Copper homeostasis in Salmonella is atypical and copper-CueP is a major periplasmic metal complex.

Salmonella enterica sv. typhimurium (S. enterica sv. Typhimurium) has two metal-transporting P(1)-type ATPases whose actions largely overlap with respect to growth in elevated copper. Mutants lacking both ATPases over-accumulate copper relative to wild-type or either single mutant. Such duplication of ATPases is unusual in bacterial copper tolerance. Both ATPases are under the control of MerR family metal-responsive transcriptional activators. Analyses of periplasmic copper complexes identified copper-CueP as one of the predominant metal pools. Expression of cueP was recently shown to be controlled by the same metal-responsive activator as one of the P(1)-type ATPase genes (copA), and copper-CueP is a further atypical feature of copper homeostasis in S. enterica sv. Typhimurium. Elevated copper is detected by a reporter construct driven by the promoter of copA in wild-type S. enterica sv. Typhimurium during infection of macrophages. Double mutants missing both ATPases also show reduced survival inside cultured macrophages. It is hypothesized that elevated copper within macrophages may have selected for specialized copper-resistance systems in pathogenic microorganism such as S. enterica sv. Typhimurium.

Differential expression from two iron-responsive promoters in Salmonella enterica serovar Typhimurium reveals the presence of iron in macrophage-phagosomes.

The metal status of macrophage-phagosomes during Salmonella infection is largely unknown. In this study, we have precisely calibrated the metal-specificities of two metal-responsive promoters, P(iroBCDE) and P(sodB), from Salmonella enterica serovar Typhimurium and used these to directly monitor iron-levels in Salmonella-containing macrophage-phagosomes. Expression from the P(iroBCDE) promoter is highly elevated in metal-depleted media but low in media supplemented with iron or cobalt, and to a lesser extent manganese. In contrast, P(sodB) shows low levels of expression in metal-depleted media but is induced in media supplemented with iron but no other metals at maximum permissive concentrations. In both cases, iron-responsive expression corresponds to changes in the number of iron atoms per bacterial cell and is unaffected by pH or the presence of reactive oxygen species (hydrogen peroxide and superoxide). Importantly, expression from P(iroBCDE) remained low while expression from P(sodB) was elevated during infection of both Nramp1(+/+) and Nramp1(-/-) macrophages. Expression from a control promoter, P(polA), unaffected by metal ions, remained unchanged. These findings are therefore consistent with the presence of iron within Salmonella-containing macrophage-phagosomes and support a model in which the toxic potential of iron may be exploited as a component of the respiratory burst killing mechanism.

Characterization of relA and codY mutants of Listeria monocytogenes: identification of the CodY regulon and its role in virulence.

Listeria monocytogenes is a Gram-positive intracellular parasite and the causative organism of human listeriosis. In this article we demonstrate that L. monocytogenes encodes a functional member of the CodY family of global regulatory proteins that is responsive to both GTP and branched chain amino acids. By transcript analyses we identified the CodY regulon in L. monocytogenes and demonstrated that it comprises genes involved in amino acid metabolism, nitrogen assimilation as well as genes involved in sugar uptake and incorporation, indicating a role for CodY in L. monocytogenes in both carbon and nitrogen assimilation. A DeltarelA mutation reduced expression of the CodY regulon in early stationary phase and introduction of a DeltacodY mutation into a DeltarelA strain restored virulence. These data indicate that the avirulence of the DeltarelA mutant can in part be explained by the continued repression of the CodY regulon. The phenotypes of DeltarelA and DeltacodY mutants were studied in J774.A1 and Caco-2 cells and the DeltarelA mutation shown to effect intracellular growth. These results provide the first direct evidence that the activity of a CodY-type protein influences pathogenesis and provides new information on the physiological adaptation of L. monocytogenes to post-exponential phase growth and virulence.

Mutations in the waaR gene of Escherichia coli which disrupt lipopolysaccharide outer core biosynthesis affect cell surface retention of group 2 capsular polysaccharides.

On the basis of increased resistance to K5 capsule-specific bacteriophage, a waaR transposon mutant defective in the biosynthesis of lipopolysaccharide outer core was isolated. In a K1-expressing strain the mutation equally affected sensitivity to K1 capsule-specific bacteriophage, indicating a general effect on group 2 capsules. The waaR mutation affected retention on the cell surface of the K5 polysaccharide, with increased polysaccharide accumulating in the culture supernatant. This indicates that interactions between the outer core of lipopolysaccharide and group 2 capsular polysaccharides are important for the stabilization of group 2 capsular polysaccharides on the cell surface.

Listeria monocytogenes relA and hpt mutants are impaired in surface-attached growth and virulence.

We describe here the identification and characterization of two Listeria monocytogenes (Tn917-LTV3) relA and hpt transposon insertion mutants that were impaired in growth after attachment to a model surface. Both mutants were unable to accumulate (p)ppGpp in response to amino acid starvation, whereas the wild-type strain accumulated (p)ppGpp within 30 min of stress induction. The induction of transcription of the relA gene after adhesion was demonstrated, suggesting that the ability to mount a stringent response and undergo physiological adaptation to nutrient deprivation is essential for the subsequent growth of the adhered bacteria. The absence of (p)ppGpp in the hpt mutant, which is blocked in the purine salvage pathway, is curious and suggests that a functional purine salvage pathway is required for the biosynthesis of (p)ppGpp. Both mutants were avirulent in a murine model of listeriosis, indicating an essential role for the stringent response in the survival and growth of L. monocytogenes in the host. Taken as a whole, this study provides new information on the role of the stringent response and the physiological adaptation of L. monocytogenes for biofilm growth and pathogenesis.