TnphoA Insertions Research Articles

Growth and colonization suppression of Salmonella enterica serovar Hadar in vitro and in vivo

Growth suppression in Salmonella enterica serovar Hadar (S. Hadar) was investigated, in vitro under strict anaerobiosis and in vivo in the intestine of the day-old chicken. Stationary-phase cultures of 20 S. Hadar field strains were tested against each other for growth suppression activity by their ability to suppress the multiplication of low counts of minority cultures inoculated into them as nalidixic acid-resistant mutants. All strains showed profound growth suppression. Four S. Hadar strains were selected and further tested for their ability to suppress growth of S. Enteritidis, S. Typhimurium, S. Virchow and S. Saintpaul. One of the four strains (S. Hadar 18) was randomly selected for further studies. Precolonization of chicken with S. Hadar 18 prevented superinfection with any of the serovars mentioned above. From more than 1000 TnphoA mutants of S. Hadar 18 screened against the parent strain anaerobically in vitro, four were non-suppressive with TnphoA insertions in dapF, aroD, sgaT or tatA. Only the dapF mutant was also non-suppressive in the chicken intestine.

Growth and colonization suppression of Salmonella enterica serovar Hadar in vitro and in vivo.

Growth suppression in Salmonella enterica serovar Hadar (S. Hadar) was investigated, in vitro under strict anaerobiosis and in vivo in the intestine of the day-old chicken. Stationary-phase cultures of 20 S. Hadar field strains were tested against each other for growth suppression activity by their ability to suppress the multiplication of low counts of minority cultures inoculated into them as nalidixic acid-resistant mutants. All strains showed profound growth suppression. Four S. Hadar strains were selected and further tested for their ability to suppress growth of S. Enteritidis, S. Typhimurium, S. Virchow and S. Saintpaul. One of the four strains (S. Hadar 18) was randomly selected for further studies. Precolonization of chicken with S. Hadar 18 prevented superinfection with any of the serovars mentioned above. From more than 1000 TnphoA mutants of S. Hadar 18 screened against the parent strain anaerobically in vitro, four were non-suppressive with TnphoA insertions in dapF, aroD, sgaT or tatA. Only the dapF mutant was also non-suppressive in the chicken intestine.

Bacterial genetic loci implicated in the Pseudomonas putida GR12-2R3--canola mutualism: identification of an exudate-inducible sugar transporter.

Pseudomonas putida GR12-2R3 promotes the emergence and growth of diverse plant species. Analyses of TnphoA insertion mutations are revealing bacterial characteristics pertinent to the plant-microbe interaction. Pseudomonas putida PG269 is a TnphoA insertion derivative of GR12-2R3 that expresses canola seed exudate-inducible alkaline phosphatase (PhoA) activity. It promoted the growth of canola roots, as well as strain GR12-2R3, and outgrew its parent when they were cocultured in the presence of canola roots or in liquid seed exudate medium. (In contrast, mutant PG126 failed to promote canola root growth and was outgrown by its parent strain.) The PhoA activity of strain PG269 was induced by glucosamine and other sugars; glucosamine inhibited the growth of strain GR12-2R3 and stimulated the growth of strain PG269. Strain PG269 contained two TnphoA insertions: seiA1::TnphoA and seiB1::TnphoA. Strain PG312, which contained only insertion seiA1::TnphoA, shared all aspects of the PG269 phenotype, except the ability to outcompete strain GR12-2R3 during coculture. Insertion seiA1::TnphoA interrupted an open reading frame related in sequence to members of the MalF family of sugar transporter subunits. The PhoA-inducing fraction of canola seed exudate was hydrophilic, low in molecular weight, and heat stable. It cochromatographed with basic amino acids and amino sugars, and was inactivated by strains GR12-2R3 and PG269. Gene seiA may encode a subunit of an ABC transporter with broad specificity for glucose and related sugars whose expression can be induced by exudate sugars.

Defective export in Escherichia coli caused by DsbA'-PhoA hybrid proteins whose DsbA' domain cannot fold into a conformation resistant to periplasmic proteases.

The disulfide bond-forming factor DsbA and the alkaline phosphatase are stable in the Escherichia coli periplasmic space and can be overproduced without significant perturbation of the cell's physiology. By contrast, DsbA'-PhoA hybrid proteins resulting from TnphoA insertions into different regions of a plasmid-borne dsbA gene could become toxic (lethal) to bacteria. Toxicity was concomitant with an impairment of some step of the export mechanism and depended on at least three parameters, i.e., (i) the rate of expression of the hybrid protein, (ii) the ability of the amino-terminal DsbA' domain of the hybrid protein to fold into a protease-resistant conformation in the periplasmic space, and (iii) the activity of the DegP periplasmic protease. Even under viable conditions of low expression, DsbA' folding-deficient hybrid proteins accumulated more than the folding-proficient ones in the insoluble material and this was aggravated in a strain lacking the DegP protease. When production was more elevated, the folding-deficient hybrid proteins became lethal, but only in strains lacking the DegP activity, while the folding-proficient ones were not. Under conditions of very high production by degP+ or degP strains, both types of hybrid proteins accumulated as insoluble preproteins. Meanwhile, the export machinery was dramatically handicapped and the cells lost viability. However, the folding-deficient hybrid proteins had a higher killing efficiency than the folding-proficient ones. Free DsbA'-truncated polypeptides, although not toxic, were processed more slowly when they could not fold into a protease-resistant form in the periplasmic space. This provides indications in E. coli for a direct or indirect influence of the folding of a protein in the periplasmic environment on export efficiency.

Expression of the exoY gene, required for exopolysaccharide synthesis in Agrobacterium, is activated by the regulatory ros gene.

Some mutants of Agrobacterium radiobacter, defective in exopolysaccharide synthesis, were phenotypically complemented by two different regions of cloned chromosomal DNA. One of these had been shown to contain a gene termed ros, a novel class of transcriptional regulator. The other contains a gene termed exoY which encodes a glycosyltransferase that is involved in one of the early steps in exopolysaccharide synthesis. Mutations in ros reduced the expression of exoY and a model to account for the complementation of certain exo alleles by both ros and exoY is presented. TnphoA insertions into exoY which expressed alkaline phosphatase activity were isolated and mapped, confirming the membrane location of the exoY gene product. Some of these mutations were dominant, causing merodiploids to be non-mucoid. exoY is linked to two genes, one encoding an omega-aminotransferase and the other encoding an aldehyde dehydrogenase.

A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus

Enteropathogenic Escherichia coli (EPEC) adhere to epithelial cells in microcolonies, a pattern termed localized adherence (LA). LA is dependent upon the presence of 50-70 MDa plasmids, termed EPEC adherence factor (EAF) plasmids. Expression of an EAF plasmid-encoded type IV fimbria, the bundle-forming pilus (BFP), is associated with the LA phenotype. TnphoA insertions in bfpA, the gene encoding the major structural subunit of the BFP, abolish LA. While bfpA::TnphoA mutants cannot be complemented for LA by plasmids carrying the bfpA gene alone in trans, this work shows that they can be complemented by plasmids carrying the bfpA gene, as well as approximately 10 kb of downstream sequence, suggesting that such mutations have polar effects on downstream genes. The identification and characterization of a cluster of 13 genes immediately downstream of bfpA are described. The introduction into a laboratory Escherichia coli strain of a plasmid containing these 14 bfp gene cluster genes, along with pJPN14, a plasmid containing another fragment derived from the EAF plasmid, confers LA ability and BFP biogenesis. However, when a mutation is introduced into the last gene of the bfp cluster, neither LA nor BFP biogenesis is conferred. This work also provides evidence to show that the fragment cloned in pJPN14 encodes a factor(s) which results in increased levels of the pilin protein. Finally, it is shown that expression of the 14 genes in the bfp cluster from an IPTG-inducible promoter, in the absence of pJPN14, is sufficient to reconstitute BFP biogenesis in a laboratory E. coli strain, but is insufficient for LA. This is the first report demonstrating the reconstitution of a type IV pilus in a laboratory E. coli strain with a defined set of genes. The BFP system should prove to be a useful model for studying the molecular mechanisms of type IV pilus biogenesis.

TnphoA-induced symbiotic mutants of Bradyrhizobium japonicum that impair cell and tissue differentiation in Glycine max nodules

Bradyrhizobium japonicum WM1, an ethylmethane-sulfonate-induced derivative of B. japonicum 110spc4 with reduced phosphatase activity but normal symbiotic properties, was randomly mutagenized using TnphoA. From about 1000 purified single colonies, approximately 300, preferentially those with enhanced phosphatase activity, were inoculated onto soybean seedlings to test their symbiotic traits. Sixteen strains were either completely Fix− or possessed markedly reduced acetylene reduction activity (Fixred). Contrary to expectations, hybridization of total DNA of these strains to a transposonspecific DNA probe showed that many contained no transposon. Apparently these strains had gained resistance towards kanamycin spontaneously rather than through the introduction of TnphoA. However, in five mutant strains, two hybridizing BamHI fragments of different sizes were detected, as expected. All strains performed acetylene reduction under ex planta conditions, indicating that mutations had not occurred in nif or fix genes. A more than 50-fold increased specific activity of alkaline phosphatase was observed in strain 132, indicating the synthesis and secretion of a polypeptide fused to 'PhoA. Light and electron-microscopic analyses showed that in nodules induced by strain 132 (Fixred) the infected cells of the central tissue were vacuolated. In some of these cells callose deposition was observed, indicating plant defense reactions. Nodules induced by mutant 184 were infected by bacteroids only in a few cells of the central tissue as isolated clusters, whereas the majority of cells remained uninfected. The concentration of phosphoenolpyruvatecarboxylase protein within the infected tissue was significantly reduced and starch granules accumulated. In both strains TnphoA insertions were identified to be the reasons for the observed phenotypes. These mutant strains should be helpful for studying the influence of the microsymbiont on the differentiation and colonization of infected cells in soybean nodules.

Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri

The rfb region of Shigella flexneri encodes the proteins required to synthesize the O-antigen component of its cell surface lipopolysaccharides (LPS). We have previously reported that a region adjacent to rfb was involved in regulating the length distribution of the O-antigen polysaccharide chains (D. F. Macpherson et al., Mol. Microbiol. 5:1491-1499, 1991). The gene responsible has been identified in Escherichia coli O75 (called rol [R. A. Batchelor et al., J. Bacteriol. 173:5699-5704, 1991]) and in E. coli O111 and Salmonella enterica serovar typhimurium strain LT2 (called cld [D. A. Bastin et al., Mol. Microbiol. 5:2223-2231, 1991]). Through a combination of subcloning, deletion, and transposon insertion analysis, we have identified a gene adjacent to the S. flexneri rfb region which encodes a protein of 36 kDa responsible for the length distribution of O-antigen chains in LPS as seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels. DNA sequence analysis identified an open reading frame (ORF) corresponding to the rol gene. The corresponding protein was almost identical in sequence to the Rol protein of E. coli O75 and was highly homologous to the functionally identical Cld proteins of E. coli O111 and S. enterica serovar typhimurium LT2. These proteins, together with ORF o349 adjacent to rfe, had almost identical hydropathy plots which predict membrane-spanning segments at the amino- and carboxy-terminal ends and a hydrophilic central region. We isolated a number of TnphoA insertions which inactivated the rol gene, and the fusion end points were determined. The PhoA+ Rol::PhoA fusion proteins had PhoA fused within the large hydrophilic central domain of Rol. These proteins were located in the whole-membrane fraction, and extraction with Triton X-100 indicated a cytoplasmic membrane location. This finding was supported by sucrose density gradient fractionation of the whole-cell membranes and of E. coli maxicells expressing L-[35S]methionine-labelled Rol protein. Hence, we interpret these data to indicate that the Rol protein is anchored into the cytoplasmic membrane via its amino- and carboxy-terminal ends but that the majority of the protein is located in the periplasmic space. To confirm that rol is responsible for the effects on O-antigen chain length observed with the cloned rfb genes in E. coli K-12, it was mutated in S. flexneri by insertion of a kanamycin resistance cartridge. The resulting strains produced LPS with O antigens of nonmodal chain length, thereby confirming the function of the rol gene product. We propose a model for the function of Rol protein in which it acts as a type of molecular chaperone to facilitate the interaction of the O-antigen ligase (RfaL) with the O-antigen polymerase (Rfc) and polymerized, acyl carrier lipid-linked, O-antigen chains. Analysis of the DNA sequence of the region identified a number of ORFs corresponding to the well-known gnd and hisIE genes. The rol gene was located immediately downstream of two ORFs with sequence similarity to the gene encoding UDPglucose dehydrogenase (HasB) of Streptococcus pyogenes. The ORFs arise because of a deletion or frameshift mutation within the gene we have termed udg (for UDPglucose dehydrogenase).

The complete hrp gene cluster of Pseudomonas syringae pv. syringae 61 includes two blocks of genes required for harpinPss secretion that are arranged colinearly with Yersinia ysc homologs.

Pseudomonas syringae pv. syringae 61 contains a 25-kb hrp cluster that is sufficient to elicit the hypersensitive response (HR) in nonhost plants. Previous studies have shown that mutations in complementation groups VIII, IX, and XI in the hrp cluster abolished the ability of the bacterium to cause the HR. The sequence of a 3.7-kb SmaI-SstI fragment covering groups VIII and IX now reveals five open reading frames (ORFs) in the same transcript, designated as hrpU, hrpW, hrpO, hrpX, and hrpY, and predicted to encode proteins of 14,795, 23,211, 9,381, 28,489, and 39,957 Da, respectively. The hrpU, hrpW, hrpO, hrpX, and hrpY genes are homologous and arranged colinearly with the yscQ/spa33/spaO, yscR/spa24/spaP, yscS/spa9/spaQ, yscT/spa29/spaR, and yscU/spa40/spaS genes of Yersinia spp., Shigella flexneri, and Salmonella typhimurium, respectively. These proteins also show similarity to Fli/Flh proteins of Bacillus and enteric bacteria. The Ysc and Spa proteins are involved in the secretion of virulence factors, like the Yop and Ipa proteins. Fli/Flh proteins are involved in flagellar biogenesis. The sequence of a 2.9-kb EcoRV-EcoRI DNA fragment containing mainly group XI revealed five ORFs, designated hrpC, hrpD, hrpE, hrpF, and hrpG, predicted to encode proteins of 29,096, 15,184, 21,525, 7,959, and 13,919 Da, respectively. The first three genes belong to an operon containing hrpZ, which encodes an extracellular protein that elicits the HR. hrpF and hrpG are two potential ORFs upstream of hrpH in the hrpH operon. HrpC is homologous to Yersinia YscJ, Pseudomonas solanacearum HrpI, Xanthomonas compestris pv. vesicatoria HrpB3, and Rhizobium fredii NolT. HrpE is similar to YscL of Yersinia spp. P. s. pv. syringae 61 Hrp proteins are most similar to Ysc proteins among those homologs. TnphoA insertions in hrpC, hrpE, hrpW, hrpX, and hrpY abolished the ability of P. s.pv. syringae 61 to secrete HrpZ (harpinPss), as determined by immunoblot analysis of cell-bound and culture supernatant fractions. Thus, many of the proteins required for flagellar biogenesis and virulence protein secretion in plant and animal pathogens may have a common ancestry.

Genetic characterization of mannose-sensitive hemagglutinin (MSHA)-negative mutants of Vibrio cholerae derived by Tn5 mutagenesis.

El Tor biotype Vibrio cholerae strains express a cell-associated mannose-sensitive hemagglutinin (MSHA) which is a putative attachment factor. Several MSHA-negative mutants from V. cholerae strain JBK70 were previously generated by Tn5 mutagenesis [Finn et al., Infect. Immun. 55 (1987) 942-946]. The chromosomal DNA regions containing the Tn5 insertions were isolated from eight strains for further analysis. Nucleotide sequencing of the insertional junctions and corresponding clones containing the intact chromosomal region from the parental strain revealed the presence of several contiguous ORFs. Only two ORFs of this region had received insertions, and these showed remarkable homology to genes involved in the general secretory pathway found in several Gram- bacterial species. Proteins corresponding to the observed ORFs were visualized with the T7 promoter/RNA polymerase expression system. Marker exchange mutagenesis was used to insert kanamycin-resistance cassettes and TnphoA insertions into different locations of this region in the chromosome of wild-type V. cholerae strains. The phenotypes of these mutants showed that this DNA region is involved in MSHA production, but is not required for general extracellular protein secretion.

Further characterization of the PhoP regulon: identification of new PhoP-activated virulence loci.

Salmonella typhimurium survival within macrophages is an essential virulence property necessary to enteric fever pathogenesis. This survival requires coordinate transcriptional activation of virulence genes within acidified macrophage phagosomes. Virulence gene transcription is regulated by a two-component system comprising the PhoP (transcriptional activator) and PhoQ (sensor-kinase) proteins. Thirteen new PhoP-activated loci (designated pagD to pagP) encoding membrane or secreted proteins have been identified by use of the transposon TnphoA. Three of these loci have a chromosomal location that was linked to the previously identified pagC locus. Strains with TnphoA insertions in pagD, pagJ, pagK, and pagM were significantly attenuated for mouse virulence (50% lethal dose greater than 1,000 times that of wild-type bacteria). No strains with pag::TnphoA insertions were found to have altered sensitivity to the cationic antimicrobial peptide NP-1 defensin. PhoP and PhoQ are pleotropic regulators of membrane or secreted proteins, suggesting that the ability to effect a global change in the expression of these proteins is required for S. typhimurium survival within acidified macrophage phagosomes.

Identification and characterization of a gene cluster mediating enteroaggregative Escherichia coli aggregative adherence fimbria I biogenesis.

The aggregative pattern of adherence (AA) exhibited by enteroaggregative Escherichia coli upon HEp-2 cells is a plasmid-associated property which correlates with aggregative adherence fimbria I (AAF/I) expression and human erythrocyte hemagglutination. By using cloning and mutagenesis strategies, two noncontiguous plasmid segments (designated regions 1 and 2) required for AA expression have previously been identified in enteroaggregative E. coli 17-2. TnphoA mutagenesis was performed on clones containing region 1, and 16 TnphoA mutants which were negative for the AA phenotype were analyzed. The TnphoA insertion site for each mutant was determined by junctional DNA sequencing. All 16 mutations occurred within a 4.6-kb span in region 1. Nucleotide sequence analysis of the region revealed four contiguous open reading frames, designated aggDCBA, in the same span. AA-negative TnphoA insertions into all open reading frames except aggB were obtained. On the basis of mutational analysis and protein homology data, it is inferred that aggA, aggC, and aggD are involved in biogenesis of AAF/I, encoding a major fimbrial subunit, outer membrane usher, and periplasmic fimbrial chaperone, respectively. By immunogold electron microscopy, polyclonal antiserum raised against the aggA gene product decorated AAF/I fimbriae, affirming that AggA encodes an AAF/I subunit.

Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent

Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1). This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa. E. amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1. Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP. The spherical mutants could be complemented by the cloned E. coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings. This suggested that the E. amylovora 69-kDa PBP is probably the functional equivalent of the E. coli PBP2 protein. Southern blot analysis using the E. coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E. amylovora equivalent of the E. coli rodA-pbpA operon. Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E. amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E. amylovora chromosome postulated to encode known virulence factors. Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants. These results indicate a possible role for cell shape in the virulence of this plant pathogen.

Nucleotide sequence of a 13.9 kb segment of the 90 kb virulence plasmid of Salmonella typhimurium: the presence of fimbriai biosynthetic genes

The 90kb plasmid resident in Salmonella typhimurium confers increased virulence in mice by promoting the spread of infection after invasion of the intestinal epithelium. The nucleotide sequence of a 13.9kb segment of this plasmid known to encode an outer membrane protein related in sequence to components of fimbrial biosynthesis in enteric bacteria was determined. This cloned segment between the repB and repC replicon regions programmed expression of abundant surface fimbriae in Escherichia coli and S. typhimurium cells. A 7kb region contained seven open reading frames, the protein products of five of which were related in sequence to regulatory, structural, and assembly proteins of adherence fimbriae/pili, such as the P and K88 pili. These five genes and two adjacent ones which were not markedly related to proteins in the data bases comprise the pef (plasmid-encoded fimbriae) locus. Transposon TnphoA insertions in four genes in the pef locus (pefA, pefC, orf5 and orf6) resulted in active PhoA fusions and blocked or reduced the surface presentation of fimbriae, indicating that the proteins encoded by these four genes are translocated at least across the cytoplasmic membrane and contribute to formation of the fimbrial structure. The differences in genetic organization and protein sequence relatedness from other fimbrial gene clusters suggest that the pef locus might encode a novel type of fimbria. Between the pef and the repB loci, there were five open reading frames, one of which (orf8) gave rise to active PhoA fusions but was not necessary for fimbrial expression. Two of the other proteins were homologous to transcription regulatory proteins and a third was the rck gene, which encodes an outer membrane protein that confers complement resistance to serum-sensitive hosts.

TnphoA Salmonella abortusovis mutants unable to adhere to epithelial cells and with reduced virulence in mice.

Salmonella abortusovis is a pathogenic bacterium highly specific to sheep, causing spontaneous abortion. In order to understand the role of genes involved in pathogenicity, we investigated S. abortusovis with the random mutagenic TnphoA transposon. A total of 95 S. abortusovis TnphoA mutants yielding alkaline phosphatase active fusion protein were obtained. In this way we created a bank of strains in order to identify any phenotypic modification which could affect the periplasmic and/or exported proteins involved in virulence. The TnphoA mutants were screened for the ability to adhere to epithelial cells: a total of 23 mutant strains lost this phenotypic feature. To detect the chromosomal TnphoA insertions, DNA was restricted by the enzyme EcoRV, which does not cleave the TnphoA sequence. Southern blotting analysis revealed the existence of four classes of integration. Colonies of adhesiveless mutants appear to be as smooth as the S. abortusovis wild type, and electrophoretic analysis indicates a normal lipopolysaccharide profile. To identify mutations affecting genes encoding for outer membrane proteins (OMPs), the alkaline phosphatase portion of the fusion proteins was revealed in TnphoA mutants by immunoblotting with specific antibodies. A mutation in OMPs was detected in seven mutants. Restriction analysis identified in four mutants a common region of 2 kb where alterations in genes coding for OMPs occur. We suggested that this region is involved in pathogenicity in mice, since a group of mutant strains has shown reduced virulence in mice and one mutant is completely avirulent. Furthermore, after mice were exposed orally to these mutants, significant protection against oral challenge with the parental virulent strain resulted.

Biogenesis and regulation of the Vibrio cholerae toxin-coregulated pilus: analogies to other virulence factor secretory systems

Biogenesis of the toxin-coregulated pilus (TCP) of Vibrio cholerae 01 is essential for successful bacterial colonization of the small intestine. Pilus assembly requires the products of at least seven genes located on the chromosome adjacent to the pilin-encoding gene, tcpA. Previously reported TnphoA insertions in the TCP-assembly-deficient V. cholerae strains, KP2.21 and KP4.2, were isolated from the chromosome for further analysis. Nucleotide sequencing of the tcpE::phoA and tcpF::phoA fusions and corresponding clones of the region containing the intact genes revealed the presence of two open reading frames (ORFs) of 340 and 338 amino acids, designated TcpE and TcpF, respectively. The partial sequence of an ORF downstream from the TcpF coding sequence was determined to correspond to the global virulence regulator, ToxT. Proteins corresponding to the observed ORFs were visualized with the T7 promoter/RNA polymerase expression system. Computer-generated alignment algorithms predict that a homology exists between TcpE and the Klebsiella pneumoniae pullulanase secretion proteins PulD and PulF, the Xanthomonas campestris extracellular enzyme secretion factor XpsF, the Bacillus subtilis DNA competence protein ComG-ORF2, and the Yersinia entercolitica Yop secretion determinant YscC. These observations provide a model to investigate further the relationship between the secretion mechanisms utilized by these seemingly diverse virulence determinants. Additionally, an extreme C-terminal segment of TcpE shows striking homology to the transmembrane segment of the eukaryotic integrin β-1 chain, which could imply a role for TcpE in not only TCP secretion, but also host cell interaction.

Topology and subcellular localization of FtsH protein in Escherichia coli.

FtsH protein in Escherichia coli is an essential protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding sequence. Western blots (immunoblots) of proteins from fractionated cell extracts and immunoelectron microscopy of the FtsH-overproducing strain showed exclusive localization of the FtsH protein in the cytoplasmic membrane. Most of the FtsH-specific labeling with gold particles was observed in the cytoplasmic membrane and the adjacent cytoplasm; much less was observed in the outer membrane and in the bulk cytoplasm. Genetic analysis by TnphoA insertions into ftsH revealed that the 25- to 95-amino-acid region, which is flanked by two hydrophobic stretchs, protrudes into the periplasmic space. From these results, we concluded that FtsH protein is an integral cytoplasmic membrane protein spanning the membrane twice and that it has a large cytoplasmic carboxy-terminal part with a putative ATP-binding domain. The average number of FtsH molecules per cell was estimated to be approximately 400.

A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence.

Enteropathogenic Escherichia coli (EPEC) form adherent microcolonies on the surface of tissue culture cells in a pattern termed localized adherence. Localized adherence requires the presence of a large EPEC adherence factor (EAF) plasmid. Recently a bundle-forming pilus has been described in EPEC possessing the EAF plasmid. An analysis of 22 non-invasive EPEC TnphoA mutants revealed that seven have insertions in the EAF plasmid and are incapable of localized adherence. We report here the mapping of the TnphoA insertions in these mutants. The nucleotide sequence of the gene interrupted in these TnphoA mutants (bfpA) was determined and found to correspond to the N-terminal amino acid sequence of the major structural protein of the bundle-forming pilus. The bfpA gene bears sequence similarities to members of the type IV fimbrial gene family and encodes a potential site for processing by a prepilin peptidase. A plasmid containing bfpA as the only open reading frame directs the synthesis of a protein recognized by antiserum raised against the bundle-forming pilus. TnphoA mutants at this locus are unable to synthesize BfpA, but synthesis is restored by introduction of a plasmid containing the cloned gene. The minimum fragment of DNA required to restore localized adherence is considerably greater than that required to restore BfpA synthesis. BfpA expression, as assessed by alkaline phosphatase activity in bfpA::TnphoA mutants, is affected by temperature and growth medium. These studies describe an EPEC plasmid-encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence.

Identification of novel loci affecting entry of Salmonella enteritidis into eukaryotic cells.

There are an estimated 2 million cases of salmonellosis in the United States every year. Unlike the incidence of many infectious diseases, the incidence of salmonellosis in the United States and other developed countries has been rising steadily over the past 30 years, and the disease now accounts for 10 to 15% of all cases of acute gastroenteritis in the United States. The infecting organism is ingested and must traverse the intestinal epithelium to reach its preferred site for multiplication, the reticuloendothelial system. Despite several recent studies, the genetic basis of the invasion process is poorly understood. An emerging theme from these studies is that wild-type Salmonella organisms probably have several chromosomal loci that are required for the most efficient level of invasion. In this study, we have identified and characterized 13 TnphoA insertion mutants of Salmonella enteritidis CDC5 that exhibit altered invasion phenotypes. The mutants were identified by screening a bank of TnphoA insertions in S. enteritidis CDC5str for their invasion phenotype in three tissue culture cell lines (HEp-2, CHO, and MDCK). These 13 mutants were separated into six classes based on their invasive phenotypes in the tissue culture cell lines. Several mutants were defective for entry of some cell lines but not for others, while two mutants (SM6 and SM7) were defective for entry into all three tissue culture cell lines. This suggests that Salmonella spp. may express more than one invasion pathway. Southern analysis and chromosomal mapping indicated that as many as nine chromosomal loci may contribute to the invasion phenotype. It is becoming clear that the invasive phenotype of Salmonella spp. is multifactorial and more complex than that of some other invasive members of the family Enterobacteriaceae.

A vir-repressed gene of Bordetella pertussis is required for virulence

Coordinate regulation of gene expression in Bordetella pertussis is controlled by the products of the vir locus, BvgA and BvgS. In the presence of modulating signals such as MgSO4 and nicotinic acid, expression of vir-activated genes (vag) is reduced, while expression of vir-repressed genes (vrg) is maximal. We have cloned one of these vir-repressed genes, vrg-6, in Escherichia coli. DNA sequencing has shown that vrg-6 is contained on a single EcoRI restriction endonuclease fragment and is predicted to code for a protein of 105 amino acids with a molecular weight of 11,441. The predicted protein product appears to have two domains, one consisting of seven hydrophobic proline-rich pentameric repeats and the other consisting of five alkaline trimeric repeats. Southern blot analysis has revealed vrg-6-homologous sequences in the chromosomes of Bordetella bronchiseptica and Bordetella parapertussis, but, unlike Bordetella pertussis, these species do not express vrg-6-homologous RNA when grown under modulating conditions. In order to assess the role of vrg gene products in B. pertussis pathogenesis, two 18323 derivatives which harbor TnphoA insertions in vrg genes were analyzed in a mouse model of respiratory infection. Strain SK6, which carries a vrg-6::TnphoA mutation, failed to induce lymphocytosis and was significantly less able to colonize lungs and trachea than its parent strain 18323 or than SK18, which harbors a TnphoA fusion in the vrg-18 locus. This is the first evidence that a vir-repressed gene may play an important role in the virulence of B. pertussis and the pathogenesis of whooping cough.