Rachel Schreiber

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.

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.

Alternative oxygen-dependent and oxygen-independent ribonucleotide reductases in Streptomyces: cross-regulation and physiological role in response to oxygen limitation

Ribonucleotide 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 oxygen‐dependent 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.

Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5’-Phosphate Production in E. coli

Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous ‘replacer’ gene rescues lethality caused by inactivation of a ‘target’ gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5’-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.

The contribution of common rpsL mutations in Escherichia coli to sensitivity to ribosome targeting antibiotics

Point mutations in the rpsL gene encoding ribosomal protein S12 can generate resistance to streptomycin, resulting in rapid emergence of resistance to this antibiotic during treatment. In this work, we demonstrate that while spontaneous rpsL mutants in Escherichia coli are resistant to streptomycin, they are more sensitive to the ribosome-targeting antibiotics chloramphenicol, tetracycline and erythromycin. Moreover, combinations of these antibiotics, even in low concentrations were enough to achieve complete growth inhibition of both wild type and rpsL mutant strains. Thus, combining ribosome-targeting drugs can be used as a new treatment strategy that may be effective against streptomycin-resistant ribosome mutants.

Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy.

BACKGROUND Transrectal ultrasound-guided (TRUS) prostate biopsy is a very common procedure that is generally considered relatively safe. However, severe sepsis can occur after TRUS prostate biopsies, with Escherichia coli being the predominant causative agent. A common perception is that the bacteria that cause post-TRUS prostate biopsy infections originate in the urinary tract, but this view has not been adequately tested. Yet other authors believe on the basis of indirect evidence that the pathogens are introduced into the bloodstream by the biopsy needle after passage through the rectal mucosa. METHODS We compared E. coli isolates from male patients with bacteremic urinary tract infection (B-UTI) to isolates of patients with post prostate biopsy sepsis (PPBS), in terms of their sequence types, determined by multi-locus sequence typing (MLST) and their virulence markers. RESULTS B-UTI isolates were much richer in virulence genes than were PPBS isolates, supporting the hypothesis that E. coli causing PPBS derive directly from the rectum. Sequence type 131 (ST131) strains and related strain from the ST131 were common (>30%) among the E. coli isolates from PPBS patients as well as from B-UTI patients and all these strains expressed extended spectrum beta-lactamases. CONCLUSIONS Our finding supports the hypothesis that E. coli causing PPBS derive directly from the rectum, bypassing the urinary tract, and therefore do not require many of the virulence capabilities necessary for an E. coli strain that must persist in the urinary tract. In light of the increasing prevalence of highly resistant E. coli strains, a new approach for prevention of PPBS is urgently required.