Bacterial Ultrastructure and inclusion bodies

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Slide 1: 

Dr. M.K.SATEESH Molecular Diagnostics Laboratory, Department of Microbiology and Biotechnology, Bangalore University Bangalore-560 056 Bacteria Ultrastructure Inclusions and other internal structures SEM3D view of E. Coli

Approximate timing of major events in the history of life on Earth. : 

2 Approximate timing of major events in the history of life on Earth.

Slide 3: 

Biology of the Prokaryotes .1999.  edited by J. Lengeler, G. Drews, H. Schlegel

Slide 5: 

The Microbial World

Slide 6: 

The Microbial World Classification of Microbes

Slide 7: 

Criteria for Classification of Prokaryotes

Slide 10: 

Image from a confocal laser-scanning-microscope: co-culture of Nanoarchaeum equitans (small cocci, red) und Ignicoccus hospitalis (large cocci, green) after sequence specific (ss rRNA) fluorescence staining 400 nm (= 0.0004 mm) 490,000 base pairs

Slide 11: 

1997 by Heide Schulz of the Max Planck Institute for Marine Microbiology in Bremen, Germany. Huber, H.; Hohn, M.J.; Rachel, R.; Fuchs, T.; Wimmer, V.C.; Stetter, K.O. 2002: A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature, 417: 63-67.

Slide 14: 

.Fluid mosaic model of a biological membrane. In aqueous environments membrane phospholipids arrange themselves in such a way that they spontaneously form a fluid bilayer. Membrane proteins, which may be structural or functional, may be permanently or transiently associated with one side or the other of the membrane, or even be permanently built into the bilayer, while other proteins span the bilayer and may form transport channels through the membrane.

Slide 15: 

080506 Möllby 15 Cytoplasmic membrane Lipid bilayer Semipermeable barrier No sterols Contains: Structural proteins Transport proteins Electron transport system Enzymes Ion pumps Flagellum anchor

Slide 16: 

Operation of bacterial transport systems. Bacterial transport systems are operated by transport proteins (sometimes called carriers, porters or permeases) in the plasma membrane. Facilitated diffusion is a carrier-mediated system that does not require energy and does not concentrate solutes against a gradient. Active transport systems such as Ion-driven transport and Binding protein-dependent transport, use energy and concentrate molecules against a concentration gradient. Group translocation systems, such as the phosphotransferase (pts) system in Escherichia coli, use energy during transport and modify the solute during its passage across the membrane.

Locomotion : 

Locomotion Outside cell wall Made of chains of flagellin Attached to a protein hook Anchored to the wall and membrane by the basal body Rotate flagella to run or tumble Move toward or away from stimuli (taxis) Flagella proteins are H antigens (e.g., E. coli O157:H7) (antigen refers to immunochemical reaction, i.e., H antigens generate an immune response if flagella are present)

Flagella : 

Flagella A) Monotrichous B) Lophotrichous C) Amphitrichous D) Peritrichous

Slide 21: 

A single flagellum can be present at one end of the cell (monotrichous); for example, in Vibrio cholerae, Pseudomonas aeruginosa, Idiomarina loihiensis (a) and Caulobacter crescentus (b). Many bacteria have numerous flagella and, if these are co-located on the surface of the cell to form a tuft, the bacterium is lophotrichous; for example, Vibrio fischeri (c) and Spirillum spp. Peritrichous flagella are distributed all over the cell; for example Escherichia coli and Salmonella enterica serovar Typhimurium (d). For spirochaetes, such as species of Borrelia (e), Treponema and Leptospira, a specialized set of flagella are located in the periplasmic space, the rotation of which causes the entire bacterium to move forward in a corkscrew-like motion.

Fig. 4.2 : 

Fig. 4.2

Slide 23: 

Rotation of bacterial flagella is powered by a proton- or sodium-motive force. The flagellar motor converts electrochemical energy into torque through an interaction between two components: the stator and the rotor. The stator consists of multiple copies of two proteins, MotA and MotB, which assemble into a structure that is associated with the inner membrane and anchored to peptidoglycan, so that it remains stationary. The rotor consists of multiple copies of FliG, which together with FliM and FliN form the C ring, mounted on the cytoplasmic face of the MS ring. Torque is transmitted from the C-ring by the MS ring to the rod (a molecular drive shaft) and from there to the hook (a universal joint) and then on to the helical flagellar filament (a molecular propeller). Rotation of this helical filament converts torque into thrust, conferring motility on the cell. The chemotaxis apparatus (not shown) integrates diverse signals to modulate the behaviour of the motor so as to propel the cell towards nutrients. Several soluble factors are involved in coordinating the assembly of the flagellar apparatus, including the flagellar factor FliA and the hook-length control protein, FliK. An excellent movie illustrating flagellar biosynthesis and structure is available, see Further information. L, lipopolysaccharide; MS, membrane/super membrane; P, peptidoglycan.

Slide 24: 

Flagellar components of Salmonella enterica serovar Typhimurium Soluble cytoplasmic components include the FliH–FliI–FliJ ATPase complex, which is thought to deliver a number of the secreted substrates and help determine the order of substrate secretion. The filament protein consists of either FliC or FljB, which are alternately transcribed. The rod cap (FlgJ) and the hook cap (FlgD) are transiently associated with the flagellum during rod and hook polymerization, respectively. FliK is secreted during rod–hook polymerization as a molecular ruler that couples rod–hook length to the flagellar secretion specificity switch at FlhB.

Slide 25: 

The flhDC operon, or flagellar master operon, is under the control of numerous global regulatory signals that lead to the expression or inhibition of flagellar gene expression. Induction of the class I flhDC operon (class I on) produces FlhD and FlhC, which form a heteromultimeric complex, FlhD4C2, that acts to direct 70-dependent transcription from class II flagellar promoters and auto-repress flhDC transcription (class I off; class II on). Class II promoters direct the transcription of genes that are necessary for the structure and assembly of the hook–basal body (HBB) substructure. Upon HBB completion, late secretion substrates are exported from the cell and their cognate chaperones are released to regulate gene expression. FliT is an FlhD4C2 factor and prevents both FlhD4C2 auto-repression and the activation of class II promoters. The 28 transcription factor directs the transcription of class III promoters, which include the filament structural genes and the genes of the chemosensory pathway (class I on; class II off; and class III on). Activation of class I transcription would re-initiate the flagellar regulon for a new round of flagellar gene expression. As drawn, the FliK and FlhA proteins are meant to reside within the C ring. The stoichiometries of Fluke, FlhA, FlhB, FliO, FliP, FliQ and FliR within the C ring are not known.

Slide 26: 

The filament of the bacterial flagellum is connected to a hook which, in turn, is attached to a rod. The basal body of the flagellum consists of a rod and a series of rings that anchor the flagellum to the cell wall and the cytoplasmic membrane. In gram-negative bacteria, the L ring anchors the flagellum to the lipopolysaccharide layer of the outer membrane while the P ring anchors the flagellum to the peptidoglycan portion of the cell wall. The MS ring is located in the cytoplasmic membrane and the C ring in the cytoplasm. The Mot proteins surround the MS and C rings of the motor and function to generate torque for rotation of the flagellum. Energy for rotation comes from proton motive force. Protons moving through the Mot proteins drives rotation. The Fli proteins act as the motor switch to trigger either clockwise or counterclockwise rotation of the flagellum and to possibly disengage the rod in order to stop motility.

Slide 27: 

Summary of steps in flagella biosynthesis. Synthesis begins with MS ring assembly in the membrane. This is followed by formation of the other rings, the hook and the cap. At this point, flagellin protein (approximately 20,000 copies are needed to make one filament) flows through the hook to form the filament. Flagellin molecules are guided into position by cap proteins to ensure that the growing filament develops evenly.

Slide 28: 

Cilia and flagella are made up of microtubules, which are composed of linear polymers of globular proteins called tubulin. The core (axoneme) contains two central fibers that are surrounded by an outer ring of nine double fibers and covered by the cellular membrane.

Axial Filament or endoflagella : 

Axial Filament or endoflagella

Cell Surfaces : 

Cell Surfaces Endoflagella In spirochetes Anchored at one end of a cell Rotation causes cell to move Fimbriae allow attachment Pili are used to transfer DNA from one cell to another Fimbriae and Pili are characteristics of gram negative staining

Slide 31: 

Some properties of pili and fimbriae

Cell Wall : 

Cell Wall Prevents osmotic lysis Made of peptidoglycan (in bacteria) Peptidoglycan Polymer of disaccharideN-acetylglucosamine (NAG) & N-acetylmuramic acid (NAM) Linked by polypeptides

Slide 35: 

Gram positive Gram negative

Peptidoglycan : 

Peptidoglycan single macromolecule highly cross-linked surrounds cell provides rigidity

Peptidoglycan : 

Peptidoglycan glycan backbone muramic acid glucosamine peptide side chain peptide cross-bridge D- and L- amino acids diaminopimelic acid

Slide 38: 

Muramic acid, D-amino acids diaminopimelic acid not synthesized by mammals

Peptidoglycan : 

Peptidoglycan Muramic acid Glucosamine L-alanine D-glutamic acid L-lysine/Diaminopimelic acid D-alanine D-alanine

Slide 40: 

Gram Positive Cell Envelope Teichoic acid polymer phosphorus ribitol or glycerol backbone Teichuronic acid polymer no phosphorus glucuronic acid backbone

Teichoic and teichuronic acids : 

Teichoic and teichuronic acids Metal ion uptake Direct autolytic enzymes holes punched in cell wall allows insertion cell wall (synthesis)

Lipoteichoic acids : 

Lipoteichoic acids cell membrane autolysins kept from cell wall

Slide 43: 

Gram Positive Cell Envelope Cytoplasm Lipoteichoic acid Peptidoglycan-teichoic acid Cytoplasmic membrane

Slide 44: 

Braun lipoprotein binds cell wall to outer membrane Gram Negative Peptidoglycan The space between the inner and the outer membranes is called the periplasmic space.

Outer Membrane : 

Outer Membrane lipopolysaccharide phospholipids Proteins porins

Lipopolysaccharide : 

Lipopolysaccharide Lipid A Glucosamine disaccharide Beta hydroxy fatty acids Core Heptoses Ketodeoxyoctonic acid O-antigen Highly variable n

Gram Negative Cell Envelope : 

Cytoplasm Inner (cytoplasmic) membrane Outer Membrane Lipopolysaccharide Porin Braun lipoprotein Gram Negative Cell Envelope Peptidoglycan

Slide 48: 

Acid Fast Cell Envelope Cytoplasm Peptidoglycan-mycolic acid-arabinogalactan Cytoplasmic membrane Mycolic acid lipids

Peptidoglycan synthesis : 

Peptidoglycan synthesis Cytoplasm Cell wall undecaprenol sugar amino acid Cell Membrane

Slide 50: 

Correlation of Grams stain with other properties of Bacteria

Lipopolysaccharide : 

Lipopolysaccharide synthesis similar to peptidoglycan also on undecaprenol carrier

Gram positive versus gram negative : 

Gram positive versus gram negative

Gram Staining : 

Gram Staining Crystal violet-iodine crystals form in cell Gram-positive Alcohol dehydrates peptidoglycan CV-I crystals do not leave Gram-negative Alcohol dissolves outer membrane and leaves holes in peptidoglycan CV-I washes out

Slide 55: 

Schematic illustration of the outer membrane, cell wall and plasma membrane of a Gram-negative bacterium. Note the structure and arrangement of molecules that constitute the outer membrane

Slide 56: 

Most procaryotes contain some sort of a polysaccharide layer outside of the cell wall polymer. In a general sense, this layer is called a capsule. A true capsule is a discrete detectable layer of polysaccharides deposited outside the cell wall. A less discrete structure or matrix which embeds the cells is a called a slime layer or a biofilm. A type of capsule found in bacteria called a glycocalyx is a thin layer of tangled polysaccharide fibers which occurs on  surface of cells growing in nature (as opposed to the laboratory). Some microbiologists refer to all capsules as glycocalyx and do not differentiate microcapsules.

Slide 58: 

Capsules have several functions and often have multiple functions in a particular organism. Like fimbriae, capsules, slime layers, and glycocalyx often mediate adherence of cells to surfaces. Capsules also protect bacterial cells from engulfment by predatory protozoa or white blood cells (phagocytes), or from attack by antimicrobial agents of plant or animal origin. Capsules in certain soil bacteria protect cells from perennial effects of drying or desiccation. Capsular materials (e.g. dextrans) may be overproduced when bacteria are fed sugars to become reserves of carbohydrate for subsequent metabolism.

Slide 61: 

Chemical composition of some bacterial capsules Gram-positive Bacteria Gram-negative Bacteria

Glycocalyx : 

Glycocalyx Outside cell wall Usually sticky A capsule is neatly organized A slime layer is unorganized & loose Extracellular polysaccharide allows cell to attach Capsules prevent phagocytosis

Slide 65: 

The structure of the muramic acid subunit of the peptidoglycan of Escherichia coli. This is the type of murein found in most Gram-negative bacteria. The glycan backbone is a repeat polymer of two amino sugars, N-acetylglucosamine (G) and N-acetylmuramic acid (M). Attached to the N-acetylmuramic acid is a tetrapeptide consisting of L-ala-D-glu-DAP-D-ala. b. Abbreviated structure of the muramic acid subunit. c. Nearby tetrapeptide side chains may be linked to one another by an interpeptide bond between DAP on one chain and D-ala on the other. d. The polymeric form of the molecule.

Fig. 4.14 : 

Fig. 4.14

Gram summary : 

Gram summary Gram-Positive vs. Gram-Negative Thick peptidoglycan Teichoic acids In acid-fast cells, contains mycolic acid Thin peptidoglycan No teichoic acids Outer membrane

Slide 69: 

Species of Archea have cell walls, but they use a different compound, called pseudopeptidoglycan. Other species in Archea use complexes of other polysaccharides, glycoproteins and proteins. The S-layer is the most common type of cell wall in Archea however. The S-layer is a paracystalline layer with generally hexagonal symmetry. It is generally composed of a identical proteins or glycoproteins, which vary between species. The nature of the molecule that composes the S-layer allows it to self assemble, however it is poorly conserved and varies widely between species. Cell Walls in Archea

Fig. 4.18 : 

Fig. 4.18

Shapes : 

Shapes Cell shapes Coccus– spherical Rod– Bacillus Vibrio Spiral Occurrences Pairs: diplococci, diplobacilli Clusters: staphylococci Chains: streptococci, streptobacilli

Slide 73: 

73 Staphylococcus aureus

Slide 75: 

Haloquadra walsbyi Square -shaped bacteria Thiopedia rosea

Atypical Cell Walls : 

Atypical Cell Walls Mycoplasma – very small size, no cell wall, smallest known “life” Archaea– often have unusual cell walls or lack entirely.

Slide 77: 

Internal structures are microscopically visible bodies in the cell that are distinguishable from the general cytoplasm. In most cases they serve some special purpose.

Slide 78: 

Inclusions are aggregates of various compounds that are normally involved in storing energy reserves or building blocks for the cell. Inclusions accumulate when a cell is grown in the presence of excess nutrients and they are often observed under laboratory conditions.

Slide 79: 

Often contained in the cytoplasm of procaryotic cells is one or another of some type of inclusion granule. Inclusions are distinct granules that may occupy a substantial part of the cytoplasm. Inclusion granules are usually reserve materials of some sort. For example, carbon and energy reserves may be stored as glycogen (a polymer of glucose) or as polybetahydroxybutyric acid (a type of fat) granules. Polyphosphate inclusions are reserves of PO4 and possibly energy; elemental sulfur (sulfur globules) are stored by some phototrophic and some lithotrophic procaryotes as reserves of energy or electrons. Some inclusion bodies are actually membranous vesicles or intrusions into the cytoplasm which contain photosynthetic pigments or enzymes.

Slide 81: 

A variety of bacterial inclusions.  a. PHB granules; b. a parasporal BT crystal in the sporangium of Bacillus thuringiensis; c. carboxysomes in Anabaena viriabilis, showing their polyhedral shape; d. sulfur globules in the cytoplasm of Beggiatoa

Phosphate globules : 

Phosphate globules Many organisms will accumulate granules of polyphosphate, since this is a limiting nutrient in the environment. The globules are long chains of phosphate. Volutin granules are an intracytoplasmic storage form of complexed inorganic polyphosphate, the production of which is used as one of the identifying criteria when attempting to isolate Corynebacterium diptheriae on Löffler's medium.

Sulfur globules : 

Sulfur globules Photosynthetic bacteria that do not evolve oxygen will often use sulfides as their source of electrons, some of them accumilate sulfur globules. These globules may later be further oxidized and disappear if the sulfide pool dries up. 30-200nm

Magnetosomes : 

Magnetosomes "magnetotactic"microorganisms use a miniature, cellular compass made of a chain of single nanomagnets, called magnetosomes Magnetosome crystals are typically 35–120 nm long (some times up to 200 nm) One large, rod-shaped organism, Magnetobacterium bavaricum, contains up to 1000 bulletshaped magnetosomes arranged in several chains traversing the cell.

Slide 85: 

Magnetotactic bacteria usually mineralize either iron oxide magnetosomes, which contain crystals of magnetite (Fe3O4), or iron sulfide magnetosomes, which contain crystals of greigite (Fe3S4). Several other iron sulfide minerals have also been identified in iron sulfide magnetosomes — including mackinawite (tetragonal FeS) and a cubic FeS — which are thought to be precursors of Fe3S4.

Glycogen : 

Glycogen Glycogen is another common carbon and energy stroage product. Glycogen is a highly branched polymer of about 60,000 glucose residues. It has a molecular weight between 106 and 107 daltons.

Gas vesicles : 

Gas vesicles Gas vesicles are the exception to the rule that all bacterial cells have one contiguous membrane. Gas vesicles are found in Cyanobacteria, which are photosynthetic and live in aquatic systems. In these lakes and oceans, the Cyanonbacteria want to control their position in the water column to obtain the optimum amount of light and nutrients. 300-1000nm X 45-120nm

Slide 89: 

Structure Gas vesicles are aggregates of hollow cylindrical structures composed of rigid proteins. They are impermeable to water, but permeable to gas. The amount of gas in the vacuole is under the control of the microorganism. Function Gas vesicles regulate the buoancy of the microbes by changing the amount of gas contained within them. Release of gas from the vesicle causes the bacteria to fall in the water column, while filling the vesicle with gas increases their height in the water.

Slide 90: 

Poly-beta-hydroxyalkanoate (PHA) One of the more common storage inclusions is PHA. It is a long polymer of repeating hydrophobic units that can have various carbon chains attached to them. The most common form of this class of polymers is poly-beta-hydroxybutyrate that has a methyl group as the side chain to the molecule. (see figure below). Some PHA polymers have plastic like qualities and there is some interest in exploiting them as a form of biodegradable plastic. The function of PHA in bacteria is as a carbon and energy storage product. Just as we store fat, bacteria store PHA

Slide 91: 

Poly-beta-hydroxybutyrate (PHB) in a Rhodospirillum species. PHB is one type of PHA. structure of a PHA monomer Poly-β-hydroxybutyrate (PHB) is a lipid-like compound. It is formed from β-hydroxybutyrate units joined by ester-linkages resulting in long PHB polymer which aggregate into granules of around 0.2-0.7 μm in diameter. PHB is accumulated by aerobic and facultative bacteria when the cells are deprived of oxygen and must carry out fermentative metabolism. On return of aerobic conditions, PHB is used as a n energy and carbon source and incorporated into the oxidative metabolism. Rhodovibrio sodomensis

Slide 92: 

Figure. Some examples of various monomers of PHAs

Slide 93: 

Figure . Cyclic biosynthesis and degradation metabolism of PHB

Slide 94: 

Dashed lines represent enzymatic steps engineered in recombinant E. coli. Solid lines represent enzymatic steps existed in wild type E. coli. A question mark denotes unidentified enzyme in E. coli. ① β-ketothiolase, PhaA ② NADPH-dependent acetoacetyl-CoA reductase, PhaB ③ PHB synthase, PhaC ④ PHB depolymerase, PhaZ ⑤ acyl-CoA synthetase or thioesterase ⑥ 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxybutyryl-CoA epimerase, FadB ⑦ acetoacetyl-CoA transferase, AtoA, AtoD ⑧ acetyl-CoA acetyltransferase, AtoB. Metabolic network between PHB biosynthesis and mobilization in recombinant E. coli DH5α (pQWQ2/pSCP-CAB).

Carboxysomes : 

Carboxysomes Carboxysomes are bacterial microcompartments that contain enzymes involved in carbon fixation. Carboxysomes are made of polyhedral protein shells about 80 to 140 nanometres in diameter. These compartments are thought to concentrate carbon dioxide to overcome the inefficiency of RuBisCo - the predominant enzyme in carbon fixation and the rate limiting enzyme in the Calvin cycle. These structures are found in all cyanobacteria and many chemotrophic bacteria that fix carbon dioxide. Bacterial microcompartmentalization will lead to the development of new materials with a wide range of applications in medicine, agriculture, and engineering. Halothiobacillus neapolitanus

Slide 96: 

(A) Electron micrograph of Halothiobacillus neapolitanus cells, arrows highlight carboxysomes. (B) Image of intact carboxysomes isolated from H. neapolitanus. Scale bars are 100 nm. Prochlorococcus marinus Rubisco Prochlorococcus may be the most abundant photosynthetic organism on Earth. carboxysomes in green

Slide 97: 

(Left, above) A thin-section electron micrograph of H. neapolitanus cells with carboxysomes inside. In one of the cells shown, arrows highlight the visible carboxysomes. (Left, below) Purified carboxysomes (material courtesy of S. Heinhorst and G. Cannon) as visualized by cryo-electron microscopy (courtesy of M. Yeager and K. Dryden). (right) Models for the structure of the carboxysome. Current data suggest that the shell is composed of several hundred hexameric protein building blocks and 12 pentameric building blocks. The three-dimensional atomic structures of the shell proteins have been determined by X-ray crystallography. RuBisCO, the main interior enzyme is shown packed inside in a regular arrangement for simplicity, though the actual organization of the enzymes is not understood yet. The other key enzyme, carbonic anhydrase, which is present in lesser amounts, is not illustrated. Scale bars are 100 nm.

Slide 98: 

Phycobilisomes: Phycobilisomes are light harvesting pigments found throughout the Cyanobacteria (and in red algae). All other photosynthetic bacteria use either light harvesting chlorophyll or Chlorosomes. Phycobilisomes, which specialize in absorbing deep penetrating green light (more than 1 meter deep, e.g., in marine environments), are capable of re-emitting light in regions which other photosynthetic pigments absorb. Phycobilisomes appear to have been very advantageous to early Cyanobacteria. Perhaps evolving in shallow marine environments protected from UV radiation, these early phycobilisomes were ideally suited for shallow marine photosynthesis. There is a central core of light harvesting complex (Allophycocyanin) which sits above the photosynthetic reaction center. There are phycocyanin and phycoerythrin subunits which radiate out from this center like thin tubes (increases surface area). The fluorescent pigments which are present in the phycobilisome, such as phycocyanobilin (Phycocyanin) and phycoerythrobilin (Phycoerythrin) re-emit the green light in regions.

Slide 99: 

Light harvesting antennae or phycobilisome Prochlorococcus Synechococcus

Slide 100: 

Anabaena

Slide 101: 

Chlorosomes are found in the green sulfur bacteria, family Chlorobiaceae). Light and excitation energy transfer is shown in red, electron transfer in blue.

Cellulosomes: plant-cell-wall-degrading enzyme complexes : 

Cellulosomes: plant-cell-wall-degrading enzyme complexes Bacterial cellulosomal systems can be categorized into 2 major types: simple cellulosome systems contain a single scaffoldin and complex cellulosome systems exhibit multiple types of interacting scaffoldins. The genes encoding for many important cellulosome subunits are organized in “enzyme-linked gene clusters” on the chromosome. In the simple cellulosome systems, the scaffoldin gene is followed downstream by a series of genes that code for dockerin-bearing enzymes.

Slide 105: 

The scaffoldin protein is shown with its cellulose-binding domain (CBD), nine cohesins, four hydrophilic domains and nine cellulosomal enzymes bound to the scaffoldin through their dockerins. EngE is believed to tether the C. cellulovorans cellulosome to the cell surface scaffoldin domain (protein)

Slide 106: 

The cellulosome system of Ruminococcus flavefaciens proved to be the most elaborate known to date, and their divergent cohesins and dockerins are classified as type III. In addition to the two major scaffoldins; a small single-cohesin scaffoldin (ScaC) serves to enrich the repertoire of enzyme subunits as a function of carbon source. Unlike all other cellulosomes, its anchoring scaffoldin exhibits an unconventional sortase-mediated covalent mode of attachment to the cell surface. A specialized cellulose-binding protein (CttA) serves to bind the cell to the cellulose substrate, instead of a CBM as a component of the primary scaffoldin.

Slide 107: 

A. One of the most common types of “free” (non-cellulosomal) cellulases consists of a catalytic module, flanked by a CBM at its N- or C-terminus. B. Cellulosomal enzymes are characterized by a dockerin module attached to a catalytic domain. C. Many cellulases contain "X modules", i.e., modules of unknown (as yet undefined) function. D. Some enzymes have more than one CBM. Often, one CBM serves to target the cellulase to the flat surface of the insoluble substrate, whereas the other acts in concert with the catalytic module by binding transiently to one cellulose or hemicellulose chain. E. Some cellulosomal cellulases have a CBM together with a dockerin in the same polypeptide chain. E. Some cellulases have more than one type of catalytic module in the same polypeptide chain, together with one or more CBMs and/or a dockerin.

Slide 108: 

Structures of a typical endoglucanase and exoglucanase. Despite the sequence similarity of both enzymes, their respective active-site architecture is different. The endoglucanase exhibits a deep cleft to accommodate the cellulose chain at any point along its length, whereas the active site of the exoglucanase bears an extended loop that forms a tunnel, through which one of the termini of a cellulose chain can be threaded.

Slide 109: 

In the simple cellulosome systems, the scaffoldins contain a single CBM, one or more X2 modules and numerous (5 to 9) cohesins. These scaffoldins are primary scaffoldins, which incorporate the dockerin-bearing enzymes into the complex. In several cases, the simple cellulosomes have been shown to be associated with the cell surface, but the molecular mechanism responsible for this is still unclear. The X2 module may play a role in attachment to the cell wall. The scaffoldins of simple cellulosome systems are given in the following bacteria are shown:

Slide 110: 

Complex cellulosome systems have been described in four different bacterial species. In these systems, more than one scaffoldin interlocks with each other in various ways to produce a complex cellulosome architecture. At least one type of scaffoldin serves as a primary scaffoldin that incorporates the enzymes directly into the cellulosome complex. In each species, another type of scaffoldin attaches the cellulosome complex to the cell surface via a specialized module or sequence, designed for this purpose. Schematic representations of complex cellulosome systems of the following bacteria are shown:

The endospore : 

Cortex (P) The endospore Exosporium Coat Inner membrane Outer membrane Core Endospores are recognized as the hardiest known form of life on Earth

Slide 112: 

Variations in endospore morphology. (1, 4) Central endospore, (2, 3, 5) terminal endospore, (6) lateral endospore Bacillus cereus B. subtilis, B. anthracis, B. licheniformis Bacillus thuringiensis B. macerans B. circulans B. sphaericus B. polymixa B. laterosporus Metabacterium polyspora

Slide 113: 

Examples of endospore-forming bacteria include the genera: Totally 22, only 2 are non-rods Bacillus Clostridium Desulfotomaculum Sporolactobacillus Sporosarcina Thermoactinomyces Oscillospira Metabacterium polyspora

Slide 114: 

Sporeforming bacteria are widespread within low-G+C subdivision of the gram+ bacteria and represent inhabitants of diverse habitats, such as aerobic heterotrophs (Bacillus, Paenibacillus and Sporosarcina spp.), halophiles (Sporosarcina halophila and the gram -ve, Sporohalobacter spp.), microaerophilic lactate fermenters (Sporolactobacillus spp.), anaerobes (Clostridium Oscillospira and Anaerobacter spp.), sufate reducers (Desulfotomaculum spp., Sulfobacillus), and even phototrophs (Heliobacterium, Heliobacillus, Heliorestis and Heliophilum spp.), symbionts (Epulopiscium) Syntrophospora, Sporomusa, Alicylobacillus,

Slide 115: 

Parasporal Crystal Many Bacillus thuringiensis (Bt) isolates produce crystalline inclusions on sporulation. These parasporal crystals may contain potent insecticidal delta endotoxins classed as either crystal toxins (Cry) or cytolytic toxins (Cyt).

Slide 116: 

The spore-forming bacterium Bacillus thuringiensis bears plasmids encoding genes for insecticidal proteins typically synthesized during sporulation. These proteins crystallize forming large polyhedral parasporal inclusions that make up as much as 30% of the cellular dry weight. When ingested by insects and certain other arthropods, these inclusions dissolve and the proteins are cleaved by proteases releasing active toxins that bind to specific receptors on the host’s midgut membrane.

Slide 117: 

Activated toxins then oligomerize, inserting into this membrane where they form cation-selective channels that cause cell lysis and host death. Two types of crystal proteins are recognized, Cry proteins, the most common, and Cyt proteins. Cry proteins are generally either 60–80 or 130–150 kDa, the former being truncated versions of the latter. Cyt proteins are ~ 28 kDa, and are unrelated to Cry proteins, having an affinity for midgut membrane lipids. Cry and Cyt proteins are the active ingredients of many commercial insecticides. Several Cry proteins are the basis of transgenic insecticidal crops such as Bt cotton and Bt corn, now a multibillion dollar global industry.

Slide 118: 

A. Sporulated culture of Bacillus thuringiensis illustrating the spore and toxin-containing parasporal body. B. Parasporal body protein inclusions containing Cry proteins produced by the HD1 isolate of B.t. kurstaki, the Bt isolate used most widely in products for control of lepidopterous pests. C. Protein inclusion characteristic of B.t. israelensis used widely to control the larvae of mosquitoes and blackflies.

Slide 119: 

Bt, is recognized by its parasporal body (known as the crystal) that is proteinaceous in nature and which possesses insecticidal properties. The parasporal body comprises of crystals varying in size, shape and morphology. The crystals are tightly packed with proteins called protoxins or-endotoxins.

Slide 122: 

The Bt toxins exert their toxicity by forming pores in the larval midgut epithelial membranes. Initially the protoxins are activated in the midgut by trypsin-like proteases to toxins (see figure below). The active toxins bind to specific receptors located on the apical brush border membrane of the columnar cells. Binding is followed by insertion of the toxin into the apical membrane leading to pore formation (see figure below). The formation of toxin-induced pores in the columnar cell apical membrane allows rapid fluxes of ions. Different studies revealed that the pores are K+ selective, permeable to cations, anions or permeable to small solutes like sucrose, irrespective of the charge. It appears that the toxin forms or activates a relatively large aqueous channel in the membrane. The disruption of gut integrity results in the death of the insect from starvation or septicemia.

Slide 123: 

More than 60 different Cry proteins and 4 Cyt proteins have been isolated over the past 20 years. All are encoded by genes carried on plasmids, circular pieces of DNA that can be transmitted from one Bt subspecies to another.

Slide 124: 

An insecticide first identified from a gram positive parasporal bacteria, Bacillus thuringiensis, capable of producing crystallized insecticidal compounds (Cry toxins)

Slide 125: 

A classification for crystal protein genes of Bacillus thuringiensis Criteria used are the insecticidal spectra and the amino acid sequences of the encoded proteins. Fourteen genes are distinguished, encoding proteins active against either Lepidoptera (cryI), Lepidoptera and Diptera (cryII), Coleoptera (cryIII), or Diptera (cryIV). One gene, cytA, encodes a general cytolytic protein and shows no structural similarities with the other genes. Toxicity studies with single purified proteins demonstrated that every described crystal protein is characterized by a highly specific, and sometimes very restricted, insect host spectrum. Comparison of the deduced amino acid sequences reveals sequence elements which are conserved for Cry proteins.

Slide 126: 

Bacillus thuringiensis is distinguished from the very closely related Bacillus cereus and Bacillus anthracis by the presence of several plasmid-encoded delta-endotoxin genes. These delta-endotoxins, synthesized as protoxins, are produced in large quantities during sporulation and are packaged into intracellular inclusions. Ingestion of the inclusions by insect larvae leads to protoxin solubilization and conversion to toxins each specific for one of several orders of insects. The toxins form cation-selective channels in the membrane of cells lining the larval midgut with subsequent lethality.

Slide 127: 

In most cases, delta-endotoxin synthesis and sporulation are closely coupled. The latter process in B. thuringiensis is probably virtually identical to that in Bacillus subtilis with the additional use of mother cell sporulation forms of RNA polymerase for the synthesis of the delta-endotoxins. There are other more subtle plasmid-encoded functions or plasmid interactions related to regulating protoxin synthesis. Consideration of both plasmid and chromosomal genes is thus critical for defining this organism.

Slide 128: 

The role Cry and Cyt proteins play in the biology of B. thuringiensis, and then focus on the synthesis and structure of the crystals they form. Their synthesis can be manipulated with recombinant DNA techniques to increase crystal size and improve insecticidal activity.

Slide 130: 

Bacillus thuringiensis (Bt) bacteria produce insecticidal Cry and Cyt proteins used in the biological control of different insect pests. In this review, we will focus on the 3d‑Cry toxins that represent the biggest group of Cry proteins and also on Cyt toxins. The 3d‑Cry toxins are pore‑forming toxins that induce cell death by forming ionic pores into the membrane of the midgut epithelial cells in their target insect. The initial steps in the mode of action include ingestion of the protoxin, activation by midgut proteases to produce the toxin fragment and the interaction with the primary cadherin receptor. The interaction of the monomeric Cry1A toxin with the cadherin receptor promotes an extra proteolytic cleavage, where helix α‑1 of domain I is eliminated and the toxin oligomerization is induced, forming a structure of 250 kDa. The oligomeric structure binds to a secondary receptor, aminopeptidase N or alkaline phosphatase. The secondary receptor drives the toxin into detergent resistant membrane microdomains forming pores that cause osmotic shock, burst of the midgut cells and insect death. Regarding to Cyt toxins, these proteins have a synergistic effect on the toxicity of some Cry toxins. Cyt proteins are also proteolytic activated in the midgut lumen of their target, they bind to some phospholipids present in the mosquito midgut cells. The proposed mechanism of synergism between Cry and Cyt toxins is that Cyt1Aa function as a receptor for Cry toxins. The Cyt1A inserts into midgut epithelium membrane and exposes protein regions that are recognized by Cry11Aa. It was demonstrated that this interaction facilitates the oligomerization of Cry11Aa and also its pore formation activity.

Slide 133: 

Proteolytic activation and mechanisms of insecticidal action of Bacillus thuringiensis endotoxins (left) and plant ureases (right). To exert their action, insecticidal proteins need to be ingested by target insects (1). Once inside the insect's midgut, the protoxins are cleaved (2) by host digestive enzymes (trypsin-or chymotrypin-like in the case of Bt toxins; cathepsin-like for plant ureases) releasing the active peptide fragment. The ∼55 kDa active Bt toxins insert into the membrane of midgut epithelial cells forming pores (3) and causing an electrolyte imbalance that leads insects to death. The ∼10 kDa fragment released from C. ensiformis ureases is cleared from the midgut and circulates in the hemolymph reaching other tissues (4) such as Malphigian tubules and the central nervous system, thereby killing the insect by mechanisms that await elucidation.

Slide 134: 

The case of Starlink corn, a plant modified with a gene that encodes the Bt protein Cry9c, was a severe test of U.S. regulatory agencies.

Slide 137: 

Another group of bacteria called Methylosinus <meth-ill-oh-sigh-nus> produces spores called exospores. The difference between endospores and exospores is mainly in how they form. Endospores form inside the original bacterial cell, as described above. Exospores form outside by growing or budding out from one end of the cell. Exospores also don’t have all the same building blocks as endospores, but they’re similarly durable. Exospores Methylosinus trichosporium Rhodomicrobium vannielii Exospores of Methylosinus trichosporium which form at the tapered end of the vegetative cell are resistant to heat and desiccation but, unlike bacterial endospores, contain no dipicolinic acid vegetative cell capsule (VC) and the characteristic groove (G) can be seen. immature exospore (IE) VC

Slide 138: 

Fig. 18 to 21. Exospores of ‘Metlzylosinus trichosporium’ strain PG losing capsule which is probably attached to outer wall of spore (see P1. 5, fig. 66) and therefore indicates that outer wall is being shed. Fig. 22 to 23. Germinated exospore of ‘Methylosinus trichosporium’ strain PG forming an exospore. Fig. 24. Preparation (Indian ink omitted) of sporing organism (‘ Methylosinus trichosporiunz’ strain PG). Fig. 25 to 27. Rosettes of organisms of ‘Methylosinus trichosporium’ strain PG in different stages of spore formation. Fig. 28. Rosette of lysed organisms of ‘ Methylosinus trichosporium ’ strain PG. Organisms appear to open at spore bearing pole. Fig. 29. Exospores, vegetative and sporulating organisms of ‘ Methylosinus sporium’ strain

Slide 139: 

Plasmids exist and replicate independently of the nucleiod. Plasmids carry between 2 and 30 genes. Plasmids have relatively few genes (fewer than 30). The genetic information of the plasmid is usually not essential to survival of the host bacteria. Plasmids are extra chromosomal elements of circular DNA in bacteria, which replicate independent of the host genome. It can be removed from the host cell in the process of curing. Curing may occur spontaneously or may be induced by treatments such as ultraviolet light. Certain plasmids, called episomes, may be integrated into the bacterial chromosome. Others contain genes for certain types of pili and are able to transfer copies of themselves to other bacteria. Such plasmids are referred to as conjugative plasmids. A special plasmid called a fertility (F) factor plays an important role in conjugation. The F factor contains genes that encourage cellular attachment during conjugation and accelerate plasmid transfer between conjugating bacterial cells. Those cells contributing DNA are called F+ (donor) cells , while those receiving DNA are the F- (recipient) cells. The F factor can exist outside the bacterial chromosome or may be integrated into the chromosome. Plasmids contain genes that impart antibiotic resistance. Up to eight genes for resisting eight different antibiotics may be found on a single plasmid. Genes that encode a series of bacteriocins are also found on plasmids. Bacteriocins are bacterial proteins capable of destroying other bacteria. Still other plasmids increase the pathogenicity of their host bacteria because the plasmid contains genes for toxin synthesis. The term "plasmid" had been coined by J. Lederberg in 1952

Slide 140: 

Charcteristics of Plasmids - 1. It should be small (40 kb), supercoiled, covalently closed circular (CCC) DNA and exist as an extrachromosomal element.2. It should carry a multiple cloning site or polycloning region for insertion of the gene of interest.3. It should possess atleast two selectable markers one of which may be an antibiotic resistance gene. 4. It should be present in large numbers per cell.5. It must have an origin of replication.6. It mayor may not contain a promoter to express the gene of interest.Plasmids are classified into various groups based upon various characteristics.Based upon the ability to take part in conjugation, plasmids are of two types. Conjugative plasmids are those which take part in conjugation. They have tra genes which help in conjugation, e.g. P. Plasmids which lack them are called as non conjugative plasmid, e.g. pBR 322. Based upon the number of copies per cell, plasmids are classified into two types.1. Stringent plasmids These plasmids exist in small numbers, i.e. <100 copies/cell. Stringent plasmid is under the control of bacterial genome for replication and segregation. Generally, conjunctive plasmids are mostly stringent plasmids. 2. Relaxed plasmids These plasmids exist in large numbers, i.e., > 100 copies/cell. Relaxed plasmid is not under the control of bacterial genome for replication and segregation. Generally, relaxed plasmids are of low molecular weight and most of them are of the non conjugative type.

Slide 141: 

The most widely used method to find the copy number of the plasmid is to estimate the amount of enzyme encoded by genes present in the plasmid. For example, β-lactamase activity can be measured if the plasmid specifies ampicillin resistance.Sometimes plasmids are also classified into compatible groups, based upon plasmid incompatibility. Plasmid incompatibility is the inability of two different plasmids to co exist in the same cell in the absence of selection pressure. But this method is not widely used. transfer gene (tra) makes a protein that binds to the origin of replication site (ori). The protein nicks the DNA, relaxing it and allowing it to be transferred to another cell. nucleiod

Slide 143: 

(a) F-Plasmids (or F-factors)These are the first described plasmids that play major role in conjugation in bacteria. It is a circular ds-DNA molecule of 99,159 base pairs. The genetic map of the F-plasmid is shown in One region of the plasmid contains genes involved in regulation of the DNA replication (rep genes), the other region contains transposable elements (IS3 Tn 1000, IS3 and IS2 gene involved in its ability to function as an episome, and the third large region, the tra region, consist of tra genes and possess ability to promote transfer of plasmids during conjugation. Example F-­plasmid of E. coli. Genetic Map of the F (Fertility Plasmid of Escherichia coli. tea region contains tra genes involves in conjugative transfer; Ori T sequences is the origin of transfer during conjugation; transposable element region responsible for functioning as episome, and the rep genes regulate DNA replication.

Slide 144: 

(b) R-Plasmids These are the most widespread and well studied group of plasmids conferring resistance (hence called resistant plasmids) to antibiotics and various other growth inhibitors. R-plasmids typically have genes that code for enzymes able to destroy and modify antibiotics. They are not usually integrated into the host chromosome. Some R-plasmids possess only a single resistant gene whereas others can have as many as eight. Plasmid R 100, for example, is a 94.3 kilobase-pair plasmid that carries resistant genes for sulfonamides streptomycin and spectinomycin, chloramphenicol, tetracyclin etc. It also carries genes conferring resistance to murcury. Many R­-plasmids are conjugative and possess drug-resistant genes as transposable elements, they play an important role in medical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections. Escherichia coli. Genetic map of the resistance plasmid R100. Cat = Chloramphenicol resistance gene; str = Streptomycin resistance gene; sul = sulfonamide resistance gene, mer = mercury ion resistance gene, IS = insertion sequences

Slide 145: 

(c) Virulence-PlasmidsThese confer pathogenicity on the host bacterium. They make the bacterium more pathogenic as the bacterium is better able to resist host defence or to produce toxins. For example, Ti-plasmids of Agrobacterium tumefaciens induce crown gall disease of angiospermic plants: enterotoxilgenic strains of E. coli cause travelers diarrhea because of a plasmid that codes for an enterotoxin which induces extensive secretion of water and salts into the bowel. (d) Col-PlasmidsThese plasmids carry genes that confer ability to the host bacterium to kill other bacteria by secreting bacteriocins, a type of proteins. Bacteriocins often kill cells by creating channels in the plasmamembrane thus increasing its permeability. They also may degrade DNA or RNA or attack peptidoglycan and weaken the cell-wall. Bacteriocins act only against closely related strains. Col E1 plasmid of E. coli code for the synthesis of bacteriocin called colicins which kill other susceptible strains of E. coli.Col plasmids of some E. coli code for the synthesis of bacteriocin, namely cloacins that kill Enterobacter species. Lactic acid bacteria produce bacteriocin NisinA which strongly inhibits the growth of a wide variety of gram (+) bacteria and is used as a preservative in the food industry.

Slide 146: 

(e) Metabolic PlasmidsMetabolic plasmids (also called degradative plasmids) possess genes to code enzymes that degrade unusual substances such as toluene (aromatic compounds), pesticides (2, 4-dichloro­phenoxy acetic acid) and sugars (lactose). TOL (= pWWO) plasmid of Pseudomonas putida is an example. However, some metabolic plasmids occurring in certain strains of Rhizobium induce nodule formation in legumes and carry out fixation bf atmospheric nitrogen Degradative Plasmid Pseudomonas putida phenol and cyanide, Alcaligenes eutrophus polychlorinated biphenyls and 2,4- dichlorophenoxyacetic acid

Slide 147: 

The uptake of mobile genetic elements (phages, virulence plasmids and pathogenicity islands), as well as the loss of chromosomal-DNA regions in different E. coli lineages, has enabled the evolution of separate clones, which belong to different E. coli pathotypes and are associated with specific disease symptoms. ;;;. LEE, locus of enterocyte effacement PAI, pathogenicity island pEAF, enteropathogenic E. coli adhesion-factor plasmid pENT, enterotoxin-encoding plasmids Stx, Shiga-toxin-encoding bacteriophage

Slide 148: 

Transposable elements Transposable elements, also known as transposons, are segments of DNA that move about within the chromosome and establish new genetic sequences. First discovered by Barbara McClintock in the 1940s, transposons behave somewhat like lysogenic viruses except that they cannot exist apart from the chromosome or reproduce themselves. The simplest transposons, insertion sequences, are short sequences of DNA bounded at both ends by identical sequences of nucleotides in reverse orientation (inverted repeats). Insertion sequences can insert within a gene and cause a rearrangement mutation of the genetic material. If the sequence carries a stop codon, it may block transcription of the DNA during protein synthesis. Insertion sequences may also encourage the movement of drug-resistance genes between plasmids and chromosomes.

Transposons and transposable elements : 

Transposons and transposable elements Dr. M.K.SATEESH Bangalore University Bangalore-56

Jumping genes : 

Jumping genes Transposable elements were first discovered by Barbara McClintock in the 1950's and recognized as genetic elements which cause unstable mutations, produce chromosomal aberrations (including breaks) and which change location in the genome.

Slide 151: 

Barbara McClintock (1902-1992), 1983 Nobel Prize in Medicine or Physiology for discovering transposons, or mobile genetic elements

Insertion sequences : 

Insertion sequences They were "re-discovered" in bacteria in the 1970's as "insertion sequences", genetic elements which move from one genetic location to another and that cause premature transcriptional termination in bacteria operons.

Transposable elements : 

Transposable elements “mobile genetic elements” comprise 45% of human chromosomal DNA “middle repetitive DNA” contribute to spontaneous mutation, genetic rearrangements, horizontal transfer of genetic material aid speciation and genomic change (in bacteria transposons are often associated with antibiotic resistance genes) cells must depress transposition to insure genetic stability

Types of transposable elements : 

Types of transposable elements DNA vs. RNA viral vs. nonviral replicative mechanism vs. excision mechanism

Slide 155: 

transposon

Slide 156: 

transposon insertion mutation

Discovery of transposons : 

Discovery of transposons Barbara McClintock 1950’s Ac -Ds system in maize influencing kernel color, unstable elements, changing map position promote chromosomal breaks Rediscovery of bacterial insertion sequencessource of polar mutations discrete change in physical length of DNA inverted repeat ends: form “lollipops” in EM after denaturation/ reannealing IS (insertion sequences) were first discovered in the gal operon of E. coli

Transposable Elements in Prokaryotes : 

Transposable Elements in Prokaryotes Prokaryotic examples include: a. Insertion sequence (IS) elements. b. Transposons (Tn). c. Bacteriophage Mu (replicated by transposition) 1. IS elements are the simplest transposable elements found in prokaryotes, encoding only genes for mobilization and insertion of its DNA. IS elements are commonly found in bacterial chromosomes and plasmids. 2. IS elements were first identified in E. coli’s galactose operon, wheresome mutations’ were shown to result from insertion of a DNA sequence now called IS1

The insertion sequence (IS) transposable element, IS1 : 

The insertion sequence (IS) transposable element, IS1 inverted terminal repeats (IR) inverted terminal repeats (IRs)

Slide 160: 

Bacterial IS elements

Slide 161: 

Prokaryotic IS elements range in size from 768 bp to over 5 kb. Known E. coli IS elements include: a. IS1 is 768 bp long, and present in 4–19 copies on the E. coli chromosome. IS2 has 0–12 copies on the chromosome, and 1 copy on the F plasmid. IS10 is found in R plasmids. 4. The ends of all sequenced IS elements show inverted terminal repeats (IRs) of 9–41 bp (e.g., IS1 has 23 bp of nearly identical sequence).

Integration of IS elements may: : 

Integration of IS elements may: a. Disrupt coding sequences or regulatory regions. b. Alter expression of nearby genes by the action of IS element promoters. c. Cause deletions and inversions in adjacent DNA. d. Serve as a site for crossing-over between duplicated IS elements.

Slide 163: 

Insertion sequences are also commonly observed in the F factor The genome of the standard WT E. coli is rich in IS elements - These elements are mobile

When an IS element transposes: : 

When an IS element transposes: a. The original copy stays in place, and a new copy inserts randomly into the chromosome. b. The IS element uses the host cell replication enzymes for precise replication. c. Transposition requires transposase, an enzyme encoded by the IS element. d. Transposase recognizes the IR sequences to initiate transposition. e. IS elements insert into the chromosome without sequence homology (illegitimate recombination) at target sites (Figure 2). i. A staggered cut is made in the target site, and the IS element inserted. ii. DNA polymerase and ligase fill the gaps, producing small direct repeats of the target site flanking the IS element (target site duplications). f. Mutational analysis shows that IR sequences are the key

Schematic of the integration of an IS element into chromosomal DNA : 

Schematic of the integration of an IS element into chromosomal DNA

Transposons : 

Transposons Transposons are similar to IS elements, but carry additional genes, and have a more complex structure. There are two types of prokaryotic transposons: a. Composite transposons carry genes (e.g., antibiotic resistance) flanked on both sides by IS elements (IS modules). i. The IS elements are of the same type, and called ISL (left) and ISR (right). ii. ISL and ISR may be in direct or inverted orientation to each other. iii. Tn10 is an example of a composite transposon (Figure 3). It is 9.3 kb, and contains: (1) 6.5 kb of central DNA with genes that include tetracycline resistance (a selectable marker). (2) 1.4 kb IS elements (IS10L and IS10R) at each end, in an inverted orientation. iv. Transposition of composite transposons results from the IS elements, which supply transposase and its recognition signals, the IRs. (1) Tn10’s transposition is rare, because transpose is produced at a rate of ,1 molecule/generation. (2) Transposons, like IS elements, produce target site duplications (e.g., a 9-bp duplication for Tn10).

Slide 168: 

Bacterial transposons ISL (left) ISR (right)

Slide 169: 

The insertion of a Tn into a plasmid RTF: resistance-transfer functions

Composite bacterial transposons : 

Composite bacterial transposons repeated ends, usually inverted, sometimes direct repeated ends themselves are IS elements and can independently transpose ends mobilize all intervening DNA often antibiotic resistance genes (examples Tn3 (ampicillin), Tn5 (kanamycin), Tn10 (tetracycline) often reside on plasmids

Structure of the composite transposon Tn10 : 

Structure of the composite transposon Tn10

Structure of Tn3 : 

Structure of Tn3 4957 bp 3 trans-acting genes: 2 cis-acting sites:

Slide 173: 

b. Noncomposite transposons also carry genes (e.g., drug resistance) but do not terminate with IS elements. i. Transposition proteins are encoded in the central region. ii. The ends are repeated sequences (but not IS elements). iii. Noncomposite transposons cause target site duplications (like composite transposons). iv. An example is Tn3. (1) Tn3’s length is about 5 kb, with 38-bp inverted terminal repeats. (2) It has three genes in its central region: (a) bla encodes β-lactamase, which breaks down ampiciliin. (b) tnpA encodes transposase, needed for insertion into a new site. (c) tnpB encodes resolvase, involved in recombinational events needed for transposition (not found in all transposons). (3) Tn3 produces a 5-bp duplication upon insertion (Figure 5).

Structure of the noncomposite transposon Tn3 : 

Structure of the noncomposite transposon Tn3

DNA sequence of a target site of Tn3 : 

DNA sequence of a target site of Tn3

Slide 176: 

Physical structure of transposons The insertion of a Tn into a plasmid RTF: resistance-transfer functions

Slide 177: 

Prokaryotic transposons Movement of transposons Each transposon can be transferred independently

Organizational maps of bacterial plasmids with transposable elements : 

Organizational maps of bacterial plasmids with transposable elements

2 types of DNA tranposons : 

2 types of DNA tranposons Excisive mechanismexamples: Tn5, Tn10, P elements Replicative mechanismexamples: Tn3, bacteriophage Mu

Replicative transposons : 

Replicative transposons copy-and-paste mechanism orignal cut of transposon is only nick and only one strand is initially ligated element replicates through itself produces as intermediate a “co-integrate” structure co-integrate is resolved by resolvase (as TnpR of Tn3) and at specific site (as res of Tn3)

Slide 181: 

Mechanism of transposition in prokaryotes Replicative transposition * A new copy of the transposable element is generated in the transposition event * The structure of transposon 3 * Transposition of Tn3 takes place through a cointegrate intermediate

Excisive transposons : 

Excisive transposons cut-and-paste mechanism cut themselves out of original site, producing double strand break cut target site and ligate to element ends, thereby inserting at new site original site break repaired usually with sister chromosome, restoring transposon at original site sometimes end healed without transposon, can also be associated with deletion at excision site

Slide 183: 

Mechanism of transposition in prokaryotes Conservative transposition Some transposons excise from the chromosome and integrate Into the target DNA Generation of heteroduplex and homoduplex Tn10 elements

Slide 185: 

Phage mu can mediate the transposition of a bacterial gene into a plasmid Phage mu can cause deletion or inversion of adjacent bacterial segment Phage mu

Slide 186: 

The proteosome proteolytic complexes are known in nuclei and the cytosol.

Systematics : 

Systematics Taxonomy The science of classifying organisms Provides universal names for organisms Provides a reference for identifying organisms Systematics or phylogeny Using “key” characteristics to classify, or The study of the evolutionary history of organisms All Species Inventory (2001-2025) To identify all species of life on Earth

Species definitions : 

Species definitions Eukaryotic species: A group of closely related organisms that breed among themselves Prokaryotic species: A population of cells with similar characteristics Clone: Population of cells derived from a single cell Strain: Genetically different cells within a clone Viral species: Population of viruses with similar characteristics that occupies a particular ecological niche

Bacterial Systematics from Bergey’s Manual : 

Bacterial Systematics from Bergey’s Manual Divide the Domain Bacteria into two Phyla Proteobacteria Mythical Greek god, Proteus, who could assume many shapes All Gram-negative Divide the phylum Proteobacteria into five classes “A, B, C, D, and E” note we use the Greek alphabet of “alpha, beta, gamma, delta and epsilon” nonProteobacteria Gram positive or gram negative Divide the phylum nonProteobacteria into Six classes

Domain Bacteria: Proteobacteria : 

Domain Bacteria: Proteobacteria

Domain Bacteria: Proteobacteria : 

Domain Bacteria: Proteobacteria

Alpha proteobacteria : 

Alpha proteobacteria Human pathogens: Bartonella Cat-scratch disease Brucella Brucellosis Obligate intracellular parasites: Rickettsia. Arthropod-borne, spotted fevers R. prowazekii Epidemic typhus R. typhi Endemic murine typhus R. rickettsii Rocky Mountain Spotted Fever

Rickettisia : 

Rickettisia

Alpha proteobacteria : 

Alpha proteobacteria Caulobacter. Stalked bacteria found in lakes Plant pathogen: Agrobacterium. Insert a plasmid into plant cells, inducing a tumor

Alpha proteobacteria, : 

Alpha proteobacteria, Nitrogen-fixing bacteria: Azospirillum Grow in soil, using nutrients excreted by plants Fix nitrogen Rhizobium Fix nitrogen in the roots of plants

BetaProteobacteria : 

BetaProteobacteria Neisseria Chemoheterotrophic, cocci N. meningitidis N. gonorrhoeae Spirillum Chemoheterotrophic, helical Bordetella B. pertussis Zoogloea. Slimy masses in aerobic sewage-treatment processes

BetaProteobacteria : 

BetaProteobacteria Pseudomonadales: Pseudomonas Opportunistic pathogens Polar flagella Legionellales: Legionella Found in streams, warm-water pipes, cooling towers L. pneumophilia Coxiella Q fever transmitted via aerosols or milk

gammaProteobacteria : 

gammaProteobacteria Vibrionales: Found in coastal water Vibrio cholerae causes cholera Enterobacteriales (enterics): Peritrichous flagella, facultatively anaerobic Enterobacter Erwinia Escherichia Klebsiella Proteus Salmonella Serratia Shigella Yersinia

gammaProteobacteria : 

gammaProteobacteria Francisella tularemia deltaProteobacteria Bdellovibrio. Prey on other bacteria epsilonProteobacteria Campylobacter Gastroenteritis Helicobacter Multiple flagella Peptic ulcers Stomach cancer

Nonproteobacteria : 

Nonproteobacteria Cyanobacteria Oxygenic photosynthesis Fix nitrogen Gram-negative

Nonproteobacteria, Firmicutes : 

Nonproteobacteria, Firmicutes all Gram-positive Clostridiales Clostridium Endospore-producing obligate anaerobes Mycoplasma No cell wall, human pathogens Pleomorphic Bacillales Bacillus Endospore-producing rods Numerous species Staphylococcus Lactobacillales Enterococcus

Nonproteobacteria : 

Nonproteobacteria Actinobacteria Corynebacterium Mycobacterium Streptomyces Chlamydiae C. trachomatis, Trachoma, STD, urethritis Spirochaetes Borrelia Treponema Fusobacteria Fusobacterium Found in mouth, characteristic “pointy” ends, may be involved in dental diseases

Fig. i4.3 : 

Fig. i4.3

Fig. 4.29 : 

Fig. 4.29

Slide 206: 

Flagellar and chemotaxis proteins and their putative locations and interactions. The protein components of the major elements of the flagellum, the external filament, the hook, the basal body, and the motor-switch complex are shown. The external flagellar proteins that form the rod, hook, and filament are secreted by a specific export apparatus forming at the cytoplasmic side of the MS ring. The proton gradient at the cytoplasmic membrane drives a proton flow that energizes flagellar motor rotation. The direction of rotation is determined by the interaction of FliM in the motor-switch complex with the chemotaxis regulator CheY, whose activity is regulated by the other components of the chemotaxis system. Symbols: H+, protons; P, phosphate; CH3, methyl group.

Slide 208: 

The lambda phage can exist as a prophage (integrated into bacterial genome, lysogenic) or as an independent genetic element (lytic). The lambda phage is a linear double-stranded bacteriophage when packaged in the phage protein coat. The lambda DNA has 13 unpaired complementary bases at each end of the linear DNA and upon infection of a cell it circularizes.

Slide 209: 

Two types of bacteriophages are commonly used, the lambda phage and the M13 family of phages. Many Phages can be detected by plating them on a bacterial lawn and looking for cleared regions (plaques) where the bacteria have been lysed (clear plaques, lambda) or the bacterial grown has been slowed (cloudy plaques, M13).

Slide 210: 

The lambda phage can exist as a prophage (integrated into bacterial genome, lysogenic) or as an independent genetic element (lytic). The lambda phage is a linear double-stranded bacteriophage when packaged in the phage protein coat. The lambda DNA has 13 unpaired complementary bases at each end of the linear DNA and upon infection of a cell it circularizes.

Slide 211: 

The M13 phage enters the bacteria through pili and mature phage buds from the cell without lysing the bacteria. M13 is a single-stranded DNA phage which produces a double-stranded replicative form. The double-stranded replicatitive form of the M13 phage can be isolated from the bacteria by standard plasmid isolation proceedures.

Slide 214: 

Bacteriophages well-known bacteriophages of the family Inoviridae, such as M13 and fd - known as Ff phages

Slide 215: 

Genome plasticity results from DNA acquisition by horizontal gene transfer (HGT; for example, through the uptake of plasmids, phages and naked DNA) and genome reduction by DNA deletions, rearrangements and point mutations. The concerted action of DNA acquisition and gene loss results in a genome-optimization process that frequently occurs in response to certain growth conditions, including host infection or colonization. Genomic fluidity and pathogenic bacteria: applications in diagnostics, epidemiology and intervention Niyaz Ahmed, Ulrich Dobrindt, Jörg Hacker & Seyed E. Hasnain Nature Reviews Microbiology 6, 387-394 (May 2008)

Slide 216: 

pBR322 was the first popular modern cloning vector. It was constructed by Ray Rodriguez and Herbert Boyer from three basic parts: The tetracycline resistance gene segment was derived from the plasmid pSC101 (constructed by Stanley Cohen) which was, in turn, derived from the broad host range conjugative plasmid, R6. An ampicillin resistance gene segment was obtained from the transposon, Tn3. The origin of replication segment was obtained from pMB9 (constructed by Mary Betlach), which was, in turn, derived from the colicin plasmid, ColEI.

Slide 217: 

In making this construction, part of the segment containing the origin of replication was deleted. As a consequence, replication of the pUC plasmids is not properly controlled so that we have a higher copy number of the plasmids in the cell. The pUC series of plasmids were all constructed by Jeffrey Vieira and Joachim Messing and all are derived from pBR322. The tetracycline resistance gene region of pBR322 has been replaced by a gene segment coding for part of the b-galactosidase enzyme into which a series of unique restriction sites have been designed (the multiple cloning site, MCS).

Slide 218: 

Shuttle Vectors Plasmids with narrow host ranges will only replicate in certain bacteria. In particular, most of the modern cloning vectors are derived from the Col E1 plasmid which has a narrow host range. Thus if you want to do molecular genetics in strains outside this range, you have to either (a) construct a new set of vectors suitable for general cloning in your chosen strain, or (b) modify one of the common vectors so that it will also replicate in your chosen organism. The second option is the easier option since all it requires is the addition of an origin of replication which will function in your chosen organism. Such vectors, with two different origins of replication suitable for two different bacteria, are called shuttle vectors.

Slide 219: 

Figure 1. Virulence determinants. This cartoon illustrates the major types of known virulence determinants. A pathogen may have one or many virulent determinants. When a pathogen loses a crucial virulence determinant it becomes avirulent. Conversely, a normally non-virulent microbe may gain virulent genes and suddenly become virulent or a mildly pathogenic strain may gain an additional virulence determinant and become virulent. Virulent genes may occur through mutations in the microbes genome, or by picking up a plasmid (#conjugation) or bacteriophage carrying virulence genes (#transduction). To see a list of the virulence determinants that pathogenic E. coli can have visit this site and click on "E.coli as a pathogen"

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