Analysis of cytoplasmic sulfur trafficking during sulfur globule oxidation in <em>Allochromatium vinosum</em>

Abstract

The present study provides significant insights into cytoplasmic sulfurtransferases and their function in <i>Alc. vinosum</i>. This thesis explores the possibility of sulfur trafficking during the process of sulfur globule degradation in <i>Alc. vinosum</i>. Potential participants in the cytoplasmic events of this mechanism should be identified. The obtained data provide the first experimental proof that the oxidation of intermediary stored sulfur includes sulfurtransferase activity and that individual sulfur atoms are passed on towards DsrAB by a pool of proteins via persulfidic intermediates. <br /> DsrEFH was verified as sulfurtransferase. Sulfane sulfur bound exclusively to Cys78 in the DsrE subunit and was transferred to DsrC and TusA. Cys111 was identified as the unique binding site for sulfur species in DsrC; apart from sulfane sulfur DsrC also binds sulfite. The results present DsrC as sulfur trap rather than a sulfurtransferase and add support to the notion that DsrC might serve as substrate donor for DsrAB in <i>Alc. vinosum</i>. The band shifts that were observed in the native PAGE upon incubation of DsrEFH with DsrC were ascribed to the formation of stable complexes between the two proteins in the stoichiometry 1:1 and 1:2 (DsrEFH:DsrC). Several independent lines of evidence indicate a function for Rhd_2599, TusA and DsrE2 in the oxidation of sulfur globules in <i>Alc. vinosum</i>. Apart from the ubiquitous presence of the genes in all major sulfur oxidizing families and their genomic link to genes encoding major components of the sulfur oxidation machinery, the transcriptomic patterns of <i>rhd_2599</i>, <i>tusA</i> and <i>dsrE2</i> paralleled the transcriptional upregulation of the <i>dsr</i> operon. The three genes form a transcriptional unit, although a secondary promoter upstream of <i>tusA</i> was detected. The disruption of the <i>rtd</i> locus led to an instable mutant with a severe and sulfur oxidizing negative phenotype. The insertion of an O-streptomycin cassette into the <i>rhd_2599</i> reading frame on the other hand was of no consequence for the mutant strain, although the enzyme accounts for the majority of rhodanese activity under sulfur oxidizing conditions. Characteristic patterns in the sequence of TusA and DsrE2 were identified and can be used to distinguish these proteins from homologues in prokaryotes with a non-sulfur based energy metabolism. TusA proteins encoded together with the rhodanese and DsrE2 contain hydrophobic residues in the “X”-position of the TusA motif. Leucine is dominating within bacterial TusA; glycine is conserved in archaeal TusA. With few exceptions all DsrE2 proteins contain two cysteine residues in short distance; Cys120 and Cys110 in DsrE2 from bacteria and Cys120 and Cys128 in DsrE2 from archaea. Rhd_2599 also carries two cysteine residues; however only the cysteine within the rhodanese motif, Cys64, was essential for sulfurtransferase activity. Rhd_2599, TusA and DsrE2 all bind sulfur, though Rhd_2599 alone was able to mobilize sulfur from an inorganic sulfur compound. Data collected from sulfur transfer experiments confirmed the flow of sulfur atoms between Rhd_2599, TusA, DsrEFH and DsrC <i>in vitro</i>.