Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae.

Gene fusions between the cholera toxin structural genes and phoA, which encodes bacterial alkaline phosphatase, were identified after TnphoA mutagenesis of the cloned genes in Escherichia coli and were then mobilized into Vibrio cholerae. The activities of the hybrid proteins were detectable in V. cholerae and suggested that, like cholera toxin, they were secreted beyond the cytoplasm. To extend the utility of TnphoA to identify additional genetic export signals in V. cholerae and other gram-negative bacteria, TnphoA delivery vectors utilizing broad-host-range plasmids were developed. By using V. cholerae as a model system, insertion mutants carrying active phoA gene fusions were identified as colonies expressing alkaline phosphatase, which appeared blue on agar containing the indicator 5-bromo-4-chloro-3-indolyl phosphate. Since alkaline phosphatase is active only upon export from the cytoplasm, PhoA+ colonies resulting from the mutagenesis procedure were enriched for insertions in genes that encode secreted proteins. Insertion mutations were identified in the gene encoding a major outer membrane protein, OmpV, and in tcpA, which encodes a pilus (fimbrial) subunit. Mutant strains harboring chromosomal insertions isolated in this manner can be used to assess the role of the corresponding inactivated gene products on survival of V. cholerae in vivo. The expression of the hybrid proteins as determined by measuring alkaline phosphatase activity also allowed the convenient study of virulence gene expression.     

Chemotactic control of the two flagellar systems of Vibrio parahaemolyticus.

Vibrio parahaemolyticus synthesizes two distinct flagellar organelles, the polar flagellum (Fla), which propels the bacterium in a liquid environment (swimming), and the lateral flagella (Laf), which are responsible for movement over surfaces (swarming). Chemotactic control of each of these flagellar systems was evaluated separately by analyzing the behavioral responses of strains defective in either motility system, i.e., Fla+ Laf- (swimming only) or Fla- Laf+ (swarming only) mutants. Capillary assays, modified by using viscous solutions to measure swarming motility, were used to quantitate chemotaxis by the Fla+ Laf- or Fla- Laf+ mutants. The behavior of the mutants was very similar with respect to the attractant compounds and the concentrations which elicited responses. The effect of chemotaxis gene defects on the operation of the two flagellar systems was also examined. A locus previously shown to encode functions required for chemotactic control of the polar flagellum was cloned and mutated by transposon Tn5 insertion in Escherichia coli, and the defects in this locus, che-4 and che-5, were then transferred to the Fla+ Laf- or Fla- Laf+ strains of V. parahaemolyticus. Introduction of the che mutations into these strains prevented chemotaxis into capillary tubes and greatly diminished movement of bacteria over the surface of agar media or through semisolid media. We conclude that the two flagellar organelles, which consist of independent motor-propeller structures, are directed by a common chemosensory control system.     

Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer

Surface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose-resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side-chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell monolayers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild-type S. choleraesuis.     

Symbiotic loci of Rhizobium meliloti identified by random TnphoA mutagenesis.

We have developed a system for using TnphoA (TnphoA is Tn5 IS50L::phoA), which generates fusions to alkaline phosphatase (C. Manoil and J. Beckwith, Proc. Natl. Acad. Sci. USA 82:8129-8133, 1985), in Rhizobium meliloti. Active fusions expressing alkaline phosphatase can arise only when this transposon inserts in genes encoding secreted or membrane-spanning proteins. By confining our screening to 1,250 TnphoA-generated mutants of R. meliloti that expressed alkaline phosphatase, we efficiently identified 25 symbiotically defective mutants, all of which formed ineffective (Fix-) nodules on alfalfa. Thirteen of the mutants were unable to synthesize an acidic exopolysaccharide (exo::TnphoA) that is required for nodule invasion. Twelve of the mutations created blocked at later stages of nodule development (fix::TnphoA) and were assigned to nine symbiotic loci. One of these appeared to be a previously undescribed locus located on the pRmeSU47a megaplasmid and to encode a membrane protein. Two others were located on the pRmeSU47b megaplasmid: one was a new locus which was induced by luteolin and encoded a membrane protein, and the other was dctA, the structural gene for dicarboxylic acid transport. The remaining six loci were located on the R. meliloti chromosome. One of these was inducible by luteolin and encoded a membrane protein which determined lipopolysaccharide structure. Three additional chromosomal loci also appeared to encode membrane proteins necessary for symbiosis. The remaining two chromosomal loci encoded periplasmic proteins required for symbiosis.     

Effect of integrating the pJFF350 vector into the 85-MDa plasmid of Azospirillum brasilense Sp245 on bacterial flagellation and mobility

Results of genetic analysis of three derivatives of Azospirillum brasilense Sp245 (strains BK570, SK051, and SK248) carrying cointegrates of plasmids 85-MDa and pJFF350 (the vector for omegon mutagenesis), which manifest abnormalities in flagellation and motility, are presented. It was shown for the first time that the integration of the suicide vector into one of Azospirillum resident plasmids is accompanied by the formation of various fusion products and changes in flagellation and motility of these bacteria, such as the loss of the polar (Fla) and lateral (Laf) flagella in SK051; inactivation of Fla and Laf in SK248; and Fla-dependent acceleration of expansion in semiliquid media in BK570.     

Export of Escherichia coli alkaline phosphatase attached to an integral membrane protein, SecY

The SecY protein is a membrane-bound factor required for bacterial protein export and embedded in the cytoplasmic membrane by its 10 transmembrane segments. We previously proposed a topology model for this protein by adapting the Manoil-Beckwith TnphoA approach, a genetic method to assign local disposition of a membrane protein from the enzymatic activity of the alkaline phosphatase (PhoA) mature sequence attached to the various regions. SecY-PhoA hybrid proteins with the PhoA domain exported to the periplasmic side of the membrane have been obtained at the five putative periplasmic domains of the SecY sequence. We now extended this method to apply it to follow export of the newly synthesized PhoA domain. Trypsin treatment of detergent-solubilized cell extracts digested the internalized (unfolded) PhoA domain but not those exported and correctly folded. One of the hybrid proteins was cleaved in vivo after export to the periplasm, providing a convenient indication for the export. Results of these analyses indicate that export of the PhoA domain attached to different periplasmic regions of SecY occurs rapidly and requires the normal functioning of the secY gene supplied in trans. Thus, this membrane protein with multiple transmembrane segments contains multiple export signals which can promote rapid and secY-dependent export of the PhoA mature sequence attached to the carboxyl-terminal sides.     

Evidence that TET protein functions as a multimer in the inner membrane of Escherichia coli.

The inner membrane TET (TetA) protein, which is involved in Tn10-mediated microbial tetracycline resistance, consists of two domains, alpha and beta, both of which are needed for tetracycline resistance and efflux (M.S. Curiale, L.M. McMurry, and S.B. Levy, J. Bacteriol. 157:211-217, 1984). Since tetracycline-sensitive mutants in one domain can partially complement sensitive mutants in the other domain and since some sensitive mutants show dominance over the wild type, a multimeric structure for TET in the membrane had been suggested. We have studied this possibility by using tetA-phoA gene fusions. We fused all but the last 40 base pairs of the tetA gene with the carboxy terminus of the phoA gene for alkaline phosphatase (PhoA), whose activity requires its dimerization in the periplasm. The tetA-phoA fusion protein was under control of the tetracycline-inducible regulatory system for the tetA gene. Induction led to the synthesis of a 78,000-dalton inner membrane protein. Tetracycline resistance was expressed at reduced levels, consistent with the terminal beta domain deletion. Alkaline phosphatase activity was also present, but at low levels, suggesting that some, but not all, of the fusion proteins had their carboxy-terminal ends in the periplasm. When wild-type or mutant TET proteins were present in the same cell with the fusion protein, the tetracycline resistance level was affected (raised or lowered); however, phosphatase activity was reduced only when TET proteins with intact or near-intact beta domains were present. These findings suggest that TET functions as a multimer and that intact beta domains, on TET molecules in the heterologous multimer, either allow fewer PhoA moieties to project into the periplasm or sterically hinder PhoA moieties from dimerizing.     

Characterization of virulence genes of enteroinvasive Escherichia coli by TnphoA mutagenesis: identification of invX, a gene required for entry into HEp-2 cells.

While enteroinvasive Escherichia coli (EIEC) and shigellae are genotypically nearly identical, a difference has been reported in the infective dose to humans: EIEC is 10,000-fold less infectious than shigellae. A possible basis for this difference lies in the inherent invasiveness of these bacteria toward epithelial cells. Thus, despite the high degree of homology between the invasion plasmids of EIEC and shigellae, substantial differences in genetic organization and/or sequence may exist. We have undertaken a systematic genetic analysis of the EIEC plasmid pSF204, using transposon mutagenesis. Congo red-negative TnphoA insertion mutants (Pcr- PhoA-) and TnphoA fusion mutants (PhoA+) were isolated and screened for the ability to invade cultured HEp-2 cells. Most invasion-negative (Inv-) mutations mapped to a 30-kb segment of the invasion plasmid, including homologs of the Shigella flexneri ipa, mxi, and spa genes. Inv- PhoA+ fusions in the EIEC ipaC, mxiG, mxiJ, mxiM, and mxiD homologs and in a proposed new gene, named invX, located downstream of the spa region were identified and characterized. This analysis indicates the presence of the ipaC, mxiG, mxiJ, mxiM, mxiD, and invX gene products in the EIEC cell envelope and demonstrates a strict requirement for these genetic loci in invasion. Overall, our results suggest a high degree of genetic, structural, and functional homology between the EIEC and S. flexneri large invasion plasmids.     

Excretion of alkaline phosphatase by Escherichia coli K-12 pho constitutive mutants transformed with plasmids carrying the alkaline phosphatase structural gene.

Escherichia coli alkaline phosphatase constitutive mutants carrying a pst or a phoS mutation and a plasmid-bearing gene phoA+ excreted into the growth medium up to 50% of the total alkaline phosphatase production. This excretion was pH dependent and did not involve drastic modifications of the cell envelope. Alkaline phosphatase accounted for 80% of total released proteins. Amplification of gene phoA+ was a necessary condition for excretion to occur. When the beta-lactamase structural gene bla+ was coamplified with gene phoA+, both enzymes were excreted. pst-transformed excretory strains did not show the pleiotrophic phenotype previously described for lky mutants.     

Domain organization of the MscS mechanosensitive channel of Escherichia coli

The major structural features of the Escherichia coli MscS mechanosensitive channel protein have been explored using alkaline phosphatase (PhoA) fusions, precise deletions and site-directed mutations. PhoA protein fusion data, combined with the positive-inside rule, strongly support a model in which MscS crosses the membrane three times, adopting an N(out)-C(in) configuration. Deletion data suggest that the C-terminal domain of the protein is essential for the stability of the MscS channel, whereas the protein will tolerate small deletions at the N-terminus. Four mutants that exhibit either gain-of-function (GOF) or loss-of-function have been identified: a double mutation I48D/S49P inactivates MscS, whereas the MscS mutants T93R, A102P and L109S cause a strong GOF phenotype. The similarity of MscS to the last two domains of MscK (formerly KefA) is reinforced by the demonstration that expression of a truncated MscK protein can substitute for MscL and MscS in downshock survival assays. The data derived from studies of the organization, conservation and the influence of mutations provide significant insights into the structure of the MscS channel.     

Topology of the membrane-bound alkane hydroxylase of Pseudomonas oleovorans.

The Pseudomonas oleovorans alkane hydroxylase is an integral cytoplasmic membrane protein that is expressed and active in both Escherichia coli and P. oleovorans. Its primary sequence contains eight hydrophobic stretches that could span the membrane as alpha-helices. The topology of alkane hydroxylase was studied in E. coli using protein fusions linking different amino-terminal fragments of the alkane hydroxylase (AlkB) to alkaline phosphatase (PhoA) and to beta-galactosidase (LacZ). Four AlkB-PhoA fusions were constructed using transposon TnphoA. Site-directed mutagenesis was used to create PstI sites at 12 positions in AlkB. These sites were used to create AlkB-PhoA and AlkB-LacZ fusions. With respect to alkaline phosphatase and beta-galactosidase activity each set of AlkB-PhoA and AlkB-LacZ fusions revealed the expected complementary activities. At three positions, PhoA fusions were highly active, whereas the corresponding LacZ fusions were the least active. At all other positions the PhoA fusions were almost completely inactive, but the corresponding LacZ fusions were highly active. These data predict a model for alkane hydroxylase containing six transmembrane segments. In this model the amino terminus, two hydrophilic loops, and a large carboxyl-terminal domain are located in the cytoplasm. Only three very short loops near amino acid positions 52, 112, and 251 are exposed to the periplasm.     

Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli.

The secY (prlA) gene product is an essential component of the Escherichia coli cytoplasmic membrane, and its function is required for the translocation of exocytoplasmic proteins across the membrane. We have analyzed the orientation of the SecY protein in the membrane by examining the hydropathic character of its amino acid sequence, by testing its susceptibility to proteases added to each side of the membrane, and by characterizing SecY-PhoA (alkaline phosphatase) hybrid proteins constructed by TnphoA transpositions. The orientation of the PhoA portion of the hybrid protein with respect to the membrane was inferred from its enzymatic activity as well as sensitivity to external proteases. The results suggest that SecY contains 10 transmembrane segments, five periplasmically exposed parts, and six cytoplasmic regions including the amino- and carboxyterminal regions.     

Formation of polar bundles of pili on a cell and the behavior of Azospirillum brasilense in semiliquid agar]

This paper describes the formation of single polar bundles of pili on Azospirillum brasilense cells, the twitching motility of cell aggregates, and a new type of social behavior--the dispersal of bacterial cells in semiliquid agar associated with the formation of granular inclusions (the so-called Gri+ phenotype)--which is an alternative to swarming (the Swa+ phenotype). The wild-type A. brasilense cells occurring in a semiliquid agar may show either the Swa+Gri-, or Swa-Gri-, or Swa-Gri+ phenotype. The formation of single polar flagella (Fla) or polar bundles of pili may reflect two alternative states of A. brasilense cells. The components of the Fla system may be involved in the regulation of the phenotypic variation of azospirilla.     

Analysis of protein localization by use of gene fusions with complementary properties.

This report describes a new transposon designed to facilitate the combined use of beta-galactosidase and alkaline phosphatase gene fusions in the analysis of protein localization. The transposon, called TnlacZ, is a Tn5 derivative that permits the generation of gene fusions encoding hybrid proteins carrying beta-galactosidase at their C termini. In tests with plasmids, TnlacZ insertions that led to high cellular beta-galactosidase activity were restricted to sequences encoding either cytoplasmic proteins or cytoplasmic segments of a membrane protein. The fusion characteristics of TnlacZ are thus complementary to those of TnphoA, a transposon able to generate alkaline phosphatase fusions whose high-activity insertion sites generally correspond to periplasmic sequences. The structure of TnlacZ allows the conversion of a TnlacZ fusion into the corresponding TnphoA fusion (and vice versa) through recombination or in vitro manipulation in a process called fusion switching. Fusion switching was used to generate the following two types of fusions with unusual properties: a low-specific-activity beta-galactosidase-alkaline phosphatase gene fusion and two toxic periplasmic-domain serine chemoreceptor-beta-galactosidase gene fusions. The generation of both beta-galactosidase and alkaline phosphatase fusions at exactly the same site in a protein permits a comparison of the two enzyme activities in evaluating the subcellular location of the site, such as in studies of membrane protein topology. In addition, fusion switching makes it possible to generate gene fusions whose properties should facilitate the isolation of mutants defective in the export or membrane anchoring of different cell envelope proteins.     

Construction of TnphoA gene fusions in Rhodobacter sphaeroides: isolation and characterization of a respiratory mutant unable to utilize dimethyl sulfoxide as a terminal electron acceptor during anaerobic growth in the dark on glucose.

We have constructed a suicide vector, pU1800, containing the transposable element TnphoA (Tn5 IS50L::phoA), for the purpose of producing protein fusions in vivo between the Escherichia coli alkaline phosphatase (APase) and proteins of the facultative photoheterotroph, Rhodobacter sphaeroides. We introduced TnphoA into the genome of R. sphaeroides at a coupled conjugation-transposition frequency of approximately 1 x 10(-6). Fusions giving rise to APase expression, as judged by blue-colony pigmentation when exconjugants were plated on growth medium containing the chromogenic indicator 5-bromo-4-chloro-3-indolyl phosphate, were observed in about 1% of the exconjugants. Numerous, distinguishable mutant phenotypes have been generated by this method, including those which lack the ability to use dimethyl sulfoxide as a terminal electron acceptor during anaerobic respiration, as well as those which are photosynthetically incompetent or altered in pigment synthesis, and others that express resistance to chlorate. The growth and spectral characteristics of several of these mutants, as well as the localization and quantitation of subcellular APase activity under different physiological conditions, have been examined. The presence of TnphoA in the host genome has been confirmed for each mutant analyzed, and specifically tagged DNA fragments containing TnphoA have been identified and localized; cosmids containing R. sphaeroides genomic DNA capable of complementing individual mutants have also been isolated. The usefulness of this approach in studying gene activity in R. sphaeroides is discussed.     

Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme.

Tn5 insertion mutations of Escherichia coli were isolated that impaired the formation of correctly folded alkaline phosphatase (PhoA) in the periplasm. The PhoA polypeptide synthesized in the mutants was translocated across the cytoplasmic membrane but not released into the periplasmic space. It was susceptible to degradation by proteases in vivo and in vitro. The wild-type counterpart of this gene (named ppfA) has been sequenced and shown to encode a periplasmic protein with a pair of potentially redox-active cysteine residues. PhoA synthesized in the mutants indeed lacked disulfide bridges. These results indicate that the folding of PhoA in vivo is not spontaneous but catalyzed at least at the disulfide bond formation step.     

Yersinia spp. HMWP2, a cytosolic protein with a cryptic internal signal sequence which can promote alkaline phosphatase export.

The iron starvation-induced, 2,042-amino-acid protein HMWP2 of Yersinia enterocolitica has two internal hydrophobic segments which might promote its export and association with the cytoplasmic membrane. To determine whether part of HMWP2 could be exported beyond the periplasmic face of the cytoplasmic membrane, we used TnphoA mutagenesis to construct 10 hybrid proteins in which periplasmic alkaline phosphatase (PhoA) was fused to the end of C-terminally truncated HMWP1 (at amino acid positions 1751 and 1753 two independent isolates]) had high alkaline phosphate activity (close to that of the native enzyme), both in Escherichia coli and in Y. pseudotuberculosis, indicating that the PhoA segment of the hybrid reached the periplasm. Deletion studies showed that the export signal resides in the second hydrophobic segment of HMWP2. This result would be compatible with the topology of the protein in the cytoplasmic membrane predicted from the distribution of charged amino acids at either end of the two hydrophobic segments. However, two hybrids in which the junction was even further toward the C terminus of HMMWP2 (at positions 1793 and 1999) had only weak alkaline phosphatase activity, suggesting that the predicted topology is incorrect. The location of HMWP2 was therefore determined by subcellular fractionation. The results indicate that HMPW2 is mainly cytoplasmic, consistent with its presumed role in the ATP-dependent, nonribosomal synthesis of an unknown peptide. We propose that the high alkaline phosphatase activity associated with some of the HMWP-2-PhoA hybrids results from the unmasking of the cryptic export signal activity in the second hydrophobic segment of HMPW2.