Dataset Information

Expression, purification and function of cysteine desulfurase from Sulfobacillus acidophilus TPY isolated from deep-sea hydrothermal vent.

ABSTRACT: The cysteine desulfurase (SufS) gene of Sulfobacillus acidophilus TPY, a Gram-positive bacterium isolated from deep-sea hydrothermal vent, was cloned and over-expressed in E. coli BL21. The recombinant SufS protein was purified by one-step affinity chromatography. The TPY SufS contained a well conserved motif RXGHHCA as found in that of other microorganisms, suggesting that it belonged to group II of cysteine desulfurase family. The recombinant TPY SufS could catalyze the conversion of l-cysteine to l-alanine and produce persulfide, and the enzyme activity was 95 ?/?L of sulfur ion per minute. The growth of E. coli BL21 was promoted by over-expressing TPY SufS in vivo or by directly adding recombinant TPY SufS in the medium (4.3-4.5 × 10⁸ cells/mL vs. 3.2-3.5 × 10⁸ cells/mL). Furthermore, the highest cell density of E. coli BL21 when the TPY SufS was over-expressed was about 3.5 times that of the control groups in the presence of sodium thiosulfate. These results indicate that the SUF system as the only assembly system of iron-sulfur clusters not only has significant roles in survival of S. acidophilus TPY, but also might be important for combating with high content of sulfide.

SUBMITTER: Wang Y

PROVIDER: S-EPMC5626668 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Expression, purification and function of cysteine desulfurase from Sulfobacillus acidophilus TPY isolated from deep-sea hydrothermal vent.

Wang Yuguang Y Liu Qian Q Zhou Hongbo H Chen Xinhua X

3 Biotech 20171003 6

The cysteine desulfurase (SufS) gene of Sulfobacillus acidophilus TPY, a Gram-positive bacterium isolated from deep-sea hydrothermal vent, was cloned and over-expressed in E. coli BL21. The recombinant SufS protein was purified by one-step affinity chromatography. The TPY SufS contained a well conserved motif RXGHHCA as found in that of other microorganisms, suggesting that it belonged to group II of cysteine desulfurase family. The recombinant TPY SufS could catalyze the conversio ...[more]

PMID: 28979833

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Project description:Sulfobacillus acidophilus TPY, isolated from a hydrothermal vent in the Pacific Ocean, is a moderately thermoacidophilic Gram-positive bacterium that can oxidize ferrous iron or sulfur compounds to obtain energy. In this study, comparative transcriptomic analyses of S. acidophilus TPY were performed under different redox conditions. Based on these results, pathways involved in sulfur metabolism were proposed. Additional evidence was obtained by analyzing mRNA abundance of selected genes involved in the sulfur metabolism of sulfur oxygenase reductase (SOR)-overexpressed S. acidophilus TPY recombinant under different redox conditions. Comparative transcriptomic analyses of S. acidophilus TPY cultured in the presence of ferrous sulfate (FeSO4) or elemental sulfur (S0) were employed to detect differentially transcribed genes and operons involved in sulfur metabolism. The mRNA abundances of genes involved in sulfur metabolism decreased in cultures containing elemental sulfur, as opposed to cultures in which FeSO4 was present where an increase in the expression of sulfur metabolism genes, particularly sulfite reductase (SiR) involved in the dissimilatory sulfate reduction, was observed. SOR, whose mRNA abundance increased in S0 culture, may play an important role in the initial sulfur oxidation. In order to confirm the pathways, SOR overexpression in S. acidophilus TPY and subsequent mRNA abundance analysis of sulfur metabolism-related genes were carried out. Conjugation-based transformation of pTrc99A derived plasmid from heterotrophic E. coli to facultative autotrophic S. acidophilus TPY was developed in this study. Transconjugation between E. coli and S. acidophilus was performed on modified solid 2:2 medium at pH 4.8 and 37°C for 72 h. The SOR-overexpressed recombinant S. acidophilus TPY-SOR had a [Formula: see text]-accumulation increase, higher oxidation/ reduction potentials (ORPs) and lower pH compared with the wild type strain in the late growth stage of S0 culture condition. The transcript level of sor gene in the recombinant strain increased in both S0 and FeSO4 culture conditions, which influenced the transcription of other genes in the proposed sulfur metabolism pathways. Overall, these results expand our understanding of sulfur metabolism within the Sulfobacillus genus and provide a successful gene-manipulation method.

Project description:Since deep-sea hydrothermal vent fluids are enriched with toxic metals, it was hypothesized that (i) the biota in the vicinity of a vent is adapted to life in the presence of toxic metals and (ii) metal toxicity is modulated by the steep physical-chemical gradients that occur when anoxic, hot fluids are mixed with cold oxygenated seawater. We collected bacterial biomass at different distances from a diffuse flow vent at 9 degrees N on the East Pacific Rise and tested these hypotheses by examining the effect of mercuric mercury [Hg(II)] on vent bacteria. Four of six moderate thermophiles, most of which were vent isolates belonging to the genus Alcanivorax, and six of eight mesophiles from the vent plume were resistant to >10 microM Hg(II) and reduced it to elemental mercury [Hg(0)]. However, four psychrophiles that were isolated from a nearby inactive sulfide structure were Hg(II) sensitive. A neighbor-joining tree constructed from the deduced amino acids of a PCR-amplified fragment of merA, the gene encoding the mercuric reductase (MR), showed that sequences obtained from the vent moderate thermophiles formed a unique cluster (bootstrap value, 100) in the MR phylogenetic tree, which expanded the known diversity of this locus. The temperature optimum for Hg(II) reduction by resting cells and MR activity in crude cell extracts of a vent moderate thermophile corresponded to its optimal growth temperature, 45 degrees C. However, the optimal temperature for activity of the MR encoded by transposon Tn501 was found to be 55 to 65 degrees C, suggesting that, in spite of its original isolation from a mesophile, this MR is a thermophilic enzyme that may represent a relic of early evolution in high-temperature environments. Results showing that there is enrichment of Hg(II) resistance among vent bacteria suggest that these bacteria have an ecological role in mercury detoxification in the vent environment and, together with the thermophilicity of MR, point to geothermal environments as a likely niche for the evolution of bacterial mercury resistance.

Dataset Information

Expression, purification and function of cysteine desulfurase from Sulfobacillus acidophilus TPY isolated from deep-sea hydrothermal vent.

Publications

Expression, purification and function of cysteine desulfurase from <i>Sulfobacillus acidophilus</i> TPY isolated from deep-sea hydrothermal vent.

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