The regulatory cysK mutant of S. typhimurium.

A triazole-resistant mutant, cysK1358, showing novel properties was isolated. Biochemical analysis of this strain suggests a regulatory character of the cysK1358 mutation. The cysK1358 mutation has a pleiotropic effect: the expression of the other gene, cysA, is also altered.     

Functional analysis of the Bacillus subtilis cysK and cysJI genes

The function of the Bacillus subtilis cysK and cysJI (previously designated yvgQR) genes, expected to be involved in the assimilatory sulfate reduction pathway, was investigated. A B. subtilis mutant with a deletion in the cysJI genes was unable to use sulfate or sulfite as sulfur source, which confirmed that these genes encode sulfite reductase. A mutant with a transposon insertion in the cysK gene, whose deduced protein sequence showed similarity to cysteine synthases, grew poorly on sulfate and butanesulfonate. A strain in which cysK and yrhA, a cysK paralog, were inactivated was unable to grow with sulfate. Whereas expression of the cysJI genes was induced by sulfate, expression of cysK was repressed both by sulfate and by cysteine.     

Engineering Escherichia coli for increased productivity of serine-rich proteins based on proteome profiling

Variations in proteome profiles of Escherichia coli in response to the overproduction of human leptin, a serine-rich (11.6% of total amino acids) protein, were examined by two-dimensional gel electrophoresis. The levels of heat shock proteins increased, while those of protein elongation factors, 30S ribosomal protein, and some enzymes involved in amino acid biosynthesis decreased, after leptin overproduction. Most notably, the levels of enzymes involved in the biosynthesis of serine family amino acids significantly decreased. Based on this information, we designed a strategy to enhance the leptin productivity by manipulating the cysK gene, encoding cysteine synthase A. By coexpression of the cysK gene, we were able to increase the cell growth rate by approximately twofold. Also, the specific leptin productivity could be increased by fourfold. In addition, we found that cysK coexpression can improve the production of another serine-rich protein, interleukin-12 beta chain, suggesting that this strategy may be useful for the production of other serine-rich proteins as well. The approach taken in this study should be useful in designing a strategy for improving recombinant protein production.     

Regulation of O-acetylserine sulfhydrylase B by L-cysteine in Salmonella typhimurium.

A technique based on resistance to azaserine was used to isolate mutants lacking O-acetylserine sulfhydrylase B, one of two enzymes in Salmonella typhimurium capable of synthesizing L-cysteine from O-acetyl-L-serine and sulfide. The mutant locus responsible for this defect has been designated cysM, and genetic mapping suggests that cysM is very close to and perhaps contiguous with cysA. Strains lacking either O-acetylserine sulfhydrylase B or the second sulfhydrylase, O-acetylserine sulfhydrylase A (coded for by cysK), are cysteine prototrophs, but cysK cysM double mutants were found to require cysteine for growth. O-Acetylserine sulfhydrylase B was depressed by growth on a poor sulfur source, and depression was dependent upon both a functional cysB regulatory gene product and the internal inducer of the cysteine biosynthetic pathway, O-acetyl-L-serine. Furthermore, a cysBc strain, in which other cysteine biosynthetic enzymes cannot be fully repressed by growth on L-cystine, was found to be constitutive for O-acetylserine sulfhydrylase B as well. Thus O-acetylserine sulfhydrylase B is regulated by the same factors that control the expression of O-acetylserine sulfhydrylase A and other activities of the cysteine regulon. It is not clear why S. typhimurium has two enzymes whose physiological function appears to be to catalyze the same step of L-cysteine biosynthesis.     

A CysB regulator positively regulates cysteine synthesis,expression of type III secretion system genes,and pathogenicity in Ralstonia solanacearum

A syringe-like type III secretion system (T3SS) plays essential roles in the pathogenicity of Ralstonia solanacearum, which is a causal agent of bacterial wilt disease on many plant species worldwide. Here, we characterized functional roles of a CysB regulator (RSc2427) in R. solanacearum OE1-1 that was demonstrated to be responsible for cysteine synthesis, expression of the T3SS genes, and pathogenicity of R. solanacearum. The cysB mutants were cysteine auxotrophs that failed to grow in minimal medium but grew slightly in host plants. Supplementary cysteine substantially restored the impaired growth of cysB mutants both in minimal medium and inside host plants. Genes of cysU and cysI regulons have been annotated to function for R. solanacearum cysteine synthesis; CysB positively regulated expression of these genes. Moreover, CysB positively regulated expression of the T3SS genes both in vitro and in planta through the PrhG to HrpB pathway, whilst impaired expression of the T3SS genes in cysB mutants was independent of growth deficiency under nutrient-limited conditions. CysB was also demonstrated to be required for exopolysaccharide production and swimming motility, which contribute jointly to the host colonization and infection process of R. solanacearum. Thus, CysB was identified here as a novel regulator on the T3SS expression in R. solanacearum. These results provide novel insights into understanding of various biological functions of CysB regulators and complex regulatory networks on the T3SS in R. solanacearum.     

Bacillus subtilis cysteine synthetase is a global regulator of the expression of genes involved in sulfur assimilation

The synthesis of L-cysteine, the major mechanism by which sulfur is incorporated into organic compounds in microorganisms, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis the cysH operon, encoding several proteins involved in cysteine biosynthesis, is induced by sulfur starvation and tightly repressed by cysteine. We show that a null mutation in the cysK gene encoding an O-acetylserine-(thiol)lyase, the enzyme that catalyzes the final step in cysteine biosynthesis, results in constitutive expression of the cysH operon. Using DNA microarrays we found that, in addition to cysH, almost all of the genes required for sulfate assimilation are constitutively expressed in cysK mutants. These results indicate that CysK, besides its enzymatic role in cysteine biosynthesis, is a global negative regulator of genes involved in sulfur metabolism.     

Identification of the CysB-regulated gene,hslJ, related to the Escherichia coli novobiocin resistance phenotype

The cysB gene product is a LysR-type regulatory protein required for expression of the cys regulon. cysB mutants of Escherichia coli and Salmonella, along with being auxotrophs for the cysteine, exhibit increased resistance to the antibiotics novobiocin (Nov) and mecillinam. In this work, by using lambdaplacMu9 insertions creating random lacZ fusions, we identify a gene, hslJ, whose expression appeared to be increased in cysB mutants and needed for Nov resistance. Measurements of the HSLJ::lacZ gene fusion expression demonstrated that the hslJ gene is negatively regulated by CysB. In addition we observe the negative autogenous control of HslJ. When the control imposed by CysB is lifted in the cysB mutant, the elevation of Nov resistance can be achieved only in the presence of wild-type hslJ allele. A double cysB hslJ mutant restores the sensitivity to Nov. Overexpression of the wild-type HslJ protein either in a cysB(+) or a cysB(-) background increases the level of Nov resistance indicating that hslJ product is indeed involved in accomplishing this phenotype. The HSLJ::OmegaKan allele encodes the C-terminaly truncated mutant protein HslJ Q121Ter which is not functional in achieving the Nov resistance but when overexpressed induces the psp operon. Finally, we found that inactivation of hslJ does not affect the increased resistance to mecillinam in cysB mutants.     

Subcellular localization of spinach cysteine synthase isoforms and regulation of their gene expression by nitrogen and sulfur.

Subcellular localization and regulation of the spinach (Spinacia oleracea) cysteine synthase (O-acetyl-L-serine[thiol]-lyase, EC 4.2.99.8) isoforms (CysA, CysB, and CysC) were determined in transgenic tobacco (Nicotiana tabacum) and in spinach cell cultures. The 5' regions of CysB and CysC encoding the chloroplastic (CysB-TP) and the putative mitochondrial (CysC-TP) transit peptide (TP) sequences were fused to a bacterial beta-glucuronidase gene (gus) and expressed in tobacco under the control of the cauliflower mosaic virus 35S promoter. Subcellular fractionation of transgenic tobacco showed transportation of beta-glucuronidase proteins to chloroplasts by CysB-TP and to mitochondria by CysC-TP, respectively, indicating that both presequences were sufficient to act specifically as chloroplastic and mitochondrial TPs in vivo. The mRNA expression patterns of CysA (cytoplasmic form), CysB, and CysC genes under nitrogen- and sulfur-starved conditions were characterized in spinach cell cultures. In sulfur-starved cells, only slight differences (approximately 1.2- to 1.5-fold) in the mRNA levels of CysA and CysB were observed during the short-term (0-24 h) cultivation periods compared with cells grown in Murashige-Skoog medium. However, under nitrogen and nitrogen/sulfur double-deficient stress conditions, mRNA levels of CysC increased up to 500% of the original level within 72 h.     

Cloning and characterization of the cysAMK region of Salmonella typhimurium.

A total of 30 kilobases of DNA comprising the cysAMK region of S. typhimurium was cloned as a series of fragments in phage lambda 1059. The genetic organization of this region was established through studies of gene expression from fragments subcloned in pBR322 and from blot hydridization analyses of restriction sites in chromosomal DNA from multisite deletion strains. The results give a gene order of cysA-cysM-crr-ptsl-ptsH-cysK over a distance of approximately 12 kilobases. cysM and cysA have been cloned and expressed in pBR322; attempts to obtain stable pBR322 derivatives carrying cysK were unsuccessful.