The stringent response is initiated by rapid (p)ppGpp synthesis, which leads to a profound reprogramming of gene expression in most bacteria. The stringent phenotype seems to be species specific and may be mediated by fundamentally different molecular mechanisms. In Staphylococcus aureus, (p)ppGpp synthesis upon amino acid deprivation is achieved through the synthase domain of the bifunctional enzyme RSH (RelA/SpoT homolog). In several firmicutes, a direct link between stringent response and the CodY regulon was proposed. Wild-type strain HG001, rsh_Syn, codY and rsh_Syn, codY double mutants were analyzed by transcriptome analysis to delineate different consequences of RSH-dependent (p)ppGpp synthesis after induction of the stringent response by amino-acid deprivation. Under these conditions genes coding for major components of the protein synthesis machinery and nucleotide metabolism were down-regulated only in rsh positive strains. Genes which became activated upon (p)ppGpp induction are mostly regulated indirectly via de-repression of the GTP-responsive repressor CodY. Only seven genes, including those coding for the cytotoxic phenol-soluble modulins (PSMs), were found to be up-regulated via RSH independently of CodY. qtRT-PCR analyses of hallmark genes of the stringent response indicate that an RSH activating stringent condition is induced after uptake of S. aureus in human polymorphonuclear neutrophils (PMNs). The RSH activity in turn is crucial for intracellular expression of psms. Accordingly, rsh_Syn and rsh_Syn, codY mutants were less able to survive after phagocytosis similar to psm mutants. Intraphagosomal induction of psmα1-4 and/or psmβ1,2 could complement the survival of the rsh_Syn mutant. Thus, an active RSH synthase is required for intracellular psm expression which contributes to survival after phagocytosis. The stringent response is a bacterial response to a multitude of different environmental stress conditions which is characterized by the synthesis of the messenger molecules (p)ppGpp. There is now growing evidence that these molecules also play a key role for pathogens to switch between specific phenotypic states within the host. This seems crucial for the adaptation to different microenvironments encountered during infection for instance after uptake by phagocytes. Killing of phagocytes as well as survival within these cells was proposed as major mechanisms for the success of the human pathogen Staphylococcus aureus to spread within the body. In the current study we demonstrate the effect of the stringent response on global gene expression in S. aureus and its impact on intracellular survival in human neutrophils. We reveal that a stringent response is induced after uptake of S. aureus in neutrophils and RSH activity is crucial for intracellular induction of psm expression, coding for cytotoxic phenol-soluble modulins (PSMs). Finally we show that this in turn mediates bacterial survival and escape after phagocytosis. These findings contribute to the understanding of how and where PSMs can act as potent cytolytic molecules and emphasise the importance of (p)ppGpp as an intracellular signalling molecule.

Low nutrient and energy availability has led to the evolution of numerous strategies for overcoming these limitations, of which symbiotic associations represent a key mechanism. Particularly striking are the associations between chemosynthetic bacteria and marine animals that thrive in nutrient-poor environments such as the deep sea because the symbionts allow their hosts to grow on inorganic energy and carbon sources such as sulfide and CO ₂ . Remarkably little is known about the physiological strategies that enable chemosynthetic symbioses to colonize oligotrophic environments. In this study, we used metaproteomics and metabolomics to investigate the intricate network of metabolic interactions in the chemosynthetic association between Olavius algarvensis , a gutless marine worm, and its bacterial symbionts. We propose previously undescribed pathways for coping with energy and nutrient limitation, some of which may be widespread in both free-living and symbiotic bacteria. These pathways include ( i ) a pathway for symbiont assimilation of the host waste products acetate, propionate, succinate and malate; ( ii ) the potential use of carbon monoxide as an energy source, a substrate previously not known to play a role in marine invertebrate symbioses; ( iii ) the potential use of hydrogen as an energy source; ( iv ) the strong expression of high-affinity uptake transporters; and ( v ) as yet undescribed energy-efficient steps in CO ₂ fixation and sulfate reduction. The high expression of proteins involved in pathways for energy and carbon uptake and conservation in the O . algarvensis symbiosis indicates that the oligotrophic nature of its environment exerted a strong selective pressure in shaping these associations.

Colonization of the human nose by Staphylococcus aureus in one-third of the population represents a major risk factor for invasive infections. The basis for adaptation of S. aureus to this specific habitat and reasons for the human predisposition to become colonized have remained largely unknown. Human nasal secretions were analyzed by metabolomics and found to contain potential nutrients in rather low amounts. No significant differences were found between S. aureus carriers and non-carriers, indicating that carriage is not associated with individual differences in nutrient supply. A synthetic nasal medium (SNM3) was composed based on the metabolomics data that permits consistent growth of S. aureus isolates. Key genes were expressed in SNM3 in a similar way as in the human nose, indicating that SNM3 represents a suitable surrogate environment for in vitro simulation studies. While the majority of S. aureus strains grew well in SNM3, most of the tested coagulase-negative staphylococci (CoNS) had major problems to multiply in SNM3 supporting the notion that CoNS are less well adapted to the nose and colonize preferentially the human skin. Global gene expression analysis revealed that, during growth in SNM3, S. aureus depends heavily on de novo synthesis of methionine. Accordingly, the methionine-biosynthesis enzyme cysteine-γ-synthase (MetI) was indispensable for growth in SNM3, and the MetI inhibitor DL-propargylglycine inhibited S. aureus growth in SNM3 but not in the presence of methionine. Of note, metI was strongly up-regulated by S. aureus in human noses, and metI mutants were strongly abrogated in their capacity to colonize the noses of cotton rats. These findings indicate that the methionine biosynthetic pathway may include promising antimicrobial targets that have previously remained unrecognized. Hence, exploring the environmental conditions facultative pathogens are exposed to during colonization can be useful for understanding niche adaptation and identifying targets for new antimicrobial strategies. Many severe bacterial infections are caused by endogenous pathogens colonizing human body surfaces. Eradication of notorious pathogens such as Staphylococcus aureus from risk patients has become an important preventive strategy. However, efficient decolonization agents are rare, and the living conditions of colonizing pathogens have hardly been studied. Using a combined metabolomics and transcriptomics approach, we explored the metabolism of S. aureus during colonization of its preferred niche, the human nose. Based on nasal metabolite profiles, a synthetic nasal medium (SNM3) was composed, enabling steady growth of S. aureus but not of staphylococcal species preferentially colonizing the human skin. Marker gene expression was similar in SNM3 and the human nose, and genome-wide expression analysis revealed that amino acid biosynthesis, in particular that of methionine, is critical for S. aureus during colonization. An inhibitor of methionine biosynthesis had anti-staphylococcal activity in SNM3 but not in complex media, and transcription of the S. aureus target enzyme was strongly up-regulated in human noses. Furthermore, mutants defective in methionine biosynthesis exhibited strongly compromised nasal colonisation capacities in a cotton rat model. Altogether, our results indicate that the elucidation of in vivo metabolism of pathogens may lead to the identification of new antimicrobial targets and compounds.

Summary: An alignment of upstream regions of anaerobically induced genes in Staphylococcus aureus revealed the presence of an inverted repeat, corresponding to Rex binding sites in Streptomyces coelicolor. Gel shift experiments of selected upstream regions demonstrated that the redox‐sensing regulator Rex of S. aureus binds to this inverted repeat. The binding sequence – TTGTGAAW₄TTCACAA – is highly conserved in S. aureus. Rex binding to this sequence leads to the repression of genes located downstream. The binding activity of Rex is enhanced by NAD⁺ while NADH, which competes with NAD⁺ for Rex binding, decreases the activity of Rex. The impact of Rex on global protein synthesis and on the activity of fermentation pathways under aerobic and anaerobic conditions was analysed by using a rex‐deficient strain. A direct regulatory effect of Rex on the expression of pathways that lead to anaerobic NAD⁺ regeneration, such as lactate, formate and ethanol formation, nitrate respiration, and ATP synthesis, is verified. Rex can be considered a central regulator of anaerobic metabolism in S. aureus. Since the activity of lactate dehydrogenase enables S. aureus to resist NO stress and thus the innate immune response, our data suggest that deactivation of Rex is a prerequisite for this phenomenon.