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.

SummaryCyclic di-GMP (c-di-GMP) is a signalling molecule that governs the transition between planktonic and biofilm states. Previously, we showed that the diguanylate cyclase HmsT and the putative c-di-GMP phosphodiesterase HmsP inversely regulate biofilm formation through control of HmsHFRS-dependent poly-b-1,6-Nacetylglucosamine synthesis. Here, we systematically examine the functionality of the genes encoding putative c-di-GMP metabolic enzymes in Yersinia pestis. We determine that, in addition to hmsT and hmsP, only the gene y3730 encodes a functional enzyme capable of synthesizing c-di-GMP. The seven remaining genes are pseudogenes or encode proteins that do not function catalytically or are not expressed. Furthermore, we show that HmsP has c-di-GMP-specific phosphodiesterase activity. We report that a mutant incapable of c-di-GMP synthesis is unaffected in virulence in plague mouse models.Conversely, an hmsP mutant, unable to degrade c-di-GMP, is defective in virulence by a subcutaneous route of infection due to poly-b-1,6-N-acetylglucosamine overproduction. This suggests that c-di-GMP signalling is not only dispensable but deleterious for Y. pestis virulence. Our results show that a key event in the evolution of Y. pestis from the ancestral Yersinia pseudotuberculosis was a significant reduction in the complexity of its c-di-GMP signalling network likely resulting from the different disease cycles of these human pathogens.

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

In Yersinia pestis, biofilm formation is stimulated by HmsT, a GGDEF-domain containing protein that synthesizes cyclic-di-GMP (c-di-GMP), and inhibited by HmsP, an EAL-domain protein. Only the EAL-domain portion of HmsP is required to inhibit biofilm formation. The EAL domain of HmsP was purified as a 6XHis-tag fusion protein and demonstrated to have phosphodiesterase activity using bis(p-nitrophenyl) phosphate (bis-pNPP) as a substrate. This enzymatic activity was strictly manganese dependent. A critical residue (E506) of HmsP within the EAL domain, that is required for inhibition of biofilm formation, is also essential for this phosphodiesterase activity. While the proposed function of EAL-domain proteins is to linearize c-di-GMP, this is a direct demonstration of the required phosphodiesterase activity of a purified EAL-domain protein.

Summary Bacterial pathogens must overcome host sequestration of zinc (Zn2+), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn2+ by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn2+-deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn2+ acquisition. Studies with the Zn2+-dependent transcriptional reporter znuA∷lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn2+. However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, that are required for Fe3+ acquisition by Ybt, are not needed for Ybt-dependent Zn2+ uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn2+ uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicemic plague mouse model.