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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 f et l inked p henotype) were required for growth of a yfe feo ybt mutant under static, iron-limiting 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 ∼90-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.

Iron acquisition from the host is an important step in the pathogenic process. While Yersinia pestis has multiple iron transporters, the yersiniabactin (Ybt) siderophore-dependent system plays a major role in iron acquisition in vitro and in vivo . In this study, we determined that the Ybt system is required for the use of iron bound by transferrin and lactoferrin and examined the importance of the Ybt system for virulence in mouse models of bubonic and pneumonic plague. Y. pestis mutants unable to either transport Ybt or synthesize the siderophore were both essentially avirulent via subcutaneous injection (bubonic plague model). Surprisingly, via intranasal instillation (pneumonic plague model), we saw a difference in the virulence of Ybt biosynthetic and transport mutants. Ybt biosynthetic mutants displayed an ∼24-fold-higher 50% lethal dose (LD 50 ) than transport mutants. In contrast, under iron-restricted conditions in vitro , a Ybt transport mutant had a more severe growth defect than the Ybt biosynthetic mutant. Finally, a Δ pgm mutant had a greater loss of virulence than the Ybt biosynthetic mutant, indicating that the 102-kb pgm locus encodes a virulence factor, in addition to Ybt, that plays a role in the pathogenesis of pneumonic plague.

In addition to the yersiniabactin (Ybt) siderophore-dependent system, two inorganic iron ABC transport systems of Yersinia pestis , Yfe and Yfu, have been characterized. Here we show that the Yfu system functions in Y. pestis : a Ybt − Yfe − Yfu − mutant exhibited a greater growth defect under iron-deficient conditions than its Ybt − Yfe − parental strain. We also demonstrate that another putative Y. pestis iron uptake system, Yiu, which potentially encodes an outer membrane receptor, YiuR, and an ABC iron transport cassette, YiuABC, is functional. The cloned yiuABC operon restored growth of an enterobactin-deficient mutant Escherichia coli strain, 1017, under iron-chelated conditions. Iron uptake by the Yiu system in Y. pestis was demonstrated only when the Ybt, Yfe, and Yfu systems were mutated. Using a yiuA :: lacZ fusion, we show that the yiuABC promoter is repressed by iron through Fur. A mouse model of bubonic plague failed to show a significant role for the Yiu system in the disease process. These results demonstrate that two additional iron transporters are functional in Y. pestis and indicate that there is a hierarchy of iron transporters, with Ybt being most effective and Yiu being the least effective of those systems which have been characterized.

We provide the first evidence for a link between polyamines and biofilm levels in Yersinia pestis , the causative agent of plague. Polyamine-deficient mutants of Y. pestis were generated with a single deletion in speA or speC and a double deletion mutant. The genes speA and speC code for the biosynthetic enzymes arginine decarboxylase and ornithine decarboxylase, respectively. The level of the polyamine putrescine compared to the parental speA + speC + strain (KIM6+) was depleted progressively, with the highest levels found in the Y. pestis Δ speC mutant (55% reduction), followed by the Δ speA mutant (95% reduction) and the Δ speA Δ speC mutant (>99% reduction). Spermidine, on the other hand, remained constant in the single mutants but was undetected in the double mutant. The growth rates of mutants with single deletions were not altered, while the Δ speA Δ speC mutant grew at 65% of the exponential growth rate of the speA + speC + strain. Biofilm levels were assayed by three independent measures: Congo red binding, crystal violet staining, and confocal laser scanning microscopy. The level of biofilm correlated to the level of putrescine as measured by high-performance liquid chromatography-mass spectrometry and as observed in a chemical complementation curve. Complementation of the Δ speA Δ speC mutant with speA showed nearly full recovery of biofilm to levels observed in the speA + speC + strain. Chemical complementation of the double mutant and recovery of the biofilm defect were only observed with the polyamine putrescine.

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 .

InYersinia pestis, the siderophore-dependent yersiniabactin (Ybt) iron transport system and heme transport system (Hmu) have putative TonB-dependent outer membrane receptors. Here we demonstrate that hemin uptake and iron utilization from Ybt are TonB dependent. However, the Yfe iron and manganese transport system does not require TonB.

Yersinia pestis possesses a heme-protein acquisition system (Hmu) that allows it to utilize heme and heme-protein complexes as the sole sources of iron. Analysis of the Y. pestis CO92 genomic sequence revealed a second heme-protein acquisition gene cluster that shares homology with the hemophore-dependent heme acquisition system (Has system) of Serratia marcescens . This locus consisted of the hasR yp receptor gene, the hasA yp hemophore gene, and genes encoding components of the HasA yp dedicated ABC transporter factor ( hasDE yp ), as well as a tonB homologue ( hasB yp ). By using a reconstituted secretion system in Escherichia coli , we showed that HasA yp is a secreted heme-binding protein and that expression of HasA yp is iron regulated in E. coli . The use of a transcriptional reporter fusion showed that the hasRADEB promoter is Fur regulated and has increased activity at 37°C. Hemoglobin utilization via the Has yp system was studied with both E. coli and Y. pestis , for which has and has hmu mutant strains were used. No contribution of the Has system to heme utilization was observed in either E. coli or Y. pestis under the conditions we tested. Previously it was shown that a deletion of the Hmu system had no effect on the virulence of Y. pestis in a mouse model of bubonic plague. An Hmu − Has − double mutant also retained full virulence in this model of infection. This report constitutes the first attempt to investigate the contribution of the hemophore-dependent heme acquisition system in bacterial pathogenicity.

In Yersinia pestis , the causative agent of plague, two inorganic iron transport systems have been partially characterized. The yersiniabactin (Ybt) system is a siderophore-dependent transport system required for full virulence. Yfe is an ABC transport system that accumulates both iron and manganese. We have identified and cloned a Y. pestis yfuABC operon. The YfuABC system is a member of the cluster of bacterial ABC iron transporters that include Sfu of Serratia , Hit of Haemophilus , and Yfu of Yersinia enterocolitica . The Y. pestis KIM6+ system is most homologous to that in Y. enterocolitica , showing identities of 84% for YfuA (periplasmic binding protein), 87% for YfuB (inner membrane permease), and 75% for YfuC (ATP hydrolase). We constructed a yfuABC promoter- lacZ fusion to examine regulation of transcription. This promoter contains a potential Fur binding sequence and is iron and Fur regulated. Significant expression from the yfuABC promoter occurred during iron-deficient growth conditions. In vitro transcription and translation of a recombinant plasmid encoding yfuABC indicates that YfuABC proteins are expressed. Escherichia coli 1017 (an enterobactin-deficient mutant) carrying this plasmid was able to grow in an iron-restrictive complex medium. We constructed a deletion encompassing the yfuABC promoter and most of yfuA . This mutation was introduced into strains with mutations in Ybt, Yfe, or both systems to examine the role of Yfu in iron acquisition in Y. pestis . Growth of the yfu mutants in a deferrated, defined medium (PMH2) at 26 and 37°C failed to identify a growth or iron transport defect due to the yfu mutation. Fifty percent lethal dose studies in mice did not demonstrate a role for the Yfu system in mammalian virulence.

One prerequisite for the virulence of Yersinia pestis , causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the pgm locus of the Y. pestis chromosome. Several gene products within the HPI have demonstrated functions in the synthesis or transport of Ybt. Here we examine the roles of ybtU and ybtT . In-frame mutations in ybtT or ybtU yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37°C but could be cross-fed by culture supernatants from a Ybt-producing strain of Y. pestis . The ybtU mutant failed to express four indicator Ybt proteins (HMWP1, HMWP2, YbtE, and Psn), a pattern similar to those for other ybt biosynthetic mutants. In contrast, strains carrying mutations in ybtT or ybtS (a previously identified gene required for Ybt biosynthesis) produced all four proteins at wild-type levels under iron-deprived conditions. To assess the effects of ybtT , - U , and - S mutations on transcription of ybt genes, reporter plasmids with ybtP or psn promoters controlling lacZ expression were introduced into these mutants. Normal iron-regulated β-galactosidase activity was observed in the ybtT and ybtS mutants, whereas a significant loss of expression occurred in the Δ ybtU strain. These results show that ybtT and ybtU genes are involved in the biosynthesis of the Ybt siderophore and that a ybtU mutation but not ybtT or ybtS mutations affects transcription from the ybtP and psn promoters.

We have identified an approximately 22-kb region of the pgm locus of Yersinia pestis KIM6+ which encodes a number of iron-regulated proteins involved in the biosynthesis of the Y. pestis cognate siderophore, yersiniabactin (Ybt), and which is located immediately upstream of the pesticin/yersiniabactin receptor gene (psn). Sequence analysis and the construction of insertion and deletion mutants allowed us to determine the putative location of the irp1 gene and the positions of irp2, ybtT, and ybtE within the ybt operon. Mutations in the irp1, irp2, or ybtE gene yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from yersiniabactin-producing strains of Y. pestis grown under iron-limiting conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell extracts from Ybt+ and Ybt- strains grown in iron-deficient media revealed that expression of ybt-encoded proteins is not only iron regulated but also influenced by the presence of the siderophore itself. Finally, Y. pestis strains with mutations in either the psn or irp2 gene were avirulent in mice when inoculated subcutaneously.

A Yersinia pestis fur mutation was constructed by insertionally disrupting the fur open reading frame. Analysis of a Fur-regulated beta-galactosidase reporter gene revealed a loss of iron regulation as a result of the fur mutation. trans complementation with the cloned Y. pestis fur gene restored iron regulation. The expression of most iron-regulated proteins was also deregulated by this mutation; however, a number of iron-repressible and two iron-inducible polypeptides retained normal regulation. Mutations in fur or hmsH, a gene encoding an 86-kDa surface protein required for hemin storage, increased the sensitivity of Y. pestis cells to the bacteriocin pesticin. Interestingly, the Y. pestis fur mutant lost temperature control of hemin storage; however, expression of the HmsH polypeptide was not deregulated. When grown with excess iron, a Y. pestis fur mutant possessing the 102-kb pigmentation locus exhibited severe growth inhibition and a dramatic increase in the number of spontaneous nonpigmented chromosomal deletion mutants present at late log phase. These results suggest that the Fur protein of Y. pestis is an important global regulator and that a separate Fur-independent iron regulatory system may exist.

The low-Ca 2+ -response (LCR) plasmid pCD1 of the plague agent Yersinia pestis KIM5 was sequenced and analyzed for its genetic structure. pCD1 (70,509 bp) has an IncFIIA-like replicon and a SopABC-like partition region. We have assigned 60 apparently intact open reading frames (ORFs) that are not contained within transposable elements. Of these, 47 are proven or possible members of the LCR, a major virulence property of human-pathogenic Yersinia spp., that had been identified previously in one or more of Y. pestis or the enteropathogenic yersiniae Yersinia enterocolitica and Yersinia pseudotuberculosis . Of these 47 LCR-related ORFs, 35 constitute a continuous LCR cluster. The other LCR-related ORFs are interspersed among three intact insertion sequence (IS) elements (IS 100 and two new IS elements, IS 1616 and IS 1617 ) and numerous defective or partial transposable elements. Regional variations in percent GC content and among ORFs encoding effector proteins of the LCR are additional evidence of a complex history for this plasmid. Our analysis suggested the possible addition of a new Syc- and Yop-encoding operon to the LCR-related pCD1 genes and gave no support for the existence of YopL. YadA likely is not expressed, as was the case for Y. pestis EV76, and the gene for the lipoprotein YlpA found in Y. enterocolitica likely is a pseudogene in Y. pestis . The yopM gene is longer than previously thought (by a sequence encoding two leucine-rich repeats), the ORF upstream of ypkA-yopJ is discussed as a potential Syc gene, and a previously undescribed ORF downstream of yopE was identified as being potentially significant. Eight other ORFs not associated with IS elements were identified and deserve future investigation into their functions.

GGDEF domain-containing proteins have been implicated in bacterial signal transduction and synthesis of the second messenger molecule cyclic-di-GMP. A number of GGDEF proteins are involved in controlling the formation of extracellular matrices. AdrA ( Salmonella enterica serovar Typhimurium) and HmsT ( Yersinia pestis ) contain GGDEF domains and are required for extracellular cellulose production and biofilm formation, respectively. Here we show that hmsT is able to restore cellulose synthesis to a Salmonella serovar Typhimurium adrA mutant and that adrA can replace hmsT in Y. pestis Hms-dependent biofilm formation. Like Y. pestis HmsT overproducers, Y. pestis cells carrying adrA under the control of an arabinose-inducible promoter produced substantial biofilms in the presence of arabinose. Finally, we demonstrate that HmsT is involved in the synthesis of cyclic di-GMP.