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.

Yersinia pestis biofilm formation causes massive adsorption of haemin or Congo red in vitro as well as colonization and eventual blockage of the flea proventriculus in vivo. This blockage allows effective transmission of plague from some fleas, like the oriental rat flea, to mammals. Four Hms proteins, HmsH, HmsF, HmsR and HmsS, are essential for biofilm formation, with HmsT and HmsP acting as positive and negative regulators, respectively. HmsH has a β-barrel structure with a large periplasmic domain while HmsF possesses polysaccharide deacetylase and COG1649 domains. HmsR is a putative glycosyltransferase while HmsS has no recognized domains. In this study, specific amino acids within conserved domains or within regions of high similarity in HmsH, HmsF, HmsR and HmsS proteins were selected for site-directed mutagenesis. Some but not all of the substitutions in HmsS and within the periplasmic domain of HmsH were critical for protein function. Substitutions within the glycosyltransferase domain of HmsR and the deacetylase domain of HmsF abolished biofilm formation in Y. pestis. Surprisingly, substitution of highly conserved residues within COG1649 did not affect HmsF function.