Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. Its ability to multiply and survive in macrophages is critical for its virulence. By screening a bank of HimarFT transposon mutants of the F. tularensis live vaccine strain (LVS) to isolate intracellular growth-deficient mutants, we selected one mutant in a gene encoding a putative γ-glutamyl transpeptidase (GGT). This gene (FTL_0766) was hence designated ggt. The mutant strain showed impaired intracellular multiplication and was strongly attenuated for virulence in mice. Here we present evidence that the GGT activity of F. tularensis allows utilization of glutathione (GSH, γ-glutamyl-cysteinyl-glycine) and γ-glutamyl-cysteine dipeptide as cysteine sources to ensure intracellular growth. This is the first demonstration of the essential role of a nutrient acquisition system in the intracellular multiplication of F. tularensis. GSH is the most abundant source of cysteine in the host cytosol. Thus, the capacity this intracellular bacterial pathogen has evolved to utilize the available GSH, as a source of cysteine in the host cytosol, constitutes a paradigm of bacteria–host adaptation.

Francisella tularensis is a highly infectious pathogen that infects animals and humans, causing tularemia. The ability to replicate within macrophages is central for virulence and relies on expression of genes located in the Francisella pathogenicity island (FPI), as well as expression of other genes. Regulation of FPI-encoded virulence gene expression in F. tularensis involves at least four regulatory proteins and is not fully understood. Here we studied the RNA-binding protein Hfq in F. tularensis and particularly the role that it plays as a global regulator of gene expression in stress tolerance and pathogenesis. We demonstrate that Hfq promotes resistance to several cellular stresses (including osmotic and membrane stresses). Furthermore, we show that Hfq is important for the ability of the F. tularensis vaccine strain LVS to induce disease and persist in organs of infected mice. We also demonstrate that Hfq is important for stress tolerance and full virulence in a virulent clinical isolate of F. tularensis, FSC200. Finally, microarray analyses revealed that Hfq regulates expression of numerous genes, including genes located in the FPI. Strikingly, Hfq negatively regulates only one of two divergently expressed putative operons in the FPI, in contrast to the other known regulators, which regulate the entire FPI. Hfq thus appears to be a new pleiotropic regulator of virulence in F. tularensis, acting mostly as a repressor, in contrast to the other regulators identified so far. Moreover, the results obtained suggest a novel regulatory mechanism for a subset of FPI genes.Regulation of gene expression by noncoding or small RNAs (sRNAs) is prevalent in both prokaryotes and eukaryotes, and recent studies have identified numerous sRNAs in different organisms. Most sRNAs encoded by bacterial chromosomes act by base pairing with mRNA targets that are encoded in trans, and these sRNAs commonly require Hfq in order to function (47). Hfq is a bacterial RNA-binding protein that was initially recognized as a host factor for replication of the Q␤ RNA phage in Escherichia coli (17). The Hfq protein is very abundant, and it belongs to the eukaryotic and archaeal families of Sm and Sm-like proteins, respectively, that form homohexameric structures (for reviews, see references 5 and 53). The importance of Hfq became clear when an E. coli hfq null mutant was created. This mutant had pleiotropic phenotypes, such as a decreased growth rate, increased sensitivity to cellular stresses, and increased cell length (51). Hfq is a posttranscriptional regulator that binds sRNAs and mRNA and facilitates RNA-RNA interaction (1,18,23,32,35,55). For the most part, sRNA-mRNA interactions result in mRNA degradation and/or inhibition of translation, but they can also increase translation (for reviews, see references 1, 20, and 48). As might be expected from the pleiotropic phenotypes of an hfq mutant, deletion of hfq has been correlated with consider-

Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. During its infectious cycle, F. tularensis is not only exposed to the intracellular environment of macrophages but also resides transiently in extracellular compartments, in particular during its systemic dissemination. The screening of a bank of F. tularensis LVS transposon insertion mutants on chemically defined medium (CDM) led us to identify a gene, designated trkH, encoding a homolog of the potassium uptake permease TrkH. Inactivation of trkH impaired bacterial growth in CDM. Normal growth of the mutant was only restored when CDM was supplemented with potassium at high concentration. Strikingly, although not required for intracellular survival in cell culture models, TrkH appeared to be essential for bacterial virulence in the mouse. In vivo kinetics of bacterial dissemination revealed a severe defect of multiplication of the trkH mutant in the blood of infected animals. The trkH mutant also showed impaired growth in blood ex vivo. Genome sequence analyses suggest that the Trk system constitutes the unique functional active potassium transporter in both tularensis and holarctica subspecies. Hence, the impaired survival of the trkH mutant in vivo is likely to be due to its inability to survive in the low potassium environment (1–5 mM range) of the blood. This work unravels thus the importance of potassium acquisition in the extracellular phase of the F. tularensis infectious cycle. More generally, potassium could constitute an important mineral nutrient involved in other diseases linked to systemic dissemination of bacterial pathogens.