Ms. W. M. N. H. Kumari

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PCR based identification of cadmium resistant determinants in Bacillus megaterium strain TWSL-4

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1 PCR based identification of cadmium resistant determinants in Bacillus megaterium strain TWSL-4 W. M. N. H. Kumari, C. D. Wijayarathna and N. V. Chandrasekharan University of Colombo, Sri Lanka

PCR based identification of cadmium resistant determinants in Bacillus megaterium strain TWSL-4:

PCR based identification of cadmium resistant determinants in Bacillus megaterium strain TWSL-4 W. M. N. H. Kumari, C. D. Wijayarathna and N. V. Chandrasekharan Department of Chemistry, Faculty of Science, University of Colombo, Colombo 03 16-Mar-17 2

Contents:

Introduction Objectives of the research Bioremediation of heavy metals Molecular characterization of bacteria Phylogenetic analysis of 16S rRNA gene Identification of Heavy metal resistance genes Conclusion Contents 16-Mar-17 3

Introduction:

Introduction 16-Mar-17 4 Heavy metals are; Hazardous to living beings. Difficult to remove from the environment. Cannot separate by physical methods and conventional methods are inefficient when metal concentration is low 1 . Thus, the use of microorganisms and plants for the decontamination of heavy metals has attracted growing attention. The use of bacteria as a bioremediation strategy is an attractive alternative to conventional methods 2 . Bioremediation ? 1 . Rajendran , P., Muthukrishnan , J., Gunasekaran , P., (2003) ; 2. H. Guo et al. (2010)

Introduction:

Introduction 16-Mar-17 5 What is Bioremediation? “Bioremediation is a process that uses living organisms, mostly microorganisms and plants, to degrade, reduce or detoxify waste products and pollutants*”. Figure 01: Schematic representation of bioremediation process *http://www.nature.com/subjects/bioremediation

Introduction:

Introduction 16-Mar-17 4 . Cannot separate by physical methods and conventional methods are inefficient when metal concentration is low 1 . Thus, the use of microorganisms and plants for the decontamination of heavy metals has attracted growing attention. The use of bacteria as a bioremediation strategy is an attractive alternative to conventional methods 2 . Suitable bacteria: Resistivity to heavy metals. Identification of the bacterial strain. Bacteria and metal resistance Bacteria have evolved with various measures to respond to heavy-metal stress 1 . Evolved biological systems such as efflux systems, uptake systems and intracellular precipitation help to withstand high concentrations of heavy metals 2 . Identification of heavy metal resistance genes ( copA, cadA , etc.) could be used as a tool in bioremediation . 6 1. Brim, H. et al (2000) ; 2. José, A et al (2013)

Objectives of the project :

Objectives of the project General objective : Isolation of genes responsible for heavy metal resistance in bacterial strain isolated from a textile dyeing industry with heavy metal tolerant ability. Specific objectives : Determination of metal tolerant ability. Molecular identification of bacterial and Phylogenetic analysis. Identification of heavy metal resistant genes. 16-Mar-17 7

Determination of heavy metal tolerance ability of isolates:

Determination of heavy metal tolerance ability of isolates Heavy metals tested in this study – Cu 2+ , Cd 2+ and Pb 2+ Metal reduction capability of the isolates in liquid media were investigated under aerobic conditions by inoculating approximately 1 x 10 5 bacteria into heavy metal containing broth and measuring the metal concentration and growth of bacteria at different time intervals (0 – 7 days)*. Heavy metal concentration - Atomic Absorption Spectrophotometer. Growth of bacteria – Absorbance at 600 nm UV/VIS Spectrophotometer. 16-Mar-17 8 * Global J. , ( 2010)

Metal tolerance ability of the isolated bacteria:

Metal tolerance ability of the isolated bacteria 16-Mar-17 Figure 02: Growth curve of bacterial isolate 9

Metal tolerance ability of the isolated bacteria:

Metal tolerance ability of the isolated bacteria 16-Mar-17 10 Figure 03: Metal removal ability of bacterial isolate

Molecular characterization of isolates:

Molecular characterization of isolates Genomic DNA isolation a Amplification of 16S rRNA gene by PCR b Nucleotide Sequencing Homology comparison to identify strains c Phylogenetic tree generation d 16-Mar-17 11 a -D. G. Pitcher et al.(1989), b - Weisburg et al.(1991), c - http:ncbi.nlm.gov, d - Sabdono et al. (2012)

PCR amplification of 16S rDNA:

PCR amplification of 16S rDNA Figure 05: Gradient PCR for determining optimal annealing temperature for primer pair fd1 and rd1. Expected size of amplified fragment is ~1500 bp. (Lane: DNA Ladder marker (100 bp), Lane 2-5: Annealing temperatures are 55 0 C, 54 0 C, 52 0 C and 56 0 C respectively. 16-Mar-17 1 2 3 4 5 1650 bp . 12

Nucleotide Sequence:

Nucleotide Sequence 16-Mar-17 16.01.2017 13

Homology comparison:

Homology comparison Figure 07: Homology comparison results after BLAST analysis 16-Mar-17 14 16.01.2017

Homology comparison:

Homology comparison Strain Homologous to % Identity TWSL-4 B. aryabhattai strain B8W22 100% B. megaterium strain NBRC 15308 99% B. megaterium strain ATCC 14581 99% B. megaterium strain IAM 13418 99% 16-Mar-17 15 Strain Homologous to % Identity TWSL-4 B. megaterium strain DSM 319 100% B. megaterium strain QM B1551 100% B. megaterium strain F4-2-27 100%

Phylogenetic Tree:

16-Mar-17 Phylogenetic Tree 16 Figure 08: Phylogenetic tree generated using MEGA 7 .

Morphological, Biochemical & Physiological Characteristics:

Morphological, Biochemical & Physiological Characteristics 16-Mar-17 Character TWSL_4 Gram staining - Shape Rods Motility Very motile Colony colour Yellow Colony shape Irregular Colony size 0.5 Cm Catalase + Hydrolysis of Gelatin + Coagulase - Hydrolysis of Starch + Citrate + Urease + +, Positive; -, Negative 17

Identification of heavy metal resistant genes :

Bacterium is capable of removing Cu 2+ , Cd 2+ and Pb 2 + . Cu 2+ , Cd 2+ and Pb 2+ resistance are conferred by P-type ATPase family ( copA, cadA ) and CDF (cation diffusion facilitator ) family ( nccA , czcD ) of transporters 1,2,3 . Bacterial genome was screened for possible heavy metal resistant determinants ( copA, copB , copC and cadA etc.) primers designed in this study ( Cad1 and Cad2 ) primers previously described 4,5,6 . PCR fragments were sequenced and analyzed. Identification of heavy metal resistant genes 16-Mar-17 18 1 -Bruins M.R., Kapil S . and Oehme F.W . (2000), 2 - Nies D.H .( 2003), 3 –Tsai et. al . (1992), 4. Kamika and Momba (2013), 5 . Nies et al. (1989 ), 6 . Abou-Shanab et. a l. (2007)

Primers used for PCR:

Primers used for PCR 16-Mar-17 19

PCR products obtained :

PCR products obtained 16-Mar-17 20 Figure 09: Gel electrophoresis of PCR products obtain from partial gene amplification using primers designed in this study. [A] Gradient PCR for amplification of cadA from Strain TWSL-4 using primer pair Cad1. [B] Gradient PCR for amplification of cadA from Strain TWSL-4 using primer pair Cad2. M – DNA ladder marker; [A]-1Kb DNA ladder, [B]-100 bp DNA ladder ; PCR control/ negative sample –Lane 1of [A] and [B]

Nucleotide sequences :

Nucleotide sequences Cad1 Cad2 16-Mar-17 21

Homology comparison :

Homology comparison Cad1 16-Mar-17 22

Homology comparison :

Homology comparison Cad 2 16-Mar-17 23

Homology comparison :

Homology comparison 16-Mar-17 24

Conclusions:

Conclusions Identified bacterial strain and its accession number: Strain Identified as Accession No . TWSL-4 Bacillus megaterium KR027922.1 Identified genes and their accession numbers: Gene Primr pair E ncoding protein Accession No . cadA 1 Cad1 ATPase KY366323 cadA 2 Cad2 ATPase KY366324 16-Mar-17 25

Conclusions:

Conclusions The isolated bacteria have the potential to be used as biological agents in the removal of Cu, Cd and Pb. The strain TWSL-4 carries two types of cadA genes code for P-type ATPases related to cadmium ion efflux mechanisms. 16-Mar-17 26

Future prospects:

Future prospects Identified heavy metal resistant determinants can be further studied to determine the mechanism of heavy metal removal. Research into further genetic manipulation of isolated strains to produce novel strains with enhanced bioremediation activity is recommended. 16-Mar-17 27

Acknowledgement:

Acknowledgement National Science Foundation for their financial assistance under Grant No. RG/2011/BT/08. 16-Mar-17 28

THANK YOU!:

THANK YOU! 16-Mar-17 29

Strain TWSL-4:

Strain TWSL-4 30 15.10.2016

Strain TWSL-4:

Strain TWSL-4 31 15.10.2016

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