Structural and functional similarities between the replication region of the Yersinia virulence plasmid and the RepFIIA replicons.

We sequenced the minimum replication region of the virulence plasmid pYVe439-80 from a serogroup O:9 Yersinia enterocolitica. This sequence is 68% homologous on a 1,873-nucleotide stretch to the sequence of the RepFIIA replicon of the resistance plasmid R100. The sequence contains two open reading frames, repA and repB, encoding proteins of 33,478 and 9,568 daltons, respectively. The amino acid sequences of the two proteins are 77 and 55% identical, respectively, to proteins RepA1 and RepA2 of the R100 replicon. Analysis of minicells transformed with a copy number mutant demonstrated that the replication region of pYVe439-80 directs the synthesis of a 33-kilodalton protein. Disruption of repA, encoding this protein, abolished replication. Two regions of pYVe439-80 are 76 and 70% homologous, respectively, to the copy number control antisense RNA and to the origin of replication region of R100. A mutation introduced in the pYVe439-80 DNA corresponding to the R100 sequence encoding the copy number control antisense RNA resulted in an increase in copy number, indicating a functional homology between the two replicons.     

Genetic map of the virulence plasmid of Salmonella enteritidis and nucleotide sequence of its replicons

Partial sequencing of a genomic library of the virulence plasmid of Salmonella enteritidis has been used to localize in the restriction map of this plasmid the genetic loci already described in other Salmonella plasmids. The comparison of the vestigial tra region with the corresponding genes in the F plasmid allowed us to define the extent of the deletions that the S. enteritidis plasmid should have suffered. The putative replicons of the plasmid, repB and repC, were isolated and both proved to be functional in Escherichia coli, but repC was segregationally unstable. The nucleotide sequence of repB showed the typical organization of RepFIIA replicons, although the similarity was lower than usual in this group of replicons. The highest homology was found with the replicon of the virulence plasmid pYVe439-80 from Yersinia enterocolitica (72.5%). Replicon repC also showed a maximum identity of 72.6% with known replicons, namely the RepFIB of pColV-K30 and P307, both virulence plasmids isolated from E. coli. We conclude that the S. enteritidis plasmid could arise from the S. typhimurium plasmid through deletions, and that they are evolutionary distant from other IncFI and IncFII plasmids.     

A new alpha-helical coiled coil protein encoded by the Salmonella typhimurium virulence plasmid.

A new protein of Salmonella typhimurium was identified and characterized. The gene (tlpA) encoding this protein (TlpA) was isolated from the large virulence-associated plasmid of S. typhimurium and sequenced in order to predict the primary structure of TlpA. tlpA encodes a 371-amino acid soluble protein with a calculated M(r) of 41600 and pI of 4.63. Secondary structure predictions and sequence statistics of TlpA indicated a predominant alpha-helical configuration and presence of heptapeptide repeat motifs characteristic of coiled coil proteins. Purified TlpA was shown to have biochemical properties similar to those of coiled coil proteins, including adoption of an alpha-helical configuration and a tendency to form homodimers. Furthermore, TlpA possessed heat resistance, evidence for a chain register and altered mobility in urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels which are characteristics of tropomyosins. TlpA shows 32% overall sequence similarity with rat cardiac myosin and 36% similarity with horse platelet beta-tropomyosin over 226 residues, whereas selected regions possessed significant sequence identities with myosins, tropomyosins, and alpha-helical surface proteins of Streptococcus pyogenes. Our results indicate that TlpA represents a new member of prokaryotic coiled coil proteins.     

Identification of contemporary plasmid virulence genes in ancestral isolates of Salmonella enteritidis and Salmonella typhimurium

Six epidemiologically distinct ancestral strains of Salmonella enteritidis and 5 of S. typhimurium from the pre-antibiotic era were examined for plasmid content, and for presence of plasmid genes implicated in mouse-virulence. Five sizes of plasmid were detected in S. enteritidis varying from 1 to 60 MDa. Two sizes of plasmid were found in S. typhimurium, 28 and 60 MDa. Plasmids of the same size were not common to both serovars. The HindIII restriction patterns of 3 of the ancestral S. enteritidis plasmids were identical to the modern 38 MDa plasmid, while all contained identical bands of 3.5, 2.7 and 1.9 kb. All the 60-MDa S. typhimurium plasmids, ancestral and contemporary, had an identical restriction pattern. Three different sized S. enteritidis plasmids and one size S. typhimurium plasmid contained a 3.5-kb DNA fragment carrying the virulence locus VirA. The VirB virulence locus was located on a 2.7-kb DNA fragment in S. enteritidis and on a 2.5-kb fragment in S. typhimurium. Both loci were precisely conserved between the ancestral strains and the modern representatives of both serovars.     

Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid

The 90-kilobase (kb) virulence plasmid of Salmonella typhimurium is responsible for invasion from the intestines to mesenteric lymph nodes and spleens of orally inoculated mice. We used Tn5 and aminoglycoside phosphotransferase (aph) gene insertion mutagenesis and deletion mutagenesis of a previously identified 14-kb virulence region to reduce this virulence region to 7.8kb. The 7.8-kb virulence region subcloned into a low copy-number vector conferred a wild-type level of splenic infection to virulence plasmid-cured S. typhimurium and conferred essentially a wild-type oral LD50. Insertion mutagenesis identified five loci essential for virulence, and DNA sequence analysis of the virulence region identified six open reading frames. Expected protein products were identified from four of the six genes, with three of the proteins identified as doublet bands in Escherichia coli minicells. Three of the five mutated genes were able to be complemented by clones containing only the corresponding wild-type gene. Only one of the five deduced amino acid sequences, that of the positive regulatory element, SpvR, possessed significant homology to other proteins. The codon usage for the virulence genes showed no codon bias, which is consistent with the low levels of expression observed for the corresponding proteins. Consensus promoters for several different sigma factors were identified upstream of several of the genes, whereas only consensus Rho-dependent termination sequences were observed between certain of the genes. The operon structure of this virulence region therefore appears to be complex. The construction of the cloned 7.8-kb virulence region and the determination of the DNA sequence will aid in the further genetic analysis of the five plasmid-encoded virulence genes of S. typhimurium.     

Isolation and characterization of plasmid mutations that enable partitioning of pSC101 replicons lacking the partition (par) locus.

Second-site mutations that allow stable inheritance of partition-defective pSC101 plasmids mapped to seven distinct sites in the 5' half of the plasmid repA gene. While the mutations also elevated pSC101 copy number, there was no correlation between copy number increase and plasmid stability. Combinations of mutations enabled pSC101 DNA replication in the absence of integration host factor and also stabilized par-deleted plasmids in cells deficient in DNA gyrase or defective in DnaA binding. Our findings suggest that repA mutations compensate for par deletion by enabling the origin region RepA-DNA-DnaA complex to form under suboptimal conditions. They also provide evidence that this complex has a role in partitioning that is separate from its known ability to promote plasmid DNA replication.     

Novel virulence properties of the Salmonella typhimurium virulence-associated plasmid: immune suppression and stimulation of splenomegaly

Mice infected subcutaneously with wild-type Salmonella typhimurium, SR11, developed a significant splenomegaly when compared with mice infected with an equal number of a plasmid-cured strain. Further, the bacterial load in the spleen at 14 days after infection, measured as colony-forming units per gram tissue, was significantly higher in mice infected with the parent strain than in mice infected with the plasmid-cured strain. These data confirm the previously reported plasmid-associated ability of Salmonella to multiply within the spleen. In addition, lymph node cells (LNC) from mice infected with the parent strain had a significantly reduced ability to proliferate in response to concanavalin A, a T-cell mitogen, and to heat-killed S. typhimurium cells when compared with LNC isolated from mice infected with the plasmid-cured strain. Finally, reintroduction of a functional Tn5-tagged 90-kb plasmid into a plasmid-free strain restored its capacity to cause a marked splenomegaly and to suppress lymph node cell proliferation in BALB/c mice. These data demonstrate that the 90-kb plasmid of highly virulent S. typhimurium strains mediates several novel pathogenic properties in infected mice: (1) enhancement of the ability of Salmonella to multiply within the spleen; (2) stimulation of a splenic inflammatory response as displayed by marked splenomegaly; and (3) a general suppression of lymphocyte responsiveness to both T-cell mitogens and specific Salmonella antigens.     

Intracellular Salmonella typhimurium induce lysis of human polymorphonuclear leukocytes which is not associated with the Salmonella virulence plasmid

The interaction between Salmonella typhimurium and human polymorphonuclear leukocytes (PMNs) was analyzed in vitro. Three S. typhimurium strains, the wild-type strain OU5043, its isogenic virulence plasmid-cured strain OU5048, and LT2, which represented the types that exhibited three mouse virulence levels, respectively, were used in this study. There was no correlation between the recovery of intracellular S. typhimurium from PMNs and the presence or absence of the virulence plasmid, or the strains' mouse virulence level. When the oxygen-dependent response of PMNs upon phagocytosis of S. typhimurium was examined by checking the intracellular reduction of nitroblue tetrazolium (NBT), the fraction of PMNs that reduced NBT on phagocytosis of the three strains was around 80%, whereas it was 58% with Escherichia coli, 95% with phorbol 12-myristate 13-acetate and 15% with a negative control. Thus there were no significant differences among the three Salmonella strains in terms of their ability to induce the oxidative response in PMNs. Microscopic analysis of Salmonella-infected PMNs indicated that the intracellular Salmonella induced lysis of PMNs. Both OU5043 and OU5048 exhibited a significant intracellular cytotoxic effect on PMNs after 24 hr of infection and this effect was not associated with the presence or absence of the virulence plasmid. On the other hand, lysis of PMNs was related to the intracellular survival of Salmnonella, as ofloxacin, an antibiotic, appeared to be able to protect human PMNs from Salmonella-induced cytotoxicity when this agent was added into the medium to inactivate the intracellular organism. The ability to induce lysis of PMNs by either wild-type or plasmid-cured strains of S. typhimurium may play a crucial role in the pathogenesis of non-typhoid Salmonella. The contribution of pSTV to human salmonellosis is likely to be limited. Furthermore, early institution of antibiotics with a high intracellular activity against Salmonella, such as fluoroquinolones, may be useful to prevent the dissemination of Salmonella infection.     

Molecular organization of genes constituting the virulence determinant on the Salmonella typhimurium 96 kilobase pair plasmid

The ability of intracellular growth is plasmid-dependent in Salmonella typhimurium. Only a small portion of this 96 kilobase pair plasmid appears essential for intracellular growth. The genetic organization of this region (the essential virulence determinant) was resolved. Fragments of the virulence determinant were cloned from the 96-kb plasmid pEX102 and transformed into minicell-producing E. coli. Plasmid-directed protein synthesis was investigated in metabolically labeled minicells. This analysis indicated the presence of at least four genes, mkaA, mkaB, mkaC and mkaD, within the virulence determinant encoding proteins of 70, 31, 30 and 29 kDa, respectively. The genes were positioned on the restriction map of the 96-kb virulence plasmid and the map locations confirmed by nucleotide sequence analysis of two new virulence genes (mkaB and mkaC).     

Viability and virulence of experimentally stressed nonculturable Salmonella typhimurium.

Maintenance of pathogenicity of viable but nonculturable Salmonella typhimurium cells experimentally stressed with UV-C and seawater, was investigated relative to the viability level of the cellular population. Pathogenicity, tested in a mouse model, was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by respiratory activity and cytoplasmic membrane and genomic integrities.     

Identification of a multimer resolution system involved in stabilization of the Salmonella dublin virulence plasmid pSDL2.

The Salmonella dublin virulence plasmid pSDL2 is a low-copy-number plasmid that is highly conserved in its host. Deletion of the 8-kb EcoRI C fragment downstream of the virulence region leads to plasmid instability and formation of multimers. We identified a multimer resolution system in the EcoRI C fragment composed of a trans-acting resolvase gene and a cis-acting resolution site. The resolvase gene, rsd, maps within a 2-kb EcoRV fragment and appears to be part of a multicistronic unit together with at least two other genes of unknown function. The derived protein, 28.7-kDa in size, is almost identical to the D protein of miniF. The C-terminal region was shown to have substantial similarity to the conserved C-terminal domains of the site-specific recombinases of the integrase family. The cis-acting resolution site, crs, is located upstream of rsd within a 628-bp SmaI-HpaI fragment. It contains eight direct incomplete 17-bp repeats followed by a segment rich in indirect repeats, the latter being homologous to the oriV1 sequence of miniF. crs contains the crossover site for specific recombination and mediates bidirectional promoter activity. A replicative function in analogy to that of oriV1 of F could not be demonstrated. The multimer resolution system was shown to stabilize pACYC184 and is dependent on the recA-mediated formation of multimeric plasmids. Screening different Salmonella serovars with a pSDL2-specific recombination assay revealed that only strains harboring a virulence plasmid encode for resolvase activity. Our results suggest that site-specific recombination contributes to the stable inheritance of pSDL2 and other Salmonella virulence plasmids.     

Two replication regions in the pJM1 virulence plasmid of the marine pathogen Vibrio anguillarum

Vibrio anguillarum is a fish pathogen that causes vibriosis, a serious hemorrhagic septicemia, in wild and cultured fish. Many serotype O1 strains of this bacterium harbor the 65kb plasmid pJM1 carrying the majority of genes encoding the siderophore anguibactin iron transport system that is one of the most important virulence factors of this bacterium. We previously identified a replication region of the pJM1 plasmid named ori1. In this work we determined that ori1 can replicate in Escherichia coli and that the chromosome-encoded proteins DnaB, DnaC and DnaG are essential for its replication whereas PolI, IHF and DnaA are not required. The copy number of the pJM1 plasmid is 1-2, albeit cloned smaller fragments of the ori1 region replicate with higher copy numbers in V. anguillarum while in E. coli we did not observe an obvious difference of the copy numbers of these constructs which were all high. Furthermore, we were able to delete the ori1 region from the pJM1 plasmid and identified a second replication region in pJM1 that we named ori2. This second replication region is located on ORF25 that is within the trans-acting factor (TAFr) region, and showed that it can only replicate in V. anguillarum.     

Characterization of the traT gene and mutants that increase outer membrane permeability from the Salmonella typhimurium virulence plasmid

The nucleotide sequence of the traT gene present in the virulence-associated plasmid of Salmonella typhimurium was determined. The predicted TraT protein encoded by this gene was found to consist of 243 amino acids and to resemble the known TraT proteins of the plasmids of the F incompatibility group. Thus it contains a signal sequence of 20 amino acids, an amino-terminal lipid attachment site, and two strongly hydrophobic regions close to each other in the mature protein. A mutation leading to increased permeability of the outer membrane to hydrophobic agents, previously localized to the traT gene, was shown to change a glycine residue to arginine within one of these hydrophobic regions. The same principle was found to apply to TraT of R6-5: the introduction, by site-directed mutagenesis, of either positively or negatively charged amino acids or the helix-disrupting proline in the corresponding hydrophobic region led to increased hydrophobic permeability of the outer membrane.     

The plasmid of Salmonella typhimurium LT2

Summary Methods of clonal analysis were applied to the study of heterogeneity of the progeny after crosses of 4 donor strains (Hfr H, Hfr C, KL 16 and KL 99) with 3 recipient strains (PC 0212, AB 712 and ECK 022). Three markers were used in each cross. The distal one was the selective marker. The inheritance of two additional proximal markers characterized the heterogeneity of clones originating from particular zygotes. In most crosses the percentage of heterogeneity exceeded 30. One of the recipient strains, obtained by conjugation of the conventional strain PC 0212 with the donor Hfr H revealed unusual properties in respect to heterogeneity. Exconjugants derived from this recipient (ECK 022) and donor Hfr H and Hfr C had a heterogeneity index of about 5%. It is shown that this unusual behavior reflects a very fast process of segregation of recombinants.In crosses with the donors KL 16 and KL 99 the same recipient revealed normal indices of heterogeneity. All these data are explained assuming that there exists a specific genetic marker which determines the process of decay of merozygotes. Tentatively it is called het. Its approximate localization was deduced from specifically designed experiments, in which the heterogeneity of the progeny was found very different, when the donor KL 16 transmitted different parts of its chromosome to the recipient ECK 022.     

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.