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 -butyl hydroperoxide, an organic peroxide. In each case the induction was independent of the general stress sigma factor 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.

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.Staphylococcus aureus is a gram-positive facultative aerobe and a major human pathogen (33,39). In common with other facultative aerobes, S. aureus can grow in the absence of oxygen either by fermentation or by using an alternative terminal electron acceptor, such as nitrate. Several studies suggest that oxygen plays a role in the pathogenesis of S. aureus, in both its capacity to produce virulence factors and its ability to persist and grow in different and often hostile environmental niches (5,6,26,37,40,53). The ability of S. aureus to adapt to extreme changes in external oxygen concentration implies the existence of one or more oxygen-sensing systems that regulate the expression of genes in the transition from aerobic to anaerobic growth. While considerable progress has been made in characterizing global regulators of anaerobic gene expression, for example, the FNR and Arc families of proteins (3, 14, 17, 42), relatively little is known about the regulatory systems that operate in S. aureus during anaerobiosis. Knowledge of these signal transduction systems is crucial for understanding how, in S. aureus, oxygen brings about changes in the expression of virulence genes. Several recent in vitro studie...

SummaryRibonucleotide reductases (RNRs) catalyse the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs. We show here that the Streptomyces coelicolor M145 nrdAB genes encoding an oxygendependent class I RNR are co-transcribed with nrdS , which encodes an AraC-like regulatory protein. Likewise, the class II oxygen-independent RNR nrdJ gene forms an operon with a likely regulatory gene, nrdR , which encodes a protein possessing an ATP-cone domain like those present in the allosteric activity site of many class Ia RNRs. Deletions in nrdB and nrdJ had no discernible effect on growth individually, but abolition of both RNR systems, using hydroxyurea to inactivate the class Ia RNR (NrdAB) in the nrdJ deletion mutant, was lethal, establishing that S. coelicolor possesses just two functional RNR systems. The class II RNR (NrdJ) may function to provide a pool of deoxyribonucleotide precursors for DNA repair during oxygen limitation and/or for immediate growth after restoration of oxygen, as the nrdJ mutant was slower in growth recovery than the nrdB mutant or the parent strain. The class Ia and class II RNR genes show complex regulation. The nrdRJ genes were transcribed some five-to sixfold higher than the nrdABS genes in vegetative growth, but when nrdJ was deleted, nrdABS transcription was upregulated by 13-fold. In a reciprocal experiment, deletion of nrdB had little effect on nrdRJ transcription. Deletion of nrdR caused a dramatic increase in transcription of nrdJ and to a less extent nrdABS , whereas disruption of cobN , a gene required for synthesis of coenzyme B12 a cofactor for the class II RNR, caused similar upregulation of transcription of nrdRJ and nrdABS . In contrast, deletion of nrdS had no detectable effect on transcription of either set of RNR genes. These results establish the existence of control mechanisms that sense and regulate overall RNR gene expression.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomycetes contain genes coding for two RNRs. The class Ia RNR is oxygen dependent, and the class II RNR is oxygen independent and requires coenzyme B12. Either RNR is sufficient for vegetative growth. We show here that the Streptomyces coelicolor M145 nrdABS genes encoding the class Ia RNR are regulated by coenzyme B12. The 5-untranslated region of nrdABS contains a 123-nucleotide B12 riboswitch. Similar B12 riboswitches are present in the corresponding regions of eight other S. coelicolor genes. The effect of B12 on growth and nrdABS transcription was examined in a mutant in which the nrdJ gene, encoding the class II RNR, was deleted. B12 concentrations of just 1 g/liter completely inhibited growth of the NrdJ mutant strain. Likewise, B12 significantly reduced nrdABS transcription. To further explore the mechanism of B12 repression, we isolated in the nrdJ deletion strain mutants that are insensitive to B12 inhibition of growth. Two classes of mutations were found to map to the B12 riboswitch. Both conferred resistance to B12 inhibition of nrdABS transcription and are likely to affect B12 binding. These results establish that B12 regulates overall RNR expression in reciprocal ways, by riboswitch regulation of the class Ia RNR nrdABS genes and by serving as a cofactor for the class II RNR.

Streptomyces clavuligerus is a potent producer of penicillin and cephalosporin antibiotics. A key step in the biosynthesis of these jl-lactam compounds is the cyclization of the thiol tripeptide b-(L-ac-aminoadipyl)-Lcysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). However, bis-ACV, the oxidized disulfide form of the tripeptide, is not a substrate for IPNS. We show here that S. clavuligerus possesses an NADPH-dependent disulfide reductase of broad substrate specificity that efficiently catalyzes the reduction of disulfide bonds in bis-ACV and in other low-molecular-weight disulfide containing compounds and proteins. The disulfide reductase comprises two protein components, a 70-kDa reductase consisting of two identical subunits, and a 12-kDa heat-stable protein reductant. The structural and functional properties of the disulfide reductase resemble those of the thioredoxin class of oxidoreductases. When the disulfide reductase system is coupled with IPNS, it quantitatively converts bis-ACV to isopenicillin N. These findings suggest that the disulfide reductase may play a role in the biosynthesis of penicillins and cephalosporins in streptomycetes. We also show here that S. clavuligerus lacks glutathione reductase and have previously reported that Streptomyces species do not contain glutathione. This disulfide reductase may therefore be important in determining the thiol-disulfide redox balance in streptomycetes.