Identification and characterization of the hemophore-dependent heme acquisition system of Yersinia pestis.

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 degrees 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.

Characterization of the Yersinia pestis Yfu ABC inorganic iron transport system.

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 degrees 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.

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.

Yersinia pestis YbtU and YbtT are involved in synthesis of the siderophore yersiniabactin but have different effects on regulation.

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 degrees 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 beta-galactosidase activity was observed in the ybtT and ybtS mutants, whereas a significant loss of expression occurred in the DeltaybtU 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.

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 degrees 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 degrees 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.

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.

DNA sequencing and analysis of the low-Ca2+-response plasmid pCD1 of Yersinia pestis KIM5.

The low-Ca2+-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 (IS100 and two new IS elements, IS1616 and IS1617) 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.

Sequence and genetic analysis of the hemin storage (hms) system of Yersinia pestis.

We have sequenced a region from the pigmentation (pgm) locus of Yersinia pestis KIM6+ that is identified with the exogenous hemin storage (Hms+) phenotype in cells grown at 26 but not at 37 degrees C. The hmsHFRS locus encodes a putative polycistronic operon, with hmsH encoding an outer membrane protein with a deduced molecular mass of 93.4/89.5 (unprocessed/processed) kDa. The mature HmsH 788 aa polypeptide has a pI of 4.99. The hmsF gene has an open reading frame of 654 aa, encoding a 74.6/72.2 kDa OM protein with a pI of 5.16 when processed. A deduced 423 aa, 52 kDa protein with a pI of 10.83 is encoded by hmsR. HmsR has a basic, hydrophilic, and alpha-helical carboxyl terminus; 13 aa at the amino-terminal end and a 'KRKRAR' sequence at the carboxy-terminal end are essential for an Hms+ phenotype. The hmsS gene encodes a hypothetical 155 aa, 17.5 kDa protein with a pI of 6.68. Hms- Y. pestis strain M23-2 transformed with the cloned hmsHFRS locus developed an Hms(c) phenotype (Hms+ at 26-37 degrees C) due to mutations in genes outside the pgm locus.

Genetic organization of the yersiniabactin biosynthetic region and construction of avirulent mutants in Yersinia pestis.

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.

Yersinia pestis--etiologic agent of plague.

Plague is a widespread zoonotic disease that is caused by Yersinia pestis and has had devastating effects on the human population throughout history. Disappearance of the disease is unlikely due to the wide range of mammalian hosts and their attendant fleas. The flea/rodent life cycle of Y. pestis, a gram-negative obligate pathogen, exposes it to very different environmental conditions and has resulted in some novel traits facilitating transmission and infection. Studies characterizing virulence determinants of Y. pestis have identified novel mechanisms for overcoming host defenses. Regulatory systems controlling the expression of some of these virulence factors have proven quite complex. These areas of research have provide new insights into the host-parasite relationship. This review will update our present understanding of the history, etiology, epidemiology, clinical aspects, and public health issues of plague.

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 degrees 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 (ybtA) with homology to the AraC family of transcriptional regulators. Insertional inactivation of ybtA 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 YbtA is a transcriptional activator for psn and the putative siderophore biosynthetic genes. YbtA also represses its own transcription.

Iron uptake and iron-repressible polypeptides in Yersinia pestis.

Pigmented (Pgm+) cells of Yersinia pestis are virulent, are sensitive to pesticin, adsorb exogenous hemin at 26 degrees C (Hms+), produce iron-repressible outer membrane proteins, and grow at 37 degrees C in iron-deficient media. These traits are lost upon spontaneous deletion of a chromosomal 102-kb pgm locus (Pgm-). Here we demonstrate that an Hms+ but pesticin-resistant (Pst(r)) mutant acquired a 5-bp deletion in the pesticin receptor gene (psn) encoding IrpB to IrpD. Growth and assimilation of iron by Pgm- and Hms+ Pst(r) mutants were markedly inhibited by ferrous chelators at 37 degrees C; inhibition by ferric and ferrous chelators was less effective at 26 degrees C. Iron-deficient growth at 26 degrees C induced iron-regulated outer membrane proteins of 34, 28.5, and 22.5 kDa and periplasmic polypeptides of 33.5 and 30 kDa. These findings provide a basis for understanding the psn-driven system of iron uptake, indicate the existence of at least one additional 26 degrees C-dependent iron assimilation system, and define over 30 iron-repressible proteins in Y. pestis.

Analysis of the pesticin receptor from Yersinia pestis: role in iron-deficient growth and possible regulation by its siderophore.

We have sequenced a region from the pgm locus of Yersinia pestis KIM6+ that confers sensitivity to the bacteriocin pesticin to certain strains of Escherichia coli and Y. pestis. The Y. pestis sequence is 98% identical to the pesticin receptor from Yersinia enterocolitica and is homologous to other TonB-dependent outer membrane proteins. Y. pestis strains with an in-frame deletion in the pesticin receptor gene (psn) were pesticin resistant and no longer expressed a group of iron-regulated outer membrane proteins, IrpB to IrpD. In addition, this strain as well as a Y. pestis strain with a mutation constructed in the gene (irp2) encoding the 190-kDa iron-regulated protein HMWP2 could not grow at 37 degrees C in a defined, iron-deficient medium. However, the irp2 mutant but not the psn mutant could be cross-fed by supernatants from various Yersinia cultures grown under iron-deficient conditions. An analysis of the proteins synthesized by the irp2 mutant suggests that HMWP2 may be indirectly required for maximal expression of the pesticin receptor. HMWP2 likely participates in synthesis of a siderophore which may induce expression of the receptor for pesticin and the siderophore.

Pleiotropic effects of a Yersinia pestis fur mutation.

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 pigmentation locus of Yersinia pestis KIM6+ is flanked by an insertion sequence and includes the structural genes for pesticin sensitivity and HMWP2.

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 subclones 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.

Orientation of the cleavage site of the herpes simplex virus glycoprotein G-2.

During the synthesis of glycoprotein G-2 (gG-2) of herpes simplex virus type 2, the 104,000-Da gG-2 precursor (104K precursor) is cleaved to generate the 72K and the 31K intermediates. The 72K product is processed to generate the mature gG-2 (molecular mass, 108,000 Da), while the 31K product is additionally processed and secreted into the extracellular medium as the 34K component (H. K. Su, R. Eberle, and R. J. Courtney, J. Virol. 61:1735-1737, 1987). In this study, the orientations of the 31K and 72K products on the 104K precursor were determined by using two antipeptide sera produced in rabbits and a monoclonal antibody, 13 alpha C6, directed against gG-2. The sera prepared against synthetic peptides corresponding to the terminal amino acid residues 67 to 78 and an internal peptide at amino acids 247 to 260 of gG-2 recognized the 104K precursor and the 31K cleavage product but not the 72K intermediate. In contrast, 13 alpha C6 detected the 72K cleavage product and the uncleaved precursor but not the 31K cleavage component. The epitope recognized by 13 alpha C6 was mapped within amino acids 486 to 566. These results suggest that the 31K cleavage product is derived from the amino-terminal portion of the 104K precursor molecule and that the 72K intermediate is derived from the carboxyl terminus. In support of our model described above for the synthesis of gG-2, antibodies recognizing either of the cleavage products reacted with the uncleaved precursor but not with the other cleavage product. By using partial endo-beta-N-acetylglucosaminidase H analysis, two N-linked glycosylation sites were found on each of the cleavage products. The distribution of the N-linked glycosylation sites and the reactivities of the antipeptide sera allowed the cleavage region on the precursor to be mapped to within amino acids 260 to 437.

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.

In vitro synthesis and processing of herpes simplex virus type 2 gG-2, using cell-free transcription and translation systems.

Translation of in vitro-synthesized herpes simplex virus type 2 (HSV-2) gG-2 mRNA in a reticulocyte lysate system was used to study the processing of HSV-2 gG-2. In the presence of canine pancreatic microsomal membranes, a single species that is protected from trypsin digestion was detected. This product comigrates with the 104,000-Mr (104K) high mannose intermediate seen in HSV-2-infected-cell lysates. Endo-beta-N-acetylglucosaminidase H treatment of the in vitro-synthesized 104K protein yielded a single product migrating at 100 K. The 72K and 31K cleavage products of gG-2 were not observed in the in vitro system. These data show that the molecular weight of the nonglycosylated form of the gG-2 protein is 100,000 and that the cotranslational processing of this protein in the endoplasmic reticulum yields the 104K high-mannose intermediate.

Transfection of normal and transformed hamster cerebral cortex glial cells with activated c-H-ras-1 results in the acquisition of a diffusely invasive phenotype.

We have examined the effect of activated c-Ha-ras oncogene on the invasive phenotype of normal and tumorigenic glial cells of Syrian hamster cerebral cortex derivation. T24 ras oncogene derived from a human bladder carcinoma was transfected into normal glial cells and minimally invasive tumorigenic glial cells produced by SV40 transformation. The normal cells acquired the capacity to grow in soft agar and become tumorigenic. With ras transfection, both the normal and minimally invasive SV40 tumor cells became diffusely invasive for adjacent normal brain. (N.B.: One cell line exhibited less extensive invasion than the others.) All of the transfected cell lines exhibited high levels of ras expression. These results indicate that ras can impart tumorigenic potential to normal glial cells and progress these or minimally invasive glial cells to full invasiveness.

Induction of the c-myc protooncogene after antigen binding to hapten-specific B cells.

The stimulation of hapten-specific B cell populations with the thymus-dependent antigen, TNP-KLH, was found to induce elevated levels of c-myc mRNA by 2 h. A similar treatment with carrier protein alone did not elevate c-myc mRNA above the level seen in the nonstimulated, resting B cells. These results indicate that antigen binding to the sIg receptor, in the absence of Th cell involvement, directly signals the antigen-binding cell and implicates the active participation of sIg during the process of antigen-mediated B cell activation.