Stress induction of the virulence proteins (SpvA, -B, and -C) from native plasmid pSDL2 of Salmonella dublin.

The virulence region of the wild-type plasmid pSDL2 contained in Salmonella dublin is highly conserved among plasmids from several nontyphoid Salmonella serotypes and is essential for the development of systemic infection in BALB/c mice. Polyclonal antibodies against three proteins (SpvA, -B, and -C) expressed from a 4.1-kb EcoRI subclone of the plasmid virulence region were generated. These antibodies were used to detect expression of the Spv proteins when S. dublin was grown in vitro under stress-inducing conditions, such as nutrient deprivation and increased temperature, that the bacteria may encounter during the course of infection within the host. Glucose starvation resulted in expression of all three proteins shortly after the lag phase. When the bacteria were grown to the late-log phase without glucose, heat shock strongly induced expression of SpvA but not SpvB or SpvC. The addition of 0.2% glucose to the medium resulted in loss of expression of the proteins until the late-log to stationary phase. Iron limitation or lowered pH induced expression of the proteins during exponential growth even in the presence of glucose. Insertion mutations into the positive regulator gene spvR upstream from spvABC and insertions into spvA and spvC resulted in loss of expression of SpvA, -B, and -C, suggesting a complex regulation of expression. These studies define a variety of environmental conditions that induce expression of the Spv virulence proteins from the wild-type plasmid pSDL2 in S. dublin in vitro.

Tn1725 transposon mutagenesis of 9-18 delta 7, an EcoRI deletion derivative of Salmonella dublin lane plasmid pSDL2.

A 37.5-kb derivative of the Salmonella dublin virulence plasmid pSDL2 was subjected to mutagenesis with the transposon Tn1725. Fifty-two insertions were mapped, and the mutants were tested for their ability to restore virulence to a plasmid-free strain of S. dublin. Twenty-nine of these inserts could not restore full virulence and thus define nine regions of the plasmid essential for virulence. Deletion of a 4.5-kb region by Bal31 nuclease resulted in a 33-kb derivative that maintained full virulence.

Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane-spanning sequences using a novel alkaline phosphatase expression vector.

Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence-encoding region. Library construction with Sau3A-digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum-AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non-cleavable hydrophobic membrane-spanning sequences. Triton X-114 detergent phase partitioning of individual T. pallidum-AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]-palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N-terminal signal sequence containing a consensus leader peptidase II recognition site. The DNA sequence of Tp75 also indicates that this is a previously unreported T. pallidum lipoprotein. T. pallidum-AP fusion proteins which partitioned into the hydrophobic detergent phase but did not incorporate palmitate were also identified. DNA and amino acid analysis of one such clone, Tp70, showed no cleavable signal but had a significant hydrophobic region of approximately 20 residues, consistent with a membrane-spanning domain. Immunoblot analysis of T. pallidum-AP fusions detected with a monoclonal antibody specific for AP identified several fusion proteins which migrated as doublets separated in apparent electrophoretic mobility by no more than 3 kDa. [35S]-methionine pulse-chase incorporation showed that the doublet AP fusions represented precursor and processed forms of the same protein. DNA and amino acid sequence analysis of clones expressing processed fusion proteins demonstrated hydrophobic N-terminal signal sequences containing consensus leader peptidase I recognition sites.

Characterization of three proteins expressed from the virulence region of plasmid pSDL2 in Salmonella dublin.

Infection of both cattle and humans with Salmonella dublin can result in septicemia and death. Like many nontyphoid Salmonella species that cause disease, S. dublin contains a cryptic plasmid (pSDL2) that is required for the full expression of virulence. Transposon mutagenesis of pSDL2 defined a 4.1-kb EcoRI region that is necessary for the development of a systemic infection in BALB/c mice. This EcoRI fragment was cloned into an expression vector (pEL11), and three proteins produced from this region with apparent molecular weights of 30,500, 76,000, and 27,000 were identified. Because bacterial proteins that play a role in virulence are often associated with the outer membrane, we were interested in establishing whether the proteins expressed from the EcoRI fragment are located in the membrane. Transposon mutagenesis of pEL11 with TnphoA defined the order of the genes along the fragment and suggested that the proteins may be exported out of the cytoplasm. Sucrose gradient cell fractionation was done to identify the cellular location of each of the three proteins. The 30-kDa protein was identified in the outer membrane fraction, and the 76-kDa protein was located in the cytosolic fraction. The 27-kDa protein was identified in both the cytosolic and the outer membrane fractions. The outer membrane contained less than 10% of the activity of enzymes known to be located in the cytoplasm, periplasm, and inner membrane. Sequence data of the 4.1-kb EcoRI region revealed that both the 30- and the 27-kDa proteins lack a typical signal sequence for export out of the cytoplasm (M. Krause, C. Roudier, J. Fierer, J. Harwood, and D. G. Guiney, Mol. Microbiol. 5:307, 1991). The outer membrane location of these proteins suggests that they may be exported out of the cytoplasm by an unusual mechanism.

Plasmid-mediated virulence in Salmonella dublin demonstrated by use of a Tn5-oriT construct.

Salmonella dublin, a serotype which causes invasive disease in cattle and humans, carries a characteristic 80-kilobase plasmid (pSDL2). We were able to cure the plasmid from a strain of S. dublin. The cured strain was avirulent for mice by either the oral or intraperitoneal route of infection. A derivative of Tn5 which contains the transfer origin of the broad-host-range plasmid RK2 (Tn5-oriT) was transposed onto pSDL2, allowing mobilization of the plasmid by an RK2 helper plasmid. Reintroduction of the pSDL2 derivative plasmid into the cured strain restored virulence, demonstrating that the plasmid is necessary for virulence. These studies also demonstrate the usefulness of the Tn5-oriT construct for genetic manipulations.

Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication.

We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolated from several different countries. Two regions of RK2, the best-studied plasmid of this group, are required for vegetative DNA replication: the origin of replication (oriV) and the trfA region, which codes for a gene product necessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA regions as shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology with the RK2 trfA, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751, R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility but differed markedly from RK2 and the other six plasmids in the GC-rich region of the origin required for replication. These data indicate that the P-group plasmids can be divided into two subgroups: IncP alpha, which includes the RK2-like plasmids, and IncP beta which includes the R751-like plasmids.