Diversity Of Arsenite-resistant Bacteria From Deep Sea Sediments Of Ocean Ridge

More evidences suggest that microbes participate in the geochemical circulation of arsenic, and play an important role in the biocirculation. However,little is know about the diversity of arsenite-resistant bacteria in marine environment, especially deep sea. Aims of this study are to isolate such bacteria and detect the diversity of these bacterial species from deep sea sediments.Seven deep sea sediment samples were collected from the Indian and Atlantic oceans, with a depth range from 1420m to 4019m. Seven arsenite-resistant bacterial communities were obtained by enrichment in PTA medium with 2 mmol/L NaAsO2 . Arsenite-resistant bacteria were isolated from the communities. Structure of bacterial communities of three samples (IR-TVG1, IR-TVG2, IR-TVG3) was analyzed by 16S rDNA library analyses and DGGE of V3 region of 16S rDNA. In arsenite-resistant bacterial community of IR-TVG2 and IR-TVG3, the dynamic changes in response to different incubation times and increasing arsenite concentration were examined further.Total of 102 strains were isolated from the communities, they were tentatively identified to be 50 species of 28 genera. Most of them belong toα-Proteobacteria,γ-Proteobacteria and Actinobacteria. The predominant genera were Microbacterium, Alcanivorax, Marinobacter, Idiomarina, Halomona and Pseudoalteromonas. One isolate possibly belonged to a novel species of novel genus, while fifty isolates possibly belonged to novel species according to the 16S rDNA sequence.DGGE and 16S rDNA library analyses results showed that the predominant bacteria in IR-TVG1 community was Pseudoalteromonas, corresponding isolate was AS-I1-3; The predominant bacteria in IR-TVG2 community were Alcanivorax and Microbacterium, corresponding isolates were CK-I2-8 and AS-I2-1; The predominant bacterium in IR-TVG3 community was Microbacterium, corresponding to isolate AS-I3-5. The two isolates, AS-I2-1 and AS-I3-5, had the same 16S rDNA sequence, but varied in BOX-PCR and arsenite tolerance. In addition, community structure of IR-TVG2 and IR-TVG3 changed dynamically in response to incubation periods and arsenite concentration, and varied from each other.Among all the 102 isolates, only 16.6% can grow in present of 10 mmol/L NaAsO2, which were mainly isolated from IR-TVG2 and IR-TVG3, and the most key bacteria are Microbacterium, occupying 52.6%.With degenerate primers, genes encoding arsenite efflux pump (ars B and ACR3) were PCR detected, showing the expected size of 750bp. Of the 102 isolates, 17 showed successful amplifications with either one or two of the specific primer sets. These fragments showed ~75% similarities to the reported arsenite transporters.Strain D8-2(Brachybacterium paraconglomeratum,100%), isolated from a deep sea shrimp, and strain CK-I1-6(Pseudomonas alcalophila,99.9%),isolated from sediment of IR-TVG1, were further studied. Both two showed high resistance but both could not oxidize arsenite to arsenate. D8-2 showed high ability of arsenite absorption, after 48h incubation arsenic accumulation in cells amounted to 2313.7μgAsg-1 (dry weight). A gene of 76% similarity to the arsenite transporter of ACR3 of Pseudomonas sp. A07 was amplified from CK-I1-6.