CysB Protein Research Articles

Upregulation of PBP1B and LpoB in cysB Mutants Confers Mecillinam (Amdinocillin) Resistance in Escherichia coli.

Mecillinam (amdinocillin) is a β-lactam antibiotic that inhibits the essential penicillin-binding protein 2 (PBP2). In clinical isolates of Escherichia coli from urinary tract infections, inactivation of the cysB gene (which encodes the main regulator of cysteine biosynthesis, CysB) is the major cause of resistance. How a nonfunctional CysB protein confers resistance is unknown, however, and in this study we wanted to examine the mechanism of resistance. Results show that cysB mutations cause a gene regulatory response that changes the expression of ∼450 genes. Among the proteins that show increased levels are the PBP1B, LpoB, and FtsZ proteins, which are known to be involved in peptidoglycan biosynthesis. Artificial overexpression of either PBP1B or LpoB in a wild-type E. coli strain conferred mecillinam resistance; conversely, inactivation of either the mrcB gene (which encodes PBP1B) or the lpoB gene (which encodes the PBP1B activator LpoB) made cysB mutants susceptible. These results show that expression of the proteins PBP1B and LpoB is both necessary and sufficient to confer mecillinam resistance. The addition of reducing agents to a cysB mutant converted it to full susceptibility, with associated downregulation of PBP1B, LpoB, and FtsZ. We propose a model in which cysB mutants confer mecillinam resistance by inducing a response that causes upregulation of the PBP1B and LpoB proteins. The higher levels of these two proteins can then rescue cells with mecillinam-inhibited PBP2. Our results also show how resistance can be modulated by external conditions such as reducing agents.

Transcriptional control of the pvdS iron starvation sigma factor gene by the master regulator of sulfur metabolism CysB in Pseudomonas aeruginosa

In the Gram-negative pathogen Pseudomonas aeruginosa, the alternative sigma factor PvdS acts as a key regulator of the response to iron starvation. PvdS also controls P. aeruginosa virulence, as it drives the expression of a large set of genes primarily implicated in biogenesis and transport of the pyoverdine siderophore and synthesis of extracellular factors, such as protease PrpL and exotoxin A. Besides the ferric uptake regulatory protein Fur, which shuts off pvdS transcription under iron-replete conditions, no additional regulatory factor(s) controlling the pvdS promoter activity have been characterized so far. Here, we used the promoter region of pvdS as bait to tentatively capture, by DNA-protein affinity purification, P. aeruginosa proteins that are able to bind specifically to the pvdS promoter. This led to the identification and functional characterization of the LysR-like transcription factor CysB as a novel regulator of pvdS transcription. The CysB protein directly binds to the pvdS promoter in vitro and acts as a positive regulator of PvdS expression in vivo. The absence of a functional CysB protein results in about 50% reduction of expression of PvdS-dependent virulence phenotypes. Given the role of CysB as master regulator of sulfur metabolism, our findings establish a novel molecular link between the iron and sulfur regulons in P. aeruginosa.

Two Transsulfurylation Pathways in Klebsiella pneumoniae

In most bacteria, inorganic sulfur is assimilated into cysteine, which provides sulfur for methionine biosynthesis via transsulfurylation. Here, cysteine is transferred to the terminal carbon of homoserine via its sulfhydryl group to form cystathionine, which is cleaved to yield homocysteine. In the enteric bacteria Escherichia coli and Salmonella enterica, these reactions are catalyzed by irreversible cystathionine-gamma-synthase and cystathionine-beta-lyase enzymes. Alternatively, yeast and some bacteria assimilate sulfur into homocysteine, which serves as a sulfhydryl group donor in the synthesis of cysteine by reverse transsulfurylation with a cystathionine-beta-synthase and cystathionine-gamma-lyase. Herein we report that the related enteric bacterium Klebsiella pneumoniae encodes genes for both transsulfurylation pathways; genetic and biochemical analyses show that they are coordinately regulated to prevent futile cycling. Klebsiella uses reverse transsulfurylation to recycle methionine to cysteine during periods of sulfate starvation. This methionine-to-cysteine (mtc) transsulfurylation pathway is activated by cysteine starvation via the CysB protein, by adenosyl-phosphosulfate starvation via the Cbl protein, and by methionine excess via the MetJ protein. While mtc mutants cannot use methionine as a sulfur source on solid medium, they will utilize methionine in liquid medium via a sulfide intermediate, suggesting that an additional nontranssulfurylation methionine-to-cysteine recycling pathway(s) operates under these conditions.

Sulfonate-sulfur metabolism and its regulation in Escherichia coli.

In the absence of sulfate and cysteine, Escherichia coli can use aliphatic sulfonates as a source of sulfur for growth. Starvation for sulfate leads to the expression of the tauABCD and ssuEADCB genes. Each of these gene clusters encodes an ABC-type transport system required for uptake of aliphatic sulfonates and a desulfonation enzyme. The TauD protein is an alpha-ketoglutarate-dependent dioxygenase that preferentially liberates sulfite from taurine (2-aminoethanesulfonic acid). SsuD is a monooxygenase that catalyzes the oxygenolytic desulfonation of a range of aliphatic sulfonates other than taurine. Its cosubstrate is FMNH2, which is provided by SsuE, an NAD(P)H-dependent FMN reductase. In contrast to many other bacteria, E. coli is unable to grow with arylsulfonates or with sulfate esters as sulfur source. The tau and ssu systems thus provide all genes for the utilization of known organosulfur sources by this organism, except the as yet unidentified gene(s) that enable some E. coli strains to grow with methanesulfonate or cysteate as a sulfur source. Expression of the tau and ssu genes requires the LysR-type transcriptional regulatory proteins CysB and Cbl. Synthesis of Cbl itself is under control of the CysB protein, and the CysB protein may therefore be regarded as the master regulator for sulfur assimilation in E. coli, while the Cbl protein functions as an accessory element specific for utilization of sulfur from organosulfur sources.

Bacterial transporters for sulfate and organosulfur compounds

Microorganisms require sulfur for growth, and obtain it either from inorganic sulfate or from organosulfur compounds such as sulfonates, sulfate esters, or sulfur-containing amino acids. Transport of sulfate into the cell is catalyzed either by ATP binding cassette (ABC)-type transporters (SulT family) or by major facilitator superfamily-type transporters (SulP family). By contrast, the sulfonate and sulfate ester transporters identified to date are all ABC-type systems, whose synthesis is tightly regulated by the sulfur supply to the cell, mediated by the CysB protein and other transcriptional regulators of the LysR-family.

A new gene, cbl, encoding a member of the LysR family of transcriptional regulators belongs to Escherichia coli cys regulon

The cbl ( cysB-like) gene has been identified in Escherichia coli. The analysis of the cloned cbl sequence revealed strict homology to an ORF of unknown function found initially in Klebsiella aerogenes [Schwacha and Bender, J. Bacteriol. 175 (1993) 2107–2115]. The predicted Cbl protein has structural features of the LysR family of transcriptional activators. It is also strongly similar to the CysB protein, the activator of the cys regulon. The position of cbl on the Ec physical map has been established at a 2070-kb (43.5 min) region between asnU and asnV. The gene is expressed in vivo as a 1-kb monocistronic transcript starting from one major transcription start point. Unexpectedly, the in vivo expression of cbl has shown dependence on CysB, belonging to the same family of proteins. The promoter region of cbl binds purified CysB protein in a manner similar to other CysB-responsive promoters. A cbl disruption mutant was constructed by insertion of a Km Rgene cartridge into the ORF on the chromosome. Phenotypes related to cbl expression suggest the involvement of the gene in an accessory regulatory circuit within the cys regulon engaging, in the last step, the function of the cysM gene encoding O-acetylserine (thiol)-lyase B.

Characterization of the CysB protein of Klebsiella aerogenes: direct evidence that N-acetylserine rather than O-acetylserine serves as the inducer of the cysteine regulon.

The cysB gene of Klebsiella aerogenes has been cloned, sequenced and shown to complement the cysteine auxotrophic phenotype of Escherichia coli cysB mutants. The K. aerogenes cysB gene is predicted to encode a protein of 324 amino acid residues that shares approx. 95% sequence similarity with the Salmonella typhimurium and E. coli CysB proteins. Gel-retardation assays demonstrate that the purified protein binds to DNA fragments containing either the K. aerogenes cysb promoter or the S. typhimurium cysJIH promoter. Acetylserine enhances CysB binding to the cysJIH promoter fragment while diminishing its binding to the cysB promoter fragment. Fluorescence-emission-spectroscopy measurements suggest strongly that N-acetylserine binds to CysB apoprotein but that O-acetylserine does not, and support the notion that N-acetylserine is the physiological inducer of cysteine biosynthesis.

Crystallization of a DNA and N-acetylserine Binding Fragment (Residues 1 to 233) of Klebsiella aerogenes CysB Protein, a Member of the LysR Family

CysB protein is a positive regulator of transcription of genes involved in cysteine biosynthesis in bacteria and a member of the LysR family of DNA binding proteins. A 233-residue N-terminal chymotryptic fragment of the protein, with DNA and N-acetylserine binding activity, has been crystallized in the presence of monomethyl-polyethylene glycol 750. The crystals diffract to 2·5 Å (1 Å = 0·1 nm) spacing on a rotating copper anode X-ray source and to 2·1 Å spacing using synchrotron radiation and are suitable for structural studies. The space group is P21212 with unit cell dimensions of a = 68·8 Å, b = 109·9 Å and c = 33·5 Å. On the assumption that the asymmetric unit comprises one monomer, the crystals have a solvent content of approximately 50%.

The molecular basis for positive regulation of cys promoters in Salmonella typhimurium and Escherichia coli.

Most genes required for cysteine biosynthesis in Salmonella typhimurium and Escherichia coli are positively regulated by cysB, which encodes a transcriptional activator belonging to the LysR family of regulatory proteins. CysB protein binds just upstream of the -35 region of positively regulated promoters, where in the presence of inducer it facilitates formation of a transcription initiation complex. CysB protein also autoregulates its own synthesis by binding to the cysB promoter as a repressor. Cysteine down-regulates the pathway by inhibiting synthesis of O-acetylserine, a direct cysteine precursor and possibly an inducer of gene expression. O-Acetylserine spontaneously isomerizes to N-acetylserine, which is clearly an inducer. Sulphide and thiosulphate provide additional regulation by acting as anti-inducers. Inducer stimulates CysB protein binding to sites involved in positive regulation, and inhibits binding to the negatively autoregulated cysB promoter. For three sites with unknown function, binding is stimulated at one and inhibited at the other two.

The cysP promoter of Salmonella typhimurium: characterization of two binding sites for CysB protein, studies of in vivo transcription initiation, and demonstration of the anti-inducer effects of thiosulfate

The cysPTWA operons of Escherichia coli and Salmonella typhimurium encode components of periplasmic transport systems for sulfate and thiosulfate and are regulated as part of the cysteine regulons. In vitro transcription initiation from the cysP promoter was shown to require both CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, which act as inducers, and was inhibited by the anti-inducer sulfide. Thiosulfate was found to be even more potent than sulfide as an anti-inducer. DNase I protection experiments showed two discrete binding sites for CysB protein in the presence of N-acetyl-L-serine. CBS-P1 is located between positions -85 and -41 relative to the major transcription start site, and CBS-P2 is located between positions -19 and +25. Without N-acetyl-L-serine, the CysB protein protected the region between positions -63 and -11, which was designated CBS-P3. In gel mobility shift assays, the mobility of CysB protein-cysP promoter complexes was increased by O-acetyl-L-serine, N-Acetyl-L-serine had no effect in gel shift experiments, presumably because its anionic charge results in its rapid removal from the complex during electrophoresis. Comparison of DNA fragments differing with respect to binding site position indicated that complexes with CysB protein contain DNA that is bent somewhere between CBS-P1 and CBS-P2 and that O-acetyl-L-serine decreases DNA bending. Binding studies with fragments containing either CBS-P2 alone, CBS-P1 alone, or the entire cysP promoter region suggest a model in which the complex of bent DNA observed in the absence of O-acetyl-L-serine contains a single CysB protein molecule bound to CBS-P3. At relatively low CysB protein concentrations, O-acetyl-L-serine would cause a single CysB protein molecule to bind tightly to CBS-P1, rather than to CBS-P3, thereby decreasing DNA bending and increasing complex electrophoretic mobility. At higher CysB protein concentrations, O-acetyl-L-serine would cause a second molecule to bind at CBS-P2, giving a more slowly migrating complex.

Negative autoregulation of cysB in Salmonella typhimurium: in vitro interactions of CysB protein with the cysB promoter

CysB protein positively regulates genes of the Salmonella typhimurium cysteine regulon and negatively autoregulates cysB. The cysB promoter was characterized by primer extension of cellular RNA, which gave products identifying a major in vivo transcription start site located 95 bp upstream of the cysB start codon and two minor sites located 9 and 10 bp downstream of the major site. Gel shift binding studies and DNase I footprinting experiments showed that CysB protein binds to the cysB promoter from position -10 to +36 relative to the major transcription start site. We have designated this binding site CBS-B. CysB protein inhibited transcription initiation at the cysB promoter in an in vitro runoff assay, indicating that cysB is negatively autoregulated by the binding of CysB protein to the cysB promoter, where it acts as a repressor. N-Acetyl-L-serine, an inducer of the cysteine regulon, inhibited the binding of CysB protein to the cysB promoter and partially reversed the ability of CysB protein to inhibit transcription initiation. These effects are in contrast to those observed in studies of positively regulated cys promoters, in which N-acetyl-L-serine stimulates binding and causes CysB protein to activate transcription initiation.

In vitro interactions of CysB protein with the cysK and cysJIH promoter regions of Salmonella typhimurium

The cysteine regulons of Salmonella typhimurium and Escherichia coli are positively regulated by CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, both of which act as inducers. Gel mobility shift assays and DNase I footprinting experiments showed that CysB protein binds to the S. typhimurium cysK promoter at two sites, one, designated CBS-K1, at positions -78 to -39 relative to the major transcription start site, and the other, designated CBS-K2, at positions -115 to -79. The S. typhimurium cysJIH promoter was found to contain a single binding site, designated CBS-JH, at positions -76 to -35. Acetyl-L-serine stimulated binding to CBS-K1 and CBS-J and inhibited binding to CBS-K2. In the absence of acetyl-L-serine, CysB protein bound to both CBS-K1 and CBS-K2 and gave a complex that migrated more slowly during gel electrophoresis than did that formed in the presence of acetyl-L-serine, in which case CysB protein bound only to CBS-K1. Complexes formed with DNA containing the two binding sites either at the middle or at one end of the fragment migrated differently, suggesting that DNA was bent in the slow complex formed in the absence of acetyl-L-serine and that DNA in the fast complex was less bent or not bent at all. An analysis of upstream deletions of the cysK promoter showed that only CBS-K1 is required for in vivo promoter activity. CBS-J is analogous in position to CBS-K1 and is probably also required for activity of the cysJIH promoter. CBS-K2 has no known function but may help sequester CysB protein at the cysK promoter.

Analysis of the [formula omitted][formula omitted] K-12 [formula omitted] gene and its product using the method of gene fusion

The amino-terminal structure and the essential functional region of the cysB gene product of Escherichiacoli K-12 were analyzed by the method of gene fusion. The translational start codon of the cysB gene was located by determining the amino-terminal sequence of a hybrid protein containing the first 31 amino acid residues of the CysB protein at the amino terminus of β-galactosidase(LacZ protein). The fact that two other CysB‘−’LacZ hybrid polypeptides expressed a normal CysB activity indicated that the functional region of the CysB protein was located within the first 215 amino acid residues of the total 324 amino acids deduced from the nucletide sequence.

Promoter elements and regulation of expression of the cysD gene of Escherichia coli K-12

The cysD gene, involved in cysteine biosynthesis in Escherichia coli and Salmonella typhimurium, is positively regulated by the CysB regulatory protein. The cysD promoter of E. coli K-12 in a 492-bp PstIEcoRI fragment was sequenced. The in vivo transcription start point (tsp) for the cysD gene was determined by the methods of T4 DNA polymerase mapping and mung-bean nuclease mapping. The -10 region of the cysD promoter (TATAGT) is closely homologous to the -10 consensus sequence (TATAAT) for E. coli promoters. The -35 region of this promoter (TTCATT) is less closely related to the -35 consensus sequence (TTGACA). Several mutants were obtained by using a chain-termination method for generating unidirectional deletions. Evidence is presented for a possible CysB protein binding site around -89, thought to be involved in regulation of expression of the cysD gene.

In vitro interactions of CysB protein with the cysJIH promoter of Salmonella typhimurium: inhibitory effects of sulfide

The cysteine regulon of Salmonella typhimurium is positively regulated by the CysB protein and an inducer, which can be either O-acetyl-L-serine or N-acetyl-L-serine. In vivo experiments confirmed that sulfide and L-cysteine (supplied as L-cystine) interfere with induction by exogenously supplied O-acetyl-L-serine and also showed the same effects when N-acetyl-L-serine was used as an inducer. In a gel shift assay, purified CysB protein bound specifically to a 278-base-pair DNA fragment containing the S. typhimurium cysJIH promoter region. Binding occurred in the absence of inducer but did not stimulate in vitro transcription initiation, indicating that binding alone is insufficient to cause formation of a transcription initiation complex. Addition of N-acetyl-L-serine or O-acetyl-L-serine was required for transcription initiation and also stimulated binding three- to eightfold. Sulfide inhibited both transcription initiation and binding by interfering with the stimulatory effects of inducer in a competitive manner. These findings indicate that sulfide is an anti-inducer and may explain why full expression of the cysteine regulon requires sulfur limitation. L-Cysteine did not affect in vitro transcription initiation or binding of CysB protein to the cysJIH promoter region. The in vivo effects of L-cysteine may be secondary to its degradation to sulfide by the inducible enzyme cysteine desulfhydrase.

Purification of the cysB protein from Salmonella typhimurium.

Using an assay based on polypeptide mobility in one- and two-dimensional polyacrylamide electropherograms, we purified the Salmonella typhimurium cysB protein from an Escherichia coli strain carrying plasmid pGBK14, in which the S. typhimurium cysB gene is under transcriptional control of the strong promoter PL from phage lambda. cysB protein constitutes approximately 4% of total soluble protein in such cells and was obtained with good yield at a purity of 85% after precipitation of nucleic acids with streptomycin sulfate, ammonium sulfate fractionation, and hydrophobic chromatography on methyl agarose. Material with 95% purity was obtained with poor yield by ion-exchange chromatography of native protein and with good yield by size exclusion chromatography of denatured protein in sodium dodecyl sulfate. Physicochemical studies of the purified cysB protein show that it is a tetramer of identical 36-kDa subunits with a pI, amino acid composition, amino-terminal sequence, and carboxyl-terminal amino acid content identical to those known from previous studies or deduced from the cysB DNA sequence. Although O-acetyl-L-serine is believed to bind to cysB protein to form a complex that stimulates transcription of genes of the cysteine regulon, we were unable to demonstrate such an interaction using methods that would have detected binding with a Kd of less than 0.1 mM.

Identification of the Salmonella typhimurium cysB gene product by two-dimensional protein electrophoresis.

Examination of two-dimensional electropherograms of proteins from wild-type Salmonella typhimurium and 16 different cysB strains permitted the identification of a single 34,500-dalton polypeptide chain with a pI of 7.6 that was the product of cysB. Exclusion chromatography indicated that the native cysB protein is a multimer of at least two and probably four or more such subunits.

Isolation of a lambdadcys transducing bacteriophage and its use in determining the regulation of cysteine messenger ribonucleic acid synthesis in Escherichia coli K-12

A defective specialized lambda transducing phage carrying the cysJ, cysI, cysH, and cysD genes has been isolated from a secondary-site lysogen. Deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization studies utilizing this phage have been carried out to detect cysteine-specific messenger RNA (cys mRNA) synthesized in vivo. A vivo. A 3.5- to 9-fold increase in the rate of synthesis of cys mRNA has been detected in the derepressed wild-type (Cys+) strain grown on glutathione compared with a repressed control grown on cystine. Pleiotropic cysE and cysB mutants grown on glutathione were found to possess rates of synthesis of cys mRNA that were significantly lower than their derepressed isogenic parent. The addition of O-acetyl-L-serine to the cysE strain produced a 5.5-fold increase in the rate of synthesis of cys mRNA. These results indicate that cysteine biosynthesis is controlled at the level of transcription by the inducer O-acetylserine, the cysB protein and cyst(e)ine.