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  • 1
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 32 (1999), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 8 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: One characteristic of pigmented (Pgm+) cells of Yersinia pestis is the adsorption of sufficient quantities of exogenous haemin during growth at 26°C to form dark-brown colonies. Carriage of the cloned haemin-storage (hms) locus in pHMS1 restores this phenotype to spontaneous Pgm− chromosomal deletion mutants of Y. pestis. We have mapped the location of the structural genes for four proteins encoded on pHMS1 using minicell, in vitro transcription/translation, and complementation analysis. The hmsH and hmsF genes encode 90kDa and 72 kDa protein precursors processed to surface-exposed, outer membrane proteins of 86kDa and 70kDa, respectively. Beta-galactosidase positive MudII1734 insertions in hmsR suggest that it encodes a protein that is also essential for haemin storage. Finally, the structural gene for a 41 kDa protein lies distal to the hmsH gene but, unlike hmsH, hmsF, and hmsR, its expression is not essential for the Hms+ phenotype in Y. pestis.
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 6 (1992), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The effects of iron have been linked with several phenomena including regulation of membrane proteins; however, the mechanism of iron regulation is not well characterized in Yersinia pestis. It is well known that in Escherichia coli, the fur gene product mediates negative transcriptional regulation of several genes in response to iron. We have cloned a Y. pestis fur gene which is highly homologous to the E coli fur regulatory gene. The sequence of the Y. pestis fur gene exhibits 75% homology to the E. coli gene at the nucleotide level, and 84% homology at the predicted amino acid level. The Y. pestis fur gene is transcribed as a single gene message of approximately 0.5 kb which encodes an approximately 16 kDa protein when expressed in E coli minicells. A Yersinia enterocolitica fur mutant exhibits hypersensitivity to the Y. pestis bacteriocin, pesticin; the cloned Y. pestis fur gene restores wild-type levels of pesticin sensitivity. Furthermore, iron regulation of at least five surface proteins in this Y. enterocolitica fur mutant is restored by transcomplementation with the Y. pestis fur gene. These data indicate that Y. pestis and Y. enterocolitica possess homologous Fur systems which regulate expression of proteins in response to iron availability.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 20 (1996), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Yersinia pestis strains utilize haem and several haem-protein complexes as sole sources of iron. In this study, the haemin uptake locus (hmu) of Y. pestis KIM6+ was selected from a genomic library by trans-duction into an Escherichia coli siderophore synthesis (entC) mutant. Recombinant plasmids containing a common 16 kb BamHI insert were isolated that allowed E. coli entC to use haemin as an iron source. An 8.6 kb region of this insert was found to be essential for haemin utilization and encoded at least five proteins with molecular masses of 79/77, 44, 37, 35, and 30/27.5 kDa. A 10.9 kb Clal fragment containing the hmu locus showed varying degrees of homology to genomic DNA from Yersinia pseudotuberculosis, Yersinia enter-ocolitica, and other genera of Enterobacteriaceae. An E. coli hemA aroB strain harbouring cloned hmu genes used haemin as both an iron and porphyrin source but only on iron-poor medium, suggesting that haemin uptake is tightly iron regulated. Additionally, haemoglobin and myoglobin were used as iron sources by an E. coli entC (pHMU2.2) strain. Deletion of the hmu locus from Y. pestis KIM6+ chromosome generated a mutant that grew poorly on iron-depleted medium containing free haemin as well as mammalian haem-protein complexes including haemoglobin, haemoglobin-haptoglobin, myoglobin, haem-haemopexin, and haem-albumin unless it was complemented with cloned hmu genes.
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  • 6
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The pigmentation (Pgm+) phenotype of Yersinia pestis includes a number of different characteristics which appear to be associated with a 102 kb segment of chromosomal DNA known as the pgm locus. In Y. pestis KIM6+, the pgm locus is flanked by direct copies of a repeated element that probably plays a role in the spontaneous deletion of this region. We have sequenced the ends of these elements and shown that they have features in common with bacterial insertion sequences, in addition we show that a clone, pSDR498, from the pgm locus of KIM6+ restores pesticin sensitivity and the iron-regulated expression of three polypeptides, 240 kDa, 190 kDa, and 68 kDa in size, to Pgm− cells. In vitro transcription/translation assays and Escherichia coli minicells were used to analyse the products encoded by various subciones of pSDR498. Pesticin sensitivity mapped to a 5.9 kb fragment that encodes a 68 kDa protein derived from a 72 kDa precursor. Synthesis of the 190 kDa protein was restored by a 19.2 kb clone, indicating that the structural gene for this protein also resides within the pgm locus of Y. pestis KIM6+. Finally, a survey of our pgm− strains indicates that 97% have also deleted the sequences encoding the 190 kDa protein and pesticin sensitivity.
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 32 (1999), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 22 (1996), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
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  • 9
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
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  • 10
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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