Yair Aharonowitz

Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the sigmaR regulon.

In the Gram‐positive, antibiotic‐producing bacterium Streptomyces coelicolor A3(2), the thiol‐disulphide status of the hyphae is controlled by a novel regulatory system consisting of a sigma factor, σR, and its cognate anti‐sigma factor, RsrA. Oxidative stress induces intramolecular disulphide bond formation in RsrA, which causes it to lose affinity for σR, thereby releasing σR to activate transcription of the thioredoxin operon, trxBA. Here, we exploit a preliminary consensus sequence for σR target promoters to identify 27 new σR target genes and operons, thereby defining the global response to disulphide stress in this organism. Target genes related to thiol metabolism encode a second thioredoxin (TrxC), a glutaredoxin‐like protein and enzymes involved in the biosynthesis of the low‐molecular‐weight thiol‐containing compounds cysteine and molybdopterin. In addition, the level of the major actinomycete thiol buffer, mycothiol, was fourfold lower in a sigR null mutant, although no candidate mycothiol biosynthetic genes were identified among the σR targets. Three σR target genes encode ribosome‐associated products (ribosomal subunit L31, ppGpp synthetase and tmRNA), suggesting that the translational machinery is modified by disulphide stress. The product of another σR target gene was found to be a novel RNA polymerase‐associated protein, RbpA, suggesting that the transcriptional machinery may also be modified in response to disulphide stress. We present DNA sequence evidence that many of the targets identified in S. coelicolor are also under the control of the σR homologue in the actinomycete pathogen Mycobacterium tuberculosis.

Transcriptional Regulation of the Staphylococcus aureus Thioredoxin and Thioredoxin Reductase Genes in Response to Oxygen and Disulfide Stress

In this report we describe the cloning, organization, and promoter analysis of the Staphylococcus aureus thioredoxin (trxA) and thioredoxin reductase (trxB) genes and their transcription in response to changes in oxygen concentration and to oxidative stress compounds. Northern analysis showed that the S. aureus trxA and trxB genes were transcribed equally well in aerobic and anaerobic conditions. Several oxidative stress compounds were found to rapidly induce transcription of the trxA and trxB genes. The most pronounced effects were seen with diamide, a thiol-specific oxidant that promotes disulfide bond formation; menadione, a redox cycling agent; and tau-butyl hydroperoxide, an organic peroxide. In each case the induction was independent of the general stress sigma factor sigma(B). These studies show that the S. aureus trxA and trxB genes are upregulated following exposure to these oxidative stress agents, resulting in increased disulfide bond formation. In contrast, no effect of hydrogen peroxide on induction of the trxA and trxB genes was seen. We also show that the S. aureus thioredoxin reductase appears to be essential for growth. This observation, coupled with structural differences between the bacterial and mammalian thioredoxin reductases, suggests that it may serve as a target for the development of new antimicrobials.

Role of a Cysteine Synthase in Staphylococcus aureus

The gram-positive human pathogen Staphylococcus aureus is often isolated with media containing potassium tellurite, to which it has a higher level of resistance than Escherichia coli. The S. aureus cysM gene was isolated in a screen for genes that would increase the level of tellurite resistance of E. coli DH5alpha. The protein encoded by S. aureus cysM is sequentially and functionally homologous to the O-acetylserine (thiol)-lyase B family of cysteine synthase proteins. An S. aureus cysM knockout mutant grows poorly in cysteine-limiting conditions, and analysis of the thiol content in cell extracts showed that the cysM mutant produced significantly less cysteine than wild-type S. aureus SH1000. S. aureus SH1000 cannot use sulfate, sulfite, or sulfonates as the source of sulfur in cysteine biosynthesis, which is explained by the absence of genes required for the uptake and reduction of these compounds in the S. aureus genome. S. aureus SH1000, however, can utilize thiosulfate, sulfide, or glutathione as the sole source of sulfur. Mutation of cysM caused increased sensitivity of S. aureus to tellurite, hydrogen peroxide, acid, and diamide and also significantly reduced the ability of S. aureus to recover from starvation in amino acid- or phosphate-limiting conditions, indicating a role for cysteine in the S. aureus stress response and survival mechanisms.

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus.

The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.

NrdR Controls Differential Expression of the Escherichia coli Ribonucleotide Reductase Genes

Escherichia coli possesses class Ia, class Ib, and class III ribonucleotide reductases (RNR). Under standard laboratory conditions, the aerobic class Ia nrdAB RNR genes are well expressed, whereas the aerobic class Ib nrdEF RNR genes are poorly expressed. The class III RNR is normally expressed under microaerophilic and anaerobic conditions. In this paper, we show that the E. coli YbaD protein differentially regulates the expression of the three sets of genes. YbaD is a homolog of the Streptomyces NrdR protein. It is not essential for growth and has been renamed NrdR. Previously, Streptomyces NrdR was shown to transcriptionally regulate RNR genes by binding to specific 16-bp sequence motifs, NrdR boxes, located in the regulatory regions of its RNR operons. All three E. coli RNR operons contain two such NrdR box motifs positioned in their regulatory regions. The NrdR boxes are located near to or overlap with the promoter elements. DNA binding experiments showed that NrdR binds to each of the upstream regulatory regions. We constructed deletions in nrdR (ybaD) and showed that they caused high-level induction of transcription of the class Ib RNR genes but had a much smaller effect on induction of transcription of the class Ia and class III RNR genes. We propose a model for differential regulation of the RNR genes based on binding of NrdR to the regulatory regions. The model assumes that differences in the positions of the NrdR binding sites, and in the sequences of the motifs themselves, determine the extent to which NrdR represses the transcription of each RNR operon.

Staphylococcus aureus ArcR Controls Expression of the Arginine Deiminase Operon

We identified a single open reading frame that is strongly similar to ArcR, a member of the Crp/Fnr family of bacterial transcriptional regulators, in all sequenced Staphylococcus aureus genomes. The arcR gene encoding ArcR forms an operon with the arginine deiminase (ADI) pathway genes arcABDC that enable the utilization of arginine as a source of energy for growth under anaerobic conditions. In this report, we show that under anaerobic conditions, S. aureus growth is subject to glucose catabolic repression and is enhanced by arginine. Likewise, glucose and arginine have reciprocal effects on the transcription of the arcABDCR genes. Furthermore, we show using a mutant deleted for arcR that the transcription of the arc operon under anaerobic conditions depends strictly on a functional ArcR. These findings are supported by proteome analyses, which showed that under anaerobic conditions the expression of the ADI catabolic proteins depends on ArcR. Bioinformatic analysis of S. aureus ArcR predicts an N-terminal nucleotide binding domain and a C-terminal helix-turn-helix DNA binding motif. ArcR binds to a conserved Crp-like sequence motif, TGTGA-N(6)-TCACA, present in the arc promoter region and thereby activates the expression of the ADI pathway genes. Crp-like sequence motifs were also found in the regulatory regions of some 30 other S. aureus genes mostly encoding anaerobic enzymatic systems, virulence factors, and regulatory systems. ArcR was tested and found to bind to the regulatory regions of four such genes, adh1, lctE, srrAB, and lukM. In one case, for lctE, encoding l-lactate dehydrogenase, ArcR was able to bind only in the presence of cyclic AMP. These observations suggest that ArcR is likely to play an important role in the expression of numerous genes required for anaerobic growth.

A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis.

Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, gamma-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate gamma-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a gamma-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.

Analysis of Transcription of the Staphylococcus aureus Aerobic Class Ib and Anaerobic Class III Ribonucleotide Reductase Genes in Response to Oxygen

Staphylococcus aureus is a gram-positive facultative aerobe that can grow in the absence of oxygen by fermentation or by using an alternative electron acceptor. To investigate the mechanism by which S. aureus is able to adapt to changes in oxygen concentration, we analyzed the transcriptional regulation of genes that encode the aerobic class Ib and anaerobic class III ribonucleotide reductase (RNR) systems that are responsible for the synthesis of deoxyribonucleotides needed for DNA synthesis. The S. aureus class Ib RNR nrdIEF and class III RNR nrdDG genes and their regulatory regions were cloned and sequenced. Inactivation of the nrdDG genes showed that the class III RNR is essential for anaerobic growth. Inhibition of aerobic growth by hydroxyurea showed that the class Ib RNR is an oxygen-dependent enzyme. Northern blot analysis and primer extension analysis demonstrated that transcription of class III nrdDG genes is regulated by oxygen concentration and was at least 10-fold higher under anaerobic than under aerobic conditions. In contrast, no significant effect of oxygen concentration was found on the transcription of class Ib nrdIEF genes. Disruption or deletion of S. aureus nrdDG genes caused up to a fivefold increase in nrdDG and nrdIEF transcription under anaerobic conditions but not under aerobic conditions. Similarly, hydroxyurea, an inhibitor of the class I RNRs, resulted in increased transcription of class Ib and class III RNR genes under aerobic conditions. These findings establish that transcription of class Ib and class III RNR genes is upregulated under conditions that cause the depletion of deoxyribonucleotide. Promoter analysis of class Ib and class III RNR operons identified several inverted-repeat elements that may account for the transcriptional response of the nrdIEF and nrdDG genes to oxygen.

Diamide Triggers Mainly S Thiolations in the Cytoplasmic Proteomes of Bacillus subtilis and Staphylococcus aureus

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.

Microbial isopenicillin N synthase genes: Structure, function, diversity and evolution

Clinically and economically, penicillins and cephalosporins are the most important class of the beta-lactam antibiotics. They are produced by a wide variety of microorganisms including numerous species of Streptomyces, some unicellular bacteria and several filamentous fungi. A key step common to their biosynthetic pathways is the conversion of a linear, cysteine-containing tripeptide to a bicyclic beta-lactam antibiotic by isopenicillin N synthase. Recent successes in the cloning and expression of isopenicillin N synthase genes now permit production of a plentiful supply of this enzyme, which may be used for structural and mechanistic studies, or for biotechnological applications in the creation of novel beta-lactam compounds from peptide analogues. New ideas concerning the evolution and prevalence of the penicillin and cephalosporin biosynthetic genes have emerged from studies of isopenicillin N synthase genes.