Staphylococcus aureus is both a commensal and a pathogen of the human host. Survival in the host environment requires resistance to host-derived nitric oxide (NO·). However, S. aureus lacks the NO·-sensing transcriptional regulator NsrR that is used by many bacteria to sense and respond to NO·. In this study, we show that S. aureus is able to sense and respond to both NO· and hypoxia by means of the SrrAB two-component system (TCS). Analysis of the S. aureus transcriptome during nitrosative stress demonstrates the expression of SrrAB-dependent genes required for cytochrome biosynthesis and assembly (qoxABCD, cydAB, hemABCX), anaerobic metabolism (pflAB, adhE, nrdDG), iron-sulfur cluster repair (scdA), and NO· detoxification (hmp). Targeted mutations in SrrAB-regulated loci show that hmp and qoxABCD are required for NO· resistance, whereas nrdDG is specifically required for anaerobic growth. We also show that SrrAB is required for survival in static biofilms, most likely due to oxygen limitation. Activation by hypoxia, NO·, or a qoxABCD quinol oxidase mutation suggests that the SrrAB TCS senses impaired electron flow in the electron transport chain rather than directly interacting with NO· in the manner of NsrR. Nevertheless, like NsrR, SrrAB achieves the physiological goals of selectively expressing hmp in the presence of NO· and minimizing the potential for Fenton chemistry. Activation of the SrrAB regulon allows S. aureus to maintain energy production and essential biosynthetic processes, repair damage, and detoxify NO· in diverse host environments.

Staphylococcus aureus is a major human pathogen that is resistant to numerous antibiotics in clinical use. We found two nonribosomal peptide secondary metabolites--the aureusimines, made by S. aureus--that are not antibiotics, but function as regulators of virulence factor expression and are necessary for productive infections. In vivo mouse models of bacteremia showed that strains of S. aureus unable to produce aureusimines were attenuated and/or cleared from major organs, including the spleen, liver, and heart. Targeting aureusimine synthesis may offer novel leads for anti-infective drugs.

Bacterial two-hybrid analysis identified the Staphylococcus aureus RNA degradosome-like complex to include RNase J1, RNase J2, RNase Y, polynucleotide phosphorylase (PNPase), enolase, phosphofructokinase, and a DEAD box RNA helicase. Results also revealed that the recently recognized RNase RnpA interacts with the S. aureus degradosome and that this interaction is conserved in other Gram-positive organisms.Escherichia coli bulk RNA degradation is mediated by a holoenzyme complex, the RNA degradosome, which includes the ribonucleases RNase E and polynucleotide phosphorylase (PNPase), an RNA helicase, RhlB, and the glycolytic enzyme enolase (3,20). RNase E is considered the key component of the E. coli degradosome; it catalyzes the initiation of mRNA degradation and serves as a scaffold for the assembly of the other degradosome components (2). By comparison, most Gram-positive bacteria do not contain an RNase E amino acid ortholog and much less is known about their mRNA degradation machinery. Nonetheless, recent studies suggest that the Gram-positive organism Bacillus subtilis produces two RNase E functional orthologs, ribonucleases J1 (RNase J1) and J2 (RNase J2) (17). Using bacterial two-hybrid analyses, both enzymes have been shown to interact with an RNA degradosome-like complex consisting of PNPase, enolase, and a DEAD box RNA helicase (CshA), as well as phosphofructokinase and RNase Y (6).Staphylococcus aureus is a Gram-positive bacterial pathogen of immense health care concern that is evolutionarily similar, but divergent, from the Bacillus genus. Herein we used bacterial two-hybrid and biochemical analyses to measure interactions between putative members of the S. aureus RNA degradosome and compare the results to those with B. subtilis. Results revealed that although many interactions are conserved between both bacterial species, differences are likely to exist. Moreover, we show that the RNase RnpA interacts with components of the S. aureus RNA degradosome and that this interaction is conserved within B. subtilis.Using bacterial two-hybrid analyses, interactions between putative members of the B. subtilis RNA degradosome were recently measured and used to develop the first model of the Gram-positive holoenzyme complex (Fig. 1A) (6, 13). According to the model, the E. coli RNase E orthologs RNase J1 and RNase J2 interact with one another; RNase J1 also interacts with two additional ribonucleases, PNPase and RNase Y, as well as with the glycolytic enzyme phosphofructokinase, each of which binds to the RNA helicase CshA (Fig. 1A). CshA also binds to a second glycolytic enzyme, enolase. Although the mechanism of B. subtilis cellular RNA degradation has yet to be established, RNases J1 and J2 are bifunctional ribonucleases with endonuclease and 5Ј33Ј exoribonuclease activities (8,16,24). In complex, the enzymes act synergistically and may initiate 5Ј mRNA digestion and/or generate endonucleolytic products that could subsequently be digested by RNA helicase-facilitated 3Ј35Ј PNPase exoribonuclease activity (14,1...

SUMMARY Eradication of persistent intracellular bacterial pathogens with antibiotic therapy is often slow or incomplete. However, strategies to augment antibiotics are hampered by our poor understanding of the nutritional environment that sustains chronic infection. Here we show that the intracellular pathogen Brucella abortus survives and replicates preferentially in alternatively activated macrophages (AAM), which are more abundant during chronic infection. A metabolic shift induced by peroxisome proliferator activated receptor γ (PPARγ), which increases intracellular glucose availability, is identified as a causal mechanism promoting enhanced bacterial survival in AAM. Glucose uptake was crucial for increased replication of B. abortus in AAM, and chronic infection, as inactivation of the bacterial glucose transporter gluP reduced both intracellular survival in AAM and persistence in mice. Thus, a shift in intracellular nutrient availability induced by PPARγ promotes chronic persistence of B. abortus within AAM and targeting this pathway may aid in eradicating chronic infection.

Summary Hfq is an RNA‐binding protein that functions in post‐transcriptional gene regulation by mediating interactions between mRNAs and small regulatory RNAs (sRNAs). Two proteins encoded by BAB1_1794 and BAB2_0612 are highly over‐produced in a Brucella abortus hfq mutant compared with the parental strain, and recently, expression of orthologues of these proteins in Agrobacterium tumefaciens was shown to be regulated by two sRNAs, called AbcR1 and AbcR2. Orthologous sRNAs (likewise designated AbcR1 and AbcR2) have been identified in B. abortus 2308. In Brucella, abcR1 and abcR2 single mutants are not defective in their ability to survive in cultured murine macrophages, but an abcR1 abcR2 double mutant exhibits significant attenuation in macrophages. Additionally, the abcR1 abcR2 double mutant displays significant attenuation in a mouse model of chronic Brucella infection. Quantitative proteomics and microarray analyses revealed that the AbcR sRNAs predominantly regulate genes predicted to be involved in amino acid and polyamine transport and metabolism, and Northern blot analyses indicate that the AbcR sRNAs accelerate the degradation of the target mRNAs. In an Escherichia coli two‐plasmid reporter system, overexpression of either AbcR1 or AbcR2 was sufficient for regulation of target mRNAs, indicating that the AbcR sRNAs from B. abortus 2308 perform redundant regulatory functions.