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:

The Yfe/Sit and Feo transport systems are important for the growth of a variety of bacteria. In Yersinia pestis, single mutations in either yfe or feo result in reduced growth under static (limited aeration), iron-chelated conditions, while a yfe feo double mutant has a more severe growth defect. These growth defects were not observed when bacteria were grown under aerobic conditions or in strains capable of producing the siderophore yersiniabactin (Ybt) and the putative ferrous transporter FetMP. Both fetP and a downstream locus (flp for fet linked phenotype) were required for growth of a yfe feo ybt mutant under static, ironlimiting conditions. An feoB mutation alone had no effect on the virulence of Y. pestis in either bubonic or pneumonic plague models. An feo yfe double mutant was still fully virulent in a pneumonic plague model but had an ϳ90-fold increase in the 50% lethal dose (LD 50 ) relative to the Yfe ؉ Feo ؉ parent strain in a bubonic plague model. Thus, Yfe and Feo, in addition to Ybt, play an important role in the progression of bubonic plague. Finally, we examined the factors affecting the expression of the feo operon in Y. pestis. Under static growth conditions, the Y. pestis feo::lacZ fusion was repressed by iron in a Fur-dependent manner but not in cells grown aerobically. Mutations in feoC, fnr, arcA, oxyR, or rstAB had no significant effect on transcription of the Y. pestis feo promoter. Thus, the factor(s) that prevents repression by Fur under aerobic growth conditions remains to be identified.

In Yersinia pestis, the Yfe and Feo systems likely function to transport ferrous iron. Both FeoA and FeoB are essential for iron acquisition activity while FeoC is not. Mutations in yfe and feo had an additive effect on microaerophilic growth under iron-chelating conditions. Y. pestis cells lacking the Ybt siderophore-dependent system, the Yfe or the Feo system grow normally in J774A.1 cells. However, a double yfeAB feoB mutant was no longer able to grow in this murine macrophage cell line. This growth defect likely resulted from iron and not manganese deprivation since a yfeAB mntH mutant grew normally in J774A.1 cells. These results suggest that the Yfe and Feo systems are somewhat redundant ferrous iron transporters capable of iron acquisition during intracellular growth of the plague bacterium.

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.

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