Isolation and characterization of Marinobacter sp.A Project Report Submitted to the Gulbarga University, Gulbargaas a partial fulfilment for the degree of

MASTER OF SCIENCEINBIOTECHNOLOGYSubmitted bySonaliBharath.N.VSupervised byDr. (Smt.) M. B. Sulochana M.Sc., M.Phil, Ph.D.

DEPARTMENT OF BIOTECHNOLOGY,GULBARGA UNIVERSITY,GULBARGA 585 106201112

GULBARGA UNIVERSITY, GULBARGA Telex: 0895-208-GULU-INJnana Ganga, Gulbarga 585 106, Karnataka, India Fax. No. 08472-245632Post-Graduate Department of Studies and Phone: 245446.Research in Biotechnology

Certificate

This is to certify that, the project work entitled Isolation and Molecular characterization of Halomonas Organivornas sp. submitted by Ashok Rathod and Srikant Simpi for the partial fulfilment for the degree of Master of Science in Biotechnology in Gulbarga University, Gulbarga under the guidance of Dr. (Smt.) M. B. Sulochana, during the academic year of 2013-2014.

Project Supervisor ChairmanDr. (Smt.) M. B. Sulochana Dr. Chandrakanth Kelmani Department of Biotechnology, Gulbarga University, Gulbarga.Examiners:1.

2.

DeclarationWe hereby declare that the present report entitled Isolation and Molecular Characterization of Halomonas Organivornas sp. submitted to Department of Biotechnology, Gulbarga University, Gulbarga, for the partial fulfilment of Masters Degree in Biotechnology is the result of the project work carried out under the guidance of Dr. (Smt.) M. B. Sulochana, Reader, Department of Biotechnology, Gulbarga University, Gulbarga. We further declare that the results of this work have not been previously submitted for any other degree or discipline.

Place: Date: Ashok Rathod Srikant Simpi

Acknowledgement

We would like to express our heartly and sincere gratefulness to the Almighty for having blessed us to undertake the academic project work in Biotechnology and to commence and successfully complete the same by his grace. We immensely express our special gratitude and deep sense of esteem to our project supervisor Dr.(Smt.) M. B. Sulochana,, Reader, Department of Biotechnology, Gulbarga University, Gulbarga for her indispensable support, encouragement and valuable suggestions throughout the project.Our grateful thanks are due to Dr. Chandrakanth Kelmani Chairman, Department of Biotechnology, Gulbarga University, Gulbarga for providing us timely facilities. It is a matter of pride to extend our deep sense of gratitude to Prof. G. R. Naik, and Dr. Ramesh Londonkar, Department of Biotechnology and Guest Lecturers who have helped us throughout the project work and non-teaching staff for their help during the work. We are grateful Mr. Jayachandra S Y., Research Scholar, Department of Biotechnology, Gulbarga University, Gulbarga for his timely help and valuable suggestions throughout the project. Further, we extend our heartfelt thanks to the Research Scholars Mr. parameshwar, Mrs. Merely D.P, Mr. Mohan Reddy, Mr. Ajay Kumar Olie, Mr. Hanumanthappa B Nayak, Department of Biotechnology, Gulbarga University, Gulbarga for their support, co-operation and encouragement during this work. We shall be failing in our duty if we do not place on record our hearty and loving gratefulness to our dearest parents for their constant inspiration without which we could not have completed our task to the subjective satisfaction.

Ashok Rathod Srikant Simpi

Contents

I. Review of Literature

1. Halophiles

1.1Moderately Halophilic Bacteria1.2Halomonadaceaea 1.3Halotolerance1.4Osmoregulation2. Extremophiles and Biomolecules

2.1Compatible Solutes 2.2Ectoine 2.3Betaine2.4Mannosylglycerate 2.5Diglycerol Phosphate2.6Trehalose

3. Production of Compatible Solutes

3.1Product Recovery and Purification

4. Heavy Metal Stress

4.1Metal Tolerance Mechanisms4.2Correlation of Metal Tolerance and Antibiotic Resistance

II. Materials and Methods Grams staining MotilityPhysicochemical Parameters Effect of Inoculums sizeEffect of pHEffect of SaltEffect of Heavy Metals IIIResults and Discussion IV. Conclusion

Two groups of prokaryotes are adapted to live under extreme hypersaline conditions, halophilic Archaea (haloarchaea) and halophilic bacteria. The halophilic bacteria are represented by the moderate halophiles (showing optimal growth at 3 to 15% NaCl) and the extreme halophiles (with optimal growth at 15 to 15% NaCl). Although haloarchaea have been considered as the most interesting models for basic research in halophiles and several important biotechnological applications have been developed, halophilic bacteria have a great biotechnological potential as a source of new compounds (compatible solutes, polymers) or for biodegradation processes. Since these bacteria are able to grow at changing environmental conditions they can be important sources of extracellular enzymes that could be used as biocatalysts under extreme conditions of salinity as well as of temperature and pH values.Halophiles are microorganisms that adapt to moderate and high salt concentrations. They are found in all three domains of life: Archaea, Bacteria and Eukarya. Halophilic bacteria grow over an extended range of salt concentrations (315% NaCl, w/v and above), unlike the truly halophilic Archaea whose growth is restricted to high saline environments(Litchfield, C. D, et al,. 2002. ). Water availability in microorganisms is inversely proportional to the concentration of dissolved salts (Galinski, E. A. et al,. 1995). Halophilic archaea usually employ a continuous influx of ions like K+ in order to balance the high salt environment outside the cell. In such cases the intracellular protein machinery is dependent on high salt concentrations for function and stability. On the other hand, halophilic bacteria usually synthesize or accumulate compatible solutes to maintain the osmotic equilibrium in response to the high-salt external environment. These organic molecules are compatible with the intracellular machinery even at molar concentrations and hence the name (Da Costa, M. S, et al,. 1998). They maintain the cell volume, turgor and electrolyte concentrations within the cell system. As a result, an appropriate hydration level of the cytoplasm is achieved and cell growth can proceed under osmotically unfavourable conditions (Bursy, J., et al,.2008). Compatible solutes are low-molecular weight osmoregulatory compounds which are highly water-soluble sugars, alcohols, amino acids, betaines, ectoines or their derivatives. In addition to their stabilizing effects, they are used as salt antagonists, stress protective agents, moisturizers and therapeutics. They stabilize enzymes, DNA and whole cells against stresses such as freezing, drying and heating. They increase freshness of foods by stabilizing components. Induction of osmolytes in cells can increase protein folding and thereby improve salt tolerance which could be useful in agriculture and xeriscaping (Roberts, M. F, 2005, Detkova, E. N. 2007)Halophiles Halophiles are a group of microorganisms that live in saline environments and in many cases require salinity to survive. Halophiles include a great diversity of organisms, like moderately halophilic aerobic bacteria, cyanobacteria, sulphur-oxidizing bacteria, heterotrophic bacteria, anaerobic bacteria, archaea, protozoa, fungi, algae and multicellular eukaryotes. Microorganisms that are able to grow in the absence as well as in the presence of salt are designated as halotolerant and those that are able to grow above approximately 15% (w/v) NaCl (2.5 M) are considered extremely halotolerant (Kushner, D. J. 1988, DasSarma, S. 2001) According to Kushner, many marine organisms are slight halophiles (with 3% w/v NaCl in sea water). Moderate halophiles optimally grow at 315% w/v NaCl, extreme halophiles at 25% w/v NaCl (halobacteria and halococci) and borderline extreme halophiles require at least 12% w/v salt

Moderately halophilic bacteria

This group of bacteria has been reviewed extensively by Ventosa et al.(1998). The occurrence of nonpigmented halotolerant bacteria is said to be first mentioned in 1919 by LeFevre and Round(1919) in their study on microbiology of cucumber fermentation brines. Halophilic bacteria are found in a variety of salt environments like marine ecosystems, salted meat, salt evaporation pools and salt mines. Most of the important groups of bacteria are able to live in concentrations up to about 15% salt and others can adapt to conditions even at higher salt concentrations (Hof, T. et al., 1935). They form a versatile group and are adapted to life at the lower range of salinities and have the ability to adjust rapidly to changes in the external salt concentration.

HaLOMONADACEAEA A dramatic increase in the number of descriptions of new species in recent years can also be observed for the second phylogenetic group that consists (almost) entirely of halophiles: the family Halomonadaceae (Gammaproteobacteria). When Halomonas elongata was isolated in 1980 few people predicted that it would be the first representative of a very large group of metabolically versatile moderate halophiles. The recognition that Halomonas and relatives deserve to be classified in a separate family came in 1988 [Galinski, E. A., et al,.1988 ], and the number of species and genera has been rising steadily since, Recently two new genera were added to the family, Halotalea and Modicisalibacter , bringing the census for March 2008 to 7 genera with 63 species whose names have been validly published. Out of these 63 species, 60 can be considered halophiles as based on the above definition. The three genera Zymomonas, Carnimonas, andCobetia each contain presently one non-halophilic or marine species. As of March 2, 2008, four new species of the genus Halomonas were listed as 'in press' in the International Journal of Systematic and Evolutionary Microbiology.Marinobacter The genus Marinobacter was proposed by Gauthier et al. (1992) with a single species, Marinobacter hydrocarbonoclasticus. The second species, Marinobacter aquaeolei, was described by Huu et al. (1999). Recently, five further species, Marinobacter litoralis (Yoon et al., 2003), Marinobacterlipolyticus (Martn et al., 2003), Marinobacter excellens (Gorshkova et al., 2003), Marinobacter lutaoensis (Shieh et al., 2003) and Marinobacter squalenivorans (Rontani et al.,2003), have been added to the genus Marinobacter. Members of the genus Marinobacter have been isolated from marine environments, saline soil and coastal hot springs. Halotolerance

Halotolerance is the adaptation of living organisms to conditions of high salinity. Proteins of halophilic microorganisms contain an excess ratio of acidic to basic amino acids and are resistant to high salt concentration. Proteins in extreme halophiles have structurefunction stability only in the presence of salt and their enzymes require salts for activity. Surface negative charges prevent denaturation and precipitation of proteins at high salt concentrations (Kushner, D. J, 1978.). Adaptation to conditions of high salinity has an evolutionary significance. The concentration of brines during prebiotic evolution suggests haloadaptation at earliest evolutionary times (Dundas, I,. et, al,. 1998). An understanding of halotolerance can be applicable to areas such as arid-zone agriculture, xeriscaping, aquaculture or remediation of salt-affected soils. In addition, knowledge of halotolerance involving osmotic changes can also be relevant to understanding tolerance to extremes in moisture or temperature (Kastritis, P. L., et al., 2007).

Osmoregulation

Many microorganisms respond to increase in osmolarity by accumulating osmotica in their cytosol, which protects them from cytoplasmic dehydration (Yancey, P. H., et al.,1982). Osmophily refers to the osmotic aspects of life at high salt concentrations, especially turgor pressure, cellular dehydration and desiccation. Halophily refers to the ionic requirements for life at high salt concentrations. According to Rothschild and Mancinelli (2001) organisms live within a range of salinities essentially from distilled water to saturated salt solutions. Halophilic microorganisms usually adopt either of the two strategies of survival in saline environments: compatible solute strategy and salt-in strategy (Ventosa, A., et al., 1998). Compatible solute strategy is employed by the majority of moderately halophilic and halotolerant bacteria, some yeasts, algae and fungi. In this strategy cells maintain low concentrations of salt in their cytoplasm by balancing osmotic potential through the synthesis or uptake of organic compatible solutes. Hence these microorganisms are able to adapt to a wide range of salt concentrations. The compatible solutes include polyols such as glycerol, sugars and their derivatives, amino acids and their derivatives, and quaternary amines such as glycine betaine and ectoines. The salt-in strategy is employed by true halophiles, including halophilic archaea and extremely Halophilic bacteria. These microorganisms are adapted to high salt concentrations and cannot survive when the salinity of the medium is lowered. They generally do not synthesize organic solutes to maintain the osmotic equilibrium. This adaptation involves the selective influx of K+ ions into the cytoplasm. All enzymes and structural cell components must be adapted to high salt concentrations for proper cell function.

Extremophiles and Biomolecules

The industrial and environmental applications of Halophilic microorganisms have been reviewed by Oren (2010). The review highlights the salient features of halophiles, including their highly successful applications like - carotene production by Dunaliella and ectoine synthesis using Halomonas and other moderately halophilic bacteria. The potential use of bacteriorhodopsin, the retinal protein proton pump of Halobacterium is being explored in photochemical processes. Other possible uses of Halophilic microorganisms, as discussed in the review, include treatment of saline and hypersaline wastewaters and productionof exopolysaccharides, poly- -hydroxyalkanoate bioplastics and biofuel. Halophilic bacteria offer potential applications in various fields of biotechnology (Margesin, R. et al., 2001). Although moderately Halophilic bacteria have many industrial applications, only a few studies have been carried out concerning this aspect. These microorganisms can be used as a source of metabolites, compatible solutes and other compounds of industrial value. Novel halophilic biomolecules may also be used for specialized applications, e.g. bacteriorhodopsin for biocomputing, pigments for food colouring and compatible solutes as stress protectants. Biodegradation of organic pollutants by halophilic bacteria and archaea has been recently reviewed. These microorganisms are good candidates for the bioremediation of hypersaline environments and treatment of saline effluents. Understanding the degradation process would also shed light on the enzymes involved and on the regulation of metabolism. Halophilic bacteria are a potential source of extracellular hydrolases like proteases with a wide array of industrial applications. These enzymes exhibit stability over a range of saline conditions (. Shivanand, P et,al., 2009). Halophilic bacteria constitute excellent models for the molecular study of osmoregulatory mechanisms (Ventosa, A., et al., 1998). Naturally halotolerant plants or microorganisms could be developed into useful agricultural crops or fermentation organisms. This has possible application in agriculture, where conventional agricultural species could be made more halotolerant by gene transfer from naturally halotolerant species. Arabidopsis thaliana transformed with a choline oxidase gene from Arthrobacter globiformis has been reported to have a significantly improved tolerance of salt stress (Arakawa, T,. et al., 1985). This field offers a lot of scope for research and development.

Compatible solutes Compatible solutes are low-molecular weight osmoregulatory compounds, including highly water-soluble sugars, alcohols, amino acids, betaines, ectoines or their derivatives ( Roberts, M. F.,2005, Ventosa, A., et al., 1998, Empadinhas, N. et, al,. 2008). Compatible solutes are useful as stabilizers of biomolecules and whole cells, salt antagonists or stress-protective agents. Due to their stabilizing effects, they can be used for various research and industrial applications. Moderate halophiles are expected to produce polar uncharged and zwitterionic solutes and halo-thermophilic microbes produce anionic compatible solutes. Compatible solutes have protein-stabilizing properties that help in the proper folding of polypeptide chains (Arakawa, T,. 1982). Due to their stabilizing effect on protein molecules they are also sometimes referred to as chemical chaperones ( Chattopadhyay, M. K.,2004). Conformational shift of protein towards folded, native like states induced by preferential exclusion of the solute is responsible for the chaperone-like effects. Compatible solutes exert their effect through changes in solvent structure and/or subtle changes in the dynamic properties of the protein rather than by changing the structure of the protein itself (Kolp, S., Pietsch, M.,2006). Compatible solutes also interact with nucleic acids and can influence proteinDNA interactions ( Lamosa, P.,et al., 2003 Pul, U., et al., 2007). Ectoine Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is one of the most common osmotic solutes in the domain bacteria. It was first discovered in the haloalkaliphilic photosynthetic sulphur bacterium, Ectothiorhodospira halochloris, but later a great variety of halophilic and halotolerant bacteria were found to produce this compound, often together with its 5-hydroxy derivative (Rothschild, L. J.et, al,. 2001). Ectoines are common in aerobic heterotrophic eubacteria. The entry molecule into ectoine biosynthesis is aspartate semialdehyde, which is an intermediate in amino acid metabolism. The aldehyde is converted to L-2,4-diaminobutyric acid, which is then acetylated to from N -acetyldiaminobutyric acid (NADA). The final step is the cyclization of this solute to form ectoine. Ectoine synthesis is carried out by the products of threegenes: ectABC. The ectA gene codes for diaminobutyric acid acetyltransferase; ectB codes for the diaminobutyric acid aminotransferase and ectC codes for ectoine synthase. Ectoines have gained much attention in biotechnology as protective agents for enzymes, DNA and whole cells against stresses such as freezing, drying and heating. Inrecent years additional properties of interest were found for ectoine. It is claimed that it counteracts the effects of UV-A-induced and accelerated skin ageing, and therefore, is being used as a dermatological cosmetic additive in moisturizers for the care of aged, dry or irritated skin. Ectoine also inhibits aggregation of Alzheimers -amyloid, and recently, a clinical trial was initiated to test its efficacy in inhalations against bronchial asthma (Oren, A., et al., 2010). Ectoines are used for increasing the stability and freshness of foods by stabilizing food components. Ectoines also find applications in the treatment of the mucous membranes of the eye. Ophthalmologic preparations containing these molecules are useful for eye treatment to decrease the dryness syndrome. Introduction of ectoine and its derivatives into preparations for oral care has also been suggested (Detkova, E. N, et al., 2007). Betaine Betaines are the compatible solutes occurring in Halophilic phototrophic bacteria, chemotrophic bacteria and archaebacteria. They have therapeutic potential for the treatment and prophylaxis of adipose infiltration of the liver, which is the initial stage of cirrhosis(Detkova, E. N, et al., 2007). Betaines decrease side effects of anti-inflammatory preparations. Their anticoagulant properties prevent thrombus formation and decrease the probability of heart attacks, infarctions and strokes (Messadek, J.,et,.al, 2005). They are useful in PCR amplification of GC-rich DNA templates to increase product yield andspecificity. Betaine was shown to be a more effective cryo-protectant than serum albumin or trehalose or dextran, particularly under conditions stimulating long-term storage (Cleland, D., et al., 2004).

Mannosylglycerate Mannosylglycerate (MG) is a novel compatible solute widely found in the halotolerant Methanothermus fervidus, Pyrococcus furiosus and Rhodothermus marinus. This compound has also been detected in many hyperthermophilic archaea, where it accumulates concomitantly with increasing salinity of the medium (Santosh, H et al., 2004). Although present in many red algae, the apparent restriction of MG to thermophilic bacteria and hyperthermophilic Archaea led to the hypothesis that it plays a major role in thermal adaptation (Empadinhas, N. et al., 2008). In R. marinus, there is a direct condensation of GDP-mannose and D-glycerate to form MG catalysed by mannosylglycerate synthase. Possible applications are utilization as protectants for enzymes against physical or chemical stress, as additive in PCR, and excipient in pharmaceuticals. Diglycerol phosphateDiglycerol phosphate accumulates under salt stress in the hyperthermophile Archaeoglobus fulgidus. This new compatible solute is a potentially useful protein stabilizer, as it exerted a considerable stabilizing effect against heat inactivation of various dehydrogenases and a strong protective effect on rubredoxins (with a fourfold increase in the half-lives) from Desulfovibrio gigas and Clostridium pasteurianum (Litchfield, C. D., et al., 2002).TrehaloseThe non-reducing glucose disaccharide, trehalose is used by organisms to counteract drying, but it also serves as an osmolyte (. Roberts, M. F., et al., 2005). It occurs in a wide variety of organisms, from bacteria and archaea to fungi, plants and invertebrates. Trehalose is not only useful as a cryoprotectant for the freezedrying of biomolecules, but also for long-term conservation of microorganisms, as the membrane structure is preserved in the presence of this disaccharide (Empadinhas, N et al., 2008). Production of compatible solutes For practical applications, reasonable quantities of compatible solutes have to be generated either in vitro or in vivo. A novel bioprocess for the production of ectoine from Halomonas elongata called bacterial milking (Guzmn, H., et al.,2009) has been the basis of the German biotechnology company Bitop, which develops products from osmolytes. After a high-cell-density fermentation, cells are fivefold concentrated using cross-flow filtration. Bacteria in high concentrations of NaCl are subjected to osmotic shock by transferring the cell biomass to low osmolarity medium, where they rapidly excrete the now excess solutes in the medium to maintain the osmotic equilibrium. After each dilution step, the medium (containing the solutes) has to be removed prior to re-exposure to high salt. Subsequent reincubation in a medium of higher salt concentration results in the resynthesis of these compatible solutes. The process could be repeated several times after a defined generation time. After nine repetitions, 155 mg ectoine/g cell dry wt/cycle was produced. A process comprising two fed-batch cultures for the production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) by a moderate halophile, Halomonas boliviensis has been reported32. The co-production process as described has been proposed for lowered production costs of the respective molecules. Application of statistical design involving response surface methodology allowed a quick optimization of medium components for ectoine production by H. Boliviensis (Van-Thuoc, D.,et al., 2003). An overall ectoine volumetric productivity of 6.3 g/l/day was obtained. The optimized medium also showed improvement in ectoine productivity when used in fed-batch fermentation. An alternative, economically viable production method was proposed by demonstrating that a metabolic bottleneck for ectoine production in the non-halophilic recombinant Escherichia coli DH5 can be relieved by coexpression of deregulated aspartate kinase from Corynebacterium glutamicum (Bestvater, T., et al., 2005).

Product recovery and purification The determination of endogenous compatible solute accumulation in a study by Teixid et al.(2005), cell suspensions were centrifuged; cells were harvested and freezedried. Subsequently, cell material was extracted using the method described by Kunte et al.(1993) for quantitative analysis with extraction mixture methanol/chloroform/water 10 : 4 : 4 by volume) by vigorous shaking followed by the addition of equal volumes of chloroform and water. Phase separation was enhanced by centrifugation. The hydrophilic top layer containing compatible solutes was recovered and analysed by HPLC. For the recovery of ectoines from the product solution (. Sauer, T. et al., 1998), the pH of the solution was lowered with HCl to permit ectoines in their cationic form. Ectoines were purified using cation exchange resin packed in a column and subsequently eluted with NaOH. The ectoine fraction was marked by increased UV absorbance (at 230 nm). The recovered ectoines can be subsequently crystallized from water. Several analytical methods have been established for the detection of compatible solutes and further quantification using HPLC analysis. Purification of compatible solutes relies on chromatographic steps. Ectoine and hydroxyectoine concentrations can be determined by HPLC analysis (Severin, J., et al., 2007). The quality of purified ectoines can be controlled using NMR measurement (Frings, E.,et al., 1993) as well as isocratic HPLC and FMOCHPLC techniques. A combination of anion-exchange chromatography and pulse amperometric detection is a sensitive method that can detect osmolytes such as ectoine after hydrolytic cleavage of the pyrimidine

Heavy Metal Stress Many mechanisms solely responsible for specific metalion resistance in prokaryotes have been described at the molecular level (recently reviewed by Nies, 1999; Xu et al., 1998). The effect of metal-ion stress on microbial cells} communities has been investigated and suggests that individual strains adapt to elevated metal-ion concentrations; however, no analysis of the resultant strains was initiated at the molecular level (reviewed by Giller et al., 1998; Kelly et al., 1999). To survive under metal-stressed conditions, bacteria have evolved several types of mechanisms to tolerate the uptake of heavy metal ions. These mechanisms include the efflux of metal ions outside the cell, accumulation and complexation of the metal ions inside the cell, and reduction of the heavy metal ions to a less toxic state (Nies, 1999)Microbes may play a large role in the biogeochemical cycling of toxic heavy metals also in cleaning up or remediating metal-contaminated environments. There is also evidence of a correlation between tolerance to heavy metals and antibiotic resistance, a global problem currently threatening the treatment of infections in plants, animals, and humansMetal Tolerance MechanismsIn high concentrations, heavy metal ions react to form toxic compounds in cells (Nies, 1999). heavy metal ions must first enter the cell. Because some heavy metals are necessary for enzymatic functions and bacterial growth, uptake mechanisms exist that allow for the entrance of metal ions into the cell. There are two general uptake systems one is quick and unspecific, driven by a chemiosmotic gradient across the cell membrane and thus requiring no ATP, and the other is slower and more substrate-specific, driven by energy from ATP hydrolysis. While the first mechanism is more energy efficient, it results in an influx of a wider variety of heavy metals, and when these metals are present in high concentrations, they are more likely to have toxic effects once inside the cell (Nies and Silver, 1995).

Correlation of Metal Tolerance and Antibiotic ResistanceBacterial resistance to antibiotics and other antimicrobial agents is an increasing problem in todays society. Resistance to antibiotics is acquired by a change in the genetic makeup of a bacterium, which can occur by either a genetic mutation or by transfer of antibiotic resistance genes between bacteria in the environment, Because our current antibiotic are becoming less useful but used more heavily against antibiotic resistant pathogenic bacteria, infectious diseases are becoming more difficult and more expensive to treat. The increased use of antibiotics in health care, as well as in agriculture and animal husbandry, is in turn contributing to the growing problem of antibiotic resistant bacteria. Products such as disinfectants, sterilants, and heavy metals used in industry and in household products are, along with antibiotics, creating a selective pressure in the environment that leads to the mutations in microorganisms that will allow them better to survive and multiply (Baquero et al., 1998). According to Jeffrey J. Lawrences (2000) discussion of the selfish Operon Theory, clustering of genes on a plasmid, if both or all genes clustered are useful to the organism, is beneficial to the survival of that organism and its species because those genes are more likely to be transferred together in the event of conjugation. Thus, in an environment with multiple stresses, for example antibiotics and heavy metals, it would be more ecologically favourable, in terms of survival, for a bacterium to acquire resistance to both stresses. If the resistance is plasmid mediated, those bacteria with clustered resistance genes are more likely to simultaneously pass on those genes to other bacteria, and those bacteria would then have a better chance at survival. In such a situation, one may suggest an association with antibiotic resistance and metal tolerance. For example, Calomiris et al. (1984) isolated bacteria were tolerant to metals were also antibiotic resistant. Although some heavy metals are important and essential trace elements, at high concentrations, such as those found in many environments today, most can be toxic to microbes. Microbes have adapted to tolerate the presence of metals or can even use them to grow. Thus, a number of interactions between microbes and metals have important environmental and health implications. Some implications are useful, such as the use of bacteria to clean up metal-contaminated sites. Other implications are not as beneficial, as the presence of metal tolerance mechanisms may contribute to the increase in antibiotic resistance. Overall, it is most important to remember that what we put into the environment can have many effects, not just on humans, but also on the environment and on the microbial community on which all other life depends. Although some heavy metals are essential trace elements, most can be, at high concentrations, toxic to all branches of life, including microbes, by forming complex compounds within the cell. Because heavy metals are increasingly found in microbial habitats due to natural and industrial processes, microbes have evolved several mechanisms to tolerate the presence of heavy metals (by either efflux, complexation, or reduction of metal ions) or to use them as terminal electron acceptors in anaerobic respiration. Thus far, tolerance mechanisms for metals such as copper, zinc, arsenic, chromium, cadmium, and nickel have been identified and described in detail. Most mechanisms studied involve the efflux of metal ions outside the cell, and genes for this general type of mechanism have been found on both chromosomes and plasmids. Because the intake and subsequent efflux of heavy metalions by microbes usually includes a redox reaction involving the metal (that some bacteria can even use for energy and growth), bacteria that are resistant to and grow on metals also play an important role in the biogeochemical cycling of those metal ions. This is an important implication of microbial heavy metal tolerance because the oxidation state of a heavy metal relates to the solubility and toxicity of the metal itself. When looking at the microbial communities of metal-contaminated environments, it has been found that among the bacteria present, there is more potential for unique forms of respiration. Also, since the oxidation state of a metal ion may determine its solubility, many scientists have been trying to use microbes that are able to oxidize or reduce heavy metals in order to remediate metal-contaminated sites. Another implication of heavy metal tolerance in the environment is that it may contribute to the maintenance of antibiotic resistance genes by increasing the selective pressure of the environment. Many have speculated and have even shown that a correlation exists between metal tolerance and antibiotic resistance in bacteria because of the likelihood that resistance genes to both (antibiotics and heavy metals) may be located closely together on the same plasmid in bacteria and are thus more likely to be transferred together in the environment. Because of the prevalence of antibiotic resistant pathogenic bacteria, infectious diseases are becoming more difficult and more expensive to treat; thus we need to not only be more careful of the drastic overuse of antibiotics in our society, but also more aware of other antimicrobials, such as heavy metals, that we put into the environment. Bacteria can be Gram-negative or Gram-positive depending on the composition of the cell wall membrane. Gram-negative cell walls are a multilayered structure with an outer membrane containing lipopolysaccharide (e.g.lipopolysaccharide layer), phospholipids and a small peptidoglycan layer. On the other hand, Gram-positive cells have as much as 90 % of the cell wall consisting of peptidoglycan in several layers, with small amounts of teichoic acid usually present (Madigan et al., 2003; Guin et al., 2007). These structures are negatively charged and can interact with metal ions (Guin et al., 2007).

Heavy metal supplemented media

Microbial counts and isolation was also made using media supplemented with Cd, Zn and As, supplied as CdCl2, ZnCl2 and KH2AsO4 from sterile stock solutions, to final concentrations ranging from 50 to 1000 mg L-1.. The metal stock solutions contained 10000 mg L-1 of CdCl2 (Sigma), ZnCl2 (Sigma) and KH2AsO4 (Sigma). Appropriate volumes of the metal solutions were mixed with the media after being autoclaved. All the other steps were as described previously for the media without metals

ResultMetal tolerance for growth

Despite the high levels of metal contamination present in this environment, the majority of isolates were sensitive to moderate to high concentrations of metals, and isolate was able to grow especially at the highest concentration. The majority of the isolates grew when Zn was present. Most of the isolates had no growth or small growth when was present. About half the isolates grew when was present. to be more tolerant to the heavy metals at the concentrations tested. strains were capable of growing at high concentration of, Zn and . were gram-negative and only four were gram-positive.

Discussion It was noticed in this study that some strains isolated from this particular environment did not grow with high concentrations of metals, or had difficulties in growing under laboratory conditions in culture media adjusted with metals, with the exception of Zn. However, this effect is somewhat common, and most of the isolates from metal contaminated environments only proliferate under laboratory conditions when no metal or small amounts of metal are used (Gadd, 1990). The fact that these isolates did not grow when metal concentrations similar to those found in their environment can also be explained probably due to the fact that the composition of the media and other conditions of incubation were decisive for the determination not only of the metal tolerance but also for the determination of the optimal pH and temperature for growth (Horikoshi, 1999; Krulwich, 2000). However, since all strains were isolated from highly contaminated soils, they should have been stress adapted and able to proliferate, or at least survive in such an environment. Although some studies have shown detailed mechanisms of heavy metal tolerance for some bacterial species, for others these mechanisms are not yet very well known (Tsai et al., 2005). The ability to cope under this metal-contaminated environment may depend on genetic and/or physiological adaptation (Gadd, 1990; Jjemba, 2004). Therefore certain bacterial strains that are not adapted to live in metal contaminated environments may be sustained or induced by the activity of other bacterial strains or at a given concentration, a metal - may be toxic to one species while serving as a growth stimulant to others (Tsai et al., 2005). Heavy metals are more than likely able to affect the microbial populations in a given environment by reducing abundance and species diversity and selecting for a resistant population (Gadd, 1990). Tolerance to heavy metals may result from intrinsic properties of the microorganism, e.g. producing extracellular mucilage or polysaccharide or an impermeable cell wall (Gadd, 1990). Likely in this type of extreme environment, one of the most important factors that explains the presence or absence of a given population may well be the relationships that exist between the whole bacterial community.

References

Arakawa, T. and Timasheff, S. N., The stabilization of proteins by osmolytes. Biochem. J., 1985, 47, 411414. Baquero, F., Negri, MC., Morosini, MI., and Blazquez, J. (1998). Antibiotic-selective environments. Clinical Infectious Diseases 27: S5-S11. Bestvater, T., Louis, P. and Galinski, E. A., Heterologous ectoine production in Escherichia coli: By-passing the metabolic bottleneck. Saline Syst., 2008, 4, 114. Borgne, S. L., Paniagua, D. and Vazquez-Duhalt, R., Biodegradation of organic pollutants by halophilic bacteria and archaea.J. Mol. Microbiol. Biotechnol., 2008, 15, 7492.Bursy, J., Kuhlmann, A. U., Pittelkow, M., Hartmann, H., Jebbar,M., Pierik, A. J. and Bremer, E., Synthesis and uptake of the compatible solutes ectoine and 5-hydroxyectoine by Streptomyces coelicolor A3(2) in response to salt and heat stresses. Appl. Environ.Microbiol., 2008, 74, 72867296.Calormiris, J., Armstrong, J.L., and Seidler, R.J. (1984). Association of metal tolerance with multiple antibiotic resistance of bacteria isolated from drinking water. Appl Environ Microbiol 47(6): 1238-1242. Chattopadhyay, M. K., Kern, R., Mistou, M. Y., Dandekar, A. M., Uratsu, S. L. and Richarme, G., The chemical chaperone prolinerelieves the thermosensitivity of a dnaK deletion mutant at 42C. J. Bacteriol., 2004, 186, 81498152. Cleland, D., Krader, P., McCree, C., Tang, J. and Emerson, D., Glycine betaine as a cryoprotectant for prokaryotes. J. Microbiol.Methods, 2004, 58, 3138.Da Costa, M. S., Santos, H. and Galinski, E. A., An overview of the role and diversity of compatible solutes in bacteria and archaea. Adv. Biochem. Eng. Biotechnol., 1998, 61, 117153. DasSarma, S., Halophiles. Encycl. Life Sci., 2001, 19. Detkova, E. N. and Boltyanskaya, Y. V., Osmoadaptation of haloalkaliphilic bacteria: role of osmoregulators and their possible practical application. Microbiology, 2007, 76, 511522. Dundas, I., Was the environment for primordial life hypersaline? Extremophiles, 1998, 2, 375377. Empadinhas, N. and da Costa, M. S., Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes. Int. Microbiol., 2008, 11, 151161. Frings, E., Kunte, H. J. and Galinski, E. A., Compatible solutes in representatives of the genera Brevibacterium and Corynebacterium: occurrence of tetrahydropyrimidines and glutamine. FEMS Microbiol. Lett., 1993, 109, 2532.Gadd, G.M. & De Rome, L. (1988). Biosorption of copper by fungal melanins. Applied Microbiology and Biotechnology 29, 610-617. Gadd, G.M. (1986). The uptake of heavy metals by fungi and yeasts: the chemistry and physiology of the process and applications for biotechnology. Immobilization of Ions by Biosorption. H. H. Eccles & S. Hunt, Ellis Horwood, 135-147. Gadd, G.M. (1990). Microbiology of Extreme Environments. Milton Keynes UK. Open Universty Press. Gadd, G.M. (1992). Microbial control of heavy metal pollution. Microbial control of pollution, 48th Symposium of the Society for General Microbiology, J.C. Fry et al., Cambridge University Press, 59-88.Gadd, G.M., Gray, D.J. & Newby, P.J. (1990). Role of melanin in fungal biosorption of tributyltin chloride. Applied Microbiology and Biotechnology 34, 116-121. Galinski, E. A., Osmoadaptation in bacteria. Adv. Microb.Physiol., 1995, 37, 272328.Giller, K. E., Witter, E. & McGrath, S. P. (1998). Toxicity of heavy metals to microorganisms and microbial processes in agricultural soils : a review. Soil Biol Biochem 30, 13891414.Guzmn, H., Van, T. D., Martn, J., Hatti-Kaul, R. and Quillaguaman, J., A process for the production of ectoine and poly(3-hydroxybutyrate) by Halomonas boliviensis. Appl. Microbiol. Biotechnol., 2009, 84, 10691077. Hiroki, M. (1992). Effects of heavy metal contamination on soil microbial populations. Soil Science and Plant Nutrition 38, 141-147.Hof, T., Investigations concerning bacterial life in strong brines. Rev. Trav. Bot. Neerl., 1935, 32, 92171.Jjemba, P.K., (2004). Envirnomental Microbiology Principles and applications. Enfield, NH. Science Publishers.Kastritis, P. L., Papandreou, N. C. and Hamodrakas, S. J., Haloadaptation: insights from comparative modeling studies ofhalophilic archaeal DHFRs. Int. J. Biol. Macromol., 2007, 41, 447453.Kelly, J. J., Haggblom, M. & Tate, R. L. (1999). Changes in soil microbial communities over time resulting from one time application of zinc : a laboratory microcosm study. Soil Biol Biochem 31, 14551456. Kolp, S., Pietsch, M., Galinski, E. A. and Gtscho, M., Compatible solutes as protectants for zymogens against proteolysis. BBA Proteins Proteomics, 2006, 1764, 12341242. Krulwich, T. A. 2000. Alkaliphilic prokaryotes. In Dworkin et al. (ed.), The prokaryotes: an evolving electronic resource for the microbiological community, 2nd ed. Springer-Verlag, New York, N.Y. Kunte, H. J., Galinski, E. A. and Truper, H. G., A modified FMOC method for the detection of amino acid-type osmolytes and tetrahydropyrimidines (ectoines). J. Microbiol. Methods, 1993, 17, 129136. Kurz, M., Compatible solute influence on nucleic acids: Many questions but few answers. Saline Syst., 2008, 4, 114. Kushner, D. J. and Kamekura, M., Physiology of halophilic eubacteria. In Halophilic bacteria (ed. Rodriguez-Valera, F.), CRCPress, Boca Raton, FL, USA, 1988. Kushner, D. J., Life in high salt and solute concentrations: Halophilic bacteria. In Microbial Life in Extreme Environments (ed. Kushner, D. J.), Academic Press, London, UK, 1978.Lamosa, P., Turner, D. L., Ventura, R., Maycock, C. and Santos, H., Protein stabilization by compatible solutes. Effect of diglycerol phosphate on the dynamics of Desulfovibrio gigas rubredoxins studied by NMR. Eur. J. Biochem., 2003, 270, 4606 4614.Lawrence, J.G. (2000). Clustering of antibiotic resistance genes: beyond the selfish operon. ASM News 66(5): 281-286. LeFevre, E. and Round, L. A., A preliminary report upon some halophilic bacteria. J. Bacteriol., 1919, 4, 177182.Litchfield, C. D., Halophiles. J. Ind. Microbiol. Biotechnol., 2002, 28, 2122. Margesin, R. and Schinner, F., Potential of halotolerant microorganisms for biotechnology. Extremophiles, 2001, 5, 7383. Messadek, J., Glycine betaine and its use. US Patent, 6855734, 2005. Nies, D. H. (1999). Microbial heavy-metal resistance. Appl Microbiol Biotechnol 51, 730750.Nies, D.H., and Silver, S. (1995). Ion efflux systems involved in bacterial metal resistances. Journal of Industrial Microbiology 14: 186-199. Oren, A., Industrial and environmental applications of Halophilic microorganisms. Environ. Technol., 2010, 31, 825834.Pul, U., Wurm, R. and Wagner, R., The role of LRP and H-NS in transcription regulation: involvement of synergism, allostery and macromolecular crowding. J. Mol. Biol., 2007, 366, 900915. Riis, V., Maskow, T. and Babel, W., Highly sensitive determination of ectoine and other compatible solutes by anion-exchange chromatography and pulsed amperometric detection. Anal. Bioanal.Chem., 2003, 377, 203207. Roberts, M. F., characterization of organic compatible solutesof halotolerant and halophilic microorganisms. Methods Microbiol., 2006, 35, 615647.Roberts, M. F., Organic compatible solutes of halotolerant and halophilic microorganisms. Saline Syst., 2005, 1, 130. Rothschild, L. J. and Mancinelli, R. L., Life in extreme environments. Nature, 2001, 409, 10921101. Santosh, H. and da Costa, M. S., Compatible solutes of organisms that live in hot saline environments. Environ. Microbiol., 2002, 4, 501509. Sauer, T. and Galinski, E. A., Bacterial milking: a novel bioprocess for production of compatible solutes. Biotechnol. Bioeng., 1998, 57, 306313. Severin, J., Wohlfarth, A. and Galinski, E. A., The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria. J. Gen. Microbiol., 1992, 138, 16291638. Shivanand, P. and Jayaraman, G., Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast. Process Biochem., 2009, 44,10881094.Teixid, N., Caams, T. P., Usall, J., Torres, R., Magan, N. and Vias, I., Accumulation of the compatible solutes, glycinebetaine and ectoine, in osmotic stress adaptation and heat shock crossprotection in the biocontrol agent Pantoea agglomerans CPA-2. Lett. Appl. Microbiol., 2005, 41, 248252. Tsai, Y.-P., You, S.-J., Pai, T.-Y. & Chen, K.-W. (2005). Effect of cadmium on composition and diversity of bacterial communities in activated sludges. International Biodeterioration & Biodegradation 55: 285-291.Van-Thuoc, D., Guzmn, H., Thi-Hang, M. and Hatti-Kaul, R., Ectoine production by Halomonas boliviensis: optimization using response surface methodology. Mar. Biotechnol., 2010, 12, 586 593. Ventosa, A., Nieto, J. J. and Oren, A., Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev., 1998, 62, 504544.Xu, C., Zhou, T. Q., Kuroda, M. & Rosen, B. P. (1998). Metalloid resistance mechanisms in prokaryotes. J Biochem -123, 1623 Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D. And Somero, G. N., Living with water stress: evolution of osmolyte systems. Science, 1982, 217, 12141216.