New antibiotics are urgently needed to control infectious diseases. Metabolic enzymes could represent attractive targets for such antibiotics, but in vivo target validation is largely lacking. Here we have obtained in vivo information about over 700 Salmonella enterica enzymes from network analysis of mutant phenotypes, genome comparisons and Salmonella proteomes from infected mice. Over 400 of these enzymes are non-essential for Salmonella virulence, reflecting extensive metabolic redundancies and access to surprisingly diverse host nutrients. The essential enzymes identified were almost exclusively associated with a small subgroup of pathways, enabling us to perform a nearly exhaustive screen. Sixty-four enzymes identified as essential in Salmonella are conserved in other important human pathogens, but almost all belong to metabolic pathways that are inhibited by current antibiotics or that have previously been considered for antimicrobial development. Our comprehensive in vivo analysis thus suggests a shortage of new metabolic targets for broad-spectrum antibiotics, and draws attention to some previously known but unexploited targets.

Vaccines effective against intracellular pathogens could save the lives of millions of people every year, but vaccine development has been hampered by the slow largely empirical search for protective antigens. In vivo highly expressed antigens might represent a small attractive antigen subset that could be rapidly evaluated, but experimental evidence supporting this rationale, as well as practical strategies for its application, is largely lacking because of technical difficulties. Here, we used Salmonella strains expressing differential amounts of a fluorescent model antigen during infection to show that, in a mouse typhoid fever model, CD4 T cells preferentially recognize abundant Salmonella antigens. To identify a large number of natural Salmonella antigens with high expression levels during infection, we used a quantitative in vivo screening strategy. Immunization studies with five particularly attractive candidates revealed two highly protective antigens that might permit the development of an improved typhoid fever vaccine. In conclusion, we have established a rationale and an experimental strategy that will substantially facilitate vaccine development for Salmonella and possibly other intracellular pathogens.

During in vitro broth culture, bacterial gene expression is typically dominated by highly expressed factors involved in protein biosynthesis, maturation, and folding, but it is unclear if this also applies to conditions in natural environments. Here, we used a promoter trap strategy with an unstable green fluorescent protein reporter that can be detected in infected mouse tissues to identify 21 Salmonella enterica promoters with high levels of activity in a mouse enteritis model. We then measured the activities of these and 31 previously identified Salmonella promoters in both the enteritis and a murine typhoid fever model. Surprisingly, the data reveal that instead of protein biosynthesis genes, disease-specific genes such as Salmonella pathogenicity island 1 (SPI-1)-associated genes and genes involved in anaerobic respiration (enteritis) or SPI-2-associated genes and genes of the PhoP regulon (typhoid fever), respectively, dominate Salmonella in vivo gene expression. The overall functional profile of highly expressed genes suggests a marked shift in major transcriptional activities to nutrient utilization during enteritis or to fighting against the host during typhoid fever. The large proportion of known and novel essential virulence factors among the identified genes suggests that high expression levels during infection may correlate with functional relevance.

In Bacillus subtilis, the alternative sigma factor B is activated in response to environmental stress or energy depletion. Bacillus subtilis has 17 different factors, which are synthesized and activated at various times during development or after changes in environmental conditions. The active factors bind to core RNA polymerase (E) to recognize specific promoter sequences and thus to catalyze gene expression that is appropriate to the conditions. If several factors are active at the same time, what mechanisms determine which of them binds to the core RNA polymerase? In particular, do they compete with one another for binding, or is core RNA polymerase present in excess, with the result that they can all be accommodated? By investigating the composition of the holoenzyme during sporulation in Bacillus subtilis, Fujita concluded that core RNA polymerase is indeed in excess in the cell, so that successive factors do not need to displace each other from the holoenzyme (14). However, this conclusion, which was based on the finding that there is twofold more E than A in the cell, is open to question, as twothirds of the molecules of E are known to be involved in transcription elongation (9) and are therefore not in a state in which they can bind any factor. Furthermore, other measurements of the intracellular concentration of E and A have suggested that the two proteins are present at approximately the same molar concentration in sporulating cells (26,36). In addition, expression studies have suggested that A and H in B. subtilis compete for binding to the core RNA polymerase, as do 70 and S in Escherichia coli, since in both systems overexpression of one factor leads to a decrease in the gene expression that is dependent on the other factor (13, 18).In this study, we wanted to understand how the general stress factor of B. subtilis, B , replaces A in the holoenzyme. E B transcribes genes whose products provide the cell with nonspecific, general, and multiple stress resistance. It is known to be activated after energy depletion or after a variety of environmental stresses such as heat, ethanol, acid, and osmotic and oxidative stress through cascades of PP2C phosphatases.B is held inactive by its anti-factor RsbW as long as the anti-anti-factor RsbV is phosphorylated. After environmental stress or energy depletion, RsbV is dephosphorylated by the PP2C phosphatases RsbU and RsbP, and the resulting RsbV binds to RsbW, which thereupon liberates B (for recent reviews, see references 16 and 33).Although this mechanism of activating B is relatively well understood, what is not known is how the activated B competes successfully with A for binding to E. In the present study, we examined whether the genetic loss of B affects the expression of A -dependent general stress genes under conditions that would normally induce B , determined the relative affinities of the two factors for E, and measured the intracellular concentrations of E and of the two factors before and during a period of ethanol stress. MATERIALS AND METHODSBac...