Search

The thioredoxin (Trx) system, found universally, is responsible for the regeneration of reversibly oxidized protein thiols in living cells. This system is made up of a Trx and a Trx reductase, and it plays a central role in maintaining thiol‐based redox homeostasis by reducing oxidized protein thiols, such as disulfide bonds in proteins. Some Trxs also possess a chaperone function that is independent of thiol‐disulfide exchange, in addition to their thiol‐disulfide reductase activity. These two activities of the Trx system are involved in numerous physiological processes in bacteria. This review describes the diverse physiological roles of the Trx system that have emerged throughout bacterial evolution. The Trx system is essential for responding to oxidative and nitrosative stress. Beyond this primary function, the Trx system also participates in redox regulation and signal transduction, and in controlling metabolism, motility, biofilm formation, and virulence. This range of functions has evolved alongside the diversity of bacterial lifestyles and their specific constraints. This evolution can be characterized by the multiplication of the systems and by the specialization of cofactors or targets to adapt to the constraints of atypical lifestyles, such as photosynthesis, insect endosymbiosis, or spore‐forming bacteria. [ABSTRACT FROM AUTHOR]

MetR (formerly Smu.1225), a regulator of the LysR family, controls key genes for methionine supply in Streptococcus mutans. An S. mutans metR mutant is unable to transport L-methionine and to grow in the absence of this amino acid. Accordingly, MetR activates transcription by binding to the promoter regions of two gene clusters and smu.1487, whose products are involved in methionine biosynthesis (MetEF and Smn.1487) and uptake (AtmBDE). Transcriptional activation by MetR requires the presence of a 17-bp palindromic sequence, the Met box. Base substitutions in the Met box hinder the formation of a MetR-DNA complex and abolish MetR-dependent activation, showing that Met boxes correspond to MetR recognition sites. Activation by MetR occurs in methionine-depleted medium and is rapidly triggered under nonactivating conditions by the addition of homocysteine. This intermediate of methionine biosynthesis increases the affinity of MetR for DNA in vitro and appears to be the MetR eoeffector in vivo. Homocysteine plays a crucial role in methionine metabolic gene regulation by controlling MetR activity. A similar mechanism of homocysteine- and MetR-dependent control of methionine biosynthetic genes operates in S. thermophilus. These data suggest a common mechanism for the regulation of the methionine supply in streptococci. However, some streptococcal species are unable to synthesize the homocysteine coeffector. This intriguing feature is discussed in the light of comparative genomies and streptococcal ecology.

Bacillus subtilis can use methionine as the sole sulfur source, indicating an efficient conversion of methionine to cysteine. To characterize this pathway, the enzymatic activities of CysK, YrhA and YrhB purified in Escherichia coli were tested. Both CysK and YrhA have an O-acetylserine-thiol-lyase activity, but YrhA was 75-fold less active than CysK. An atypical cystathionine [beta]-synthase activity using O-acetylserine and homocysteine as substrates was observed for YrhA but not for CysK. The YrhB protein had both cystathionine lyase and homocysteine [gamma]-lyase activities in vitro. Due to their activity, we propose that YrhA and YrhB should be renamed MccA and MccB for methionine-to-cysteine conversion. Mutants inactivated for cysK or yrhB grew similarly to the wild-type strain in the presence of methionine. In contrast, the growth of an [DELTA]yrhA mutant or a luxS mutant, inactivated for the S-ribosyl-homocysteinase step of the S-adenosylmethionine recycling pathway, was strongly reduced with methionine, whereas a [DELTA]yrhA [DELTA]cysK or cysE mutant did not grow at all under the same conditions. The yrhB and yrhA genes form an operon together with yrrT, mtnN, and yrhC. The expression of the yrrT operon was repressed in the presence of sulfate or cysteine. Both purified CysK and CymR, the global repressor of cysteine metabolism, were required to observe the formation of a protein-DNA complex with the yrrT promoter region in gel-shift experiments. The addition of O-acetyl-serine prevented the formation of this protein-DNA complex.

YrzC has previously been identified as a repressor controlling ytml expression via its regulation of YtlI activator synthesis in Bacillus subtilis. We identified YrzC as a master regulator of sulfur metabolism. Gene expression profiles of B. subtilis [DELTA]yrzC mutant and wild-type strains grown in minimal medium with sulfate as the sole sulfur source were compared. In the mutant, increased expression was observed for 24 genes previously identified as repressed in the presence of sulfate. Since several genes involved in the pathways leading to cysteine formation were found, we propose to rename YrzC CymR, for 'cysteine metabolism repressor.' A CymR-dependent binding to the promoter region of the ytll, ssuB, tcyP, yrrT, yxeK, cysK, or ydbM gene was demonstrated using gel shift experiments. A potential CymR target site, [TAAWNCN.sub.2][ANTWNAN.sub.3]ATMGGAA TTW, was found in the promoter region of these genes. In a DNase footprint experiment, the protected region in the ytll promoter region contained this consensus sequence. Partial deletion or introduction of point mutations in this sequence confirmed its involvement in ytll, yrrT, and yxeK regulation. The addition of O-acetylserine in gel shift experiments prevented CymR-dependent binding to DNA for all of the targets characterized. Transcriptome analysis of a [DELTA]cymR mutant and the wild-type strain also brought out significant changes in the expression level of a large set of genes related to stress response or to transition toward anaerobiosis.

The YtlI regulator of Bacillus subtilis activates the transcription of the ytmI operon encoding an L-cystine ABC transporter, a riboflavin kinase, and proteins of unknown function. The expression of the ytlI gene and the ytmI operon was high with methionine and reduced with sulfate. Using deletions and site-directed mutagenesis, a cis-acting DNA sequence important for YtlI-dependent regulation was identified upstream from the -35 box of ytmI. Gel mobility shift assays confirmed that YtlI specifically interacted with this sequence. The replacement of the sulfur-regulated ytlI promoter by the xyl4 promoter led to constitutive expression of a ytmI'-lacZ fusion in a ytlI mutant, suggesting that the repression of ytmI expression by sulfate was mainly at the level of YtlI synthesis. We further showed that the YrzC regulator negatively controlled ytlI expression while this repressor also acted on ytmI expression via YtlI. The cascade of regulation observed in B. subtilis is conserved in Listeria spp. Both a YtlI-like regulator and a ytmI-type operon are present in Listeria spp. Indeed, the Lmo2352 protein from Listeria monocytogenes was able to replace YtlI for the activation of ytmI expression and a lmo2352'-lacZ fusion was repressed in the presence of sulfate via YrzC in B. subtilis. A common motif, AT(A/T)ATTCCTAT, was found in the promoter region of the ytlI and lmo2352 genes. Deletion of part of this motif or the introduction of point mutations in this sequence confirmed its involvement in ytlI regulation.

The symporter YhcL and two ATP binding cassette transporters, YtmJKLMN and YckKJI, were shown to mediate L-cystine uptake in Bacillus subtilis. A triple [DELTA]yhcL [DELTA]ytmJKLMN [DELTA]yckK mutant was unable to grow ixn the presence of L-cystine and to take up L-cystine. We propose that yhcL, ytmJKLMN, and yckKJI should be renamed tcyP, tcyJKLMN, and tcyABC, respectively. The L-cystine uptake by YhcL ([K.sub.m] = 0.6 [micro]M) was strongly inhibited by seleno-DL-cystine, while the transport due to the YtmJKLMN system ([K.sub.m] = 2.5 [micro]M) also drastically decreased in the presence of DL-cystathionine, L-djenkolic acid, or S-methyl-L-cysteine. Accordingly, a [DELTA]ytmJKLMN mutant did not grow in the presence of 100 [micro]M DL-cystathionine, 100 [micro]M L-djenkolic acid, or 100 [micro]M S-methyl-L-cysteine. The expression of the ytmI operon and the yhcL gene was regulated in response to sulfur availability, while the level of expression of the yckK gene remained low under all the conditions tested.

Research has been conducted on Bacillus subtilis. The use of the DNA arrays in investigating the global transcriptional profile of this bacterium grown in the presence of methionine or sulfate is described.

Research has been conducted on Bacillus subtilis cysteine biosynthesis. Results demonstrate that the genes involved in this biosynthesis are regulated according to the available sulfur source.

Research has been conducted on the pathways for the methionine biosynthesis in microorganisms. The involvement of the metI and metC gene products in Bacillus subtilis methionine biosynthesis has been investigated and the results are reported.

Research has been conducted on the spore coat protein CotA of Bacillus subtilis which is similar to multicopper oxidases. Results demonstrate that this protein is a laccase.

A two-dimensional (2D) gel electrophoresis study of sulfur-limitation-regulated proteins has been undertaken in Bacillus subtilis to compare the proteome of a wild-type strain where sulfate or glutathione is the only sulfur source. The ytml gene is within a locus of 12 genes co-regulated in response to sulfur availability. This putative operon is activated by a LysR-like regulator, YtIl. This is the first regulator involved in control of expression in response to sulfur availability to be identified in B. subtilis.

Research was conducted to examine any changes in the catalytic and regulatory functions of catabolite repression HPr (Crh) protein by substituting glutamine-15 to a histidine residue. Results show that the modification, although it enables Crh carry out phosphorylation, it disables sugar transport function to Crh.

Results demonstrate that pta gene is involved in the carbon excretion and encoding for phosphotransacetylase in Bacillus subtilis. The data also indicate that two proteins influence pta expression by catabolite activation.

The role of P-Ser-HPr and P-Ser Crh in carbon catabolite repression (CCR) of the Bacillus subtilis levanase operon was investigated by constructing a ptsH1 crh1 double mutant and examining the interaction of the CcpA/P-Ser-HPr and CcpA/P-Ser Crh complexes with the cre sequence of the operon. The involvement of HPr kinase in the regulation of the levanase operon was also examined. The expression of the levanase operon was found to be totally relieved from CCR in ptsH1 crh1 and hprK mutants. It was also observed that both P-Ser-HPr and P-Ser Crh induced the binding of CcpA to the cre sequence of the operon.