Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis.

The temperature-dependent absorption of sufficient exogenous hemin or Congo red to form pigmented colonies of Yersinia pestis has been termed the pigmentation phenotype (Pgm+). Spontaneous mutation to a Pgm- phenotype results in the loss of a number of divergent physiological characteristics, including the ability to store hemin and to bind Congo red at 26 degrees C. In this study, we generated and isolated transposon insertion mutants that are hemin storage negative (Hms-) and therefore unable to form pigmented colonies. These mutations are due to single mini-kan insertions within a 19.5-kilobase (kb) SalI fragment of chromosomal DNA. Restriction site analysis of eight mutants identified a minimum of six potentially different insertion sites spanning an approximately 10-kb hemin storage (hms) locus. The 19.5-kb SalI fragment (containing approximately 18 kb of Y. pestis DNA and the mini-kan insert) was cloned from one of these mutants, KIM6-2012. By using this cloned fragment as a DNA probe, the mechanism of spontaneous mutation to a Pgm- phenotype was identified as a massive deletion event. The deletion spans at least 18 kb of genomic DNA in spontaneous Pgm- mutants from nine separate strains of Y. pestis. DNA adjacent to the mini-kan insert was used to identify a clone containing a wild-type hms locus. A spontaneous Pgm- mutant of Y pestis KIM containing this clone exhibits an Hms+ phenotype. The hms::mini-kan mutations and cloned wild-type hms locus generated in this study will greatly aid in identifying the function of hemin storage in Y. pestis.     

Transport of haemin across the cytoplasmic membrane through a haemin-specific periplasmic binding-protein-dependent transport system in Yersinia enterocolitica

The Yersinia enterocolitica O:8 periplasmic binding-protein-dependent transport (PBT) system for haemin was cloned and characterized. It consisted of four proteins: the periplasmic haemin-binding protein HemT, the haemin permease protein HemU, the ATP-binding hydrophilic protein HemV and the putative haemin-degrading protein HemS. Y. enterocolitica strains mutated in hemU or hemV genes were unable to use haemin as an iron source whereas those mutated in the hemT gene were able to use haemin as an iron source. As Escherichia coli strains expressing only the haemin outer membrane receptor protein HemR from Y. enterocolitica were capable of using haemin as an iron source the existence of an E. coli K-12 haemin-specific PBT system is postulated. The first gene in the Y. enterocolitica haemin-specific PBT system encoded a protein, HemS, which is probably involved in the degradation of haemin in the cytoplasm. The presence of the hemS gene was necessary to prevent haemin toxicity in E. coli strains that accumulate large amounts of haemin in the cytoplasm. We propose a model of haemin utilization in Y. enterocolitica in which HemT, HemU and HemV proteins transport haemin into the cytoplasm where it is degraded by HemS thereby liberating the iron.     

Proteomic analysis of iron acquisition,metabolic and regulatory responses of <Emphasis Type="Italic">Yersinia pestis</Emphasis> to iron starvation

Background  

The Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26°C and 37°C).     

Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin

Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild-type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS-PAGE of protein fractions indicate that pHUT10 (a subclone of p>HUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem-iron utilization genes allow transport of the entire haem moiety into the cell.     

Identification and cloning of a fur regulatory gene in Yersinia pestis.

Yersinia pestis is one of many microorganisms responding to environmental iron concentrations by regulating the synthesis of proteins and an iron transport system(s). In a number of bacteria, expression of iron uptake systems and other virulence determinants is controlled by the Fur regulatory protein. DNA hybridization analysis revealed that both pigmented and nonpigmented cells of Y. pestis possess a DNA locus homologous to the Escherichia coli fur gene. Introduction of a Fur-regulated beta-galactosidase reporter gene into Y. pestis KIM resulted in iron-responsive beta-galactosidase activity, indicating that Y. pestis KIM expresses a functional Fur regulatory protein. A cloned 1.9-kb ClaI fragment of Y. pestis chromosomal DNA hybridized specifically to the fur gene of E. coli. The coding region of the E. coli fur gene hybridized to a 1.1-kb region at one end of the cloned Y. pestis fragment. The failure of this clone to complement an E. coli fur mutant suggests that the 1.9-kb clone does not contain a functional promoter. Subcloning of this fragment into an inducible expression vector restored Fur regulation in an E. coli fur mutant. In addition, a larger 4.8-kb Y. pestis clone containing the putative promoter region complemented the Fur- phenotype. These results suggest that Y. pestis possesses a functional Fur regulatory protein capable of interacting with the E. coli Fur system. In Y. pestis Fur may regulate the expression of iron transport systems and other virulence factors in response to iron limitation in the environment. Possible candidates for Fur regulation in Y. pestis include genes involved in ferric iron transport as well as hemin, heme/hemopexin, heme/albumin, ferritin, hemoglobin, and hemoglobin/haptoglobin utilization.     

Identification and characterization of autotransporter proteins of Yersinia pestis KIM

Yersinia pestis is a Gram-negative bacterium that causes plague. Currently, plague is considered a re-emerging infectious disease and Y. pestis a potential bioterrorism agent. Autotransporters (ATs) are virulence proteins translocated by a variety of pathogenic Gram-negative bacteria across the cell envelope to the cell surface or extracellular environment. In this study, we screened the genome of Yersinia pestis KIM for AT genes whose expression might be relevant for the pathogenicity of this plague-causing organism. By in silico analyses, we identified ten putative AT genes in the genomic sequence of Y. pestis KIM; two of these genes are located within known pathogenicity islands. The expression of all ten putative AT genes in Y. pestis KIM was confirmed by RT-PCR. Five genes, designated yapA, yapC, yapG, yapK and yapN, were subsequently cloned and expressed in Escherichia coli K12 for protein secretion studies. Two forms of the YapA protein (130 kDa and 115 kDa) were found secreted into the culture medium. Protease cleavage at the C terminus of YapA released the protein from the cell surface. Outer membrane localization of YapC (65 kDa), YapG (100 kDa), YapK (130 kDa), and YapN (60 kDa) was established by cell fractionation, and cell surface localization of YapC and YapN was demonstrated by protease accessibility experiments. In functional studies, YapN and YapK showed hemagglutination activity and YapC exhibited autoagglutination activity. Data reported here represent the first study on Y. pestis ATs.     

The Neisseria meningitidis haemoglobin receptor: its role in iron utilization and virulence

The Neisseris meningitidis haemoglobin receptor gene, hmbR, was cloned by complementation in a porphyrin-requiring Escherichia coli mutant. hmbR encodes an 89.5 kDa outer membrane protein which shares amino acid homology with the TonB-dependent receptors of Gram-negative bacteria. HmbR had the highest similarity to Neisseria transferrin and lactoferrin receptors. The utilization of haemoglobin as an iron source required internalization of the haemin moiety by the cell. The mechanism of haemin internalization via the haemoglobin receptor was TonB-dependent in E. coli. A N. meningitidis hmbR mutant was unable to use haemoglobin but could still use haemin as a sole iron source. The existence of a second N. meningitidis receptor gene, specific for haemin, was shown by the isolation of cosmids which did not hybridize with the hmbR probe, but which were able to complement an E. coli hemA aroB mutant on haemin-supplemented plates. The N. meningitidis hmbR mutant was attenuated in an infant rat model for meningococcal infection, indicating that haemoglobin utilization is important for N. meningitidis virulence.     

Characterization of a hemin-storage locus ofYersinia pestis

Summary The pigmentation phenotype (Pgm⁺) ofYersinia pestis refers to temperature-dependent storage of hemin as well as expression of a number of other physiological characteristics. Spontaneous mutation to a Pgm^– phenotype occurs via a large chromosomal deletion event and results in the inability to express the Pgm⁺ characteristics. In this study, we have used transposon insertion mutants to define two regions of a hemin-storage (hms) locus. A clone (pHMSI) encompassing this locus reinstates expression of hemin storage (Hms⁺) inY. pestis spontaneous Pgm^– strains KIM and Kuma but not inEscherichia coli. Complementation analysis using subclones of pHMS1 inY. pestis transposon mutants indicates that both regions (hmsA andhmsB), which are separated by about 4 kb of intervening DNA, are essential for expression of the Hms⁺ phenotype. The 9.1-kb insert of pHMS1 contains structural genes encoding 90-kDa, 72-kDa, and 37-kDa polypeptides. Two-dimensional gel electrophoresis analysis of cells from Pgm⁺, spontaneous Pgm^–, and Hms^– transposon strains, as well as a spontaneous Pgm^– strain transformed with pHMS1, indicated that two families of surface-exposed polypeptides (of about 87 and 69-73 kDa) are associated with the Hms⁺ phenotype.     

Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7

In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene ( chuA ) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependentFur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis . A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.     

Genomic comparison of Yersinia pestis and Yersinia pseudotuberculosis by combination of suppression subtractive hybridization and DNA microarray

In order to further figure out the genetic differences between Yersinia pestis and Yersinia pseudotuberculosis, and to provide novel insights into the evolution of Y. pestis, we compared the genomes of Y. pseudotuberculosis serogroup I strain ATCC29833 and Y. pestis Antiqua strain 49006 using a combination of suppression subtractive hybridization (SSH) and comparative genomic hybridization with DNAs from a diverse panel of Y. pestis and Y. pseudotuberculosis strains. SSH followed by BLAST analysis revealed 112 SSH fragments specific to strain ATCC29833, compared to the genomic sequence data of Y. pestis strains CO92, KIM and 91001. We identified 17 SSH fragments that appeared to be newly determined genetic contents of Y. pseudotuberculosis. The combination of SSH and microarray analysis showed that the parallel loss of genes contributed greatly not only to the significant genomic divergence between Y. pestis and Y. pseudotuberculosis but also to the intra-species microevolution of both of species. The results confirmed our earlier hypothesis that Y. pestis Antiqua isolates from the natural plague focus B in China represented the most ancestral strains in China, hence phylogenetically the closest isolates to Y. pseudotuberculosis.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Xiaoyi Wang and Dongsheng Zhou contributed equally to this work.     

Yersinia pestis Intracellular Parasitism of Macrophages from Hosts Exhibiting High and Low Severity of Plague

Background

Yersinia pestis causes severe disease in natural rodent hosts, but mild to inapparent disease in certain rodent predators such as dogs. Y. pestis initiates infection in susceptible hosts by parasitizing and multiplying intracellularly in local macrophages prior to systemic dissemination. Thus, we hypothesize that Y. pestis disease severity may depend on the degree to which intracellular Y. pestis overcomes the initial host macrophage imposed stress.

Methodology/Principal Findings

To test this hypothesis, the progression of in vitro infection by Y. pestis {"type":"entrez-protein","attrs":{"text":"KIM62053.1","term_id":"751713025","term_text":"KIM62053.1"}}KIM62053.1+ of mouse splenic and RAW264.7 tissue culture macrophages and dog peripheral blood-derived and DH82 tissue culture macrophages was studied using microscopy and various parameters of infection. The study showed that during the early stage of infection, intracellular Y. pestis assumed filamentous cellular morphology with multiple copies of the genome per bacterium in both mouse and dog macrophages. Later, in mouse macrophages, the infection elicited spacious vacuolar extension of Yersinia containing vacuoles (YCV), and the filamentous Y. pestis reverted to coccobacillary morphology with genomic equivalents approximately equaling colony forming units. In contrast, Y. pestis infected dog macrophages did not show noticeable extension of YCV, and intracellular Y. pestis retained the filamentous cellular morphology for the entire experiment in DH82 cells or were killed by blood-derived macrophages. In addition, during the later stage of infection, Y. pestis infected mouse macrophages exhibited cell lysis whereas dog macrophages did not.

Conclusion/Significance

Overall, these results support our hypothesis that Y. pestis in mouse macrophages can overcome the initial intracellular stress necessary for subsequent systemic infection. However, in dogs, failure of Y. pestis to overcome macrophage imposed stress may result in mild or in apparent disease in dogs.     

Yersinia ironomics: comparison of iron transporters among Yersiniapestis biotypes and its nearest neighbor, Yersinia pseudotuberculosis

Although Yersinia pestis epidemic biovars and Yersinia pseudotuberculosis are recently diverged, highly related species, they cause different diseases via disparate transmission routes. Since iron transport systems are important for iron acquisition from hosts and for survival in the environment, we have analyzed potential iron transport systems encoded by epidemic and non-epidemic or endemic strains of Y. pestis as well as two virulent Y. pseudotuberculosis strains. Computational biology analysis of these genomes showed a high degree of identity/similarity among 16 proven or possible iron/heme transporters identified. Of these, 7 systems were essentially the same in all seven genomes analyzed. The remaining 9 loci had 2–6 genetic variations among these genomes. Two untested, potential siderophore-dependent systems appear intact in Y. pseudotuberculosis but are disrupted or absent in all the endemic Y. pestis strains as well as the epidemic strains from the antiqua and mediaevalis biovars. Only one of these two loci are obviously disrupted in Y. pestis CO92 (epidemic orientalis biovar). Experimental studies failed to identify a role for hemin uptake systems in the virulence of pneumonic plague and suggest that Y. pestis CO92 does not make a siderophore other than Ybt.     

Curing of four different plasmids in Yersinia pestis using plasmid incompatibility

Aims: Plasmids are critical for the pathogenicity of Yersinia pestis. In order to carry out a systematic investigation of their role in pathogenesis, we cured plasmids from Y. pestis. Methods and Results: Each plasmid’s replicon of Y. pestis was cloned into plasmid pEX18Gm containing a counter‐selectable sacB gene, and was then introduced into Y. pestis strain 201 by electroporation. Strains containing recombinant plasmids were cultivated under antibiotic selection. The resultant plasmid‐curing colonies, identified by specific polymerase chain reactions, were then cured off pEX18Gm under sucrose pressure. This method was used to successfully cure all four plasmids of Y. pestis, singly or in different combinations. Conclusions: Naturally evolving plasmids in Y. pestis are difficult to remove by conventional curing methods. We employed a method based on plasmid incompatibility to cure the plasmids from Y. pestis, which confirmed the efficacy of this method for curing plasmids with different types of replicons from one bacterium. Significance and Impact of the Study: There have been no reports on the curing of multiple plasmids by using replication mechanisms from one bacterium with this technique. In the present study, we were able to successfully apply this methodology to cure four plasmids from Y. pestis, confirming its feasibility.     

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 pseudotuberculosis 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.     

Yersinia pestis lacZ expresses a beta-galactosidase with low enzymatic activity

Although very little, if any, beta-galactosidase activity is detected in Yersinia pestis by a standard Miller assay, we found that Y. pestis KIM6+ cells formed blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). Searches of the Y. pestis genome databases revealed the presence of noncontiguous sequences highly homologous to Escherichia coli lacZ, lacY, and lacI. Yersinia pestis lacZ is predicted to encode a 1060 amino-acid protein with 62% identity and 72% similarity to beta-galactosidase from E. coli. A deletion in the Y. pestis lacZ gene caused the formation of white colonies on X-gal-containing plates and beta-galactosidase activity was at background levels in the KIM6+lacZ mutant, while the complemented strain expressed about 190 Miller units. The Y. pestis lacZ promoter was not regulated by isopropylthiogalactoside or glucose. Finally, uptake of lactose by Y. pestis may be impaired.     

Corynebacterium diphtheriae genes required for acquisition of iron from haemin and haemoglobin are homologous to ABC haemin transporters

Corynebacterium diphtheriae and Corynebacterium ulcerans use haemin and haemoglobin as essential sources of iron during growth in iron-depleted medium. C. diphtheriae and C. ulcerans mutants defective in haemin iron utilization were isolated and characterized. Four clones from a C. diphtheriae genomic library complemented several of the Corynebacteria haemin utilization mutants. The complementing plasmids shared an approximately 3 kb region, and the nucleotide sequence of one of the plasmids revealed five open reading frames that appeared to be organized in a single operon. The first three genes, which we have termed hmuT, hmuU and hmuV, shared striking homology with genes that are known to be required for haemin transport in Gram-negative bacteria and are proposed to be part of an ABC (ATP-binding cassette) transport system. The hmuT gene encodes a 37 kDa lipoprotein that is associated with the cytoplasmic membrane when expressed in Escherichi coli and C. diphtheriae. HmuT binds in vitro to haemin- and haemoglobin-agarose, suggesting that it is capable of binding both haemin and haemoglobin and may function as the haemin receptor in C. diphtheriae. This study reports the first genetic characterization of a transport system that is involved in the utilization of haemin and haemoglobin as iron sources by a Gram-positive bacterium.     

Acquisition of omptin reveals cryptic virulence function of autotransporter YapE in Yersinia pestis

Autotransporters, the largest family of secreted proteins in Gram‐negative bacteria, perform a variety of functions, including adherence, cytotoxicity and immune evasion. In Yersinia pestis the autotransporter YapE has adhesive properties and contributes to disease in the mouse model of bubonic plague. Here, we demonstrate that omptin cleavage of Y. pestis YapE is required to mediate bacterial aggregation and adherence to eukaryotic cells. We demonstrate that omptin cleavage is specific for the Y. pestis and Y. pseudotuberculosis YapE orthologues but is not conserved in the Yersinia enterocolitica protein. We also show that cleavage of YapE occurs in Y. pestis but not in the enteric Yersinia species, and requires the omptin Pla (plasminogen activator protease), which is encoded on the Y. pestis‐specific plasmid pPCP1. Together, these data show that post‐translation modification of YapE appears to be specific to Y. pestis, was acquired along with the acquisition of pPCP1 during the divergence of Y. pestis from Y. pseudotuberculosis, and are the first evidence of a novel mechanism to regulate bacterial adherence.