungi in another. In the pro­ tozoan phase they hunt for and engulf food particles consum­ ing decaying vegetation and other microbes. Water molds are protists that grow on the surface of freshwater and moist soil. Tey feed on decaying vegetation such as logs and mulch. Some water molds have produced devastating plant infections includ­ ing the Great Potato Famine of 1846-1847 in Ireland .. I Te protists chapter 25 Fungi are a diverse group of microorganisms that range from unicellular forms yeasts to molds and mushrooms. Molds and mushrooms are multicellular fungi that form thin thread­ like structures called hyphae. Tey absorb nutrients from their environment including the organic molecules they use as sources of carbon and energy. Because of their metabolic capa­ bilities many fungi play benefcial roles including making bread rise producing antibiotics and decomposing dead organ­ isms. Some fungi associate with plant roots to form mycorrhi­ zae. Mycorrhizal fungi transfer nutrients to the roots improving growth of the plants especially in poor soils. Other fungi cause plant diseases e.g. rusts powdery mildews and smuts and diseases in humans and other animals. .I Te Fungi chapter 26 Te microbial world also includes numerous acellular infec­ tious agents. Viruses are acellular entities that must invade a host cell to multiply. The simplest viruses are composed only of proteins and a nucleic acid and can be extremely small the smallest is 10000 times smaller than a typical bacterium. How­ ever their small size belies their power: they cause many animal and plant diseases and have caused epidemics that have shaped human history. Viral diseases include smallpox rabies infu­ enza AIDS the common cold and some cancers. Viruses also play important roles in aquatic environments and their role in shaping aquatic microbial communities is currently being ex­ plored. Viroids and satellites are infectious agents composed only of ribonucleic acid RNA. Viroids cause numerous plant diseases whereas satellites cause plant diseases and some im­ portant animal diseases such as hepatitis. Finally prions infec­ tious agents composed only of protein are responsible for causing a variety of spongiform encephalopathies such as scra­ pie and "mad cow disease." . I Viruses and other acellular in­ fectious agents chapter 6 Retrieve Infer Apply 1. How did the methods used to classify microbes change particularly in the last half of the twentieth century What was the result of these technological advances 2. Identify one characteristic for each of these types of microbes that distinguishes it from the other types: bacteria archaea protists fungi viruses viroids satellites and prions. 1.2 Microbial Evolution After reading this section you should be able to: • Propose a time line of the origin and history of microbial life and integrate supporting evidence into it • Design a set of experiments that could be used to place a newly discovered cellular microbe on a phylogenetic tree based on small subunit SSU rRNA sequences • Compare and contrast the defnitions of plant and animal species microbial species and microbial strains A review of fgure 1.2 reminds us that in terms of the number of taxa microbes are the dominant organisms on Earth. How has microbial life been able to radiate to such an astonishing level of diversity To answer this question we must consider microbial evolution. Te feld of microbial evolution like any other scien­ tifc endeavor is based on the formulation of hypotheses the gathering and analysis of data and the reformation of hypotheses based on newly acquired evidence. Tat is to say the study of microbial evolution is based on the scientifc method see ww .mhhe.com/willey. To be sure it is sometimes more difcult to amass evidence when considering events that occurred millions and ofen billions of years ago but the advent of molecular meth­ ods has ofered scientists a living record of lifes ancient history. Tis section describes the outcome of this scientifc research. Evidence for the Origin of Life Dating meteorites through the use of radioisotopes places our planet at an estimated 4.5 to 4.6 billion years old. However con­ ditions on Earth for the frst 100 million years or so were far too harsh to sustain any type of life. Eventually bombardment by meteorites decreased water appeared on the planet in liquid form and gases were released by geological activity to form Earths atmosphere. Tese conditions were amenable to the ori­ gin of the frst life forms. But how did this occur and what did these life forms look like Clearly in order to fnd evidence of life and to develop hypotheses about its origin and subsequent evolution scien­ tists must be able to defne life. Although even very young children can examine an object and correctly determine whether it is living or not defning life succinctly has proven elusive for scientists. Tus most defnitions of life consist of a set of attributes. Te attributes of particular importance to paleobiologists are an orderly structure the ability to obtain

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and use energy i.e. metabolism and the ability to reproduce. Just as NASA scientists are using the characteristics of mi­ crobes on Earth today to search for life elsewhere p. 1 so too are scientists examining extant organisms those organisms present today to explore the origin of life. Some extant organ­ isms have structures and molecules that represent "relics" of ancient life forms. Furthermore they can provide scientists with ideas about the type of evidence to seek when testing hypotheses. The frst direct evidence of primitive cellular life was the 1977 discovery of microbial fossils in the Swartkoppie chert. Chert is a type of granular sedimentary rock rich in silica. The Swartkoppie chert fossils as well as those from the Archaean Apex chert of Australia have been dated at about 3.5 billion years old fgures 1.3 and 1.4. Despite these fndings the mi­ crobial fossil record is understandably sparse. Thus to piece to­ gether the very early events that led to the origin of life biologists must rely primarily on indirect evidence. Each piece of evidence must ft together as in a jigsaw puzzle for a coherent picture to emerge. RNA World The origin of life rests on a single question: How did early cells arise At a minimum modern cells consist of a plasma membrane enclosing water in which numer­ ous chemicals are dissolved and subcellular structures foat. I seems likely that the frst self- replicating entity was much sim­ pler than even the most primitive modern living cells. Before there was life most evidence suggests that Earth was a very diferent place: hot and anoxic with an atmosphere rich in water vapor carbon di- oxide and nitrogen. In the oceans hydrogen methane and carboxylic acids were formed by geological and chemical processes. Areas near hydrothermal vents or in shallow pools may have provided the conditions that allowed chemicals to react with one another randomly "testing" the usefulness of the reaction and the stabili ty of its products. Some reac- tions released energy and would eventually become the basis of modern cellular Figure 1.3 Microfossils of the Archaeon Apex Chert of Australia. These microfossils are similar to modern flamentous cyanobacteria. 1.2 Microbial Evolution 5 metabolism. Other reactions generated molecules that could function as catalysts some aggregated with other molecules to form the predecessors of modern cell structures and others were able to replicate and act as units of hereditary information. In modern cells three diferent molecules fulfll the roles of catalysts structural molecules and hereditary molecules fgure 1.5. Proteins have two major roles in modern cells: structural and catalytic. Catalytic proteins are called enzymes and they speed up the myriad of chemical reactions that occur in cells. DNA stores hereditary information and can be repli­ cated to pass the information on to the next generation. RNA is involved in converting the information stored in DNA into pro­ tein. Any hypothesis about the origin of life must account for the evolution of these molecules but the very nature of their relationships to each other in modern cells complicates attempts to imagine how they evolved. As demonstrated in fgure 1.5 proteins can do cellular work but their synthesis in­ volves other proteins and RNA and uses information stored in DNA. DNA cant do cellular work. It stores genetic information and serves as the template for its own replication a process that requires proteins. RNA is synthesized using DNA as the tem­ plate and proteins as the catalysts for the reaction. Based on these considerations it is hypothesized that at some time in the evolution of life there must have been a single molecule that could do both cellular work and replicate itself. A possible molecule was suggested in 1981 when Thomas Cech discovered a catalytic RNA molecule in a protist Tetrhymena sp. that could cut out an interal section of itself and splice the re­ maining sections back together. Since then other catalytic RNA molecules have been discovered including an RNA found in ribosomes that is responsible for forming peptide bonds­ the bonds that hold together amino acids the building blocks of proteins. Catalytic RNA molecules are now called ribozymes. The discovery of ribozymes suggested that RNA at some time had the ability to catalyze its own replication using itself as the template. In 1986 Walter Gilbert coined the term RNA world to describe a precellular stage in the evolution of life in which RNA was capable of storing copying and expressing genetic information as well as catalyzing other chemical reac­ tions. However for this precellular stage to proceed to the evo­ lution of cellular life forms a lipid membrane must have formed around the RNA fgure 1.6. This important evolutionary step is easier to imagine than other events in the origin of cellular life forms because lipids major structural components of the membranes of modern organisms spontaneously form liposomes-vesicles bounded by a lipid bilayer. A fascinating experiment performed by Marin Hanczyc Shelly Fujikawa and Jack Szostak in 2003 showed that clay triggers the forma­ tion of liposomes that actually grow and divide. Together with the data on ribozymes these data suggest that early cells may have been liposomes containing RNA molecules fgure 1.6. �I Lipids appendix I Apart from its ability to perform catalytic activities the function of RNA suggests its ancient origin. Consider that much of the cellular pool of RNA in modern cells exists in the

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6 CHAPTER 1 I The Evolution of Microorganisms and Microbiology Millions of Years Ago 0 1.8 65 144 206 248 290 354 417 443 490 543 900 1600 2500 3000 3400 3800 4550 � a Il � w a 0. U U c: f 0 w w u i N 0 z w u u i u N 0 i e N w 0 : a w z : 0. u i N 0 w . i �w : w . u c i c N : 0 a w f 0 a 0. � a w - w . z w w : . c u �i a � a w �z w c U 0 · Il D Quaternary Teriary Cretaceous Jurassic Triassic Permian Carboniferous Devonian Silurian Ordovician Cambrian + 7 mya-Hominids first appear. + 225 mya-Dinosaurs and mammals first appear. + 300 mya-Reptiles first appear. + 450 mya-Large terrestrial colonization by plants and animals. �1 520 mya-First verebrates first land plants. 533-525 mya-ambrian explosion creates diverse animal life. + 1.5 bya--ulticellular eukaryotic organisms first appear. I + 2.5-2.0 bya-Eukaryotic cells first appear. I + 3.5 bya-Fossils of primitive filamentous microbes. + 3.8-3.5 bya-First cells appear. Figure 1.4 An Overview ofthe History of Life on Earth. mya million years ago bya billion years ago.

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Serves as template for synthesis of new Encodes sequence of nucleotides in DNA RNA Catalyzes synthesis of Regulates expression of Functions in Catalyzes synthesis of synthesis of Forms Encodes sequence of amino acids in Protein Catalyzes Figure 1.5 Functions of DNA RNA and Protein and Their Relationships to Each Other in Modern Cells. Involved in synthesis of more ribosome a structure that consists largely of rRNA and uses messenger RNA mRNA and transfer RNA tRNA to construct proteins. Also recall that rRNA itself catalyzes peptide bond for­ mation during protein synthesis. Tus RNA seems to be well poised for its importance in the development of proteins. Be­ cause RNA and DNA are structurally similar RNA could have given rise to double-stranded DNA. It is suggested that once DNA evolved it became the storage facility for genetic informa­ tion because it provided a more chemically stable structure. Two other pieces of evidence support the RNA world hypothesis: the fact that the energy currency of the cell ATP is a ribonucleotide and the more recent discovery that RNA can regulate gene ex­ pression. So it would seem that proteins DNA and cellular en­ ergy can be traced back to RNA. �I ATP section 10.2 Riboswitches sections 14.3 and 14.4 Despite the evidence supporting the hypothesis of an RNA world it is not without problems and many argue against it. Another area of research is also fraught with considerable de- 1.2 Microbial Evolution 7 E e E E E E Prebiotic soup E E � Liposome \\RNA Probiont: RNA only Probiont: RNA and proteins Cellular life: RNA DNA and proteins Figure 1.6 The RNA World Hypothesis for the Origin of Life. MIcRO INQUIRY Why are the prbionts pictured above not considered cellular life bate: the evolution of metabolism in particular the evolution of energy-conserving metabolic processes. Recall that early Earth was a hot environment that lacked oxygen. Tus the cells that arose there must have been able to use the available energy sources under these harsh conditions. Today there are heat-loving archaea capable of using inorganic molecules such as FeS as a source of energy. Some suggest that this interesting metabolic capability is a remnant of the frst form of energy metabolism. Another metabolic strategy oxygen-releasing photosynthesis appears to have evolved perhaps as early as 2.5 billion years ago. Fossils of cyanobacteria­ like cels found in rocks dating to that time support this hypothesis

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8 CHAPTER 1 I The Evolution of Microorganisms and Microbiology a b Figure 1.7 Stromatolites. a Section of a fossilized stromatolite. Evolutionary biologists think the layers of material were formed when mats of cyanobacteria layered one on top of each other became mineralized. b Modern stromatolites from Western Australia. Each stromatolite is a rocklike structure typically 1 min diameter containing layers of cyanobacteria. as does the discovery of ancient stromatolites fgure 1.7a. Stro­ matolites are layered rocks ofen domed that are formed by the incorporation of mineral sediments into layers of microorganisms growing as thick mats on surfaces fgure 1.7b. Te appearance of cyanobacteria-like cells was an important step in the evolution of life on Earth. Te oxygen they released is thought to have altered Earths atmosphere to its current oxygen-rich state allowing the evolution of additional energy-capturing strategies such as aerobic respiration the oxygen-consuming metabolic process that is used by many microbes and animals. Evolution ofthe Three Domains of Life As noted in section 1.1 rRNA comparisons were an important breakthrough in the classifcation of microbes this analysis also provides insights into the evolutionary history of all life. What began with the examination of rRNA from relatively few organisms has been expanded by the work of many others including Nor­ man Pace. Dr. Pace has developed a universal phylogenetic tree fgure 1.2 based on comparisons of small subunit rRNA mole­ cules SSU rRNA the rRNA found in the small subunit of the ribosome. Here we examine how these comparisons are made and what the universal phylogenetic tree tells us. 111 Bacterial ribosomes section 3.6 Exploring microbial tax­ onomy and phylogeny section 19.3 Comparing SSU rRNA Molecules Te details of phylogenetic tree construction are discussed in chapter 19. However the general concept is not difcult to under­ stand. In one approach the sequences of nucleotides in the genes that encode SSU rRNAs from diverse organisms are aligned and pair-wise comparisons of the sequences are made. For each pair of SSU rRNA gene sequences the number of diferences in the nucleotide sequences is counted fgure 1.8. Tis value serves as a measure of the evolutionary distance between the organisms the more diferences counted the greater the evolutionary dis- tance. Te evolutionary distances from many comparisons are used by sophisticated computer programs to construct the tree. Each branch in the tree represents one of the organisms used in the comparison. Te distance from the tip of one branch to the tip of another is the evolutionary distance between the two organ­ isms represented by the branches. Two things should be kept in mind when examining phylogenetic trees developed in this way. Te frst is that they are molecular trees not organismal trees. In other words they rep­ resent as accurately as possible the evolutionary history of a molecule and the gene that encodes it. Second the distance be­ tween branch tips is a measure of relatedness not of time. If the distance along the lines is very long then the two organisms are more evolutionarily diverged i.e. less related. However we do not know when they diverged from each other. Tis concept is analogous to a map that accurately shows the distance between two cities but because of many factors trafc road conditions etc. cannot show the time needed to travel that distance. LUCA What does the universal phylogenetic tree tell us about the evo­ lution of life At the center of the tree is a line labeled "Origin" fgure 1.2. Tis is where the data indicate the last universal common ancestor LUCA to all three domains should be placed. LUCA is on the bacterial branch which means that Archaea and Eukarya evolved independently separate from Bacteria. Tus the universal phylogenetic tree presents a picture in which all life regardless of eventual domain arose from a single common ancestor. One can envision the universal tree of life as a real tree that grows from a single seed. Te evolutionary relationship of Archaea and Eukarya is still the matter of considerable debate. According to the univer­ sal phylogenetic tree we show here Archae a and Eukarya shared common ancestry but diverged and became separate domains. Other versions suggest that Eukarya evolved out of Archaea. Te

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Cells from organism 1 Lyse cells to release contents and isolate DNA. � DNA I Use polymerase chain reaction to amplify • and purify SSU rRNA genes. � SSU rRNA genes + Sequence genes. ATGCTCAAGTCA + Repeat process for other organisms. + Align sequences to be compared. Organism SSU rRNA sequence ATGCTCAAGTCA TAGCTCG TGTAA AAGCTCTAGTA AACCTCAT GTA 1 - 3 4 Count the number of nucleotide diferences between each pair of sequences and calculate evolutionary distance E0. For organisms 1 and 2 5 of the 12 �.....� nucleotides are diferent: Pair compar 1-2 1-3 1-4 2-3 2-4 3-4 0.44 0.33 0.44 0.33 0.44 0.25 0.30 E0 5/12 0.42 . The initial ED calculated is corrected using a statistical method that considers for each site the prbability of a mutaion back to the original nucleotide or of additional forard mutations. I Feed data into computer and use appropriate sofware T to construct phylogenetic tree. 3 Unrooted phylogenetic tree. Note that distance from one tip to another is proporional to the E0. 0.08 I :: ::: � 08 0 23 : o.3o :: 2 4 Figure 1.8 The Construction of Phylogenetic Trees Using a Distance Method. MICRO INQuIRY Why does the brnch length indicate amount of evolutionary change but not the time it took for that change to occur 1.2 Microbial Evolution 9 close evolutionary relationship of these two forms of life is still evident in the manner in which they process genetic informa­ tion. For instance certain protein subunits of archaeal and eu­ karyotic RNA polymerases the enzymes that catalyze RNA synthesis resemble each other to the exclusion of those of bacte­ ria. However archaea have other features that are most similar to their counterparts in bacteria e.g. mechanisms for conserv­ ing energy. Tis has further complicated and fueled the debate. Te evolution of the nucleus and endoplasmic reticulum is also at the center of many controversies. However hypotheses re­ garding the evolution of other membrane-bound organelles are more widely accepted and are considered next. Endosymbiotic Origin of Mitochondria Chloroplasts and Hydrogenosomes The endosymbiotic hypothesis is generally accepted as the origin of three eukaryotic organelles: mitochondria chloro­ plasts and hydrogenosomes. Endosymbiosis is an interaction between two organisms in which one organism lives inside the other. Te initial statement of the endosymbiotic hypothesis proposed that over time a bacterial endosymbiont of an ances­ tral cell in the eukaryotic lineage lost its ability to live indepen­ dently becoming either a mitochondrion if the intracellular bacterium used aerobic respiration or a chloroplast if the en­ dosymbiont was a photosynthetic bacterium see fgure 19.11. Although the mechanism by which the endosymbiotic rela­ tionship was established is unknown there is considerable evi­ dence to support the hypothesis. Mitochondria and chloroplasts contain DNA and ribosomes both are similar to bacterial DNA and ribosomes. Indeed inspection of fgure 1.2 shows that both organelles belong to the bacterial lineage based on SSU rRNA analysis. Further evidence for the origin of mitochondria comes from the genome sequence of the bacterium Rickettsia prowazekii an obligate intracellular parasite and the cause of epidemic lice-borne typhus. Its genome is more similar to that of modern mitochondrial genomes than to any other bacterium. Te chloro­ plasts of plants and green algae are thought to have descended from an ancestor of the cyanobacterial genus Prochloron which con­ tains species that live within marine invertebrates. Recently the endosymbiotic hypothesis for mitochondria has been modifed by the hydrogen hypothesis. Tis asserts that the endosymbiont was an anaerobic bacterium that produced H2 and C02 as end products of its metabolism. Over time the host became dependent on the H2 produced by the endosymbiont. Ultimately the endosymbiont evolved into one of two organelles. If the endosymbiont developed the capacity to perform aerobic respiration it evolved into a mito chondrion. However if the en­ dosymbiont did not develop this capaci ty it evolved into a hydrogenosome-an organelle found in some extant protists that produce ATP by a process called fermen tation see fgure 5.16.

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10 CHAPTER 1 I The Evolution of Microorganisms and Microbiology Evolution of Cellular Microbes Although the history of early cellular life forms may never be known we know that once they arose they were subjected to the same evolutionary processes as modern organisms. Te ances­ tral bacteria archaea and eukaryotes possessed genetic infor­ mation that could be duplicated lost or mutated. Tese mutations could have many outcomes. Some led to the death of the mutant microbe but others allowed new functions and char­ acteristics to evolve. Tose mutations that allowed the organism to increase its reproductive ability were selected for and passed on to subsequent generations. In addition to selective forces iso­ lation of populations allowed some groups to evolve separately from others. Tus selection and isolation led to the eventual de­ velopment of new collections of genes i.e. genotypes and many new species. In addition to mutation other mechanisms exist for re­ configuring the genotypes of a species and therefore creating genetic diversity. Most eukaryotic species increase their ge­ netic diversity by reproducing sexually. Thus each offspring of the two parents has a mixture of parental genes and a unique genotype. Bacterial and archaeal species do not repro­ duce sexually. They increase their genetic diversity by hori­ zontal lateral gene transfer HGT. During HGT genetic information from a donor organism is transferred to a recipi­ ent creating a new genotype. Thus genetic information can be passed from one generation to the next as well as between individuals of the same generation and even between differ­ ent microbial species. Genome sequencing has revealed that HGT has played an important role in the evolution of bacte­ rial and archaeal species. Importantly HGT still occurs and continues to shape their genomes leading to the evolution of species with antibiotic resistance new virulence properties and novel metabolic capabilities. The outcome of HGT is that many bacterial and archaeal species have mosaic ge­ nomes composed of bits and pieces of the genomes of other organisms fgure 1.9 . �I Microbial evolutionary processes section 19.5 Microbial Species All students of biology are introduced early in their careers to the concept of a species. But the term has diferent meanings depending on whether the organism is sexual or not. Ta xono­ mists working with plants and animals defne a species as a group of interbreeding or potentially interbreeding natural populations that is reproductively isolated from other groups. Tis defnition also is appropriate for the many eukaryotic microbes that reproduce sexually. However bacterial and archaeal species cannot be defned by this criterion since they do not reproduce sexually. An appropriate defnition is currently the topic of considerable discussion. A common definition is that bacterial and archaeal species are a collec­ tion of strains that share many stable properties and differ 000 yy 00 y 00 0 Microbial genomes Y Horizontal gene transfer events Figure 1.9 The Mosaic Nature of Bacterial and Archaeal Genomes. Horizontal gene transfer HGT events move pieces of the genome of one organism to another. Over time HGT creates organisms having mosaic genomes composed of portions of the genomes of other microbes. The length of segments drawn is arbitrary and is not meant to represent the actual size of the portion of genome transferred. significantly from other groups of strains. A strain consists of the descendants of a single pure microbial culture. Strains within a species may be described in a number of diferent ways. Biovars are variant strains characterized by biochemical or physiological diferences morphovars difer morphologi­ cally serovars have distinctive properties that can be detected by antibodies p. 17 and pathovars are pathogenic strains distinguished by the plants in which they cause disease. �I Evolutionary processes and the concept of a microbial species section 19.5 Microbiologists name microbes using the binomial system of the eighteenth-century biologist and physician Carl Lin­ naeus. Te Latinized italicized name consists of two parts. Te frst part which is capitalized is the generic name i.e. the name of the genus to which the microbe belongs and the second is the uncapitalized species epithet. For example the bacterium that causes plague is called Yersinia pestis. Ofen the name of an organism will be shortened by abbreviating the genus name with a single capital letter e.g. Y pestis. Retrieve Infer Apply 1. Why is RNA thought to be the frst self-replicating biomolecule 2. Explain the endosymbiotic hypothesis of the origin of mitochondria hydrogenosomes and chloroplasts. List two pieces of evidence that support this hypothesis. 3. What is the diference between a microbial species and a strain 4. What is the correct way to write this microbes name: bacillus subtilis Bacillus subtilis Bacillus Subtilis or Bacilus subtilis Identify the genus name and the species epithet.

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1.3 Microbiology and Its Origins After reading this section you should be able to: • Evaluate the importance of the contributions to microbiology made by Hooke Leeuwenhoek Pasteur Koch Cohn Beijerinck von Behring Kitasato Metchnikof and Winogradsky • Outline a set of experiments that might be used to decide if a particular microbe is the causative agent of a disease • Predict the difculties that might arise when using Kochs postulates to determine if a microbe causes a disease unique to humans Even before microorganisms were seen some investigators sus­ pected their existence and role in disease. Among others the Roman philosopher Lucretius about 98-55 BCE and the physi­ cian Girolamo Fracastoro 1478-1553 suggested that disease was caused by invisible living creatures. However until microbes could actually be seen or studied in some other way their exis­ tence remained a matter of conjecture. Terefore microbiology is defned not only by the organisms it studies but also by the tools used to study them. Te development of microscopes was the critical frst step in the evolution of the discipline. However microscopy alone is unable to answer the many questions micro­ biologists ask about microbes. A distinct feature of microbiology is that microbiologists ofen remove microorganisms from their normal habitats and culture them isolated from other microbes. Tis is called a pure or axenic culture. Te development of tech­ niques for isolating microbes in pure culture was another criti­ cal step in microbiologys history. However it is now recognized as having limitations. Microbes in pure culture are in some ways like animals in a zoo just as a zoologist cannot fully understand the ecology of animals by studying them in zoos microbiolo­ gists cannot fully understand the ecology of microbes by study­ ing them in pure culture. Today molecular genetic techniques and genomic analyses are providing new insights into the lives of microbes . .I Methods in microbial ecology chapter 29 Microbial genomics chapter 18 Here we describe how the tools used by microbiologists have infuenced the development of the feld. As microbiology evolved as a science it contributed greatly to the well-being of humans. Tis is exemplifed by the number of microbiologists who have won the Nobel Prize see www.mhhe.com/willey9. Te historical context of some of the important discoveries in microbiology is shown in fgure 1.10 . Microscopy and the Discovery of Microorganisms Te earliest microscopic observations of organisms appear to have been made between 1625 and 1630 on bees and weevils by the Italian Francesco Stelluti 1577-1652 using a microscope probably supplied by Galileo 1564-1642. Robert Hooke 1635-1703 is credited with publishing the frst drawings of 1.3 Microbiology and Its Origins 11 microorganisms in the scientifc literature. In 1665 he published a highly detailed drawing of the fungus Mucor in his book Micrographia. Micrographia is important not only for its exqui­ site drawings but also for the information it provided on build­ ing microscopes. One design discussed in Micrographia was probably a prototype for the microscopes built and used by the amateur microscopist Antony van Leeuwenhoek 1632-1723 of Delf the Netherlands fgure l.lla. Leeuwenhoek earned his living as a draper and haberdasher a dealer in mens cloth­ ing and accessories but spent much of his spare time construct­ ing simple microscopes composed of double convex glass lenses held between two silver plates fgure 1.11b. His microscopes could magnif about 50 to 300 times and he may have illumi­ nated his liquid specimens by placing them between two pieces of glass and shining light on them at a 45° angle to the specimen plane. Tis would have provided a form of dark-feld illumina­ tion whereby organisms appeared as bright objects against a dark background fgure 1.11c. Beginning in 1673 Leeuwen­ hoek sent detailed letters describing his discoveries to the Royal Society of London. It is clear from his descriptions that he saw both bacteria and protists. Culture-Based Methods for Studying Microorganisms As important as Leeuwenhoeks observations were the develop­ ment of microbiology essentially languished for the next 200 years until techniques for isolating and culturing microbes in the labora­ tory were formulated. Many of these techniques began to be devel­ oped as scientists grappled with the conflict over the theory of spontaneous generation. Tis confict and the subsequent studies on the role played by microorganisms in causing disease ultimately led to what is now called the golden age of microbiology. Spontaneous Generation From earliest times people had believed in spontaneous generation-that living organisms could develop from nonliving matter. Tis view fnally was challenged by the Italian physician Francesco Redi 1626-1697 who carried out a series of experiments on decaying meat and its ability to produce maggots spontaneously. Redi placed meat in three containers. One was uncovered a second was covered with paper and the third was covered with fne gauze that would exclude fies. Flies laid their eggs on the uncovered meat and maggots developed. Te other two pieces of meat did not produce maggots spontaneously. How­ ever fies were attracted to the gauze-covered container and laid their eggs on the gauze these eggs produced maggots. Tus the generation of maggots by decaying meat resulted from the pres­ ence of fly eggs and meat did not spontaneously generate mag­ gots as previously believed. Similar experiments by others helped discredit the theory for larger organisms. Leeuwenhoeks communications on microorganisms re­ newed the controversy. Some proposed that microbes arose by spontaneous generation even though larger organisms did not.

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1665 Hooke publishes Micrgrphia. 1668 Redi refutes spontaneous generation of maggots. 1765-1776 Spallanzani �: ::: - . .. � � � � . . � � � � � � � : : � � : a : tac ks spontaneous 167 4-1676 Leeuwenhoek generation. discovers "animacules." 1798 Jenner introduces 1543 Publication of cowpox vaccination for Copernicuss work smallpox. on heliocentric solar system 1620 Francis Bacon arues for importance of inductive reasoning in scientific method. 1687 Newtons Prncipia published. 1775 American Revolution begins. 1859 Darins Orgin of Species 1861-1865 ------- 1854 Snow traces cholera source to water pump. 1861 Pasteur disproves spontaneous generation. 1893 Munsch --1 paints The Scream. 1898 Spanish­ American War American Civil War 1879 Edisons ----. first light bulb 1876 Koch demonstrates that Bacilus anthrcis causes anthrax. ----- 1884 Kochs postulates 1887-1890 Winogradsky studies sulfur and nitrifying bacteria. 1885 Pasteur develops rabies vaccine. published Metchnikof describes phagocytosis autoclave developed Gram stain developed. . _ _ 1899 Beijerinck proves 1911 Rous discovers a virus can cause cancer. 1900 Planck-- � � virus causes tobacco mosaic disease. develops quantum theory. 1908 First --- Model T Ford 1945 Atomic bomb dropped on Hirshima. 1950 Korean War -- -- W begins. 1961 First --- human in space 1917 Russian Revolution 1918 Influenza pandemic ----- kills over 50 million people. 1927 Lindbergs 1937 Krbs discovers citric acid cycle. transAtlantic flight 1933 Hitler 1929 Stock 1928 Grifith discovers bacterial transformation. � :e::� ����� ������--- 1932 Knoll and Ruska - build first electron 1953 Watson and Crick propose DNA double helix. microscope. 1990 First human gene therapy testing begun. 1983-1984 HIV isolated and identified by Gallo and Montagnier Mullis develops PCR technique. 2001 Anthrax bioterrrism attacks in New York Washington D.C. and Florida 1969 Neil Armstrong -- --� -l 1977 Woese divides prokaryotes into Bacteria and Archaea. 1992 First human trials of antisense therapy f 2005 Genome of 1918 influenza walks on the moon. 1973 Vietnam War ends. 1980 First home computers 2003 Second war with Iraq 2001 World Trade 2010 H1 N1 Center attack influenza outbrak Figure 1.10 Some Important Events in the Development of Microbiology. Milestones in microbiology are marked in red other historical events are in black.

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a Lens Specimen holder b c Figure 1.11 Antony van Leeuwenhoek. a An oil painting of Leeuwenhoek. b A brass replica of the Leeuwenhoek microscope. Inset photo shows how it is held. c Leeuwenhoeks drawings of bacteria from the human mouth. 1.3 Microbiology and Its Origins 13 Tey pointed out that boiled extracts of hay or meat gave rise to microorganisms afer sitting for a while. Indeed such extracts were the forerunners of the culture media still used today in many microbiology laboratories. In 1748 the English priest John Needham 1713-1781 reported the results of his experiments on spontaneous gen­ eration. Needham boiled mutton broth in fasks that he then tightly stoppered. Eventually many of the fasks became cloudy and contained microorganisms. He thought organic matter contained a vital force that could confer the properties of life on nonliving matter. A few years later the Italian priest and naturalist Lazzaro Spallanzani 1729-1799 improved on Needhams experimental design by frst sealing glass fasks that contained water and seeds. If the sealed fasks were placed in boiling water for about 45 min­ utes no growth took place as long as the fasks remained sealed. He proposed that air carried germs to the culture medium but also commented that the external air might be required for growth of animals already in the medium. Te supporters of spontaneous generation maintained that heating the air in sealed fasks destroyed its ability to support life. Several investigators attempted to counter such arguments. Teodore Schwann 1810-1882 allowed air to enter a fask containing a sterile nutrient solution afer the air had passed through a red-hot tube. Te fask remained sterile. Subsequently Georg Friedrich Schroder 1810-1885 and Teodor von Dusch 1824-1890 allowed air to enter a fask of heat-sterilized medium afer it had passed through sterile cotton wool. No growth occurred in the medium even though the air had not been heated. Despite these experiments the French naturalist Felix Pouchet 1800-1872 claimed in 1859 to have carried out experi­ ments conclusively proving that microbial growth could occur without air contamination. Pouchets claim provoked Louis Pasteur 1822-1895 to set­ tle the matter of spontaneous generation. Pasteur fgure 1.12 frst fltered air through cotton and found that objects resembling plant spores had been trapped. If a piece of the cotton was placed in sterile medium afer air had been fltered through it microbial growth occurred. Next he placed nutrient solutions in fasks heated their necks in a fame and drew them out into a variety of curves. Te swan-neck fasks that he pro­ duced in this way had necks open to the atmosphere. Pas­ teur then boiled the solutions for a few minutes and allowed them to cool. No growth took place even though the con­ tents of the fasks were ex­ posed to the air fgure 1.13. Pasteur pointed out that growth did not occur because dust and germs had been Figure 1.12 Louis Pasteur.

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14 CHAPTER 1 I The Evolution of Microorganisms and Microbiology Microbes being destroyed Vigorous heat is applied. Broth free of live cells sterile / Neck on second sterile flask is broken growth occurs. Neck intact airborne microbes are trapped at base and broth is sterile. Figure 1.13 Pasteurs Experiments with Swan-Neck Flasks. trapped on the walls of the curved necks. If the necks were bro­ ken growth commenced immediately. Pasteur had not only re­ solved the controversy by 1861 but also had shown how to keep solutions sterile. Te English physicist John Tyndall 1820-1893 and the Ger­ man botanist Ferdinand Cohn 1828-1898 dealt a fnal blow to spontaneous generation. In 1877 Tyndall demonstrated that dust did indeed carry germs and that if dust was absent broth remained sterile even if directly exposed to air. During the course of his studies Tyndall provided evidence for the existence of exception­ ally heat-resistant forms of bacteria. Working independently Cohn discovered that the heat-resistant bacteria recognized by Tyndall were species capable of producing bacterial endospores. Cohn later played an instrumental role in establishing a classifca­ tion system for bacteria based on their morphology and physiology. �I Bacterial endospores section 3.9 Clearly these early microbiologists not only disproved spontaneous generation but also contributed to the rebirth of microbiology. Tey developed liquid media for culturing mi­ crobes. Tey also developed methods for sterilizing media and maintaining their sterility. Tese techniques were next applied to understanding the role of microorganisms in disease. Retrieve Infer Apply 1. What does the theory of spontaneous generation propose How did Pasteur Tyndall and Cohn fnally settle the spontaneous generation controversy 2. What did Pasteur prove when he showed that a cotton plug that had fltered air would trigger microbial growth when transferred to the medium What argument made previously was he addressing Microorganisms and Disease Although Fracastoro and a few others had suggested that invisi­ ble organisms produced disease most people believed that dis­ ease was caused by supernatural forces poisonous vapors called miasmas and imbalances among the four humors thought to be present in the body. Te role of the four humors blood phlegm yellow bile choler and black bile melancholy in disease had been widely accepted since the time of the Greek physician Galen 129-199. Support for the idea that microorganisms cause disease-that is the germ theory of disease-began to accumulate in the early nineteenth century from diverse felds. Agostino Bassi 1773-1856 demonstrated in 1835 that a silk­ worm disease was due to a fungal infection. He also suggested that many diseases were due to microbial infections. In 1845 M. J. Berkeley 1803-1889 proved that the great potato blight of Ireland was caused by a water mold then thought to be a fungus and in 1853 Heinrich deBary 1831-1888 showed that smut and rust fungi caused cereal crop diseases. Pasteur also contributed to this area of research in several ways. His contributions began in what may seem an unlikely way. Pasteur was trained as a chemist and spent many years studying the alcoholic fermentations that yield ethanol and are used in the production of wine and other alcoholic beverages. When he began his work the leading chemists were convinced that fermentation was due to a chemical instability that degraded the sugars in grape juice and other substances to alcohol. Pasteur did not agree he believed that fermentations were carried out by living organisms. In 1856 M. Bigo an industrialist in Lille France where Pasteur worked requested Pasteurs assistance. His business produced ethanol from the fermentation of beet sugars and the alcohol yields had recently declined and the product had be­ come sour. Pasteur discovered that the fermentation was failing because the yeast normally responsible for alcohol formation had been replaced by bacteria that produced acid rather than ethanol. In solving this practical problem Pasteur demon­ strated that all fermentations were due to the activities of spe­ cifc yeasts and bacteria and he published several papers on fermentation between 1857 and 1860. Pasteur was also called upon by the wine industry in France for help. For several years poor-quality wines had been pro­ duced. Pasteur referred to the wines as diseased and demon­ strated that particular wine diseases were linked to particular microbes contaminating the wine. He eventually suggested a method for heating the wines to destroy the undesirable mi­ crobes. Te process is now called pasteurization. Indirect evidence for the germ theory of disease came from the work of the English surgeon Joseph Lister 1827-1912 on the prevention of wound infections. Lister impressed with Pasteurs studies on fermentation and putrefaction developed a system of antiseptic surgery designed to prevent microorganisms from en­ tering wounds. Instruments were heat sterilized and phenol was used on surgical dressings and at times sprayed over the surgical area. Te approach was remarkably successful and transformed surgery. It also provided strong indirect evidence for the role of

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Figure 1.14 Robert Koch. Koch examining a specimen in his laboratory. microorganisms in disease because phenol which kills bacteria also prevented wound infections. Kochs Postulates Te frst direct demonstration that bacteria cause disease came from the study of anthrax by the German physician Robert Koch 1843- 1910. Koch fgre 1.14 used the criteria proposed by his former teacher Jacob Henle 1809-1885 and others to establish the relationship between Bacillus anthracis and anthrax he pub­ lished his fndings in 1876. Koch injected healthy mice with ma­ terial from diseased animals and the mice became ill. Afer transferring anthrax by inoculation through a series of 20 mice he incubated a piece of spleen containing the anthrax bacillus in beef serum. Te bacteria grew reproduced and produced endo­ spores. When isolated bacteria or their spores were injected into healthy mice anthrax developed. His criteria for proving the causal relationship between a microorganism and a specifc dis­ ease are known as Kochs postates. Kochs proof that B. anthrcis caused anthrax was independently confrmed by Pasteur and his coworkers. Tey discovered that afer burial of dead animals an­ thrax spores survived and were brought to the surface by earth­ worms. Healthy animals then ingested the spores and became ill. Afer completing his anthrax studies Koch fully outlined his postulates in his work on the cause of tuberculosis fgre 1.15 . In 1884 he reported that this disease was caused by the rod-shaped bacterium Mycobacterium tuberculosis and in 1905 he was awarded the Nobel Prize in Physiology or Medicine. Kochs pos­ tulates were quickly adopted by others and used to connect many diseases to their causative agent. While Kochs postulates are still widely used their applica­ tion is at times not feasible. For instance organisms such as 1.3 Microbiology and Its Origins 15 Mycobacterium lepre the causative agent of leprosy cannot be isolated in pure culture. Some human diseases are so deadly e.g. Ebola hemorrhagic fever that it would be unethical to use hu­ mans as the experimental organism if an appropriate animal model does not exist the postulates cannot be fully met. To avoid some of these difculties microbiologists sometimes use molec­ ular and genetic evidence. For instance molecular methods might be used to detect the nucleic acid of a virus in body tissues rather than isolating the virus or the genes thought to be associ­ ated with the virulence of a pathogen might be mutated. In this case the mutant organism should have decreased ability to cause disease. Introduction of the normal gene back into the mutant should restore the pathogens virulence. Pure Culture Methods During Kochs studies on bacterial diseases it became necessary to isolate suspected bacterial pathogens in pure culture p. 11. At frst Koch cultured bacteria on the sterile surfaces of cut boiled potatoes but the bacteria did not always grow well. Even­ tually he developed culture media using meat extracts and pro­ tein digests reasoning these were similar to body fuids. Initially he tried to solidify the media by adding gelatin. Separate bacte­ rial colonies developed afer the surface of the solidifed medium had been streaked with a bacterial sample. Te sample could also be mixed with liquefed gelatin medium. When the medium hardened individual bacteria produced separate colonies. Despite its advantages gelatin was not an ideal solidifying agent because it can be digested by many microbes and melts at temperatures above 28°C. A better alternative was provided by Fanny Eilshemius Hesse 1850-1934 the wife of Walther Hesse 1846-1911 one of Kochs assistants. She suggested the use of agar which she used to make jellies as a solidifying agent. Agar was not attacked by most bacteria. Furthermore it did not melt until reaching a temperature of 100°C and once melted did not solidify until reaching a temperature of 50°C this eliminated the need to handle boiling liquid. Some of the media developed by Koch and his associates such as nutrient broth and nutrient agar are still widely used. Another important tool developed in Kochs laboratory was a container for holding solidifed media­ the Petri dish plate named afer Richard Petri 1852-1921 who devised it. Tese developments directly stimulated progress in all areas of microbiology. 1 Culture media section 75 Enrichment and isolation of pure cultures section 75 Our focus thus far has been on the development of methods for culturing bacteria. But viral pathogens were also being studied during this time and methods for culturing them were also being developed. Te discovery of viruses and their role in disease was made possible when Charles Chamberland 1851-1908 one of Pasteurs associates constructed a porcelain bacterial flter in 1884. Dimitri Ivanowski 1864-1920 and Martinus Beijerinck pronounced "by-a-rink 1851-1931 used the flter to study to­ bacco mosaic disease. Tey found that plant extracts and sap from diseased plants were infectious even afer being fltered with Chamberlands flter. Because the infectious agent passed through

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16 CHAPTER 1 I The Evolution of Microorganisms and Microbiology Postulate Experimentation 1. The microorganism must be present in every case of the disease but absent from healthy organisms. Koch developed a staining technique to examine human tissue. Mycobacterium tuberculosis could be identified in diseased tissue. 2. The suspected microorganisms must be isolated and grown in a pure culture. Koch grew M. tuberculosis in pure culture on coagulated blood serum. M. tuberculosis colonies 3. The same disease must result when the isolated microorganism is inoculated into a healthy host. Koch injected cells from the pure culture of M. tuberculosis into guinea pigs. The guinea pigs subsequently died of tuberculosis. 4. The same microorganisms must be isolated again from the diseased host. Koch isolated M. tuberculosis in pure culture on coagulated blood serum from the dead guinea pigs. M. tuberculosis colonies Figure 1.15 Kochs Postulates Applied to Tuberculosis. a flter that was designed to trap bacterial cells they reasoned that the agent must be something smaller than a bacterium. Beijerinck proposed that the agent was a "flterable virus: Eventually viruses were shown to be tiny acellular infectious agents. Retrieve Infer Apply 1. Discuss the contributions of Lister Pasteur and Koch to the germ theory of disease and the treatment or prevention of diseases. What other contributions did Koch make to microbiology 2. Describe Kochs postulates. What is a pure culture Why are pure cultures important to Kochs postulates Immunology Te ability to culture microbes also played an important role in early immunological studies. During studies on the bacterium that causes chicken cholera Pasteur and Pierre Roux 1853-1933 discovered that incubating the cultures for long intervals between transfers resulted in cultures that had lost their ability to cause the disease. Tese cultures were said to be attenuated. When the chickens were injected with attenuated cultures they not only remained healthy but also were able to resist the disease when exposed to virulent cultures. Pasteur called the attenuated culture a vaccine Latin vacca cow in honor of Edward Jenner 1749-1823 because many years earlier Jenner had used mate­ rial from cowpox lesions to protect people against smallpox see Historical Highlights 37.5. Shortly afer this Pasteur and Cham­ berland developed an attenuated anthrax vaccine . . I Vaccines and immunizations section 37.7 Pasteur also prepared a rabies vaccine using an attenuated strain of rabies virus. During the course of these studies Joseph Meister a nine-year-old boy who had been bitten by a rabid dog was brought to Pasteur. Since the boys death was certain in the absence of treatment Pasteur agreed to try vaccination. Joseph was injected 13 times over the next 10 days with increasingly virulent preparations of the attenuated virus. He survived. In gratitude for Pasteurs development of vaccines people from around the world contributed to the construction of the Pasteur

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Institute in Paris France. One of the initial tasks of the institute was vaccine production. Tese early advances in immunology were made without any concrete knowledge about how the immune system works. Immunologists now know that the immune system uses chemi­ cals produced by several types of blood cells to provide protec­ tion. Among the chemicals are soluble proteins called antibodies which can be found in blood lymph and other body fuids. Te role of soluble substances in preventing disease was recognized by Emil von Behring 1854-1917 and Shibasaburo Kitasato 1852-1931. Afer the discovery that diphtheria was caused by a bacterial toxin they injected inactivated diphtheria toxin into rabbits. Te inactivated toxin induced rabbits to produce an antitoxin which protected against the disease. Antitoxins are now known to be antibodies that specifcally bind toxins neutraliz­ ing them. Te frst immune system cells were discovered when Elie Metchnikof 1845-1916 found that some white blood cells could engulf disease-causing bacteria. He called these cells phagocytes and the process phagocytosis Greek phagein eating. Microbial Ecology Culture-based techniques were also applied to the study of mi­ crobes in soil and aquatic habitats. Early microbial ecologists studied microbial involvement in the carbon nitrogen and sul­ fur cycles. Te Russian microbiologist Sergei Winogradsky 1856-1953 made many contributions to soil microbiology. He discovered that soil bacteria could oxidize iron sulfur and am­ monia to obtain energy and that many of these bacteria could incorporate C02 into organic matter much as photosynthetic organisms do. Winogradsky also isolated anaerobic nitrogen­ fxing soil bacteria and studied the decomposition of cellulose. Martinus Beijerinck was one of the great general microbiolo­ gists who made fundamental contributions not only to virology but to microbial ecology as well. He isolated aerobic nitrogen­ fxing bacteria Azotobacter spp. a root nodule bacterium also capable of fxing nitrogen genus Rhizobium and sulfate­ reducing bacteria. Beijerinck and Winogradsky also developed the enrichment culture techniques and the use of selective media which have been of great importance in microbiology. �I Biogeoche mical cycling section 28.1 Culture media section 7.5 Retrieve Infer Apply 1. How did Jenner Pasteur von Behring Kitasato and Metchnikof contribute to the development of immunology How was the ability to culture microbes important to their studies 2. How did Winogradsky and Beijerinck contribute to the study of microbial ecology What new culturing techniques did they develop in their studies 3. How might the work ofWinogradsky and Beijerinck have contributed to research on bacterial pathogens Conversely how might Koch and Pasteur have infuenced Winogradskys and Beijerincks study of microbial ecology 1.4 Microbiology Today 17 1.4 Microbiology Today After reading this section you should be able to: • Construct a concept map table or drawing that illustrates the diverse nature of microbiology and how it has improved human conditions • Support the belief held by many microbiologists that microbiology is experiencing its second golden age Microbiology today is as diverse as the organisms it studies. It has both basic and applied aspects. Te basic aspects are con­ cerned with the biology of microorganisms themselves. Te ap­ plied aspects are concerned with practical problems such as disease water and wastewater treatment food spoilage and food production and industrial uses of microbes. Te basic and applied aspects of microbiology are intertwined. Basic research is ofen conducted in applied felds and applications ofen arise out of basic research. An important recent development in microbiology is the in­ creasing use of molecular and genomic methods to study microbes and their interactions with other organisms. Tese methods have led to a time of rapid advancement that rivals the golden age of microbiology. Indeed many feel that microbiology is in its second golden age. Here we describe some of the important advances that have enabled microbiologists to use molecular and genomic tech­ niques. We then discuss some of the important research being done in the numerous subdisciplines of microbiology. Molecular and Genomic Methods for Studying Microbes Molecular and genomic methods for studying microbes rely on the ability of scientists to manipulate the genes and genomes of the organisms being studied. An organisms genome is all the genetic information that organism contains. To study single genes or the entire genome microbiologists must be able to iso­ late DNA and RNA cut DNA into smaller pieces insert one piece of DNA into another and determine the sequence of nu­ cleotides in DNA. Cutting double-stranded DNA into smaller pieces was ac­ complished using bacterial enzymes now known as restriction endonucleases or simply restriction enzymes. Tese enzymes were discovered by Werner Arber and Hamilton Smith in the 1960s. Teir discovery was followed by the report in 1972 that David Jackson Robert Symons and Paul Berg had successfully generated recombinant DNA molecules-molecules made by combining two or more diferent DNA molecules together. Tey did this by cutting DNA from two diferent organisms with the same restriction enzyme mixing the two DNA molecules to­ gether and linking them together with an enzyme called DNA ligase. �I Key developments in recombinant DNA technology section 171 Te next major breakthrough was the development of meth­ ods to determine the sequence of nucleotides in DNA. In the late

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18 CHAPTER 1 I The Evolution of Microorganisms and Microbiology 1970s Frederick Sanger introduced a method that has since been modifed and adapted for use in automated systems. Today en­ tire genomes of organisms can be sequenced in a matter of days. In addition newer even more rapid sequencing methods have been devised. "I Genome sequencing section 18.2 Genome sequencing is the frst step in genomic anal y sis. Once the genome sequence is in hand microbiologists must decipher the information found in the genome. Tis involves identifying potential protein-coding genes determining what they code for and identify ing other regions of the genome that may have other important functions e.g. genes encoding tRNA and rRNA or sequences playing a role in regulating the function of genes. Tis work requires the use of computers which has given rise to the scientifc discipline bioinformatics. Bioinformaticists manage the ever-increasing amount of ge­ netic information available for analysis. Tey also determine the function of genes and generate hypotheses that can be tested either in silica i.e. in the computer or in the laboratory. "I Bioinformatics section 18.3 Major Fields in Microbiology As noted in section 1.1 pathogenic microbes though relatively few in number have had and continue to have considerable im­ pact on humans. Tus one of the most active and important felds in microbiology is medical microbiology which deals with dis­ eases of humans and animals. Medical microbiologists identif the agents causing infectious diseases and help plan measures for their control and elimination. Frequently they are involved in tracking down new unidentifed pathogens such as those causing variant Creutzfeldt-Jakob disease the human version of "mad cow disease hantavirus pulmonary syndrome and West Nile encephalitis. Tese microbiologists also study the ways microor­ ganisms cause disease. As described in section 1.3 our under­ standing of the role of microbes in disease began to crystallize when we were able to isolate them in pure culture. Today clinical laboratory scientists the microbiologists who work in hospital and other clinical laboratories use a variety of techniques to pro­ vide information needed by physicians to diagnose infectious dis­ ease. Increasingly molecular genetic techniques are also being used. Major epidemics have regularly afected human history. Te 1918 infuenza pandemic is of particular note it killed more than 50 million people in about a year. Public health microbiology is concerned with the control and spread of such communicable diseases. Public health microbiologists and epidemiologists mon­ itor the amount of disease in populations. Based on their observa­ tions they can detect outbreaks and developing epidemics and implement appropriate control measures. Tey also conduct sur­ veillance for new diseases as well as bioterrorism events. Public health microbiologists working for local governments monitor community food establishments and water supplies to ensure they are safe and free from pathogens. To understand treat and control infectious disease it is important to understand how the immune system protects the body from pathogens this question is the concern of immunol­ ogy. Immunology is one of the fastest growing areas in science. Much of the growth began with the discovery of the human immunodeficiency virus HIV which specifcally targets cells of the immune system. Immunology also deals with the nature and treatment of allergies and autoimmune diseases such as rheumatoid arthritis. " Innate host resistance chapter 33 Adaptive immunity chapter 34 Microbial ecology is another important feld in microbiol­ ogy. Microbial ecology developed when early microbiologists such as Winogradsky and Beijerinck chose to investigate the eco­ logical role of microorganisms rather than their role in disease. Today a variety of approaches including non-culture-based tech­ niques are used to describe the vast diversity of microbes in terms of their morphology physiology and relationships with organisms and the components of their habitats. Te importance of microbes in global and local cycling of carbon nitrogen and sulfur is well documented however many questions are still un­ answered. Of particular interest is the role of microbes in both the production and removal of greenhouse gases such as carbon di­ oxide and methane. Microbial ecologists also are employing mi­ croorganisms in bioremediation to reduce pollution. A new frontier in microbial ecology is the study of the microbes nor­ mally associated with the human body-so-called human micro­ biota. Scientists are currently trying to identify all members of the human microbiota using molecular techniques that grew out of Woese s pioneering work to establish the phylogeny of microbes. "I Global climate change section 28.2 Biodegradation and bioremediation section 43.4 Agricultural microbiology is a feld related to both medical microbiology and microbial ecology. Agricultural microbiology is concerned with the impact of microorganisms on agriculture. Microbes such as nitrogen-fxing bacteria play critical roles in the nitrogen cycle and afect soil fertility. Other microbes live in the digestive tracts of ruminants such as cattle and break down the plant materials these animals ingest. Tere are also plant and ani­ mal pathogens that have signifcant economic impact if not con­ trolled. Furthermore some pathogens of domestic animals also can cause human disease. Agricultural microbiologists work on methods to increase soil fertility and crop yields study rumen microorganisms in order to increase meat and milk production and try to combat plant and animal diseases. Currently many agricultural microbiologists are studying the use of bacterial and viral insect pathogens as substitutes for chemical pesticides. "I Agricultural biotechnology section 42.4 Agricultural microbiology has contributed to the ready supply of high-quality foods as has the discipline of food and dairy microbiology. Numerous foods are made using micro­ organisms. On the other hand some microbes cause food spoilage or are pathogens that are spread through food. Ex­ cellent examples of the latter are the rare Escherichia coli 0104:H4 which in 2011 caused a widespread outbreak of dis­ ease in Europe thought to have been spread by bean sprouts and also in 2011 contaminated ground turkey was implicated in a Salmonella outbreak in the United States. Food and dairy

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microbiologists explore the use of microbes in food produc­ tion. They also work to prevent microbial spoilage of food and the transmission of food-b orne diseases. Tis involves monitoring the food industry for the presence of pathogens. Increasingly molecular methods are being used to detect pathogens in meat and other foods. Food and dairy microbi­ ologists also conduct research on the use of microorganisms as nutrient sources for livestock and humans. _1 Microbi­ ology of food chapter 41 Humans unknowingly exploited microbes for thousands of years. However the systematic and conscious use of microbes in industrial microbiology did not begin until the 1800s. Industrial microbiology developed in large part from Pasteurs work on alco­ holic fermentations as described in section 1.3. His success led to the development of pasteurization to preserve wine during storage. Pasteurs studies on fermentation continued for almost 20 years. One of his most important discoveries was that some fermentative microorganisms were anaerobic and could live only in the absence of oxygen whereas others were able to live either aerobically or anaerobically. _1 Controlling food spoilage section 41.2 Another important advance in industrial microbiology occurred in 1929 when Alexander Fleming discovered that the fungus Penicillium sp. produced what he called penicillin the frst antibiotic that could successfully control bacterial infec­ tions. Although it took World War II for scientists to learn how to mass-produce penicillin scientists soon found other micro­ organisms capable of producing additional antibiotics. Today industrial microbiologists also use microorganisms to make products such as vaccines steroids alcohols and other sol­ vents vitamins amino acids and enzymes. Microbes are also being used to produce biofuels such as ethanol. Tese alterna­ tive fuels are renewable and may help decrease pollution associ­ ated with burning fossil fuels. _1 Major products of industrial microbiology section 42.1 Biofuel production section 42.2 Industrial microbiologists identify or genetically engineer microbes of use to industrial processes medicine agriculture and other commercial enterprises. Tey also utilize techniques to improve production by microbes and devise systems for cultur­ ing them and isolating the products they make. Members ofthe Microbial World • Microbiology studies microscopic cellular organisms that are ofen unicellular or if multicellular do not have highly diferentiated tissues. Microbiology also focuses on biological entities that are acellular fgure 1.1. • Microbiologists divide cellular organisms into three domains: Bacteria Archaea and Eukarya fgure 1.2. • Domains Bacteria and Archaea consist of prokaryotic microorganisms. Te eukaryotic microbes protists and Key Concepts 19 Te advances in medical microbiology agricultural micro­ biology food and dairy microbiology and industrial microbiol­ ogy are in many ways outgrowths of the labor of many microbiologists doing basic research in areas such as microbial physiology microbial genetics molecular biology and bioinfor­ matics. Microbes are metabolically diverse and can employ a wide variety of energy sources including organic matter inor­ ganic molecules e.g. H2 and NH3 and sunlight. Microbial phys­ iologists study many aspects of the biology of microorganisms including their metabolic capabilities. Tey also study the synthesis of antibiotics and toxins the ways in which microorganisms survive harsh environmental conditions and the efects of chemical and physical agents on microbial growth and survival. Microbial geneticists molecular biologists and bioinformaticists study the nature of genetic information and how it regulates the develop­ ment and function of cells and organisms. Te bacteria E. coli and Bacillus subtilis the yeast Saccharomyces cerevisiae bakers yeast and bacterial viruses such as T4 and lambda continue to be important model organisms used to understand biological phenomena. Clearly the future of microbiology is bright. Genomics in par­ ticular is revolutionizing microbiology as scientists are now begin­ ning to understand organisms in toto rather than in a reductionist piecemeal manner. How the genomes of microbes evolve the na­ ture of host-pathogen interactions the minimum set of genes re­ quired for an organism to survive and many more topics are aggressively being examined by molecular and genomic analyses. Tis is an exciting time to be a microbiologist. Enjoy the journey. Retrieve Infer Apply 1. Since the 1970s microbiologists have been able to study individual genes and whole genomes at the molecular level. What advances made this possible 2. Briefy describe the major subdisciplines in microbiology. Which do you consider to be applied felds Which are basic 3. Log all the microbial products you use in a week. Be sure to consider all foods and medications including vitamins. 4. List all the activities or businesses you can think of in your community that directly depend on microbiology. fngi are placed in Eukarya. Viruses viroids satellites and prions are acellular entities that are not placed in any of the domains but are classifed by a separate system. 1.2 Microbial Evolution • Evolutionary biologists and others interested in the origin of life must rely on many types of evidence. • Earth is approximately 4.5 billion years old. Within the frst 1 billion years of its existence life arose fgure 1.4.

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20 CHAPTER 1 I The Evolution of Microorganisms and Microbiology • Te RNA world hypothesis posits that the earliest self­ replicating entity on the planet used RNA both to store genetic information and to conduct celular processes fgure 1.6. • Comparisons of small subunit SSU rRNA genes have been useful in creating universal phylogenetic trees. Tese trees provide information about the evolution of life afer it arose fgure 1.8. • Te last universal common ancestor LUCA is placed on the bacterial branch of the universal phylogenetic tree. Tus Bacteria diverged frst and Archaea and Eukar y a arose later. • Mitochondria chloroplasts and hydrogenosomes are thought to have evolved from bacterial endosymbionts of ancestral cells in the eukaryotic lineage. • Te concept of a bacterial or archaeal species is difcult to defne and is the source of considerable debate because these microbes do not reproduce sexually. Species are named using the binomial system of Linnaeus. 1.3 Microbiology and Its Origins • Microbiology is defned not only by the organisms it studies but also by the tools it uses. Microscopy and culture-based techniques have played and continue to play important roles in the evolution of the discipline. • Antony van Leeuwenhoek used simple microscopes and was the frst person to extensively describe microorganisms fgure 1.11. • Culture-based techniques for studying microbes began to develop as scientists debated the theory of spontaneous generation. Experiments by Redi and others disproved the theory of spontaneous generation of larger organisms. Te spontaneous generation of microorganisms was disproved by Spallanzani Pasteur Tyndall Cohn and others fgure 1.13. esize Invent Microscopic organisms such as rotifers are not studied by microbiologists. Why is this so 2. Why arent viruses viroids satellites and prions included in the three domain system 3. Why was the belief in spontaneous generation an obstacle to the development of microbiology as a scientifc discipline 4. Would microbiology have developed more slowly if Fanny Hesse had not suggested the use of agar Give your reasoning. 5. Some individuals can be infected by a pathogen yet not develop disease. In fact some become chronic carriers of the pathogen. How does this observation afect Kochs postulates How might the postulates be modifed to account for the existence of chronic carriers • Te availability of culture-based techniques played an important role in the study of the microbes as the causative agents of disease. Support for the germ theory of disease came from the work of Bassi Pasteur Koch Lister and others. • Kochs postulates are used to prove a direct relationship between a suspected pathogen and a disease. Koch and his coworkers developed the techniques required to grow bacteria on solid media and to isolate pure cultures of pathogens fgure 1.15. • Viruses were discovered following the invention of a bacterial flter by Chamberland. Dimitri Ivanowski and Martinus Beijerinck were important contributors to the feld of virology. • Te feld of immunology developed as early microbiologists created vaccines and discovered antibodies and phagocytic cells. Pasteur von Behring Kitasato and Mechnikof made important contributions to this feld. • Microbial ecology grew out of the work of Winogradsky and Beijerinck. Tey studied the role of microorganisms in carbon nitrogen and sulfur cycles and developed enrichment culture techniques and selective media. 1.4 Microbiology Today • Today molecular and genomic analyses have paved the way for understanding microbes as biological systems. Tese methods include recombinant DNA techniques nucleic acid sequencing methods and genome sequencing. • Tere are many felds in microbiology. Tese include medical public health industrial and food and dairy microbiology. Microbial ecology physiology and genetics are important subdisciplines of microbiology. 6. Develop a list of justifcations for the usefulness of microorganisms as experimental models. 7. History is full of examples in which one group of people lost a struggle against another. a. Choose an example of a battle or other human activity such as exploration of new territory and determine the impact of microorganisms either indigenous or transported to the region on that activity. b. Discuss the efect that the microbes had on the outcome in your example. c. Suggest whether the advent of antibiotics food storage and preparation technology or sterilization technology would have made a diference in the outcome.

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8. Antony van Leeuwenhoek is ofen referred to as the father of microbiology. However many historians feel that Louis Pasteur Robert Koch or perhaps both deserve that honor. Decide who should be considered the father of microbiology and justify your decision. 9. Consider the discoveries described in sections 1.3 and 1.4. Which do you think were the most important to the development of microbiology Why 10. Support this statement: "Vaccinations against various childhood diseases have contributed to the entry of women particularly mothers into the full-time workplace: 11. Scientists are very interested in understanding when cyanobacteria frst emerged because as the frst organisms capable of oxygenic photosynthesis it is thought that they triggered a sharp rise in atmospheric oxygen. For many years certain lipid biomarkers have served as "molecular fossils" to date the frst appearance of cyanobacteria. However a 2010 study questioned whether these lipids called 2-methylhopanoids provide accurate information in Learn More 21 light of a 2007 discovery that they also are produced by an anoxygenic phototrophic bacterium-a bacterium that does not produce oxygen as it uses light energy. Te authors of the 2010 study identifed genes in extant bacteria involved in synthesis of the lipid biomarkers and then constructed phylogenetic trees based on comparisons of these genes. Tey also identifed the phyla to which the bacteria belonged based on SSU rRNA analysis and noted the habitats and metabolic capabilities of the bacteria used in the study. Discuss the specifc challenges encountered in the study of microbial evolution. What results from the phylogenetic analysis would support their claim that 2-methylhopanoids are not reliable biomarkers Why were habitat and metabolic characteristics also part of their analysis Read the original paper: Welander P. V. et al. 2010. Identifcation of a methylase required for 2-methylhopanoid production and implications for the interpretation of sedimentary hopanes. Proc. Natl. Acad. Sci. 10719:8537. ww . :cgrawhillcon nect.com Enhance your study of this chapter with interactive study tools and practice tests. Also ask your instructor about the resources available through ConnectPlus including the media-rich eBook adaptive learning tools and animations. • cn S 1 LearnSmart· I MICROBIOLOGY

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