SummaryThe Hms + phenotype of Yersinia pestis promotes the binding of haemin or Congo red (CR) to the cell surface at temperatures below 34 ∞ ∞ ∞ ∞ C. We previously demonstrated that temperature regulation of the Hms + phenotype is not controlled at the level of transcription. Instead, HmsH, HmsR and HmsT are degraded upon a temperature shift from 26 ∞ ∞ ∞ ∞ C to 37 ∞ ∞ ∞ ∞ C. We used random transposon mutagenesis to identify new genes involved in the temperature-regulated expression of the Hms phenotype. One of these genes, which we designated hmsP , encodes a putative phosphodiesterase with a conserved EAL motif. Mutations in hmsP caused formation of red colonies on CR plates at 26 ∞ ∞ ∞ ∞ C and 37 ∞ ∞ ∞ ∞ C. Strains complemented with hmsP + on a plasmid form white colonies at both temperatures. We used a crystal violet assay and confocal laser scanning microscopy to demonstrate Hmsdependent biofilm formation by Y. pestis cells. Y. pestis Hms + strains grown at 26 ∞ ∞ ∞ ∞ C but not at 37 ∞ ∞ ∞ ∞ C form a biofilm on borosilicate glass surfaces. Strains that either overexpress HmsT (a GGDEF domain protein) or have a mutation in hmsP produced an extremely thick biofilm. Alanine substitutions for each of the GGEE residues (amino acids 296-299) of HmsT as well as the E506 and L508 residues of HmsP caused a loss of function. We propose that HmsT and HmsP together control the amount of biofilm produced in Y. pestis . Degradation of HmsT at 37 ∞ ∞ ∞ ∞ C may be a critical factor in controlling the temperature-dependent expression of the Hms biofilm.

a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis . Mol Microbiol 54: 74-87.

Little is known about Zn homeostasis inZinc (Zn) is an essential trace metal required to preserve the biological function and/or structural integrity of numerous enzymes and proteins in all eukaryotic and prokaryotic cells (3,49,99). Procuring sufficient Zn to sustain growth during mammalian infection is a considerable challenge for bacterial pathogens (56). Serum levels of Zn are in the micromolar range, and the metal's bioavailability is restricted further because it is tightly bound to proteins and not freely exchangeable (35,85,88,103). In addition, as with iron (Fe), mammals sequester Zn systemically and locally in an attempt to deprive invading pathogens of this critical micronutrient (35,85,88,103). Bacteria, therefore, must depend upon the expression of high-affinity Zn uptake systems to compete successfully with the mammalian host for this metal. Although Zn is essential, high concentrations are toxic because of its proclivity to occupy ligand sites intended for other transition metals, such as Mn and Fe (39); consequently, bacteria must strictly control intracellular Zn levels to avoid disruption of physiological processes (49). Two major mechanisms by which Zn homeostasis is achieved are metal effluxers and regulation of Zn uptake systems.The discovery of the cluster 9 (C9) family of transition metal ATP-binding cassette (ABC) transporters significantly advanced our understanding of bacterial Zn metabolism (19). The function of the C9 family was revealed primarily through genetic studies in which the growth defects of mutants under metal-limiting conditions were reversed by supplementation with Zn (27,77) or Mn (8,27,59). Bioinformatic analyses of the C9 solute-binding protein (SBP) components, which capture metals within the periplasmic space and ferry them to the cytoplasmic membrane-bound permease complex, revealed a bimodal clustering pattern appearing to correlate with experimentally proven metal specificities (19). One subcluster con-* Corresponding author. Mailing address:

In Yersinia pestis, the Congo red (and hemin) binding that is characteristic of the Hms؉ phenotype occurs at temperatures up to 34°C but not at higher temperatures. Manifestation of the Hms ؉ phenotype requires at least five proteins (HmsH, -F, -R, -S, and -T) that are organized into two separate operons: hmsHFRS and hmsT. HmsH and HmsF are outer membrane proteins, while HmsR, HmsS, and HmsT are predicted to be inner membrane proteins. We have used transcriptional reporter constructs, RNA dot blots, and Western blots to examine the expression of hms operons and proteins. Our studies indicate that transcription from the hmsHFRS and hmsT promoters is not regulated by the iron status of the cells, growth temperature, or any of the Hms proteins. In addition, the level of mRNA for both operons is not significantly affected by growth temperature. However, protein levels of HmsH, HmsR, and HmsT in cells grown at 37°C are very low compared to those in cells grown at 26°C, while the amounts of HmsF and HmsS show only a moderate reduction at the higher growth temperature. Neither the Pla protease nor a putative endopeptidase (Y2360) encoded upstream of hmsH is essential for temperature regulation of the Hms ؉ phenotype. However, HmsT at 37°C is sensitive to degradation by Lon and/or ClpPX. Thus, the stability of HmsH, HmsR, and HmsT proteins likely plays a role in temperature regulation of the Hms ؉ phenotype of Y. pestis.

Yersinia pestis has a flea-mammal-flea transmission cycle, and is a zoonotic pathogen that causes the systemic diseases bubonic and septicaemic plague in rodents and humans, as well as pneumonic plague in humans and non-human primates. Bubonic and pneumonic plague are quite different diseases that result from different routes of infection. Manganese (Mn) acquisition is critical for the growth and pathogenesis of a number of bacteria. The Yfe/Sit and/or MntH systems are the two prominent Mn transporters in Gram-negative bacteria. Previously we showed that the Y. pestis Yfe system transports Fe and Mn. Here we demonstrate that a mutation in yfe or mntH did not significantly affect in vitro aerobic growth under Mn-deficient conditions. A yfe mntH double mutant did exhibit a moderate growth defect which was alleviated by supplementation with Mn. No short-term energy-dependent uptake of 54 Bearden & Perry, 1999;Desrosiers et al., 2010;Hazlett et al., 2003;Janakiraman & Slauch, 2000;Janulczyk et al., 1999Janulczyk et al., , 2003Kehres et al., 2002a;Paik et al., 2003;Runyen-Janecky et al., 2006Sabri et al., 2006).In this study we examine the Mn regulation of the Y. pestis mntH and yfe promoters as well as the role of these systems in Mn uptake and virulence. Our in vitro analyses indicate that Yfe and MntH serve semi-redundant functions in Mn acquisition. Mutation of both systems results in a modest growth inhibition and complete loss of short-term, energydependent 54 Mn uptake. Like the yfeABCD promoter, the mntH promoter is repressed by both Fe and Mn through Fur. Both promoters show similarity to each other in their FBSs and sequences immediately upstream of the FBS. Transfer of a small region of the yfeA promoter converted the Fur-regulated hmuP9 promoter, which is repressed by Fe but not Mn, to a chimeric promoter that is repressed by both cations. In virulence studies, the yfeAB mntH double mutant had an~133-fold loss of virulence in a mouse model of bubonic plague compared with its Yfe + MntH + parent. This loss of virulence is greater than would be predicted from our in vitro Mn-deficient growth results. Intriguingly, the yfeAB mntH mutant was fully virulent in a mouse model of pneumonic plague.