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Abstract
In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine de novo. Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5′-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA \((tRNA_i^{fMet})\)while binding of elongator \(tRNA^{Met}\) proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5′ met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence \((tRNA_i^{fMet})\) becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development.
Author Summary
Prokaryote metabolism is key for our understanding of bacterial virulence and pathogenesis and it is also an area with huge opportunity to identify novel targets for antibiotic drugs. Here, we have addressed the so far poorly characterized regulation of methionine biosynthesis in S. aureus. We demonstrate that methionine biosynthesis control in staphylococci significantly differs from that predicted for other Bacillales. Notably, involvement of a T-box instead of an S-box riboswitch separates staphylococci from other bacteria in the order. We provide, for the first time, direct experimental proof for an interaction of a methionyl-tRNA-specific T-box with its cognate tRNA, and the identification of initiator \((tRNA_i^{fMet})\) as the specific binding partner is an unexpected finding whose exact function in Staphylococcus metabolism remains to be established. The data further suggest that in staphylococci a range of regulatory elements are integrated to form a hierarchical network that elegantly limits costly (excess) methionine biosynthesis and, at the same time, reliably ensures production of the amino acid in a highly selective manner. Our findings open a perspective to exploit methionine biosynthesis and especially its T-box-mediated control as putative target(s) for the development of future anti-staphylococcal therapeutics.
Altering gene expression by aminocoumarins: the role of DNA supercoiling in Staphylococcus aureus
(2014)
BACKGROUND:
It has been shown previously that aminocoumarin antibiotics such as novobiocin lead to immediate downregulation of recA expression and thereby inhibit the SOS response, mutation frequency and recombination capacity in Staphylococcus aureus. Aminocoumarins function by inhibiting the ATPase activity of DNA gyrase subunit B with a severe impact on DNA supercoiling.
RESULTS:
Here, we have analysed the global impact of the DNA relaxing agent novobiocin on gene expression in S. aureus. Using a novobiocin-resistant mutant, it became evident that the change in recA expression is due to gyrase inhibition. Microarray analysis and northern blot hybridisation revealed that the expression levels of a distinct set of genes were increased (e.g., recF-gyrB-gyrA, the rib operon and the ure operon) or decreased (e.g., arlRS, recA, lukA, hlgC and fnbA) by novobiocin. The two-component ArlRS system was previously found to decrease the level of supercoiling in S. aureus. Thus, downregulation of arlRS might partially compensate for the relaxing effect of novobiocin. Global analysis and gene mapping of supercoiling-sensitive genes did not provide any indication that they are clustered in the genome. Promoter fusion assays confirmed that the responsiveness of a given gene is intrinsic to the promoter region but independent of the chromosomal location.
CONCLUSIONS:
The results indicate that the molecular properties of a given promoter, rather than the chromosomal topology, dictate the responsiveness to changes in supercoiling in the pathogen Staphylococcus aureus.
Staphylococcus aureus asymptomatically colonizes the nasal cavity of mammals, but it is also a leading cause of life-threatening infections. Most human nasal isolates carry Sa3 phages, which integrate into the bacterial hlb gene encoding a sphingomyelinase. The virulence factor-encoding genes carried by the Sa3-phages are highly human-specific, and most animal strains are Sa3 negative. Thus, both insertion and excision of the prophage could potentially confer a fitness advantage to S. aureus. Here, we analyzed the phage life cycle of two Sa3 phages, Φ13 and ΦN315, in different phage-cured S. aureus strains. Based on phage transfer experiments, strains could be classified into low (8325-4, SH1000, and USA300c) and high (MW2c and Newman-c) transfer strains. High-transfer strains promoted the replication of phages, whereas phage adsorption, integration, excision, or recA transcription was not significantly different between strains. RNASeq analyses of replication-deficient lysogens revealed no strain-specific differences in the CI/Mor regulatory switch. However, lytic genes were significantly upregulated in the high transfer strain MW2c Φ13 compared to strain 8325-4 Φ13. By transcriptional start site prediction, new promoter regions within the lytic modules were identified, which are likely targeted by specific host factors. Such host-phage interaction probably accounts for the strain-specific differences in phage replication and transfer frequency. Thus, the genetic makeup of the host strains may determine the rate of phage mobilization, a feature that might impact the speed at which certain strains can achieve host adaptation.
Stapylococcus aureus colonises the nose of healthy individuals but can also cause a wide range of infections. Amino acid (AA) synthesis and their availability is crucial to adapt to conditions encountered in vivo. Most S. aureus genomes comprise all genes required for AA biosynthesis. Nevertheless, different strains require specific sets of AAs for growth. In this study we show that regulation inactivates pathways under certain conditions which result in these observed auxotrophies. We analyzed in vitro and modeled in silico in a Boolean semiquantitative model (195 nodes, 320 edges) the regulatory impact of stringent response (SR) on AA requirement in S. aureus HG001 (wild-type) and in mutant strains lacking the metabolic regulators RSH, CodY and CcpA, respectively. Growth in medium lacking single AAs was analyzed. Results correlated qualitatively to the in silico predictions of the final model in 92% and quantitatively in 81%. Remaining gaps in our knowledge are evaluated and discussed. This in silico model is made fully available and explains how integration of different inputs is achieved in SR and AA metabolism of S. aureus. The in vitro data and in silico modeling stress the role of SR and central regulators such as CodY for AA metabolisms in S. aureus.
Sepsis caused by Gram-positive bacterial pathogens is a major fatal disease but its molecular basis remains elusive. Toll-like receptor 2 (TLR2) has been implicated in the orchestration of inflammation and sepsis but its role appears to vary for different pathogen species and clones. Accordingly, Staphylococcus aureus clinical isolates differ substantially in their capacity to activate TLR2. Here we show that strong TLR2 stimulation depends on high-level production of phenol-soluble modulin (PSM) peptides in response to the global virulence activator Agr. PSMs are required for mobilizing lipoproteins, the TLR2 agonists, from the staphylococcal cytoplasmic membrane. Notably, the course of sepsis caused by PSM-deficient S. aureus is similar in wild-type and TLR2-deficient mice, but TLR2 is required for protection of mice against PSM-producing S. aureus. Thus, a crucial role of TLR2 depends on agonist release by bacterial surfactants. Modulation of this process may lead to new therapeutic strategies against Gram-positive infections.