Hydrogen formation by an arsenate-reducing Pseudomonas putida, isolated from arsenic-contaminated groundwater in West Bengal, India

Anaerobic growth of a newly isolated Pseudomonas putida strain WB from an arsenic-contaminated soil in West Bengal, India on glucose, l-lactate, and acetate required the presence of arsenate, which was reduced to arsenite. During aerobic growth in the presence of arsenite arsenate was formed. Anaerobic growth of P. putida WB on glucose was made possible presumably by the non-energy-conserving arsenate reductase ArsC with energy derived only from substrate level phosphorylation. Two moles of acetate were generated intermediarily and the reducing equivalents of glycolysis and pyruvate decarboxylation served for arsenate reduction or were released as H₂. Anaerobic growth on acetate and lactate was apparently made possible by arsenate reductase ArrA coupled to respiratory electron chain energy conservation. In the presence of arsenate, both substrates were totally oxidized to CO₂ and H₂ with part of the H₂ serving for respiratory arsenate reduction to deliver energy for growth. The growth yield for anaerobic glucose degradation to acetate was Y _Glucose = 20 g/mol, leading to an energy coefficient of Y _ATP = 10 g/mol adenosine-5'-triphosphate (ATP), if the Emden–Meyerhof–Parnas pathway with generation of 2 mol ATP/mol glucose was used. During growth on lactate and acetate no substrate chain phosphorylation was possible. The energy gain by reduction of arsenate was Y _Arsenate = 6.9 g/mol, which would be little less than one ATP/mol of arsenate.     

Characterization of As efflux from the roots of As hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L.

In some plant species, various arsenic (As) species have been reported to efflux from the roots. However, the details of As efflux by the As hyperaccumulator Pteris vittata remain unknown. In this study, root As efflux was investigated for different phosphorus (P) supply conditions during or after a 24-h arsenate uptake experiment under hydroponic growth conditions. During an 8-h arsenate uptake experiment, P-supplied (P+) P. vittata exhibited much greater arsenite efflux relative to arsenate uptake when compared with P-deprived (P–) P. vittata, indicating that arsenite efflux was not proportional to arsenate uptake. In the As efflux experiment following 24 h of arsenate uptake, arsenate efflux was also observed with arsenite efflux in the external solution. All the results showed relatively low rates of arsenate efflux, ranging from 5.4 to 16.1% of the previously absorbed As, indicating that a low rate of arsenate efflux to the external solution is also a characteristic of P. vittata, as was reported with arsenite efflux. In conclusion, after 24 h of arsenate uptake, both P+ and P– P. vittata loaded/effluxed similar amounts of arsenite to the fronds and the external solution, indicating a similar process of xylem loading and efflux for arsenite, with the order of the arsenite concentrations being solution ≪ roots ≪ fronds.     

Arsenic-resistant bacteria isolated from agricultural soils of Bangladesh and characterization of arsenate-reducing strains

Aims:  To analyse the arsenic-resistant bacterial communities of two agricultural soils of Bangladesh, to isolate arsenic-resistant bacteria, to study their potential role in arsenic transformation and to investigate the genetic determinants for arsenic resistance among the isolates.
Methods and Results:  Enrichment cultures were performed in a minimal medium in the presence of As(III) and As(V) to isolate resistant bacteria. Twenty-one arsenic-resistant bacteria belonging to different genera of Gram-positive and Gram-negative bacteria were isolated. The isolates, with the exception of Oceanimonas doudoroffii Dhal Rw, reduced 2 mmol l⁻¹ As(V) completely to As(III) in aerobic conditions. Putative gene fragments for arsenite efflux pumps were amplified in isolates from Dhal soil and a putative arsenate reductase gene fragment was amplified from a Bacillus sp. from Rice soil.
Conclusions:  Phylogenetically diverse arsenic-resistant bacteria present in agricultural soils of Bangladesh are capable of reducing arsenate to arsenite under aerobic conditions apparently for detoxification purpose.
Significance and Impact of the Study:  This study provides results on identification, levels of arsenic resistance and reduction of arsenate by the bacterial isolates which could play an important role in arsenic cycling in the two arsenic-contaminated soils in Bangladesh.     

Isolation and Characterization of Arsenate-Reducing Bacteria from Arsenic-Contaminated Sites in New Zealand

Two environmental sites in New Zealand were sampled (e.g., water and sediment) for bacterial isolates that could use either arsenite as an electron donor or arsenate as an electron acceptor under aerobic and anaerobic growth conditions, respectively. These two sites were subjected to widespread arsenic contamination from mine tailings generated from historic gold mining activities or from geothermal effluent. No bacteria were isolated from these sites that could utilize arsenite or arsenate under the respective growth conditions tested, but a number of chemoheterotrophic bacteria were isolated that could grow in the presence of high concentrations of arsenic species. In total, 17 morphologically distinct arsenic-resistant heterotrophic bacteria isolates were enriched from the sediment samples, and analysis of the 16S rRNA gene sequence of these bacteria revealed them to be members of the genera Exiguobacterium, Aeromonas, Bacillus, Pseudomonas, Escherichia, and Acinetobacter. Two isolates, Exiguobacterium sp. WK6 and Aeromonas sp. CA1, were of particular interest because they appeared to gain metabolic energy from arsenate under aerobic growth conditions, as demonstrated by an increase in cellular growth yield and growth rate in the presence of arsenate. Both bacteria were capable of reducing arsenate to arsenite via a non-respiratory mechanism. Strain WK6 was positive for arsB, but the pathway of arsenate reduction for isolate CA1 was via a hitherto unknown mechanism. These isolates were not gaining an energetic advantage from arsenate or arsenite utilization, but were instead detoxifying arsenate to arsenite. As a subsidiary process to arsenate reduction, the external pH of the growth medium increased (i.e., became more alkaline), allowing these bacteria to grow for extended periods of time.     

Desulfurispira natronophila gen. nov. sp. nov.: an obligately anaerobic dissimilatory sulfur-reducing bacterium from soda lakes

Anaerobic enrichment cultures with elemental sulfur as electron acceptor and either acetate or propionate as electron donor and carbon source at pH 10 and moderate salinity inoculated with sediments from soda lakes in Kulunda Steppe (Altai, Russia) resulted in the isolation of two novel members of the bacterial phylum Chrysiogenetes. The isolates, AHT11 and AHT19, represent the first specialized obligate anaerobic dissimilatory sulfur respirers from soda lakes. They use either elemental sulfur/polysulfide or arsenate as electron acceptor and a few simple organic compounds as electron donor and carbon source. Elemental sulfur is reduced to sulfide through intermediate polysulfide, while arsenate is reduced to arsenite. The bacteria belong to the obligate haloalkaliphiles, with a pH growth optimum from 10 to 10.2 and a salt range from 0.2 to 3.0 M Na⁺ (optimum 0.4–0.6 M). According to the phylogenetic analysis, the two strains were close to each other, but distinct from the nearest relative, the haloalkaliphilic sulfur-reducing bacterium Desulfurispirillum alkaliphilum, which was isolated from a bioreactor. On the basis of distinct phenotype and phylogeny, the soda lake isolates are proposed as a new genus and species, Desulfurispira natronophila (type strain AHT11^T = DSM22071^T = UNIQEM U758^T).     

Arsenic-resistant bacteria associated with roots of the wild Cirsium arvense (L.) plant from an arsenic polluted soil,and screening of potential plant growth-promoting characteristics

A rhizobacterial community, associated with the roots of wild thistle Cirsium arvense (L.) growing in an arsenic polluted soil, was studied by fluorescence in situ hybridization (FISH) analysis in conjunction with cultivation-based methods. In the bulk, rhizosphere, and rhizoplane fractions of the soil, the qualitative picture obtained by FISH analysis of the main phylogenetic bacterial groups was similar and was predominantly comprised of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. The arsenic-resistant isolates belonged to 13 genera, the most abundant being those of Bacillus, Achromobacter, Brevundimonas, Microbacterium, and Ochrobactrum. Most bacteria grew in the presence of high arsenic concentrations (over 100 mM arsenate and 10 mM arsenite). Most strains possessed the ArsC, ArsB and ACR3 genes homologous to arsenate reductase and to the two classes of arsenite efflux pumps, respectively, peculiar to the ars operon of the arsenic detoxification system. ArsB and ACR3 were present simultaneously in highly resistant strains. An inconsistency between 16S rRNA phylogenetic affiliations and the arsenate reductase sequences of the strains was observed, indicating possible horizontal transfer of arsenic resistance genes in the soil bacterial community. Several isolates were able to reduce arsenate and to oxidise arsenite. In particular, Ancylobacter dichloromethanicum strain As3-1b possessed both characteristics, and arsenite oxidation occurred in the strain also under chemoautotrophic conditions. Some rhizobacteria produced siderophores, indole acetic acid and 1-amino-cyclopropane-1-carboxylic acid deaminase, thus possessing potential plant growth-promoting traits.     

Biological characterization of Bacillus flexus strain SSAI1 transforming highly toxic arsenite to less toxic arsenate mediated by periplasmic arsenite oxidase enzyme encoded by aioAB genes

Bacillus flexus strain SSAI1 isolated from agro-industry waste, Tuem, Goa, India displayed high arsenite resistance as minimal inhibitory concentration was 25 mM in mineral salts medium. This bacterial strain exposed to 10 mM arsenite demonstrated rapid arsenite oxidation and internalization of 7 mM arsenate within 24 h. The Fourier transformed infrared (FTIR) spectroscopy of cells exposed to arsenite revealed important functional groups on the cell surface interacting with arsenite. Furthermore, scanning electron microscopy combined with electron dispersive X-ray spectroscopy (SEM-EDAX) of cells exposed to arsenite revealed clumping of cells with no surface adsorption of arsenite. Transmission electron microscopy coupled with electron dispersive X-ray spectroscopic (TEM-EDAX) analysis of arsenite exposed cells clearly demonstrated ultra-structural changes and intracellular accumulation of arsenic. Whole-genome sequence analysis of this bacterial strain interestingly revealed the presence of large number of metal(loid) resistance genes, including aioAB genes encoding arsenite oxidase responsible for the oxidation of highly toxic arsenite to less toxic arsenate. Enzyme assay further confirmed that arsenite oxidase is a periplasmic enzyme. The genome of strain SSAI1 also carried glpF, aioS and aioE genes conferring resistance to arsenite. Therefore, multi-metal(loid) resistant arsenite oxidizing Bacillus flexus strain SSAI1 has potential to bioremediate arsenite contaminated environmental sites and is the first report of its kind.

     

Members of <Emphasis Type="Italic">Gammaproteobacteria</Emphasis> and <Emphasis Type="Italic">Bacilli</Emphasis> represent the culturable diversity of chitinolytic bacteria in chitin-enriched soils

Culturable chitinolytic bacterial diversity was studied in chitin-rich soils collected from two industries involved in chitin production. A total of 27 chitinolytic isolates were isolated among which only 10 showed zone of clearance ≥4 mm on colloidal chitin agar plate. Using morphological, biochemical and 16S rDNA analysis, isolates were identified as Bacillus, Paenibacillus, Stenotrophomonas and Pseudomonas. Molecular phylogenetic analysis revealed that Gammaproteobacteria and Bacilli were found to be the predominant classes in these chitin-enriched soils. Chitinolytic bacterial population densities were significantly high and showed a rather simple community composition dominated by genus Bacillus and Stenotrophomonas (74%). This is the first report on assessing the chitinolytic bacterial diversity of soils from industries involved in chitin production.     

Isolation and characterization of an arsenate-reducing bacterium and its application for arsenic extraction from contaminated soil

A Gram-negative anaerobic bacterium, Citrobacter sp. NC-1, was isolated from soil contaminated with arsenic at levels as high as 5,000 mg As kg⁻¹. Strain NC-1 completely reduced 20 mM arsenate within 24 h and exhibited arsenate-reducing activity at concentrations as high as 60 mM. These results indicate that strain NC-1 is superior to other dissimilatory arsenate-reducing bacteria with respect to arsenate reduction, particularly at high concentrations. Strain NC-1 was also able to effectively extract arsenic from contaminated soils via the reduction of solid-phase arsenate to arsenite, which is much less adsorptive than arsenate. To characterize the reductase systems in strain NC-1, arsenate and nitrate reduction activities were investigated using washed-cell suspensions and crude cell extracts from cells grown on arsenate or nitrate. These reductase activities were induced individually by the two electron acceptors. This may be advantageous during bioremediation processes in which both contaminants are present.     

Validation of Arsenic Resistance in <Emphasis Type="Italic">Bacillus cereus</Emphasis> Strain AG27 by Comparative Protein Modeling of <Emphasis Type="Italic">ars</Emphasis>C Gene Product

The ars gene system provides arsenic resistance to a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. Therefore, arsC gene from Bacillus cereus strain AG27 isolated from soil was amplified, cloned and sequenced. The strain exhibited a minimum inhibitory concentration of 40 and 35 mM to sodium arsenate and sodium arsenite, respectively. Homology of the sequence, when compared with available database using BLASTn search showed that 300 bp amplicons obtained possess partial arsC gene sequence which codes for arsenate reductase, an enzyme involved in the reduction of arsenate to arsenite which is then effluxed out of the cell, thereby indicating the presence of efflux mechanism of resistance in strain. The efflux mechanism was further confirmed by atomic absorption spectroscopy and scanning electron microscopy studies. Moreover, three dimensional structure of modeled arsC from Bacillus cereus strain shares significant structural similarity with arsenate reductase protein of B.subtilis, consisting of, highly similar overall fold with single α/β domain containing a central four stranded, parallel, open-twisted β-sheet flanked by α-helices on both sides. The structure harbors the arsenic binding motif AB loop or P-loop that is highly conserved in arsenate reductase family.     

Brevibacterium sp. strain CS2: A potential candidate for arsenic bioremediation from industrial wastewater

A multiple metal-resistant Brevibacterium sp. strain CS2, isolated from an industrial wastewater, resisted arsenate and arsenate upto 280 and 40 mM. The order of resistance against multiple metals was Arsenate > Arsenite > Selenium = Cobalt > Lead = Nickel > Cadmium = Chromium = Mercury. The bacterium was characterized as per morphological and biochemical characteristics at optimum conditions (37 ℃ and 7 pH). The appearance of brownish color precipitation was due to the interaction of silver nitrate confirming its oxidizing ability against arsenic. The strain showed arsenic processing ability at different temperatures, pH, and initial arsenic concentration which was 37% after 72 h and 48% after 96 h of incubation at optimum conditions with arsenite 250 mM/L (initial arsenic concentration). The maximum arsenic removal ability of strain CS2 was determined for 8 days, which was 32 and 46% in wastewater and distilled water, respectively. The heat-inactivated cells of the isolated strain showed a bioremediation efficiency (E) of 96% after 10 h. Genes cluster (9.6 kb) related to arsenite oxidation was found in Brevibacterium sp. strain CS2 after the genome analysis of isolated bacteria through illumine and nanopore sequencing technology. The arsenite oxidizing gene smaller subunit (aioB) on chromosomal DNA locus (Prokka_01508) was identified which plays a role in arsenite oxidation for energy metabolism. The presence of arsenic oxidizing genes and an efficient arsenic oxidizing potential of Brevibacterium sp. strain CS2 make it a potential candidate for green chemistry to eradicate arsenic from arsenic-contaminated wastewater.     

Arsenic resistance in Pteris vittata L.: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli

Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) grows well in arsenic-contaminated media, with an extraordinary ability to tolerate high levels of arsenic. An expression cloning strategy was employed to identify cDNAs for the genes involved in arsenic resistance in P. vittata. Excised plasmids from the cDNA library of P. vittata fronds were introduced into Escherichia coli XL-1 Blue and plated on medium containing 4 mM of arsenate, a common form of arsenic in the environment. The deduced amino acid sequence of an arsenate-resistant clone, PV4-8, had cDNA highly homologous to plant cytosolic triosephosphate isomerases (cTPI). Cell-free extracts of PV4-8 had 3-fold higher level of triosephosphate isomerase (TPI) specific activities than that found in E. coli XL-1 Blue and had a 42 kD fusion protein immunoreactive to polyclonal antibodies raised against recombinant Solanum chacoense cTPI. The PV4-8 cDNA complemented a TPI-deficient E. coli mutant. PV4-8 expression improved arsenate resistance in E. coli WC3110, a strain deficient in arsenate reductase but not in AW3110 deficient for the whole ars operon. This is consistent with the hypothesis that PV4-8 TPI increased arsenate resistance in E. coli by directly or indirectly functioning as an arsenate reductase. When E. coli tpi gene was expressed in the same vector, bacterial arsenate resistance was not altered, indicating that arsenate tolerance was specific to P. vittata TPI. Paradoxically, P. vittata TPI activity was not more resistant to inhibition by arsenate in vitro than its bacterial counterpart suggesting that arsenate resistance of conventional TPI reaction was not the basis for the cellular arsenate resistance. P. vittata TPI activity was inhibited by incubation with reduced glutathione while bacterial TPI was unaffected. Consistent with cTPI’s role in arsenate reduction, bacterial cells expressing fern TPI had significantly greater per cent of cellular arsenic as arsenite compared to cells expressing E. coli TPI. Excised frond tissue infiltrated with arsenate reduced arsenate significantly more under light than dark. This research highlights a novel role for P. vittata cTPI in arsenate reduction.     

A comparison of arsenic mobility in Phaseolus vulgaris, Mentha aquatica, and Pteris cretica rhizosphere

The ability of Phaseolus vulgaris, Mentha aquatica, and Pteris cretica to release arsenic (As) species from contaminated soil was tested in rhizobox experiments in three soils differing in their physicochemical parameters and total and mobile As concentration. Relatively low uptake of arsenic by P. vulgaris and M. aquatica resulted in very low and ambiguous changes in rhizosphere soil compared to bulk soil. However, there were observed differences in the distribution of the mobile As portion in soil to individual As species as affected by plant species and/or plantation conditions of these plants. Higher percentage of mobile arsenite in mint rhizosphere seems to be related to more reducing conditions during cultivation of these wetland plants. P. cretica planted in the soils containing between 36 and 1436 mg As kg⁻¹ was able to accumulate between 80 and 500 mg As kg⁻¹ in aboveground biomass. The extractable concentrations of As compounds in rhizosphere soil of P. cretica showed a clear depletion of arsenate (representing more than 90% of extractable arsenic) with the distance from plant roots. However, the As uptake mechanisms, as well as As transformation within hyperaccumulating fern plants, differ substantially from those in higher plants. Therefore the finding of suitable higher plant tolerant to the As soil contamination with good ability to accumulate As in aboveground biomass remains for the further research.     

Arsenate, arsenite and dimethyl arsinic acid (DMA) uptake and tolerance in maize (Zea mays L.)

The influx of arsenate, arsenite and dimethyl arsinic acid (DMA) were studied in 7-day-old excised maize roots (Zea mays L.), and then related to arsenate, arsenite and DMA toxicity. Arsenate, arsenite and DMA influx was all found concentration dependent with significant genotypic differences for arsenite and DMA. Arsenate influx in phosphate starved plants best fitted the four-parameter Michaelis–Menten model corresponding to an additive high and low affinity uptake system, while the uptake of phosphate replete plants followed the two parameter model of Michaelis–Menten kinetics. Arsenite influx was well described by the two parameter model of ‘Michaelis–Menten’ kinetics. DMA influx was comprised of linear phase and a hyperbolic phase. DMA influx was much lower than that for arsenite and arsenate. Arsenate and DMA influx decreased when phosphate was given as a pre-treatment as opposed to phosphate starved plants. The +P treatment tended to decrease influx by 50% for arsenate while this figure was 90% for DMA. Arsenite influx increasing slightly at higher arsenite concentrations in P starved plants but at lower arsenite concentrations, there was little or no difference in arsenite uptake. Low toxicity was found for DMA on maize compared with arsenate and arsenite and the relative toxicity of arsenic species was As(V) > As(III) >> DMA.     

Studies on non-symbiotic diazotrophic bacterial populations of coastal arable saline soils of India

The effect of fluctuations of salinity in three different seasons on diazotrophic populations and N₂ fixation in six mono cropped rice field soils of the coastal region of the Gangetic delta of West Bengal, India, was studied. The average pH, ECe, organic carbon and total nitrogen of the soils ranged from 4.99–7.08, 2.02–19.58 dSm⁻¹, 4.68–12.03 g kg⁻¹ and 0.44–1.70 g kg ⁻¹, respectively. The average log colony forming units of the bacterial populations and N₂-fixation in the soils varied from 4.61 to 5.86 and 2.74 to 4.52 mg N₂ fixed 50 ml ⁻¹ culture media respectively, with the lowest value recorded in summer. Recovery of microorganisms and N₂- fixation gradually decreased with extraneous addition of NaCl in the culture media. All the eight isolates were Gram positive, spore and capsule formers. They could utilize glucose, sucrose, mannitol, starch, citrate and nitrate, and were catalase and gelatinase positive, but indole, methyl red and Vogues Proskauer reaction negative. The organisms produced alkaline reaction on TSI agar slant. The acetylene reduction assay of the isolates at 0 and 1% NaCl in the culture media were 4.51–164.52 and 1.72–100.6 nmole C₂H₄ ml⁻¹ culture media in 72 h, respectively. The isolates could fix 2.42–4.45 and 2.04–4.08 mg N₂ fixed 50 ml⁻¹ culture media at 0 and 1% NaCl in the culture media respectively. 16S rDNA sequences of the isolates were similar to the species: Bacillus sp. isolate 28A, Bacillus sp. MOLA 87, Bacillus sp. By113 (B)Ydz-dh, Bacillus sp. PN13, Bacillus licheniformis strain RH101, Bacterium Antarctica 14, Bacillus sp. PN13 and Bacillus megaterium.     

Productivity of greengram in tebuconazole-stressed soil, by using a tolerant and plant growth-promoting Bradyrhizobium sp. MRM6 strain

In this study, a total of 50 rhizobial isolates were recovered from the root nodules of greengram plants. Of the 50 isolates, 9 bradyrhizobial strains namely, MRM1, MRM2, MRM3, MRM4, MRM5, MRM6, MRM7, MRM8, and MRM9, exhibiting a higher tolerance levels of 600, 800, 1,200, 1,000, 1,000, 1,600, 1,400, 1,400, and 1,000 μg ml⁻¹, respectively, to triazole fungicide tebuconazole (chromatographically pure) were selected and tested for plant growth-promoting activities. Generally, the rhizobial strain with maximum fungicide-tolerance ability produced higher amounts of plant growth-promoting substances. Among the nine bacterial strains, Bradyrhizobium strain MRM6 was preferably selected due to its ability to tolerate tebuconazole maximally (up to 1,600 μg ml⁻¹) on minimal salt agar medium. In addition, the strain MRM6 grew well in minimal salts medium supplemented with 100 (recommended), 200 (two times of the recommended), and 300 μg tebuconazole l⁻¹ (three times of the recommended rate) and synthesized highest amounts of plant growth-promoting substances like indole acetic acid, siderophores, exopolysaccharides, hydrogen cyanate, and ammonia, both in the absence and presence of 100, 200, and 300 μg l⁻¹ of tebuconazole. Following these properties, the strain MRM6 was used as inoculant and the inoculated greengram plants was raised in soils treated separately with recommended, two and three times the recommended dose of tebuconazole. Generally, tebuconazole at recommended and the higher rates decreased biomass, nodulation, nutrient-uptake, and grain yield of uninoculated greengram plants. Interestingly, Bradyrhizobium sp. (vigna) strain MRM6 when used with any concentration of tebuconazole, significantly increased the measured phyto-chemical-parameters of greengram plants when compared with those grown in soils treated exclusively (without inoculant) with tebuconazole. This study inferred that the strain MRM6 of Bradyrhizobium sp. (vigna) was compatible with tebuconazole and may be co-inoculated with this fungicide for enhancing the production of legumes especially greengram in soils poisoned with fungicides.     

Actinomycetes from solitary wasp mud nest and swallow bird mud nest: isolation and screening for their antibacterial activity

The aim of this study was to isolate and screen actinomycetes from solitary wasp and swallow bird mud nests for antimicrobial activity. The actinomycetes were isolated from soil of nests of solitary wasp and swallow bird, and identified on the basis of morphological characteristics and molecular biological methods. A total of 109 actinomycetal isolates were obtained from 12 soil samples (6 from each habitat) using two media. The highest number of actinomycetes were recovered on Humic acid vitamin agar media (65.13%, n = 71) as compared to actinomycetes isolation agar media (34.86%, n = 38). The antimicrobial activity of actinomycetes isolates was determined using the agar plug method. Among 109 isolates, 51 isolates (46.78%) showed antibacterial activity by agar plug assay. The morphological and molecular characteristics confirmed that the most of active isolates in both sample belonged to the genus Streptomyces, the other potential genera like Streptosporangium, Actinomadura, Saccharopolyspora, Thermoactinomycetes and Nocardia were also recovered, but in a low frequency. The isolates designated as 8(1)*, BN-6, MN 2(6), MN 2(7) and MN 9(V) showed most promising activity against various drug resistant bacterial pathogens. It seems that the promising isolates from these unusual/unexplored habitats may prove to be an important step in development of drug for treating multi-drug resistant bacterial pathogens.     

Isolation and characterization of arsenic resistant bacteria from tannery wastes and agricultural soils in Thailand

Highly arsenic resistant bacteria (27 isolates), which had a minimum inhibitory concentrations (MICs) for arsenite and arsenate of ? 40 mM and > 400 mM, respectively, were isolated from tannery wastes and agricultural soils collected in Central Thailand. On the basis of the morphological, cultural, physiological and biochemical characteristics, and on the principal ubiquinone component and 16S rRNA gene sequence analyses, they were identified as nine isolates each ofKlebsiella (Groups 1 and 8) andAcinetobacter (Groups 2, 3 and 7), four isolates each ofPseudomonas (Groups 4 and 6) andComamonas (Group 5), and one isolate ofEnterobacter (Group 9). From these 27 isolates, only one isolate, A3-3 from the genusComamonas, appeared potentially capable of oxidizing arsenite to arsenate, as determined by silver nitrate staining of arsenite agar plates after colony growth.     

Purification and characterization of arsenite oxidase from Arthrobacter sp.

The chemolithoautotroph, Arthrobacter sp.15b oxidizes arsenite to arsenate using a membrane bound arsenite oxidase. The enzyme arsenite oxidase is purified to its homogeneity and identified using MALDI-TOF MS analysis. Upon further characterization, it was observed that the enzyme is a heterodimer showing native molecular mass as ~100 kDa and appeared as two subunits of ~85 kDa LSU and 14 kDa SSU on SDS–PAGE. The V _max and K _m values of the enzyme was found to be 2.45 μM (AsIII)/min/mg) and 26 μM, respectively. The purified enzyme could withstand wide range of pH and temperature changes. The enzyme, however, gets deactivated in the presence of 1 mM of DEPC suggesting the involvement of histidine at the binding site of the enzyme. The peptide analysis of large sub unit of the enzyme showed close match with the arsenite oxidases of Burkholderia sp. YI019A and arsenite oxidase, Mo-pterin containing subunit of Alcaligenes faecalis. The small subunit, however, differed from other arsenite oxidases and matched only with 2Fe–2S binding protein of Anaplasma phagocytophilum. This indicates that Rieske subunits containing the iron–sulfur clusters present in the large as well as small subunits of the enzyme are integral part of the protein.     

Isolation and characterization of rhizobacteria associated with coastal agricultural ecosystem of rhizosphere soils of cultivated vegetable crops

In this study, a total of 80 rhizobacteria was isolated from coastal agricultural ecosystem of cultivated vegetable rhizosphere soils. The isolates were screened for antagonistic activity against Sclerotium rolfsii and Colletotrichum capsici and plant growth promoting traits. The results revealed that 15.0 and 43.7% isolates showed statistically significant inhibition of mycelial growth of S. rolfsii and C. capsici respectively, while 48.7% isolates produced siderophore, 57.5% isolates solubilized phosphate and 21.1% isolates produced indole-3-acetic acid more than 20 μg/mL. However, only three isolates PfS1, PfR2 and BL5 were found positive to all properties tested. The identification of potential bacterial isolates through Microbial Identification System (BIOLOG) and 16S rDNA sequencing of the isolates revealed Bacillus species were dominant in the cultivated vegetable rhizosphere soil of Neil and Havelock Islands, India.