Biological Characterization of Arsenate Resistant Bacteria from Different Econiches of Goa

BIOLOGICAL CHARACTERIZATION OF ARSENATE RESISTANT BACTERIA FROM

DIFFERENT ECONICHES OF GOA

Thesis submitted to the Goa University for the degree of

DOCTOR OF PHILOSOPHY in

MICROBIOLOGY

By

Neelam Singh

579.3

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Department Of Microbiology 's

Goa University 0 Goa - 403206

India

2007

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Goa University

Taleigao Plateau, Goa - 403 206 Tel : 0832-2451345-48/2456480-85 Fax : + 091- 832-2451184/2452889 E.mail : [email protected] Website: www.goauniversity.org (Accredited by NAAC with a rating of Four Stars)

Certificate

This is to certify that Miss Neelam Singh has worked on the thesis entitled "Biological characterization of arsenate resistant bacteria from different eco-niches of Goa" under my supervision and guidance. This thesis being submitted to the Goa University, Taleigao plateau, Goa, for the award of degree of Doctor of Philosophy in Microbiology, is an original record of the work carried out by the candidate herself and has not been submitted for the award of any other degree or diploma of this or any other university in India or abroad .

Head Dr.Santosh umar Dubey

Department of Microbiology, Reader and Research Guide Goa University

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I hereby state that this thesis for the Ph.D. Degree on

"Biological characterization of arsenate resistant bacteria from different eco-niches of Goa" is my original contribution and that the thesis or any part of it has not been previously submitted for the award of any degree or diploma of any university or institute. To the best of my knowledge, the present study is the first comprehensive work of its kind from this area.

Neelam Singh Dept of Microbiology

Goa. University

Goa

Acknowledgement

It gives 'me a great pleasure and deep feeling of satisfaction to take this opportunity to thank all those who have helped me directly or indirectly in making my thesis possible.

First and foremost, I thank my guide Dr. S.K. Dubey for being a source of infinite inspiration, his excellent and timely guidance and extraordinary patience during the entire period of research. His scientific experience,vast knowledge of the subject, innovative ideas and constructive criticism have contributed immensely to my research progress. I am grateful to Prof. D.J. Bhatt, Dr. J. D'Souza and Dr. S. Bhosle;

Heads, Dept. of Microbiology, for providing necessary facilities required for my work.

I also want to thank all faculty members of the department especially Dr. S. Nazarath for helping me in all possible ways during my time of crisis. My thanks also go to Dr.

I. Furtado and Dr. S. Garg for their help and support. I thank sincerely, Dr. Sanjeev C. 6hadi, Prof. U.M.X. Sangodkar, U. Barros and Dr. Savita S. Kerkar along with all the faculty members of Dept. of Biotechnology, Goa University.

I am thankful to Mr. 5.6. Vengerlekar, Director Mormugao Port Trust for his kind permission , Capt. S.S. Karnad (Harbour Master, MPT, Goa) and Comm.

K.M.Bhoj for assistance in the collection of water samples in the harbour area. I'm also thankful to Prof. Geeta Sharma, Director and C.S.O., Magene Life Sciences Pvt.

Ltd., Hyderabad for her timely help. Thanks are due for Dr. Yogesh Shouche, Scientist in Charge, Molecular Biology lab and Mr. Pankaj Verma (Research Fellow) at NCCS, Pune who helped me with the nucleotide sequencing and data analysis. My heartiest thanks to Dr.Rakesh Tuli, Scientist '6' and Director, and Dr. Samir V.

Sawant, Scientist, National Botanical Research Institute, Lucknow, for their guidance and providing lab facility so as to enable me perform Genome Walking experiments. I always acknowledge their caring strictness which had inspired me to work harder. I'm also grateful to my caring senior, Dr. Safdar Jawed, Ex- Scientist Bharat Biotech.

L Ltd. Hyderabad, Scientist RANBAXY labs, New Delhi. I'm greatly indebted to Dr. PA Loka Bharathi, Scientist -in-Charge, BOO NIO, Dr. Shanta A. Kutty, Dr. Judith and Dr. Krishnan; of Biological Oceanography Division, NIO, Goa. My special thanks to Miss Neetha Joseph, SRF,NIO-RC Cochin for doing the FAME analysis of my cultures. Also my sincere thanks go to Miss Divya, Miss Febby, research students, NIO. I want to show my gratitude towards Dr. K.S. Rane H.O.D. Dept. of Chemistry, Dr.R Shirsat, Dr. S.G. Tilve, Dr. DasGupta, Dr. Harsh°, Dr. Aditi and all the students and staff of Chemistry Dept for their timely help. Thanks to Dr. P.K. Sharma, Dr. Janardanam and Or. Vijay° Kerkar for their kind cooperation and help as and when required. My gratitude never fades for my brother Dr. - Utpal Pandya, Scientist, Dept of Microbiology, University of Texas, Medical Branch, Galveston(USA). I am immensely grateful to Dr. A.K. Girl, Asst. Director for helping me with AAS and Dr. Siddharth Ray, Director, IICB, Kolkata for allowing his lab facility.

I extend my indebtedness towards all the non-teaching staff of the dept. of microbiology. I can never forget the help rendered by Mr. Anant Gawde, Ana, Deep,

works. Also, I would like to express my gratiture towards the security staff of the university, especially Sharmaji, Mahesh 011, Ganpat, Kirtan, Subhash, Umashankar ji and Susheela.

I gratefully acknowledge CSIR for the financial su•pport as ,TRF and SRF.

Words seem to be inadequate to express my indebtedness towards my Jay whose encouragement and endless support helped me overcome all odds, provided zeal and brought success in my endeavor.

I thank all the research fellows, past and present, of the department of Microbiology for their help. My friends; Dr. Shweta Srivastava, Dr. Meenal Kowshik, Dr. Pritha Ghosh, Dr. Suphala Pujari, Ms. Ila, Mr. Prakash Munouli, Ms. Hema- Pramila, Mrs. Anju, Ms. Lakshangy, Mrs. Aureen, Ms. Vidya, Ms. Celisa, Ms. Nimali, Ms. Lorna and Ms. Christina have always been a source of internal energy. I acknowledge their helping hand and pray for their bright future. I thank my seniors Dr. Upal, Dr. Bramhachari, Mr. R. Krishnamurthy, Dr. Judith Braganca, Dr.T. Madhan Raghavan, Mrs. Rasika and Mr. Naveen. Heartiest thanks to Mrs. Tabitha for her prayers for my good health.

I wish to dedicate this work to my family, especially to my loving brother Ashok (ashu), who took away all my homely responsibilities and kept me free to do my research. My each and every achievement bows in front of my parents whose selfless love and blessings brought me through.

I have successfully compiled my creative and thoughtful research due to genuine concern and painstaking efforts of many more friends and well wishers, whose names are not mentioned, but they are still in my heart.

Last but not the least I thank the Almighty God for giving me strength, 1 courage, good health and wisdom to accomplish this work successfully.

Neelam

a AAS Abs Arr AO+

As 5+

As As(III) As(V) As203 Aox Ars ARMs ATPase b.p.

°C CCA Cfu DM/

DARPs DMM DSMA EDTA FAME FeAsS FeS2 Fig.

gm GC GSH h HAs04- HGAAS HCI H2SO4 Hg2+

alpha

atomic absorption spectrometry absorbance

respiratory arsenate reductase arsenite ion

arsenate ion

symbol for arsenic, except at the ,e beginning of a sentence

sum of concentrations of H3As03 and H2As03

sum of concentrations of H3As04, H2As04, HAs04 2

^,

and As043--

arsenic trioxide, arsenolite, claudetite

Arsenite oxidase

Arsenate resistance system

arsenate-resistant microorganisms adenosine triphosphatase

beta

boiling point degree celcius

copper chrome arsenate colony forming unit distilled water

Dissimilatory arsenate-reducing prokaryotes

defined minimal medium disodium methyl arsonate

ethylene diamine tetra acitic acid fatty acid methyl ester

Arsenopyrite Pyrite

figure gram(s)

gas chromatography glutathione

hour/s

Arsenate ion

Hydride generation atomic absorption spectrometry

Hydrochloric acid

Sulphuric acid

Mercuric ion

HPLC high pressure liquid chromatography

ICP-AES inductively coupled plasma atomic

emission spectrometry

ICP-MS inductively coupled plasma mass

spectrometry

IR Infra red

K+ Potassium ion

Kb kilobase pairs

KNO3 potassium nitrate

L litre

LB Luria Bertani Broth

LA Luria Bertani agar

lbs pounds

A lambda

M molar

mg milligram(s)

MMA monomethylarsonic acid

MSM mineral salts medium

MSMA Mono sodium methyl arsonate

mg2+

magnesium ion

mg milligram(s)

mg/L milligrams per liter

min minute(s)

ml milliliter

mM millimolar

mRNA messenger RNA

pg microgram

pl microlitre

PM micromolar

p g /g micrograms per gram

pg/I micrograms per liter

micrometer, micron

NA nutrient agar

NaCI sodium chloride

NCCLS National Committee for Clinical

Laboratory Standards

NH4NO3 ammonium nitrate

NH4CI ammonium chloride

NaOH sodium hydroxide

nm nanometer

nM nanomolar

NMR nuclear magnetic resonance

O.D. optical density

ORFs open reading frames

PAGE poly-acrylamide gel electrophoresis

RBS ribosome binding site

rpm revolutions per minute

- RT • room temperature

SBMLP Sea Water based minimal medium

with limiting phosphate

SD standard deviation

SDS Sodium Dodecyl Sulfate

SDW sterile distilled water

SE standard error of mean

sec second/s

SEM scanning electron microscopy

sp. species

a Standard deviation

TGHP Tris/Glucose High Posphate medium

TGLP Tris/ Glucose Low Phosphate(TGLP)

medium

TEMED tetra methyl ethylene diamine

TLC thin layer chromatography

USEPA United States Environmental

Protection Agency

UV ultra violet

V volts

v/v volume/volume

w.r.t. with respect to

w/v weight/volume

Zn2+ zinc ion

percentage

%o parts per thousand

List of Tables

CHAPTER- I Table 1:

Table 2:

Table 3:

CHAPTER-I11 'Table 4:

Table 5:

Table 6:

Table 7:

Table 8:

Naturally occurring inorganic and organic As species Other Arsenic compounds of environmental significance referred to in the text

Length of ars C gene in various bacterial species.

Geographical location of the sampling site

Physicochemical characteristics of water samples Total viable count of bacteria in various samples Viable count of Arsenate resistant bacteria

Effect of temperature on the growth of selected arsenate resistant isolates

CHAPTER- IV

Table 9: Morphological Characteristics of arsenate resistant isolates Table 10: Biochemical tests of some selected arsenate resistant

bacteria isolated from different econiches of Goa Table 11: Identification of the arsenate resistant isolates

(Based on Biochemical and morphological characteristics) Table 12: Identification of arsenate resistant isolates using

PIBWIN Software

Table13: Identification on the basis of FAME analysis

Table 14: Molecular identification of the six highly arsenate resistant bacterial strains(SI9, BL9, MPT4, Maj4, Man1, and Man2) by 16s rDNA sequencing

Table 15: Antibiotic Sensitivity Tests for Arsenate resistant Bacterial Isolates(519, BL9, MPT4, Maj4, Mani, and Man2)

Tablel6 Sequences of 16S ribosomal DNA gene of the six (a-f): selected arsenate resistant bacterial isolates(SI9, BL9,

MPT4, Maj4, Mani, and Man2)

Table 17.1 NCB! — BLAST results of the 16s rDNA sequences of the six -17.6: selected arsenate resistant bacterial isolates(SI9, BL9, MPT4,

Maj4, Mani, and Man2)

Table 18: Arsenate uptake by Vibrio sp. SI9 and Vibrio sp. Maj4 by

spectrophotometric method (Improved Molybdenum blue method) in Mineral medium (MSM+0.4% glucose+1.5% NaCI) containing 50mM Arsenate.

Table 19: Arsenate uptake in best selected Phosphate Limiting medium (SBMLP with 65pM Phosphate) at 28 ± 2°C

Table 20: Total arsenic in water samples collected from different sites determined by HG-AAS

Table 21: Arsenic uptake by the two selected strains Vibrio sp. SI9 and Vibrio sp. Maj4 Hydride generation atomic absorption spectrometry (HG-AAS)

Table 22: Rf values of arsenate and arsenite in different solvent

systems as obtained by paper chromatography and Silica gel Thin layer chromatography

CHAPTER- VI

Table 23: NCBI- BLAST hits and RDP Blast hits of the six selected strains (SI9, BL9, MPT4, Maj4, Man1 and Man2) with Type and other strains in the database.

Table 24: Multiple Sequence Alignments of the 16s rDNA gene sequences of six selected arsenate/arsenite resistant

bacterial strains (SI9, BL9, MPT4, Maj4, Man1 and Man2)using CLUSTALW (1.83)

Table 25 NCBI- BLAST search results of the arsC sequences of five (a-e): PCR positive strains, viz. SI9, BL9, Maj4, Man1 and Man2 Table 25(f): Sequences of the arsC amplicons of five arsenate resistant

strains amplified by amlt 42F/376R primers

Table 26: Multiple Sequence Alignments of the arsenate reductase

(arsC) gene sequences of five selected arsenate/arsenite resistant bacterial strains (SI9, BL9, Maj4, Man1 and Man2 using

CLUSTALW (1.83)

Table 27: Maximum similarity hits of arsC sequences of the arsenate

resistant isolates with the arsC sequences of other bacteria in the NCBI database.

Table 28: Accession numbers of the arsC sequences from arsenate resistant bacteria

Table 29: Web Cutter analysis of the plasmid borne arsC sequence of Vibrio sp. strain SI9

List of Figures

CHAPTER- I Fig.. 1:

Fig. 2:

Fig. 3.1:

Fig. 3.2:

Fig. 3.3:

CHAPTER- III

Arsenic cycle in nature (Mukhopadhyay et al. 2002)

Structures of naturally occurring inorganic and organic arsenic species

Different families of

ars

operons

Genes and products for arsenic resistance in gram positive and gram negative bacteria .

Pathways of arsenic detoxification in prokaryotes and.

eukaryotes

Fig 4: Map of Goa showing location of sampling sites.

Fig. 5.1: Levels of arsenate resistance among the bacterial

isolates from sewage water (St.Inez Nallah, Panaji, Goa).

Fig. 5.2: Levels of arsenate resistance among the bacterial

Isolates from estuarine waters (Zuari and Mandovi, Goa).

Fig. 5.3: Levels of arsenate resistance among the bacterial isolates from sea water samples (Goa).

Fig. 5.4: MIC of Sodium arsenite in Mineral Salts Medium for the six

selected bacterial isolates tolerating high levels of sodium arsenate Fig 6: Plasmid profile of six selected arsenate resistant strains.

Fig 7: Effect of temperature on growth (total protein) of six selected arsenate resistant isolates viz. SI9, BI9, MPT4, Maj4, Mani and Man2.

Figure 8.1-8.6:

Effect of pH on growth (total protein) of six highly arsenate resistant bacterial isolates SI9, BI9, MPT4, Maj4, Mani and Man2 Figure 9.1— 9.6: Salinity

Effect of frs("On growth (total protein) of six highly arsenate

resistant bacterial isolates SI9, BI9, MPT4, Maj4, Mani and Man2

CHAPTER- IV

Fig.10: Some arsenate resistant bacterial strains growing on MSM agar (with 1.5% NaCI) incorporated with 50 mM sodium arsenate Fig.1 1(a to f):

The six selected bacterial isolates showing high levels of

resistance towards arsenic.

Arsenic resistant

^Vibrio

spp. growing on TCBS agar+ 3% NaCI Fig 13: Some other arsenic resistant

Vibrio

spp. (a to e) isolates growing on

TCBS agar + 3% NaCI Fig. 14(a &b):

a.

Vibrio

sp.SI9 growing on VP8 Agar and b.

Vibrio

sp. Maj4 growing on VP8 Agar

Fig. 15.1-15.6:

Light microscopy of six highly arsenate resistant bacterial isolates after Gram staining.

Fig. 16.(i)-16(xii):

Scanning Electron micrographs of six selected arsenic resistant strains

Fig. 17.1-17.6:

PIBWIN Identification of the six arsenate resistant strains Fig. 18.1- 18.6:

Gas Chromatograms of Fatty Acid Methyl Esters extracted from the six arsenate resistant strains.

CHAPTER- V

Fig. 19.1-19.6:

Growth of six selected strains (SI9, BL9, MPT4, Maj4, Man1, and Man2) in presence of sodium arsenate in MSM + 0.4% glucose.

Fig. 20.1-20.6:

Growth of

Vibrio

sp. S19 in presence of sodium arsenite(0-2mM) in MSM + 0.4% glucose.

Fig. 21(i — vi):

Effect of carbon source on growth of six selected

arsenate resistant strains SI9, BL9, MPT4, Maj4, Mani and Man2 Fig.22.1(a-e):

Growth pattern of

Vibrio

sp. S19 in four different minimal media with limiting phosphate concentrations. (a) growth profile in PLM1, (b) growth profile in TGLP medium, (c) growth profile in DMM, (d) growth profile in SBMLP medium;& (e) growth profile in SBMLP + 50mM sodium arsenate

Fig. 22.2(a-e):

Growth pattern of

Vibrio

sp. Maj4 in four different minimal media with limiting phosphate concentrations. (a) growth profile in PLM1, (b) growth profilein TGLP medium, (c) growth profile in DMM, (d) growth profile in SBMLP medium;& (e) growth profile in SBMLP+

50mM sodium arsenate

Fig 23: Effect of sodium arsenate on total protein of

Vibrio

spp. strains SI9 and Maj4 in MSM + 0.4% glucose + 1.5% NaCI

Fig. 24: Thin layer chromatogram showing Arsenate reduction (into

arsenite) by

Vibrio

sp. Maj4

Fig. 25(GEL 1-6):

SDS-PAGE analysis of Arsenate induced proteins of the six selected arsenate resistant isolates after 24 hrs.

CHAPTER- VI

Fig. 26: Phylogenetic tree based on 16S rRNA gene sequence comparisons over 1,392 bases showing the relationship between members of genus

Vibrio, Aeromonas

and

Pseudomonas

with different isolates Maj4, BI9, SI9, Man2, MPT4 and Man1.

Fig. 27: Phylogenetic tree based on 16S rRNA gene sequence comparisons over 1,381 bases showing the relationship between members of genus Vibrio and the selected isolates Maj4, BI9, SI9 and Man2 (all Vibrios).

r

Fig. 28: Phylogenetic tree of the arsC sequences of five selected arsenate resistant bacterial strains (SI9, BL9, Maj4, Mani and Man2).

Fig. 29: PCR amplification of arsC gene using SI9 plasmid DNA and the plasmid DNA of five other isolates with E. coil arsC1 F and arsC1 R primers.

Fig.30-a: PCR amplified

arsC

fragment from Chromosomal DNA of

Vibrio

sp.

Maj4

and plasmid of

Vibrio

sp. SI9 using E.coli R773 based arsC1F and arsC1 R primers.

Fig 30-b: PCR amplified arsC fragment from SI9 plasmid using amIt42F/376R primers

Fig. 31: PCR amplification of arsC gene from chromosomal DNAs of BL9, Maj4, Mani and Man2 using amlt 42F and 376R primers(Sun et al.

2004).

Fig. 32: Restriction profile of pSI9 with four different blunt cutter enzymes.

Fig. 33: Diagrammatic sketch of Genome Walking Technique Fig 34.1: Primary PCR using AP1-NS3

Fig 34.2: Secondary PCR using AP2 - NS2 primers

(template:NS3-AP1 amplified primary PCR product) Fig 34.3: Tertiary PCR using AP2 — NS1 primers (template: AP2-

NS2amplified secondary PCR product) Fig. 34.4: Map of the cloning vector pBluescript SK+

Fig. 35.1: Agarose gel profile of plasmid of positive clone with the 2.5 Kb ars operon (partial) fragment of pSI9 in pBlueScript SK(+)

Fig 35.2: Pvull digestion of the recombinant plasmid of positive clone

pSK(+) (containing 2.5 Kb EcoRV fragment)

CHAPTER-I Photographs of diseased persons due to arsenic poisoning(downloaded from Internet). The

symptoms include leucomelanosis, melanosis dorsum, lesions, keratosis of palms and soles and dark pigmentation of skin.

CHAPTER-II Some of the sampling sites Dona Paula,

Miramar, Kakra and,

The Atomic Absorption Spectrometer.

CHAPTER-III Some of the sampling sites Dona Paula,

Majorda and Miramar

CHAPTER-IV Some methods used to identify bacteria:

Vibrio

sp. SI9 growing on VP8 medium,

Electron micrograph of

Pseudomanas

sp. Man1, A few

Vibrio

isolates growing on TCBS agar,

Chromatogram of Fatty acids (FAME) of

Vibrio

sp. BL9, Part of

arsC

gene sequence chromatogram of

Vibrio

sp. SI9, Gram stained

Aeromonas

sp. and

Vibrio

spp. SI9 and Maj4 growing on TCBS agar + 3NaCI

CHAPTER-V Scan of the American Society for Microbiology,

(ASM) 2006, News Letter citing the news of Best Poster Award, won in the Association of Microbiologists of India (AMI)

Conference,2005.

CHAPTER-VI Structure of the

ars

operon in bacteria,

Diagrammatic representation of Genome walking technique &

Model of the proposed luminescent biosensor for arsenic.

INDEX

Certificate Statement

Acknowledgement Dedication

Abbreviations List of Tables List of Figures

Legends for Chapter's Cover Pages Nature and Scope of the problem Aim and objective of the study

( I) - (ii ) (iii)

CHAPTER- I 1.1 1.2 1.3 1.4 1.5 1. 6 1.7 1.8

INTRODUCTION

Arsenic in the environment

Chemistry of Arsenic Compounds Uses of Arsenic

Abiotic factors affecting Biogeochemical Cycling of arsenic

Biological activity of arsenicals Arsenic Resistant Bacteria

Antibiotic resistance in arsenic resistant bacteria

Biotransformation

Page No.

1 4 5 6 7 8 8 - 9 9 -11 1.9

Bioaccumulation of arsenic by

microorganisms and bacteria

12

1.10

Biochemical Basis of Resistance

12

1.11

Arsenate reductases

13

1.12

Protein profile of arsenate resistant bacteria

13 - 15 1.13

Molecular basis of Arsenic Resistance

15 -18 1.14

Primers designed so far to study the ars

operon genes

18 - 20 1.15

Genome walking approach for cloning of

ars operon

20

1.16

Biosensors for arsenic

20-23

CHAPTER- II

MATERIALS AND METHODS

2.1

Collection of water samples

24

samples

2.2.1 Determination of salinity 25 25 2.2.2 Determination of Nitrite Nitrogen

(NO3-N)

26 2.2.3 Determination of Nitrate Nitrogen

(NO3-N)

26 2.2.4 Determination of Phosphate

Phosphorous (PO4-P)

2.2.5 Dissolved Oxygen 27

27 2.3 Determination of viable count; and

screening, isolation, purification and

maintenance of marine and other Arsenate resistant bacterial strains

2.4 2.4 Determination of environmental optimas 28 for the growth of Arsenate resistant isolates

2.4.1 pH optima 28

2.4.2 Temperature optima 28 2.4.3 Salinity optima 28 2.5 Identification of Arsenate resistant bacterial 29

isolates

2.5.1 Biochemical tests 29 — 32 2.5.2 Scanning Electron Microscopy (SEM) 33 2.5.3 Identification of six highly resistant 33 -34 isolates by using PIBWIN (Probabilistic

Identification of Bacteria for Windows)(Version 1.9.2)

2.5.4 Fatty Acid Methyl Ester(FAME) 34 -35 Analysis

2.5.5 16S rDNA sequence analysis of • 35 -36 selected strains

Antibiotic Sensitivity assays 36 -37 2.6

2.7 Selection of arsenate resistant bacterial 37 strains for arsenate biotransformation

molecular genetic studies

2.8 Study of growth behaviour of arsenate 37

of arsenate and arsenite

2.9 Selection of best Carbon source for the 37 -38 growth of arsenate resistant isolates

2.10 Designing of a phosphate limiting medium 38 for arsenate uptake studies

2.11 Arsenate uptake studies by 38 -39

spectrophotometric method (Improved molybdenum blue method).

2.12 Arsenate uptake studies using hydride 39- 40 generation atomic absorption spectrometry

(HG-AAS)

2.13 Biotransformation by 41- 43

a) Thin layer chromatography and Paper Chromatography

b) Microtitre plate method

2.14 SDS- PAGE analysis of the total protein 43 — 44

2.15 Plasmid Isolation 45

2.16 Restriction pattern analysis of the plasmids 46 of two arsenate resistant isolates viz. 519

and Maj4

2.17 Chromosomal DNA Isolation 46

2.18 PCR amplification of arsenate reductase 47 gene (arsC) from the plasmid/

chromosomal DNA of the selected arsenate resistant strains

2.19 Phylogenetic analysis of six selected 47 arsenate /arsenite resistant strains.

2.20 Sequence analysis of the ars C sequences 48 of five PCR positive strains of bacteria

using NCBI blast and ClustalW.

2.21 Cloning of the ars operon (partial) by 48-50 means of Genome walking

CHAPTER- III RESULTS AND DISCUSSION

3.1 Sampling sites 51

3.2 Physicochemical characteristics of water 51 -53 samples

3.3 Viable count of bacteria in water sample 53 — 54 3.4 Screening, purification and maintenance of 54 -55

arsenate resistant bacterial strains

3.5 Environmental optima of selected arsenate 56 tolerant strains

3.5.2 Optimum pH for growth 56 — 57

3.5.3 Optimum Salinity 58

CHAPTER- IV RESULTS AND DISCUSSION

4.1 Identification of arsenate resistant bacteria 59 by biochemical tests

4.2 Identification of six highly resistant isolates 60 by using PIBWIN

4.3 Identification of highly resistant isolates by 61 using FAME analysis

4.4 Molecular Identification of arsenate 61 — 62 resistant bacterial isolates by 16s ribosomal

DNA sequencing

4.5 Selection of six highly resistant strains for 62 -63 arsenate biotransformation studies and

molecular biological characterization

4. 6 Antibiotic Sensitivity Profile of Arsenate 63 -65 resistant isolates

CHAPTER- V RESULTS AND DISCUSSION

5.1 Growth of arsenate resistant strains at 66 various concentrations of sodium arsenate

5.2 Growth of Arsenate resistant strains at 67 various concentrations of arsenite

5.3 Growth in presence of different carbon 67 — 68 sources glucose, lactose, sucrose and

sorbitol in Mineral medium + 1.5% NaCI

5.4 Designing of a phosphate limiting medium 68 for arsenate uptake and biotransformation

studies

5.5 Arsenate uptake by spectrophotometric 69 — 70 method (Improved Molybdenum blue

method)

5.6 Determination of total arsenic in water 70 -72 samples and intracellular arsenic (uptake)

by the resistant strains by HG-AAS

5.7 Arsenate biotransformation by thin layer 72 chromatography

5.8 SDS- PAGE analysis of the arsenate 73 — 74 induced proteins in the selected strains

CHAPTER- VI RESULTS AND DISCUSSION

6.1 Phylogenetic analysis of six selected arsenate/ arsenite resistant strains using Clustal W, MEGA 3.1 and RDP programs 6.2 PCR amplification of arsenate reductase

gene (arsC) from the plasmid/

chromosomal DNA of the selected arsenate resistant strains

6.3 Analysis of the ars C sequences of five PCR positive strains of bacteria by NCBI- BLAST search and phylogenetic analysis of the ars C sequences of five PCR positive strains of bacteria

75 -76

77

77 — 78

6.4 Cloning of the ars operon (partial) of 78 -80 plasmid pSl9 by means of Genome

walking

SUMMARY 81- 83

Future Prospects 84-85

APPENDIX 86-106

ERRATUM E1-E2 (107

— 108)

BIBLIOGRAPHY 109-138

LIST OF PUBLICATIONS 139 LIST OF CONFERENCES ATTENDED 140

AWARDS 141

It is well known that West Bengal(India) and Bangladesh have high levels of arsenic in groundwater but slowly the problem is spreading to other states like Uttar Pradesh and Bihar. This is confirmed by the reports of All India Institute of Medical Sciences, New Delhi that people living in Ballia district of U.P. also have high levels of arsenic in their blood, hair, nails, etc. According to union ministry of water resources eight districts of West Bengal and one district of Bihar (Semria Ojha patti) are arsenic contaminated. The fact is that arsenic is increasingly found in the districts of Bihar, Terai, U.P. and even Assam. Scientists have an opinion that Arsenic came with the silt deposited by the mighty rivers centuries ago. Arsenic happened to be the theme of the XXVII annual conference of Environmental mutagen Society of India and a special symposium was held on Feb 14-16 ,2003 at the Indian Institute of Chemical Biology, Kolkata, in collaboration with Indo-US Science and technology forum, New Delhi on "Arsenic Contamination in Ground Water and its Health Effects". The conference was sponsored by national and international agencies like C.S.I.R.(India), D.B.T.(lndia), UNICEF, I.C.M.R.(India), D.A.E.(India), EMSI(lndia) and Indo — U.S. Forum. Prof. Barry P. Rosen a pioneer in the field of arsenic research was personally present in the conference.

Bacteria can detect the presence of arsenic and detoxify it efficiently. The limited availability of scientific reports about biodiversity, biotransformation of arsenate/ arsenite, structure and function of genetic determinants of arsenate resistant bacteria from our country and no such reports from Goa, in particular creates the need to carry out a detailed study including screening and identification; uptake and biotransformation of arsenate; physiological, biochemical and molecular biological characterization and phylogenetic analysis of the bacterial isolates highly resistant to arsenic, from different econiches of Goa viz. marine, estuarine and sewage water habitats. E. coli, Bacillus spp.,

(i)

S.aureus, Pseudomonas

spp.,

Acidiphilium multivorum, Alcaligenes

sp.,

Desulphotomaculum sp., .Aeromonas sp., Exiguobacterium sp.

etc. are some important bacteria in which arsenic resistance genes have been reported but new taxonomic groups are being added every day to this ever increasing list.

Therefore, there is a need to explore the possible presence of new bacterial genera from various econiches of Goa, resistant to arsenic. Many bacteria have an inherent capability to detoxify inorganic arsenic and the genes responsible for detoxification are inducible by arsenate and arsenite (Osborne and Ehrlich, 1976;

Wu and Rosen,1993). This property can be made use of in the construction of whole cell bacterial biosensors (Petanen and Romantschuk 2003). Till now there are no bacreria known which can accumulate arsenic intracellularly. This gives

1

us enough reasons to carry out a systematic study in the search of a suitable bacterium which can accumulate inorganic arsenic intracellularly and help in bioremediation of this biocide (arsenic).

Genetic studies on arsenate resistant/ detoxifying bacteria isolated in India are extremely limited with no reports demonstrating the presence of plasmids and their correlation with arsenate/ arsenite resistance.

These studies will make the foundation of developing arsenate

hyperaccumulating strains for bioremediation and can also help us in the

development of microbial biosensors utilizing the inducible promoter of the

ars

operon from the highly arsenate resistant bacteria.

1. Screening, isolation and identification of arsenate resistant bacteria from various econiches of Goa.

2. Physiological and biochemical characterization of arsenate resistant bacterial isolates with reference to growth behaviour, arsenate tolerance and arsenate biotransformation.

3. Molecular biological studies involving localization and cloning of the genes responsible for arsenate resistance into a suitable vector.

4. Cloning of arsenate resistance genes upstream to lux structural genes (lux CDABE)in the reporter plasmid pUCD615, which is expected to result in a luminescent biosensor for arsenate pollution monitoring.

GENERAL INTRODUCTION

1.1 Arsenic in the environment

Arsenic (Atomic number 33, Atomic weight 74.92) is the third element in the group VA of the periodic table. This metalloid is a member of the same family as Phosphorous. It is the 20 th most abundant element in the earth's crust found at a concentration of 1.8 ppm. It occurs in phosphate rocks and in many industrial phosphates and mine tailings as an impurity, and also as a by-product in metallurgy of copper and other metals (Vircikova and Havlik1999, Bailey et al.

2002). Its concentration in soil ranges from 5.5 -13 ppm, in streams —2 ppb and in groundwater it is generally less than 100 ppb. Arsenic is a ubiquitous element present in various compounds in the earth's crust. The most common oxidation states are: -3, 0, +3 and +5. Arsenate and arsenite are also present in the soil solutions. It occurs naturally in sulphide minerals such as pyrite. In nature, arsenic (As) can be found in insoluble forms in combination with sulfur, such as AsS and As2S3, or as arsenopyrite (FeAsS), an iron-sulfur compound (Fig.1). The oxidation of these compounds gives rise to chemical forms that are more toxic to human life, such as arsenate (As(V)) and arsenite (As(III)). Arsenate is present in oxide environments, and it binds strongly to sediments. Arsenite can be obtained from arsenate under anaerobic conditions, and it is more toxic (Carepo et al. 2004). In most environments, arsenite is generally thought to be the more soluble and mobile form, which increases its potential toxicity. However, arsenate is the thermodynamically favorable form in most aerobic systems (Ferguson and Gavis,1972; Tamaki,1992). Estimated levels of arsenic in different sources are:

sea water, 2-5 ppb; public water supplies, 5ppb (recommended limit 10ppb);

uncontaminated soil, 5ppm; human food from plant sources, <0.5ppm. Fish and sea foods may contain much higher. With the exception of fish, most sea foods contain less than 0.25 pg/g arsenic. Many species of fish contain between 1 and

Introduction

10 pg/g. Arsenic levels at or above100 pg/g have been found in bottom feeders and shellfish. Marine organisms, such as shrimp, mussels contain naturally high concentrations of this element, typically ranging from 1 to 100 mg As/kg wet weight (Lau et al. 1987, DeGieter et al. 2002, NIFES Archive, 2004). An estimated average dietary intake in U.S. is about 0.9 mg/day and total body burden in adult is about 15-20 mg. An estimated 6 million people in West Bengal are presently drinking water contaminated with arsenic > 50 pg/L in an area of 38,865 km 2 (Chowdhury et al. 2001).According to WHO, intake of 1.0 mg of inorganic arsenic per day may give rise to skin lesions within a few years (RoyChowdhury et al. 2003). Contamination of the drinking water supplies with the inorganic forms (arsenite and arsenate) has often been reported and arsenic has been identified as major risk for human health in different parts of the world (Muller et al. 2003). The organic forms of arsenic are less toxic. As (III) is 100 times more toxic than As(V) (Neff,1997). In certain types of aquatic environments, such as the hypersaline Monolake of California, USA the dissolved arsenic concentration is extremely high (0.3mM) owing to the concentration effects of hydrologic and climatic factors and an abundance of hydrothermally based sources (Dowdle et al.1996). The predominant form of arsenic in water is usually arsenate (V) (Callahan et al. 1979, Wakao et al. 1988), but aquatic microorganisms may reduce the arsenate to arsenite and a variety of methylated arsenicals. Within anoxic soils, sediments and waters arsenic occurs primarily as As(III). Arsenic can be emitted into the environment from several natural sources, including volcanic eruptions. Weathering and sedimentation leads to wide natural distribution as weathering of sulphide rich rocks results in the forthation of highly acidic (pH 3.0) and heavy metal laden effluents. At the abandoned Pb-Zn mining sites the pyrite rich tailings are subject to bioleaching which leads to the formation of acid waters heavily loaded with arsenic (Average conc. 250 ppm). Dissolved arsenic present in the seepage waters precipitates within a few meters from the bottom of the tailing dam in the presence of micro- organisms eg. Acidithiobacillus ferrooxidans (Duquesne et al. 2003).

2

Thought to be pollution free and environment friendly, geothermal wells, used as a source of energy are also a source of arsenic into surface waters. Arsenite is often the predominant valence state of inorganic arsenic in geothermal source waters, although As(V) can be present with As(III)/As(V) ratios varying among different springs due to mixing with meteoric surface waters prior to discharge.

These ratios are significantly influenced by redox transformations of different arsenic species by microorganisms (Nicholson, 1993; Mukhopadhyay et al.

2002). Hot spring waters typically contain 1-10 ppm arsenic and have been reported to bear upto 50ppm, which implies that geothermal fluids are a Significant source of Arsenic (Gihring and Banfield, 2001). Forest fires can also disperse arsenicals to the wind. The multiplicity of industrial, agricultural and anthropogenic activities has enhanced the mobilization of heavy metals above the rate manageable by biogeochemical cycles (Fig.1), thus resulting in an increased release of heavy metals in the environment. Among the anthropogenic sources of arsenic main are combustion of fossil fuels and smelting of non- ferrous metals. The above processes release arsenic as arsenic trioxide. Arsenic occurs in most coals in association with sulphur, when burned it accumulates on fly ash particles. The amount present on fly ash is significant. In the past 100 years commercially produced metal arsenites have been deliberately added to the biosphere as pesticides (Phillips and Tailor 1976),In arsenic rich environments a major concern is the potential for mobilization and transport of this toxic element to groundwater and drinking water supplies. In Bangladesh —57 million people have been exposed to arsenic through contaminated wells. This needs to understand the factors controlling the mobility and solubility of arsenic in aquatic system (Niggemyer et al. 2001). 330 million people in the Indian subcontinent are at risk of As exposure and consequently disease through contaminated drinking water (Esquivel et al., 1998).

441/1 / if//

gar! t.g J J

if HP/

it :ft rd.

arsenobetaine (C14 3⁾³As' CH 2COO

All' -40

(CI13)2As

-011

Air

.,.••••••••••

(CH 3)3AS

anthropo-

• .4"...0 •

Arsenic Cycle

oxidation

,--- M methylation

As (Ili) As (V) by algae

t t_zeduc1lon_, A

i

reduction I oxidation

I

r

^{-- ...„.. . 1} ...

As (0) As (HI) As (V) Sediment

...oxidatiolL) t„reduction ..,,

Fig. 1:

Arsenic cycle in nature (Mukhopadhyay et al. 2002)

1.2 Chemistry of Arsenic Compounds

Arsenic, the 3rd member of group VA (Nitrogen family) of the periodic table after Nitrogen and Phosphorous was discovered by Albertus Magnus (Germany) in 1250.The origin of the name comes from the greek word 'arsenikon' meaning yellow orpiment. The pure element is a steel gray crystalline solid that sublimes on heating and oxidizes readily in air. This element occurs as two modifications, yellow (sp. gr.1.97) and grey (sp. gr . 5.73).Grey arsenic is the usual stable form with a melting point of 817°C and sublimation point of 613°C. Grey arsenic is a very brittle semi metallic solid. It is steel grey in colour, crystalline and tarnishes readily in air. When heated in air, arsenic readily forms arsenious oxide, As203 (also known as arsenic trioxide), which has a garlic like odour. Arsenic occurs in minerals combined with Sulphur, like As4S3 (orpiment) and realgar (As4S4).The lemon colour of orpiment and orange colour of realgar lead to their use in pigment and cosmetics in the past. Commercially arsenic is obtained as a byproduct of gold, silver and copper metallurgy. Also by heating the ore prsenopyrite (FeAsS) from which Arsenic sublimes on heating.

600°C

FeAsS FeS+As

Arsenic and its compounds are poisonous. Arsenic compounds can be classified into three broad groups:

i) Inorganic arsenic eg. arsenate and arsenite ii) Organic arsenic e.g. lewisite

iii) Arsine gas

Elemental arsenic resists water, acid and alkalis, tarnishes in air and burns in oxygen.Organic arsenicals such as lewisite, ethyl dichloroarsine(ED), methyldichloroarsine (MD) and phenyldichloroarsine(PD) are well known chemical weapons or vesicants, quite potent in their action after mustards and

Table 1: Naturally occurring inorganic and organic As species

(see Fig. 2 for structures [1]—[22])

CAS No. Name Synonyms Structure

arsenate [1]

1--- arsenite [2]

124-58-3 methylarsonic acid monomethylarsonic acid, MMA

[3]

75-60-5 dimethylarsinic acid cacodylic acid, DMA [4]

4964-14-1 trimethylarsine oxide [5]

27742-38-7 tetramethylarsonium ion [6]

64436-13-1 arsenobetaine [7]

39895-81-3 arsenocholine [8]

T dimethylarsinoylribosides [9]—[19]

trialkylarsonioribosides [20], [21]

dimethylarsinoylribitol sulfate [22]

0

1

-o-As-o- 2

CH3-As-OH OH

0 CH3 4s- OH

CH3

4

(CH3)3Alv=0 (CH3)4As÷

5 6

(CH3)3As÷CH2Co0-

7

(cH3)A8-1-cH2cH2oH B

9-19 (cH2)2As

OH OH

9

0

OH

S031-I

10

R 0

OH

OH

11

0

OH

OSO3H

12

NH2

SO3H

13 R = OCH3 14

R ••■ 0

OH

COOH

15 ^{0 CH2}

—OH

—OH

16

R 0 0

COOH HO

HO

CI-120H

17

0 CV.. OH

OH OH

10

R 0 OCO(CH2)14CH3

OH OCO(CH2)1 4CH3

19 NH2

R

20 (CH3)3A6.- ^{0 OS03-}

OH

OH OH

21 CH3

HOm

I I

0

OH COOH CH3

T ~OS03 OH

OH OH

22 OH

II

(CH3)2A4 OSO3H

OH OH

Fig. 2: Structures of naturally occurring inorganic and organic arsenic species

Table 2: Other Arsenic compounds of environmental significance referred to in the text

CAS No. Name Synonyms Formula

Inorganic As, trivalent 1327-53-3 As(III) oxide

.

As trioxide, arsenous oxide, white As

As203 (or As406)

13768-07-5 arsenenous acid arsenious acid HAs02

7784-34-1 As(III) chloride As trichloride, arsenous trichloride

AsCI 3

1303-33-9 As(III) sulfide As trisulfide orpiment, auripigment

As2S3

Inorganic As, pentavalent

1303-28-2 As(V) oxide As pentoxide As205

7778-39-4 arsenic acid ortho-arsenic acid H3As04

10102-53-1 arsenenic acid meta-arsenic acid HAs03

arsenates, salts of ortho- arsenic acid

H2As04 , HAs042-, AsOt

Organic As

593-52-2 methylarsine CH3AsH2

593-57-7 dimethylarsine

r

^(CH3)2AsH

593-88-4 trimethylarsine (CH3)3As

98-50-0 (4-aminophenyl)-arsonic acid arsanilic acid, p-

aminobenzene-arsonic H2N AsO(OH)2 acid

139-93-5 4,4-arsenobis(2-aminophenol) dihydrochloride

arsphenamine, salvarsan

HCIH3N NH2HCI

0 H /26=A5-0-0 H

121-59-5 [4-[aminocarbonyl-

amino]phenyl] arsonic acid

carbarsone, N-

carbamoylarsanilic acid ^{NH2CONH --01--/260(OH)2} 554-72-3 [4-[2-amino-2-oxoethyl)amino]-

phenyl] arsonic acid

tryparsamide NN2cocH2NN-0—Aso( 0102

contd.

121-19-7 3-nitro-4-hydroxy- phenylarsonic acid

o2N

HO—b—Pe(OH)12

98-72-6 4-nitrophenylarsonic acid p-nitrophenylarsonic

acid o ^N

•

^{p6(0 ii)2}

dialkylchloroarsine R2AsCI

alkyldichloroarsine RasCl2

Introduction

phosgene oximes. DNA alkylation and/or inhibition of glutathione-scavenging pathways are two postulated mechanisms of its killer action. The onset of symptoms after 'exposure occurs in seconds(Table.1, Table2 and Fig.2).

1.3 Uses of Arsenic

Present day uses of arsenic are mainly in electronics e.g. solar cells, optoelectronic devices, semiconductor applications, light emitting diodes and digital watches. Among the industrial uses are glassware, electrophotography, catalysts, pyrotechnics, antifouling agents, dyes and soaps. Arsenic is also used in alloys with lead, in storage batteries and in ammunitions, automatic body solders and radiators, battery plates (hardening agents).Other uses include pigments and dyes, preservatives of animal hides, glass manufacture and wood preservatives. Currently veterinarians employ an organic arsenical, sodium capasolate, for the treatment of heartworms in dogs. In 2005, the United states was again the world's leading consumer of arsenic, mainly for CCA inspite of the voluntary ban on the consumption of CCA (Brooks, 2006).Arsenic is still not totally banned and is being used in some developing countries((Bentley and Chasteen, 2002; Mukhopadhyay et al., 2002; Lloyd, 2003; Rodriguez et al. 2003,

; Katz and Salem, 2005; Nachman et al. 2005; Jones,2007). At present arsenic is being used increasingly to make Gallium Arsenide (GaAs) semiconductors for use in semiconductor diodes. As compounds had been widely used as pesticides and wood preservatives. The first antiseptic Salvorsan 606 and the African sleeping sickness drug Melarsen (Clesceri et al. 1998) also contain arsenic.

During 18th , 19th and 20th centuries arsenicals were preferred for the control of agricultural pests before the widespread use of organochlorines. e.g. Paris green (CH3)2Cu.3Cu(As02)2 and Lead arsenate (PbHAs0 4 ) were used in insecticides.

White arsenic As203 was used as rodenticide, alkaline slution of As 203 as an insecticide and herbicide and methyl arsenic sulphide (CH3AsS) as a fungicide etc. The use in veterinary medicine as nutritional suppliments and in the

5

treatment of various diseases dates back to 15 th century. For the past centuries chronic feeding of small doses of various arsenic preparations has been reported to increase appetite, improve the level of activity, correct anaemia and improve the coats of animals. Arsenic was used as a feed additive which control enteric diseases of swine and poultry and to improve weight and feed efficiency of livestock. In the late 19 th century, a preparation known as Fowler's solution was in great demand which contains water, As203, KHCO3 and alcohol as an accepted treatment for leukemia and dermatitis. Organic arsenicals such as Lewicite (L), ethyldichloroarsine(ED), methyldichloroarsine (MD) and phenyldichloroarsine (PD) are well known chemical weapons or vesicants quite potent in their action after mustards and phosgene oxime.

1.4 Abiotic factors affecting Biogeochemical Cycling of arsenic

Speciation determines how arsenic compounds interact with their environment.

For example, the behaviour of arsenate and arsenite in soil differs considerably.

Movement in environmental matrices is a strong function of speciation and soil type. In a non-absorbing sandy loam, arsenite is 5-8 times more mobile than arsenate (Gulens et al. 1979). Soil pH also influences arsenic mobility. At a pH of 5.8 arsenate is slightly more mobile than arsenite, but when pH changes from acidic to neutral to basic, arsenite increasingly tends to become the more mobile species, though mobility of both arsenite and arsenate increases with increasing pH (Gulens et al., 1979). In strongly adsorbing soils, transport rate and speciation are influenced by organic carbon content and microbial population. Both arsenite and arsenate are transported at a slower rate in strongly adsorbing soils than in sandy soils.

Introduction

1.5 Biological activity of arsenicals

Till early 1990's it was believed that arsenic is not a mutagen (Rossman et al.

1980; Lee et al. 1985) by itself but it can act synergistically to enhance the mutagenic and clastogenic effect of knOwn potent mutagens like MMS, EMS etc.(Jan et al. 1991).Arsenic is a weak inducer of chromosome aberrations.and sister chromatid exchanges (Larramendy et al.1981, Nakamuro et al. 1981, Wen et al.1981, Wan et at 1982,Lee et al ,1985). Now, Arsenic has been classified by

USEPA as a human carcinogen. Arsenic can effect biochemical reactions.

Trivalent arsenic can bind to the thiol groups of critically important proteins and pentavalent arsenic can replace phosphate in biochemical reactions and disrupt the formation of ATP in vitro.The toxicity of arsenate ion lies in its ability to mimic the PO4 ion.Traversing into the molecular mechanism of arsenate toxicity reveals that arsenate resembles the ion phosphate both in size and valency, hence it gets preferably encorporated into ADP and gives ADP arsenate(ADP-As) instead of ATP. This molecule of ADP-As undergoes a futile cycle of hydrolysis where the cleavage of ADP-As bond is totally a wasteful process, yielding no energy for the cellular metabolic activities.Also abundance of arsenate in media leads to phosphate starvation. Arsenite on the other hand acts mainly by interacting with proteins and enzymes usually denaturing them or inhibiting their function. Arsenite (As0 2" and As033") has been shown to inhibit dehydrogenases such as pyruvate dehydrogenase, a-ketoglutarate dehydrogenase and dihydrolipoate dehydrogenases (Mahler and Cordes,1966). As reported by Da Costa, 1972 As(III) uncouples the oxidative phosphorylation i.e. inhibition of oxidative phosphorylation by chemiosmosis.

What happens to arsenic when it enters the human body? The toxicity of arsenic to mammals is related to its absorption and retention in the body and varies with chemical form. The toxicity of arsenicals in decreasing order is; inorganic arsenites>organic trivalent compounds (arseoxides) > inorganic arsenates>

arsonium compounds>elemental arsenic. Toxicity appears to be related to the solubility of arsenical in water. The low toxicity of elemental arsenic is attributed to

its near insolubility in water and body fluids. Inorganic arsenic is a potent mutagen as well as carcinogen. It is associated mainly with skin and lung cancers. It induces micronucleii, chromosomal aberrations (Oya-Ohta et al. 1996) and Sister chromatid exchanges in vitro in human lymphocytes as well as in cultured chinease hamster cell lines.

1.6 Arsenic Resistant Bacteria

Arsenic resistance in bacteria is a widespread phenomenon. The resistant bacteria fall into diverse taxonomic groups. E.coli, Pseudomonas spp., Acidiphilium multivorum, Alcaligenes sp., Desulphotomaculum sp., .Aeromonas sp., Exiguobacterium sp. etc. represent the gram negative community, while Bacillus spp. , Staphylococcus aureus, Thiobacillus sp., Acenetobacter sp., Clostridium sp., Sulfurosprilliim barnesii (Stolz et al. 2002) are among the important members of gram negative group which show high levels of resistance towards arsenic. Some archebacteria have also been found to possess arsenic resistance, e.g.

Ferroplasma acidarmanus' Fer1 is an arsenic-hypertolerant acidophilic archaeon isolated from the Iron Mountain mine, California; a site characterized by heavy metals contamination (Austin et al.2007). Halobacterium sp. NRC-1, which is an extremely halophilic archaeon possess arsenic resistance genes on its plasmid pNRC-100 (DasSarma et al. 2006).

1.7 Antibiotic resistance in arsenic resistant bacteria

Heavy metal resistance and drug resistance are often linked and are present on the same plasmid, e.g. mercury is frequently specified by drug resistance plasmids and is also common in soil Pseudomonas and Bacilli. Plasmid determined copper resistance has been reported on an antibiotic resistance plasmid, in E. coli isolated from pig fed copper supplements as growth stimulants. Arsenic resistances are governed by plasmids that also code for antibiotic and other heavy metal resistances. For example, in Tokyo in the late 1970s both heavy metal resistances

Introduction

and antibiotic resistances were found with high frequencies in Escherichia colt isolated from hospital patients ,where as heavy metal resistance plasmids without antibiotic resistance determinants were found in E. colt from an industrially polluted river. Selection occurs for resistances to both types of agents in the hospital, but only for resistance to toxic heavy metals in the river environment (Shukla et al.

2006). Virdi et al., 2001 have reported an arsenic resistant strain of Yersenia enterocolitica which was resistant to five antibiotics.MIC of five antibiotics namely amikacin, gentamicin, tetracycline, ciprofloxacin, and nitrofurantoin for pork isolates of Yersinia enterocolitica increased two- to eightfold after bacteria were grown in the presence of 5 mm arsenite. For Y. enterocolitica isolates obtained from wastewater (sewage effluents), an unequivocal increase in MICs was seen with amikacin and gentamicin. Rajini Rani et al. (1992) studied a Pseudomonas sp.

isolated from the Bay of 'Bengal (Madras coast) contained a single large plasmid (pMR1) of 146 kb. Plasmid curing was not successful with mitomycin C, sodium dodecyl sulfate, acridine orange, nalidixic acid or heat. Transfer of mercury resistance from marine Pseudomonas to Escherichia coli occurred during mixed culture incubation in liquid broth at 104 to 10 -5 m1-1 . However, transconjugants lacked the plasmid pMR1 and lost their ability to resist mercury. Transformation of pMR1 into E. colt competent cells was successful; however, the efficiency of transformation (1.49A-10 2 Hg r transformants pg -1 pMR1 DNA) was low. E. coli transformants containing the plasmid pMR1 conferred inducible resistance to mercury, arsenic and cadmium compounds similar to the parental strain, but with increased expression. The mercury resistant transformants exhibited mercury volatilization activity. A correlation existed between metal and antibiotic resistance in the plasmid pMR1.

1.8 Biotransformation

In aqueous environment bacteria and other microorganisms interact with arsenic compounds in different ways. Some bacteria, fungi and algae are able to reduce

9

arsenate into arsenite and finally into trimethylarsine (Woodfolk and Whitelay 1962, Sehlin and Lindstrom 1992). Studies with Methanobacterium M.O.H.(McBride and Wolfe 1971) have shown that the reaction is as follows :

0

0

2e- RCH3 I I RCH3 11 +2e-

As043_" As02 CH 3—AS—OH CH3—AS—OH CH 3—AS—H

OH CH CH

3 3

arsenate arsenite methylarsonic acid Dimethyl arsinic acid Dimethyl arsine (cacodylic acid)

Dissimilatory reduction of arsenic (V) has been shown to occur in at least nine different genera scattered throughout . the bacterial domain (Newman et aI.1998, Stolz and Oremland 1999, Gihring and banfield 2001 and Niggemyer et al. 2001) and has also been observed in hyperthermophilic archea (Huber et al. 2000).

These microorganisms are either strict anaerobes, facultative anaerobes or microaerophiles, capable of utilizing arsenate as the terminal electron acceptors.

However sufficient evidence is present in favour of aerobic bacteria being involved in the reduction of arsenate (Jones et al. 2000, Macy et al. 2000, Macur et al.

2001, 2004). Pseudomonas spp. and Alcaligenes spp. are able to reduce arsenate to arsenite and both to arsine (AsH 3) anaerobically (Cheng and Focht 1979).0n the other hand some microbes can oxidize reduced arsenic, i.e. arsenite into arsenate (Sehlin and Lindstrom 1992, Macur et al. 2004). In Alcaligenes faecalis the arsenite oxidising activity was found to be inducible by arsenite or arsenate (Osborne and Ehrlich 1976). Normally the bacteria which are involved in biotransformation are them selves resistant to arsenic to certain levels. The occurrence of arsenate resistant bacteria has been reported across oxic-anoxic boundaries (Saltikov et al. 2003). In that context it is necessary to mention that arsenate resistant bacteria are not necessarily arsenate respiring, which are till

r

Introduction

now reported to be isolated from anoxic waters and sediments and show a very high tolerance towards arsenite( --10mM - 60mM), which is only upto 1 mM for non-arsenate respiring arsenate resistant bacteria. Microorganisms can mediate a variety of reactions including reduction, oxidation and methylation. A number of bacteria reduce As(V) to As(III) as a detoxification mechanism based on the enhanced outward mobility from the cell of As(III)(Dowdle et al.1996). In 1994-95 first reports of some novel strains of bacteria (Ahmann et al.1994 and Laverman et al.1995) capable of respiratory growth by coupling the reduction of As(V) to As(III) with the oxidation of lactate were published. Thermodynamic calculations showed that this reduction is sufficiently exergonic to sustain growth. Reduction of As(V) to As(III) in anoxic sediments is carried out by bacterial dissimilatory arsenic reduction (DAsR). The biogeochemical cycle of this element depends on microbial transformation which affects the mobility and distribution of arsenic species in the environment. Several bacteria involved in the transformation process consisting reduction, oxidation and methylation of arsenic species have been described(Muller et al . 2003). Knowledge of bacterial biotransformation has led to the exploration of alternative methods for atrsenic remediation based on its biological oxidation. Several arsenite oxidising bacteria have also been isolated, starting with an Achromobacter strain. Since then different arsenite oxidising bacteria including several Pseudomonas strains, Alcaligenes faecalis, Thiobacillus ferrooxidans and Thiobacillus acidophilus; bacteria from the Agrobacterium /Rhizobium branch of p-Proteobacteria and bacteria of the Thermus genus have been described. Recently a bacterium belonging to the Zoogloae branch of ^[3- Proteobacteria was isolated from an Arsenic contaminated environment. This strain ULPAs1 is able to efficiently oxidize arsenite to arsenate. Due to its increased resistance to As(III) as well as other heavy metals this strain is a good candidate for bioremediation of environments heavily contaminated with arsenic (Weeger et al. 1999).

■ 11