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.     

Pesticin displays muramidase activity.

Pesticin of Yersinia pestis is the only bacteriocin that converts sensitive cells to stable spheroplasts. The amino acid sequence of pesticin as derived from the nucleotide sequence shows no similarity to those of any of the bacteriocins. The unique properties of pesticin prompted an investigation of its mode of action. Since the pesticin plasmid does not encode a lysis protein for release of pesticin into the culture medium, pesticin was isolated from cells and purified to electrophoretic homogeneity. Highly purified pesticin degraded murein and murein glycan strands lacking the peptide side chains to products that were similar to those obtained by lysozyme, as revealed by high-resolution high-pressure liquid chromatography. After reduction of the murein degradation products with tritium-labeled sodium borohydride, acid hydrolysis, and separation of the products by thin-layer chromatography, radiolabeled muraminitol was identified. This indicates that pesticin is a muramidase, and not an N-acetyl-glucosaminidase, that converts cells into stable spheroplasts by slowly degrading murein.     

Restriction map of pesticinogenicity plasmid pYP1 of Yersinia pestis

The restriction map of Yersinia pestis pesticinogenicity plasmid pYP1 has been constructed with the use of 18 restriction endonucleases. Plasmid dimensions (6.3 Md) have been specified, the genes for pesticin synthesis, for pesticin immunity protein, fibrinolysin and plasmocoagulase have been localized by molecular cloning of single plasmid DNA fragments in vector plasmid pBR322.     

Detection and characterization of the plasmids of the plague microbe which determine the synthesis of pesticin I, fraction I antigen and "mouse" toxin exotoxin

Plasmid DNA was isolated from Yersinia pestis strains containing pesticin I or fraction I antigen and "mouse" toxin determinants. Specificity of DNA preparations was studied by using them for transformation of plague agent strains carrying no plasmids. pPstI plasmid (molecular weight 7,0-7,8 MD) encoded pesticin I, fibrinolysin and plasmacoagulase synthesis. Fraction I antigen and "mouse" toxin production determinants were borne on pFraI/Tox plasmid (molecular weight about 50 MD). The observation that some Y. pestis cultures, having lost the ability to synthesize one of pFraI/Tox products, still retained this plasmid in their cells, is regarded as an evidence for a complicated regulation of pFraI/Tox function.     

Homology with a repeated Yersinia pestis DNA sequence IS100 correlates with pesticin sensitivity in Yersinia pseudotuberculosis.

We have identified IS100 sequences in a specific subset of Yersinia pseudotuberculosis isolates that were also sensitive to the Y. pestis-produced bacteriocin, pesticin. In contrast, Y. pseudotuberculosis strains which did not contain IS100 sequences were not sensitive to pesticin. We propose that IS100 serves as a molecular marker that identifies a subset of Y. pseudotuberculosis isolates that have a particularly close evolutionary and/or ecological relationship with Y. pestis.     

Periplasmic location of the pesticin immunity protein suggests inactivation of pesticin in the periplasm.

The pesticin activity and immunity genes on plasmid pPCP1 of Yersinia pestis were sequenced. They encoded proteins of 40 kDa (pesticin) and 16 kDa (immunity protein); the latter was found in the periplasm. The location of the immunity protein suggests that imported pesticin is inactivated in the periplasm before it hydrolyzes murein. Pesticin contains a TonB box close to the N-terminal end that is identical to the TonB box of colicin B. The DNA sequences flanking the pesticin determinant were highly homologous to those flanking the colicin 10 determinant. It is proposed that through these highly homologous DNA sequences, genes encoding bacteriocins may be exchanged between plasmids by recombination. In the case of pesticin, recombination may have destroyed the lysis gene, of which only a rudimentary fragment exists on pPCP1.     

Cloning and study of the function of recA-like gene of Yersinia pestis in Escherichia coli cells

A 2 kb fragment of Yersinia pestis genome cloned in Escherichia coli cells of the strain HB101 contains a gene able to complement the recA-dependent deficiency of E. coli cells in UV-resistance, resistance to alkylating agents, UV- and MNNG-induced mutability. Cellular capability for homologous recombination in crosses with HfrH donor, derepressed synthesis of bacteriocins (colicin E1 and pesticin 1) is also complemented by the fragment in E. coli recA-strains. The obtained data suggest the functional homology of the cloned recA-like gene product with the product of E. coli recA-gene.     

Structural and mechanistic studies of pesticin, a bacterial homolog of phage lysozymes

Yersinia pestis produces and secretes a toxin named pesticin that kills related bacteria of the same niche. Uptake of the bacteriocin is required for activity in the periplasm leading to hydrolysis of peptidoglycan. To understand the uptake mechanism and to investigate the function of pesticin, we combined crystal structures of the wild type enzyme, active site mutants, and a chimera protein with in vivo and in vitro activity assays. Wild type pesticin comprises an elongated N-terminal translocation domain, the intermediate receptor binding domain, and a C-terminal activity domain with structural analogy to lysozyme homologs. The full-length protein is toxic to bacteria when taken up to the target site via the outer or the inner membrane. Uptake studies of deletion mutants in the translocation domain demonstrate their critical size for import. To further test the plasticity of pesticin during uptake into bacterial cells, the activity domain was replaced by T4 lysozyme. Surprisingly, this replacement resulted in an active chimera protein that is not inhibited by the immunity protein Pim. Activity of pesticin and the chimera protein was blocked through introduction of disulfide bonds, which suggests unfolding as the prerequisite to gain access to the periplasm. Pesticin, a muramidase, was characterized by active site mutations demonstrating a similar but not identical residue pattern in comparison with T4 lysozyme.     

Connection of outer membrane protein composition in Yersinia pestis cells with intrinsic plasmids]

A set of isogenic derivatives of Yersinia pestis EV strain was obtained including the variants harbouring the different compositions of Yersinia own plasmids. The protein profiles of outer membranes of the set of strains were defined. The polyacrylamide gel electrophoresis has shown the small 6.1 Md plasmid to code an outer membrane protein with mol mass 29 kDa, different from pesticin I, while the heavy 60.0 Md plasmid encodes the 15-16 kDa polypeptide different from monomers of F1 and T-antigens of plague microbe.     

Evidence for two evolutionary lineages of highly pathogenic Yersinia species.

Sensitivity to Yersinia pestis bacteriocin pesticin correlates with the existence of two groups of human pathogenic yersiniae, mouse lethal and mouse nonlethal. The presence of the outer membrane pesticin receptor (FyuA) in mouse-lethal yersiniae is a prerequisite for pesticin sensitivity. Genes that code for FyuA (fyuA) were identified and sequenced from pesticin-sensitive bacteria, including Y. enterocolitica biotype 1B (serotypes O8; O13, O20, and O21), Y. pseudotuberculosis serotype O1, Y. pestis, two known pesticin-sensitive Escherichia coli isolates (E. coli Phi and E. coli CA42), and two newly discovered pesticin-sensitive isolates, E. coli K49 and K235. A 2,318-bp fyuA sequence was shown to be highly conserved in all pesticin-sensitive bacteria, including E. coli strains (DNA sequence homology was 98.5 to 99.9%). The same degree of DNA homology (97.8 to 100%) was established for the sequenced 276-bp fragment of the irp2 gene that encodes high-molecular-weight protein 2, which is also thought to be involved in the expression of virulence by Yersinia species. Highly conserved irp2 was also found in all pesticin-sensitive E. coli strains. On the basis of the fyuA and irp2 sequence homologies, two evolutionary groups of highly pathogenic Yersinia species can be established. One group includes Y. enterocolitica biotype 1B strains, while the second includes Y. pestis, Y. pseudotuberculosis serotype O1, and irp2-positive Y. pseudotuberculosis serotype O3 strains. E. coli Phi, CA42, K49, and K235 belong to the second group. The possible proximity of these two iron-regulated genes (fyuA and irp2), as well as their high levels of sequence conservation and similar G+C contents (56.2 and 59.8 mol%), leads to the assumption that these two genes may represent part of an unstable pathogenicity island that has been acquired by pesticin-sensitive bacteria as a result of a horizontal transfer.     

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.     

Specific proteolysis by fibrinolysin-coagulase from Yersinia pestis of Yersinia pseudotuberculosis outer membrane proteins coded by the Ca(2+)-dependence plasmid]

The pesticinogenicity 9.5 kb plasmid from Yersinia pestis strain EV76 has been marked by the kanamycin phosphotransferase gene inserted into PstI site and designated pP3. The obtained plasmid pP3 determines the synthesis of 45 kd pesticin, alpha and beta-forms of fibrinolysin coagulase (37 and 35 kd) and the 29, 19 and 13 kd proteins in Escherichia coli mini cells. When transferred into Yersinia pseudotuberculosis strain 6933 the plasmid causes the proteolysis of outer membrane proteins. The 150 kd protein is reduced to 138 kd, the 48.5 kd protein is reduced to 45 kd. The proteins secreted into the cultural medium (51 and 38 kd) are also cleaved. The proteolysis of the 150 kd protein was found to occur at the stage of secretion via the inner membrane. The purified fibrinolysin coagulase from Escherichia coli strain JM83 harbouring the plasmid pP3 induces the proteolysis in vitro of the isolated membrane proteins from Yersinia pseudotuberculosis strain 6953 similar to the proteolysis registered in vivo.     

Mode of action of pesticin: N-acetylglucosaminidase activity.

Homogeneous preparations of pesticin, a bacteriocin produced by Yersinia pestis, neither significantly inhibited net synthesis of deoxyribonucleic acid, ribonucleic acid, or protein in Escherichia coli phi nor caused detectable degradation of deoxyribonucleic acid in vivo. Accordingly, its mode of action does not resemble that of colicin E2 as suggested by others. However, incorporation of cell wall-specific label ([14C]diaminopimelic acid) into trichloroacetic acid-insoluble material of growing cells was inhibited by pesticin which also promoted release of such radioactivity from both resting cells and purified mureinlipoprotein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reaction mixtures containing appropriately labeled mureinlipoprotein showed that [3H]N-acetylglucosamine comigrated either with [14C]diaminopimelic acid in the murein peptide or with [14C]isoleucine of the Braun lipoprotein. As judged by these findings and pesticin-dependent release of reducing equivalents but not 4-hydroxy-2-acetamido sugars, the bacteriocin possesses N-acetylglucosaminidase activity. Hydrolysis of murein-lipoprotein occurred over a broad pH, with an optimum of 4.7. Mureinlipoproteins from a variety of pesticin-sensitive and -resistant organisms were hydrolyzed by the bacteriocin, indicating that its antibacterial specificity resides at the level of absorption.     

Plasmid-like properties of the four virulence-associated factors of Yersinia pestis

The virulence-associated factors of Yersinia pestis, which determine the abilities to produce pesticin I (Pst+), capsular fraction I antigen (Fra+), V and W antigen complex (Vwa+) and a cell-surface component for adsorption of exogenous pigments (Pgm+), were independently eliminated by cultivation of the cells in the presence of acridine orange, ethidium bromide or sodium dodecyl sulfate at a subinhibitory concentration. A virulent Y. pestis strain, Yreka, harbored at least five extrachromosomal DNA molecules of different sizes. In these molecules, a novel 13-megadalton DNA which was cured concomitantly with the elimination of the Fra factor was found, in addition to the known species of 7 and 44 megadaltons which were lost with the conversions to Pst- and Vwa-, respectively. Although the conversion to Pgm- could not be correlated with the lack of any proper extra-chromosomal DNA, the factor was transmitted to Pgm- cells with the aid of self-conjugative RP4 plasmid. The cells acquiring the Pgm factor regained virulence for mice.     

The effect of the bacterial genome on the expression and behavior of the plasmid determining the Ca2+-dependence of the plague pathogen]

The conjugative cointegrate containing the 47 Md plasmid of Yersinia pestis has been transferred into the strains of the different Yersinia (Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica) and Escherichia coli CA. There appeared in the populations of recombinant Yersinia under the conditions of Ca2+ deficit at 37 degrees C the cells coming into the stasis stage or dying. It was shown on the model of Yersinia enterocolitica that bacterial lethality might be prevented by exclusion of the sheep blood from Ca2+ deficient medium. Ca(2+)-dependence was not expressed in Escherichia coli cells in which the cointegrates were prone to deletions although the cad-genes were preserved intact. The latter conclusion is based on the positive reciprocal transfer of the Cad(+)-marker into Yersinia pestis cells.     

Protein expression of Yersinia pestis during changes due to long-term cultivation in vivo]

The electrophoretic study of Yersinia pestis proteins made possible to find the significant modification of Yersinia pestis polypeptide specters when the bacteria were cultivated in semi-penetrable cells implanted into the guinea pigs peritoneum. The proteinogramms of the isolates from the implanted cells lacked the stained bands characteristic of Yersinia pestis cells grown in vitro and contained the new polypeptides absent from the bacteria grown on the Hottinger agar plates. The difference was found at the late stage of bacteria incubation in implanted cells and had the predominantly reversible characteristics. The protein of Yersinia pestis being changed in vivo is proposed to be the species specific fraction I.     

Antigenic profiling of yersinia pestis infection in the Wyoming coyote (Canis latrans)

Although Yersinia pestis is classified as a "high-virulence" pathogen, some host species are variably susceptible to disease. Coyotes (Canis latrans) exhibit mild, if any, symptoms during infection, but antibody production occurs postinfection. This immune response has been reported to be against the F1 capsule, although little subsequent characterization has been conducted. To further define the nature of coyote humoral immunity to plague, qualitative serology was conducted to assess the antiplague antibody repertoire. Humoral responses to six plasmid-encoded Y. pestis virulence factors were first examined. Of 20 individual immune coyotes, 90% were reactive to at least one other antigen in the panel other than F1. The frequency of reactivity to low calcium response plasmid (pLcr)-encoded Yersinia protein kinase A (YpkA) and Yersinia outer protein D (YopD) was significantly greater than that previously observed in a murine model for plague. Additionally, both V antigen and plasminogen activator were reactive with over half of the serum samples tested. Reactivity to F1 was markedly less frequent in coyotes (35%). Twenty previously tested antibody-negative samples were also examined. While the majority were negative across the panel, 15% were positive for 1-3 non-F1 antigens. In vivo-induced antigen technology employed to identify novel chromosomal genes of Y. pestis that are up-regulated during infection resulted in the identification of five proteins, including a flagellar component (FliP) that was uniquely reactive with the coyote serum compared with immune serum from two other host species. Collectively, these data suggest that humoral immunity to pLcr-encoded antigens and the pesticin plasmid (pPst)-encoded Pla antigen may be relevant to plague resistance in coyotes. The serologic profile of Y. pestis chromosomal antigens up-regulated in vivo specific to C. latrans may provide insight into the differences in the pathogen-host responses during Y. pestis infection.     

The effect of Yersinia pestis EV76 6 MD plasmid on the composition of outer membrane proteins of Yersinia pseudotuberculosis YPIII]

On the basis of Yersinia pseudotuberculosis strain YPIII the isogenic variants containing the different combinations of 47 Md plasmids from Yersinia pestis or Yersinia pseudotuberculosis cells with the 6 Md pYP plasmid from Yersinia pestis EV (intact or having impaired the pla gene determining the synthesis of plasmocoagulase). The degradation of the secreted proteins encoded by the 47 Md plasmids of Yersinia pestis and Yersinia pseudotuberculosis in the cells harbouring the 6Md pYP plasmid has been registered. Yersinia pseudotuberculosis strain YPIII carrying its own 47Md and pYP plasmids also contained no YOP1 protein, in contract to the parent strain. The damage of the pla gene eliminated the destructive effect on the outer membrane proteins. Imposition of the 47Md and 6Md plasmids from Yersinia pestis in Yersinia pseudotuberculosis cells may be used for obtaining and study of the physiological role of low molecular mass proteins resulting from proteolysis of proteins encoded by the 47Md virulence plasmid of Yersinia.