Robert D. Perry

The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague

Iron acquisition in Yersinia pestis is fundamental to the success of plague pathogenesis. We have previously identified an ≈5.6 kb region (yfe) of Y. pestis genomic DNA, capable of restoring iron‐deficient growth but not siderophore production to an Escherichia coli mutant (SAB11) incapable of synthesizing the siderophore, enterobactin. The yfe locus of Y. pestis, found in both pigmented (Pgm+) and nonpigmented (Pgm−) strains, comprises five genes arranged in two distinct operons (yfeA–D and yfeE ). The larger of these, yfeABCD, encodes an ABC transport system, whose expression is iron and Fur regulated and is repressed in cells grown in the presence of manganese. Cells from a Pgm−, Yfe− (ΔyfeAB ) mutant strain of Y. pestis exhibited reduced transport of both 55Fe and 54Mn. Furthermore, cells containing an intact yfe locus showed reduced 55Fe uptake when competing amounts of MnCl2 or ZnCl2 were present, whereas 54Mn uptake was inhibited by FeCl3 but not by ZnCl2. Similarly, yfe mutants of Y. pestis exhibited growth defects on media supplemented with the iron chelators 2,2′‐dipyridyl or conalbumin. These growth defects were not relieved by supplementation with MnCl2. A ybt−, ΔyfeAB mutant of Y. pestis was completely avirulent in mice infected intravenously (LD50 > 1.7 × 107 cfu) compared with its parental ybt−, yfe+ strain, which had an LD50 of < 12. In addition, compared with its ybt+, yfe+ parent, a ybt+, ΔyfeAB mutant of Y. pestis had an ≈100‐fold increase in the LD50 from a subcutaneous route of infection. These data suggest that the Yfe and Ybt systems may function effectively to accumulate iron during different stages of the infectious process of bubonic plague.

Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element

The pigmentation. (Pgm+) phenotype of Yersinia pestis encompasses a variety of different physiological traits, all of which are missing in Pgm‐ mutants. We have previously shown that loss of the Pgm+ phenotype is accompanied by the spontaneous deletion of at least 45 kb of chromosomal DNA, referred to as the pgm locus. Using chromosomal walking, we have now mapped the full extent of the pgm locus in Y. pestis strain KIM6+. Our results indicate that the locus spans 102 kb of DNA which is absent in the spontaneous Pgm‐ mutant, KIM6. Yersinia pseudo‐tuberculosis PB1/0 contains sequences homologous to the entire pgm locus while only part of this region hybridized to Yersinia enterocolitica WA‐LOX DNA. Restriction enzyme mapping and hybridization studies revealed the presence of a repetitive element at both ends of the pgm locus and in multiple copies elsewhere in the Y. pestis genome. This element may be responsible for generating the deletion.

HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis.

The 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 Hms‐dependent 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.

Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis

BACKGROUND Virulence in the pathogenic bacterium Yersinia pestis, causative agent of bubonic plague, has been correlated with the biosynthesis and transport of an iron-chelating siderophore, yersiniabactin, which is induced under iron-starvation conditions. Initial DNA sequencing suggested that this system is highly conserved among the pathogenic Yersinia. Yersiniabactin contains a phenolic group and three five-membered thiazole heterocycles that serve as iron ligands. RESULTS The entire Y. pestis yersiniabactin region has been sequenced. Sequence analysis of yersiniabactin biosynthetic regions (irp2-ybtE and ybtS) reveals a strategy for siderophore production using a mixed polyketide synthase/nonribosomal peptide synthetase complex formed between HMWP1 and HMWP2 (encoded by irp1 and irp2). The complex contains 16 domains, five of them variants of phosphopantetheine-modified peptidyl carrier protein or acyl carrier protein domains. HMWP1 and HMWP2 also contain methyltransferase and heterocyclization domains. Mutating ybtS revealed that this gene encodes a protein essential for yersiniabactin synthesis. CONCLUSIONS The HMWP1 and HMWP2 domain organization suggests that the yersiniabactin siderophore is assembled in a modular fashion, in which a series of covalent intermediates are passed from the amino terminus of HMWP2 to the carboxyl terminus of HMWP1. Biosynthetic labeling studies indicate that the three yersiniabactin methyl moieties are donated by S-adenosylmethionine and that the linker between the thiazoline and thiazolidine rings is derived from malonyl-CoA. The salicylate moiety is probably synthesized using the aromatic amino-acid biosynthetic pathway, the final step of which converts chorismate to salicylate. YbtS might be necessary for converting chorismate to salicylate.

The tc genes of Photorhabdus: a growing family.

The toxin complex (tc) genes of Photorhabdus encode insecticidal, high molecular weight Tc toxins. These toxins have been suggested as useful alternatives to those derived from Bacillus thuringiensis for expression in insect-resistant transgenic plants. Although Photorhabdus luminescens is symbiotic with nematodes that kill insects, tc genes have recently been described from other insect-associated bacteria such as Serratia entomophila, an insect pathogen, and Yersinia pestis, the causative agent of bubonic plague, which has a flea vector. Here, recent advances in our understanding of the tc gene family are reviewed in view of their potential development as insect-control agents.

Environmental modulation of gene expression and pathogenesis in Yersinia

The yersiniae are a useful model for understanding how environmental modulation of gene expression allows pathogens to inhabit a wide range of niches. This review follows the enteropathogenic yersiniae, Yersinia enterocolitica and Yersinia pseudotuberculosis, and the agent of plague, Yersinia pestis, through their life cycles, describing how adaptive gene expression may promote successful pathogenesis.

Polyamines Are Essential for the Formation of Plague Biofilm

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 DeltaspeC mutant (55% reduction), followed by the DeltaspeA mutant (95% reduction) and the DeltaspeA DeltaspeC 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 DeltaspeA DeltaspeC 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 DeltaspeA DeltaspeC 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.

YbtP and YbtQ: two ABC transporters required for iron uptake in Yersinia pestis.

Yersinia pestis, the causative agent of plague, makes a siderophore termed yersiniabactin (Ybt), which it uses to obtain iron during growth at 37°C. The genes required for the synthesis and utilization of Ybt are located within a large, unstable region of the Y. pestis chromosome called the pgm locus. Within the pgm locus, just upstream of a gene (ybtA) that regulates expression of the Ybt receptor and biosynthetic genes, is an operon consisting of 4 genes —ybtP, ybtQ, ybtX and ybtS. Transcription of the ybtPQXS operon is repressed by Fur and activated by YbtA. The product of ybtX is predicted to be an exceedingly hydrophobic cytoplasmic membrane protein that does not appear to contribute any vital function to Ybt biosynthesis or utilization in vitro. ybtP and ybtQ encode putative members of the traffic ATPase/ABC transporter family. YbtP and YbtQ are structurally unique among the subfamily of ABC transporters associated with iron transport, in that they both contain an amino‐terminal membrane‐spanning domain and a carboxy‐terminal ATPase. Cells with mutations in ybtP or ybtQ still produced Ybt but were impaired in their ability to grow at 37°C under iron‐deficient conditions, indicating that YbtP and YbtQ are needed for iron uptake. In addition, a ybtP mutant showed reduced iron accumulation and was avirulent in mice by a subcutaneous route of infection that mimics flea transmission of bubonic plague.

Yersiniabactin from Yersinia pestis: biochemical characterization of the siderophore and its role in iron transport and regulation

A siderophore-dependent iron transport system of the pathogenic yersiniae plays a role in the pathogenesis of these organisms. The structure of the yersiniabactin (Ybt) siderophore produced by Yersinia enterocolitica has been elucidated. This paper reports the purification of Ybt from Yersinia pestis and demonstrates that it has the same structure as Ybt from Y. enterocolitica. Purified Ybt had a formation constant for Fe3+ of approximately 4x10(-36). Addition of purified Ybt from Y. pestis enhanced iron uptake by a siderophore-negative (irp2) strain of Y. pestis. Maximal expression of the Ybt outer-membrane receptor, Psn, in this strain was dependent upon exogenously supplied Ybt. Regulation of Psn expression by Ybt occurred at the transcriptional level. Y. pestis DNA was used to construct irp2 and psn mutations in Yersinia pseudotuberculosis. The irp2 mutant strain no longer synthesized Ybt and the psn mutant strain could not use exogenously supplied Ybt. As in Y. pestis, Ybt was required for maximal expression of Psn. Regulation by Ybt occurred at the transcriptional level. In contrast to Y. pestis, in which a psn mutation does not repress synthesis of Ybt siderophore or expression of the iron-regulated HMWP1 and HMWP2 proteins, the same mutation in Y. pseudotuberculosis partially repressed these products.

YBTA, AN ARAC-TYPE REGULATOR OF THE YERSINIA PESTIS PESTICIN/YERSINIABACTIN RECEPTOR

The pesticin receptor (Psn) of Yersinia pestis confers sensitivity to the bacteriocin, pesticin, and is an integral component of an inorganic‐iron‐transport system that functions at 37°C. Synthesis of Psn is under the control of its own promoter and is regulated by iron and probably by the presence of its cognate siderophore. We have used a psn promoter fusion with lacZ to identify cis‐ and trans‐acting factors which affect transcription of the psn gene. As expected, expression of lacZ from this construct was iron regulated and repressed by Fur. Mutations within a putative siderophore biosynthetic gene (irp2 ) also decreased expression. A set of repeats adjacent to the −35 region of the psn promoter was required for maximum expression of the psn::lacZ gene. Sequence analysis of the region upstream of irp2 revealed the presence of a gene (ybt A) with homology to the AraC family of transcriptional regulators. Insertional inactivation of ybt A resulted in decreased synthesis of Psn and proteins encoded by the irp2 operon as well as decreased expression from the psn::lacZ promoter fusion, indicating that Ybt A is a transcriptional activator for psn and the putative siderophore biosynthetic genes. Ybt A also represses its own transcription.