Abstract
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Abstract
Extracellular electron transfer (EET) – the process by which microorganisms transfer electrons across their membrane(s) to/from solid-phase materials – has implications for a wide range of biogeochemically important processes in marine environments. Though EET is thought to play an important role in the oxidation of inorganic minerals by lithotrophic organisms, the mechanisms involved in the oxidation of solid particles are poorly understood. To explore the genetic basis of oxidative EET, we utilized genomic analyses and transposon insertion mutagenesis screens (Tn-seq) in the metabolically flexible, lithotrophic Alphaproteobacterium Thioclava electrotropha ElOx9T. The finished genome of this strain is 4.3 MB, and consists of 4,139 predicted ORFs, 54 contain heme binding motifs, and 33 of those 54 are predicted to localize to the cell envelope or have unknown localizations. To begin to understand the genetic basis of oxidative EET in ElOx9T, we constructed a transposon mutant library in semi-rich media which was comprised of >91,000 individual mutants encompassing >69,000 unique TA dinucleotide insertion sites. The library was subjected to heterotrophic growth on minimal media with acetate and autotrophic oxidative EET conditions on indium tin oxide coated glass electrodes poised at –278 mV vs. SHE or un-poised in an open circuit condition. We identified 528 genes classified as essential under these growth conditions. With respect to electrochemical conditions, 25 genes were essential under oxidative EET conditions, and 29 genes were essential in both the open circuit control and oxidative EET conditions. Though many of the genes identified under electrochemical conditions are predicted to be localized in the cytoplasm and lack heme binding motifs and/or homology to known EET proteins, we identified several hypothetical proteins and poorly characterized oxidoreductases that implicate a novel mechanism(s) for EET that warrants further study. Our results provide a starting point to explore the genetic basis of novel oxidative EET in this marine sediment microbe.
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Abstract
A taxonomic and physiologic characterization was carried out on Thioclava strain ElOx9T, which was isolated from a bacterial consortium enriched on electrodes poised at electron donating potentials. The isolate is Gram-negative, catalase-positive and oxidase-positive; the cells are motile short rods. The bacterium is facultatively anaerobic with the ability to utilize nitrate as an electron acceptor. Autotrophic growth with H2 and S0 (oxidized to sulfate) was observed. The isolate also grows heterotrophically with organic acids and sugars. Growth was observed at salinities from 0 to 10% NaCl and at temperatures from 15 to 41 °C. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain belongs in the genus Thioclava ; it had the highest sequence similarity of 98.8 % to Thioclava atlantica 13D2W-2T, followed by Thioclava dalianensis DLFJ1-1T with 98.5 % similarity, Thioclava pacifica TL 2T with 97.7 % similarity, and then Thioclava indica DT23-4T with 96.9 %. All other sequence similarities were below 97 % to characterized strains. The digital DNA–DNA hybridization estimated when compared to T. atlantica 13D2W-2T, T. dalianensis DLFJ1-1T, T. pacifica TL 2T and T. indica DT23-4T were 15.8±2.1, 16.7+2.1, 14.3±1.9 and 18.3±2.1 %. The corresponding average nucleotide identity values between these strains were determined to be 65.1, 67.8, 68.4 and 64.4 %, respectively. The G+C content of the chromosomal DNA is 63.4 mol%. Based on these results, a novel species Thioclava electrotropha sp. nov. is proposed, with the type strain ElOx9T (=DSM 103712T=ATCC TSD-100T).