Rapid arsenite oxidation by Paenarthrobacter nicotinovorans strain SSBW5: unravelling the role of GlpF, aioAB and aioE genes.

A novel arsenite resistant bacterial strain SSBW5 was isolated from the battery waste site of Corlim, Goa, India. This strain interestingly exhibited rapid arsenite oxidation with an accumulation of 5 mM arsenate within 24 h and a minimum inhibitory concentration (MIC) of 18 mM. The strain SSBW5 was identified as Paenarthrobacter nicotinovorans using 16S rDNA sequence analysis. Fourier-transformed infrared (FTIR) spectroscopy of arsenite-exposed cells revealed the interaction of arsenite with several important functional groups present on the cell surface, possibly involved in the resistance mechanism. Interestingly, the whole genome sequence analysis also clearly elucidated the presence of genes, such as GlpF, aioAB and aioE encoding transporter, arsenite oxidase and oxidoreductase enzyme, respectively, conferring their role in arsenite resistance. Furthermore, this strain also revealed the presence of several other genes conferring resistance to various metals, drugs, antibiotics and disinfectants. Further suggesting the probable direct or indirect involvement of these genes in the detoxification of arsenite thereby increasing its tolerance limit. In addition, clumping of bacterial cells was observed through microscopic analysis which could also be a strategy to reduce arsenite toxicity thus indicating the existence of multiple resistance mechanisms in strain SSBW5. In the present communication, we are reporting for the first time the potential of P. nicotinovorans strain SSBW5 to be used in the bioremediation of arsenite via arsenite oxidation along with other toxic metals and metalloids.

Biogenesis of selenium nanospheres using Halomonas venusta strain GUSDM4 exhibiting potent environmental applications.

Selenite reducing bacterial strain (GUSDM4) isolated from Mandovi estuary of Goa, India was identified as Halomonas venusta based on 16S rRNA gene sequence analysis. Its maximum tolerance level for sodium selenite (Na2SeO3) was 100 mM. The 2, 3-diaminonaphthalene-based spectroscopic analysis demonstrated 96 and 93% reduction of 2 and 4 mM Na2SeO3 respectively to elemental selenium (Se0) during the late stationary growth phase. Biosynthesis of Se nanoparticles (SeNPs) commenced within 4 h during the log phase, which was evident from the brick red color in the growth medium and a characteristic peak at 265 nm revealed by UV-Vis spectrophotometry. The intracellular periplasmic synthesis of SeNPs in GUSDM4 was confirmed by transmission electron microscopy (TEM). Characterization of SeNPs by X-ray crystallography, TEM and energy-dispersive X-ray analysis (EDAX) clearly demonstrated spherical SeNPs of 20-80 nm diameter with hexagonal crystal lattice. SeNPs (0.8 and 1 mg/L) primed seeds under arsenate [As(V)] stress showed increase in shoot length, root length and biomass by 1.4-, 1.5- and 1.1-fold respectively, as compared to As(V) primed seeds alone. The proline and phenolic content in seeds primed with SeNPs under arsenate stress showed alleviated levels proving its ameliorative potential. SeNPs also demonstrated anti-biofilm activity at 20 µg/mL against human pathogens which was evident by scanning electron microscopic (SEM) analysis. SeNPs interestingly revealed mosquito larvicidal activity also. Therefore, these studies have clearly demonstrated amazing potential of the marine bacterium, Halomonas venusta in biosynthesis of SeNPs and their applications as ameliorative, anti-biofilm and mosquito larvicidal agents which is the first report of its kind.

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.

Arsenite biotransformation and bioaccumulation by Klebsiella pneumoniae strain SSSW7 possessing arsenite oxidase (aioA) gene.

Arsenite oxidizing Klebsiella pneumoniae strain SSSW7 isolated from shipyard waste Goa, India showed a minimum inhibitory concentration of 21 mM in mineral salts medium. The strain possessed a small supercoiled plasmid and PCR amplification of arsenite oxidase gene (aioA) was observed on plasmid as well as chromosomal DNA. It was confirmed that arsenite oxidase enzyme was a periplasmic protein with a 47% increase in arsenite oxidase activity at 1 mM sodium arsenite. Scanning electron microscopy coupled with electron dispersive X-ray spectroscopic (SEM-EDS) analysis of 15 mM arsenite exposed cells revealed long chains of cells with no surface adsorption of arsenic. Transmission electron microscopy combined with electron dispersive X-ray spectroscopic (TEM-EDS) analysis demonstrated plasma membrane disruption, cytoplasmic condensation and periplasmic accumulation of arsenic. The bacterial strain oxidized 10 mM of highly toxic arsenite to less toxic arsenate after 24 h of incubation. Fourier transformed infrared (FTIR) spectroscopy confirmed the interaction of arsenite with functional groups present on the bacterial cell surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of 5 mM arsenite exposed cells demonstrated over-expression of 87 kDa and 14 kDa proteins of two subunits aioA and aioB of heterodimer arsenite oxidase enzyme as compared to control cells. Therefore, this bacterial strain might be employed as a potential candidate for bioremediation of arsenite contaminated environmental sites.

Tellurite biotransformation and detoxification by Shewanella baltica with simultaneous synthesis of tellurium nanorods exhibiting photo-catalytic and anti-biofilm activity.

Tellurite reducing bacterial strain was isolated from Zuari estuary, Goa India which could tolerate 5.5 mM potassium tellurite with a minimum inhibitory concentration of 6 mM. This strain was designated as GUSDZ9 and was identified as Shewanella baltica (accession number: MF350629) based on 16S rRNA gene sequencing and BLAST analysis. The Diethyl-dithiocarbamate based colorimetric analysis clearly demonstrated a complete reduction of 2 mM tellurite to elemental tellurium during the late stationary phase. Te Nanoparticles (TeNPs) biosynthesis which initiated at early log phase (i.e. 4 h) was evidently monitored through colour change and a peak due to surface plasmon resonance at 210 nm using UV-Vis spectroscopic analysis. X-ray crystallographic studies and transmission electron microscopy revealed unique nano-rods with a diameter ranging from 8 to 75 nm. Energy dispersive X-ray analysis further confirmed the presence of pure tellurium. The biogenic TeNPs at 10 and 5 µg/mL evidently demonstrated 90% degradation of methylene blue dye and anti-biofilm activity against potential Gram-positive and Gram-negative human pathogens respectively. The alkaline comet assay revealed time and dose-dependent genotoxicity at concentrations higher than 15 µg/mL of TeNPs. This study clearly demonstrated the potential of Shewanella baltica strain GUSDZ9 in bioremediation of toxic tellurite through bio-reduction into elemental tellurium and involvement of biogenic TeNPs in the photo-catalytic reduction of methylene blue and anti-biofilm activity. This is the first report of its kind on the synthesis of biogenic TeNPs from Shewanella baltica demonstrating photo-catalytic, anti-biofilm activity as well as genotoxicity.