Detection of the ability to dissimilate sulfate reduction in representatives of colorless sulfur bacteria of the genus Thiothrix using RNA isolated by sorption
Abstract
For the first time, the ability to anaerobic respiration using thiosulfate as a terminal electron acceptor was discovered for representatives of filamentous colorless sulfur bacteria of the genus Thiothrix, such as T. litoralis AST, T. unzii A1T and T. nivea JP2T. Previously, the ability to lithotrophic growth in the presence of thiosulfate as an electron donor for energy metabolism was shown for these bacteria.
To establish the mechanism of thiosulfate reduction during anaerobic growth, the participation of thiosulfate reductase or the disproportionation of thiosulfate, an analysis of the end products of its transformation was carried out. In all studied representatives of the genus Thiothrix, products formed during the conversion of thiosulfate, sulfite and sulfide, characteristic of the functioning of thiosulfate reductase, were identified, whereas products characteristic of the process of thiosulfate disproportionation (sulfate and sulfide) were not identified.
During the study, the genes phsA, phsB and phsC encoding thiosulfate reductase (quinone, EC 1.8.5.5) were identified, and the expression of these genes under anaerobic conditions was shown. It was found that during anaerobic growth on thiosulfate, compared with aerobic cultivation, the expression of the phsA, phsB, phsC genes in T. litoralis AST, T. unzii A1T and T. nivea JP2T in the first passage increases by 6-15 times, whereas in the second passage in T. unzii A1T there is a multiple increase in gene expression on average by 180 time. Sorbing columns were used to extract the total RNA. The samples were homogenized and lysed in the presence of a chaotropic salt, and then applied to a spin column, washed from contamination. DNA impurities were removed and pure RNA was obtained by subjecting the sample to DNAse treatment directly on the spin column. The RNA quality was assessed by electrophoresis of at least 2% agarose gel with the addition of 2.2 M formaldehyde solution.
In hydrogen sulfide biotopes, where anaerobic conditions are sporadically created, representatives of the genus Thiothrix, leading an attached lifestyle, need to switch from aerobic to anaerobic respiration to survive in dramatically changing conditions. The low yield of T. litoralis AST, T. unzii A1T and T. nivea JP2T cells
(15 mg/dm3) during anaerobic growth on thiosulfate indicates that the process of anaerobic respiration on thiosulfate has the character of a supportive metabolism.
The data obtained allow us to approach the solution of how lithotrophic sulfur bacteria of the genus Thiothrix, leading an attached lifestyle, effectively use thiosulfate in their metabolic strategy, adapting to changing environmental conditions, and explain their dominant position in flowing hydrogen sulfide biotopes.
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References
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