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Virulence factors generally enhance a pathogen's fitness and thereby foster transmission. However, most studies of pathogen fitness have been performed by averaging the phenotypes over large populations. Here, we have analyzed the fitness costs of virulence factor expression by Salmonella enterica subspecies I serovar Typhimurium in simple culture experiments. The type III secretion system ttss-1, a cardinal virulence factor for eliciting Salmonella diarrhea, is expressed by just a fraction of the S. Typhimurium population, yielding a mixture of cells that either express ttss-1 (TTSS-1+ phenotype) or not (TTSS-1− phenotype). Here, we studied in vitro the TTSS-1+ phenotype at the single cell level using fluorescent protein reporters. The regulator hilA controlled the fraction of TTSS-1+ individuals and their ttss-1 expression level. Strikingly, cells of the TTSS-1+ phenotype grew slower than cells of the TTSS-1− phenotype. The growth retardation was at least partially attributable to the expression of TTSS-1 effector and/or translocon proteins. In spite of this growth penalty, the TTSS-1+ subpopulation increased from <10% to approx. 60% during the late logarithmic growth phase of an LB batch culture. This was attributable to an increasing initiation rate of ttss-1 expression, in response to environmental cues accumulating during this growth phase, as shown by experimental data and mathematical modeling. Finally, hilA and hilD mutants, which form only fast-growing TTSS-1− cells, outcompeted wild type S. Typhimurium in mixed cultures. Our data demonstrated that virulence factor expression imposes a growth penalty in a non-host environment. This raises important questions about compensating mechanisms during host infection which ensure successful propagation of the genotype.

The intestinal microbiota features intricate metabolic interactions involving the breakdown and reuse of host- and diet-derived nutrients. The competition for these resources can limit pathogen growth. Nevertheless, some enteropathogenic bacteria can invade this niche through mechanisms that remain largely unclear. Using a mouse model for Salmonella diarrhea and a transposon mutant screen, we discovered that initial growth of Salmonella Typhimurium (S. Tm) in the unperturbed gut is powered by S. Tm hyb hydrogenase, which facilitates consumption of hydrogen (H2), a central intermediate of microbiota metabolism. In competitive infection experiments, a hyb mutant exhibited reduced growth early in infection compared to wild-type S. Tm, but these differences were lost upon antibiotic-mediated disruption of the host microbiota. Additionally, introducing H2-consuming bacteria into the microbiota interfered with hyb-dependent S. Tm growth. Thus, H2 is an Achilles' heel of microbiota metabolism that can be subverted by pathogens and might offer opportunities to prevent infection.

The Tat (twin-arginine translocation) system of Escherichia coli serves to translocate folded proteins across the cytoplasmic membrane. The reasons established so far for the Tat dependence are cytoplasmic cofactor assembly and/or heterodimerization of the respective proteins. We were interested in the reasons for the Tat dependence of novel Tat substrates and focused on two uncharacterized proteins, YcdO and YcdB. Both proteins contain predicted Tat signal sequences. However, we found that only YcdB was indeed Tat-dependently translocated, whereas YcdO was equally well translocated in a Tat-deficient strain. YcdB is a dimeric protein and contains a heme cofactor that was identified to be a high-spin Fe IIIprotoporphyrin IX complex. In contrast to all other periplasmic hemoproteins analyzed so far, heme was assembled into YcdB in the cytoplasm, suggesting that heme assembly could take place prior to translocation. The function of YcdB in the periplasm may be related to a detoxification reaction under specific conditions because YcdB had peroxidase activity at acidic pH, which coincides well with the known acid-induced expression of the gene. The data demonstrate the existence of a class of heme-containing Tat substrates, the first member of which is YcdB.

SUMMARY Microorganisms can facilitate their survival in stressful environments by entering a state of metabolic inactivity or dormancy [1]. However, this state impairs the function of the very sensory systems necessary to detect favorable growth conditions. Thus, how can a metabolically quiescent cell accurately monitor environmental conditions in order to best decide when to exit dormancy? One strategy employed by microbes to deal with changing environments is the generation of phenotypes that may be less well adapted to a current condition but might confer an advantage in the future [2, 3]. This bet-hedging depends on phenotypic diversity in the population [4], which itself can derive from naturally occurring stochastic differences in gene expression [5, 6]. In the case of metabolic dormancy, a bet-hedging strategy that has been proposed is the “scout model” where cells comprising a fraction of the dormant population reinitiate growth stochastically, independent of environmental cues [7, 8]. Here, we provide experimental evidence that such a mechanism exists in dormant spores produced by the ubiquitous soil bacterium Bacillus subtilis. We observe that these spores reinitiate growth at a low but measureable frequency even in the absence of an inducing signal. This phenomenon is the result of phenotypic variation in the propensity of individual spores to reinitiate growth spontaneously. Since this bet-hedging mechanism produces individuals that will either grow under favorable conditions or die under unfavorable conditions, a population can properly respond to environmental changes despite the impaired sensory ability of individual cells.