Use of TnphoA to detect genes for exported proteins in Escherichia coli: identification of the plasmid-encoded gene for a periplasmic acid phosphatase.

The structural gene (appA) for the periplasmic acid phosphatase (optimum pH 2.5) of Escherichia coli was cloned into a plasmid by using a combination of in vivo and in vitro techniques. The position and orientation of the appA gene within the cloned DNA fragment were identified by using fusions to the alkaline phosphatase gene (phoA) generated by Tn5 IS50L::phoA (TnphoA) insertions. For TnphoA-generated hybrid proteins to have high enzymatic activity, it appears that the phoA gene must be fused to a target gene coding for a signal which promotes protein export. The approach used to identify the appA gene thus appears to provide a simple general means of selectively identifying genes encoding membrane and secreted proteins.     

A gene fusion approach to the study of pullulanase export and secretion in Eschenchia coli

A series of fusions between the gene for the Klebsiella pneumoniae secreted lipoprotein pullulanase (pulA) and the genes for cytoplasmic beta-galactosidase (lacZ) or periplasmic alkaline phosphatase (phoA) were created by transposon mutagenesis using mini-MudII1681 or TnphoA, respectively. The hybrid genes were expressed in Escherichia coli K-12 with or without the K. pneumoniae genes that promote pullulanase secretion in E. coli. We characterized seven different pulA-lacZ gene fusions encoding hybrid polypeptides containing from 14 to c. 1060 residues of pro-pullulanase. All but the smallest hybrid were fatty acylated and were toxic to producing cells, causing the accumulation of precursors of other exported proteins. Four different pulA-phoA gene fusions encoded hybrids with alkaline phosphatase activity. All four hybrids were fatty acylated, but were not toxic. Although the hybrids were apparently membrane-associated, they were not secreted into the medium either by E. coli carrying pullulanase secretion genes or by K. pneumoniae. Immunofluorescence tests indicated that the pullulanase secretion genes promoted the localization of one of these hybrids to the outer face of the E. coli outer membrane, which may have important implications for the design of live vaccine strains and of immobilized enzymes.     

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.     

Construction of transposon Tn3phoA: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane proteins. To study the topology of the Agrobacterium T-DNA transfer proteins, we constructed a transposon, Tn 3phoA . The transposon mobilizes into plasmids at a high frequency, is stable after transposition, can produce phoA translational fusions and can be used for the analysis of protein topology directly in the bacterium of interest. For studies on the DNA transfer proteins, an Agrobacterium strain deficient in phoA under our experimental conditions was constructed by chemical mutagenesis. A plasmid containing virB and virD4 was used as a target for mutagenesis. Twenty-eight unique phoA -positive clones that mapped to eight virB genes were isolated. Multiple insertions throughout VirB1, VirB5, VirB7, VirB9 and VirB10 indicated that these proteins primarily face the periplasm. Insertions in VirB2, VirB6 and VirB8 allowed the identification of their periplasmic domains. No insertions were found in VirB3, VirB4 and VirB11. These proteins either lack or have a short periplasmic domain. No insertions mapped to VirD4 either. To study VirD4 topology, targeted phoA fusions and random lacZ fusions were constructed. Analysis of the fusion proteins indicated that VirD4 contains a single periplasmic domain near the N-terminus, and most of the protein lies in the cytoplasm. A hypothetical model for the T-DNA transport pore is presented.     

PhoA gene fusions in Legionella pneumophila generated in vivo using a new transposon, MudphoA

To enable effective use of phoA gene fusions in Legionella pneumophila, we constructed MudphoA, a derivative of the mini-Mu phage Mu dII4041, which is capable of generating gene fusions to the Escherichia coli alkaline phosphatase gene (EC 3.1.3.1). Although an existing fusion-generating transposon, TnphoA, has been a useful tool for studying secreted proteins in other bacteria, this transposon and other Tn5 derivatives transpose inefficiently in Legionella pneumophila, necessitating the construction of a more effective vector for use in this pathogen. Using MudphoA we generated fusions to an E. coli gene encoding a periplasmic protein and to an L. pneumophila gene encoding an outer membrane protein; both sets of fusions resulted in alkaline phosphatase activity. We have begun to use MudphoA to mutate secreted proteins of L. pneumophila specifically, since this subset of bacterial proteins is most likely to be involved in host-bacterial interactions. This modified transposon may be useful for studies of other bacteria that support transposition of Mu, but not Tn5, derivatives.     

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.     

The first gene in the Escherichia coli secA operon, gene X, encodes a nonessential secretory protein.

TnphoA insertions in the first gene of the Escherichia coli secA operon, gene X, were isolated and analyzed. Studies of the Gene X-PhoA fusion proteins showed that gene X encodes a secretory protein, since the fusion proteins possessed normal alkaline phosphatase activity and a substantial portion of this activity was found in the periplasm. In addition, the Gene X-PhoA fusion proteins were initially synthesized with a cleavable signal peptide. A gene X::TnphoA insertion was used to construct a strain containing a disrupted chromosomal copy of gene X. Analysis of this strain indicated that gene X is nonessential for cell growth and viability and does not appear to play an essential role in the process of protein export.     

TnblaM: a transposon for directly tagging bacterial genes encoding cell envelope and secreted proteins.

A transposon, TnblaM, designed for the direct selection of bacterial mutants with insertions in genes encoding cell envelope and secreted proteins, was constructed and subcloned into plasmid and bacteriophage lambda delivery vectors. TnblaM is a spectinomycin-resistant derivative of Tn5 with an unexpressed open reading frame encoding mature beta-lactamase (BlaM) at its left end. Therefore, when it inserts into genes in the correct orientation and reading frame, gene fusions encoding hybrid proteins are generated. By introducing TnblaM into bacterial cells and selecting ampicillin-resistant (ApR) colonies, the subset of isolates producing extracytoplasmic BlaM, and hence containing TnblaM inserted in genes encoding secreted proteins and cell envelope proteins, can be directly selected. TnblaM, like TnphoA, can therefore be used to preferentially mutagenise genes encoding extracytoplasmic proteins, but it has the advantage over TnphoA that the desired mutants can be isolated by direct selection (as ApR colonies) rather than by phenotypic screening. Isolates in which TnblaM occupies sites in the chromosome from which it can transpose at high frequency are readily identifiable, and constitute TnblaM donors, with which to simply and efficiently generate rare types of insertion mutants. Moreover, the ApR selection that is used with TnblaM can be fine-tuned to obtain blaM fusions to poorly or well-expressed genes.     

Topology of the Escherichia coli uhpT sugar-phosphate transporter analyzed by using TnphoA fusions.

The Escherichia coli uhpT protein catalyzes the active transport of sugar-phosphates by an obligatory exchange mechanism. To examine its transmembrane topology, we isolated a collection of uhpT-phoA fusions encoding hybrid proteins of different lengths from the N terminus of UhpT fused to alkaline phosphatase by using transposon TnphoA. These fusions displayed different levels of alkaline phosphatase activity, although comparable levels of full-length UhpT-PhoA proteins were produced in maxicells of both high- and low-activity fusions. The full-length protein species were unstable and were degraded to the size of the alkaline phosphatase moiety in the case of a high-activity fusion or to small fragments in the case of a low-activity fusion. The enzyme activity present in low-activity fusions appeared to result from export of a small proportion of the fusion proteins to the periplasmic space. Although fusions were not obtained in all predicted extramembranous loops, the deduced topology of UhpT was consistent with a model of 12 membrane-spanning regions oriented with the amino and carboxyl termini in the cytoplasm.     

Identification of new genes regulated by the marRAB operon in Escherichia coli.

Random TnphoA and TnlacZ translational fusions were introduced into an Escherichia coli strain with a deletion of the multiple antibiotic resistance (mar) locus, complemented in trans by a temperature-sensitive plasmid bearing the mar locus with a constitutively expressed mar operon. Five gene fusions (two with lacZ and three with phoA) regulated by the mar operon were identified by increased or decreased marker enzyme activity following loss of the complementary plasmid at the restrictive temperature. Expression of LacZ from both lacZ fusions increased in the presence of the mar operon; expression from the three phoA fusions was represented by the mar operon. The lacZ fusions were mapped at 31.5 and 14 min on the Escherichia coli chromosome. One of the phoA fusions was located at 51.6 min while the two others mapped at 77 min. Cloning and sequencing of a portion of the fused genes showed all of them to be different. The phoA fusions at 77 min were located in a recently identified gene, slp, a lipoprotein of unknown function (D.M. Alexander and A. C. St. John, Mol. Microb. 11:1059-1071, 1994). The others showed no homology with any known genes of E. coli. The insertions caused small but reproducible changes in the antibiotic susceptibility profile. This approach has enabled the identification of new genes in E. coli which are regulated by the marRAB operon and involved in the Mar phenotype.     

Alkaline phosphatase reporter transposon for identification of genes encoding secreted proteins in gram-positive microorganisms

We describe the construction of TnFuZ, a genetic tool for the discovery and mutagenesis of proteins exported from gram-positive bacteria. This tool combines a transposable element (Tn4001) of broad host range in gram-positive bacteria and an alkaline phosphatase gene (phoZ) derived from a gram-positive bacterium that has been modified by removal of the region encoding its export signal. Mutagenesis of Streptococcus pyogenes with TnFuZ ("FuZ" stands for fusions to phoZ) identified genes encoding secreted proteins whose expression was enhanced during growth in an aerobic environment. Thus, TnFuZ should be valuable for analysis of protein secretion, gene regulation, and virulence in gram-positive bacteria.     

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation

Cholera toxin secretion is dependent upon the extracellular protein secretion apparatus encoded by the eps gene locus of Vibrio cholerae . Although the eps gene locus encodes several type four prepilin-like proteins, the peptidase responsible for processing these proteins has not been identified. This report describes the identification of a prepilin peptidase from the V. cholerae genomic database by virtue of its homology with the PilD prepilin peptidase of Pseudomonas aeruginosa . Plasmid disruption or deletion of this peptidase gene in either El Tor or classical V. cholerae O1 biotype strains results in a dramatic decrease in cholera toxin secretion. In the case of the El Tor biotype mutants, surface expression of the type 4 pilus responsible for mannose-sensitive haemagglutination is abolished. The cloned V. cholerae peptidase processes either EpsI or MshA preproteins when co-expressed in E. coli . Mutation of the V. cholerae peptidase gene also results in a defect in virulence and decreased levels of OmpU. The V. cholerae peptidase gene sequence shows 80% homology with the Vibrio vulnificus VvpD type 4 prepilin peptidase required for pilus assembly and cytolysin secretion in V. vulnificus . Accordingly, the V. cholerae type 4 prepilin peptidase required for pilus assembly and cholera toxin secretion has been designated VcpD.     

Genetic toggling of alkaline phosphatase folding reveals signal peptides for all major modes of transport across the inner membrane of bacteria

Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen.     

Genetic analysis of an MDR-like export system: the secretion of colicin V.

The extracellular secretion of the antibacterial toxin colicin V is mediated via a signal sequence independent process which requires the products of two linked genes: cvaA and cvaB. The nucleotide sequence of cvaB reveals that its product is a member of a subfamily of proteins, involved in the export of diverse molecules, found in both eukaryotes and prokaryotes. This group of proteins, here referred to as the 'MDR-like' subfamily, is characterized by the presence of a hydrophobic region followed by a highly conserved ATP binding fold. By constructing fusions between the structural gene for colicin V, cvaC, and a gene for alkaline phosphatase, phoA, lacking its signal sequence, it was determined that 39 codons in the N-terminus of cvaC contained the structural information to allow CvaC-PhoA fusion proteins to be efficiently translocated across the plasma membrane of Escherichia coli in a CvaA/CvaB dependent fashion. This result is consistent with the location of point mutations in the cvaC gene which yielded export deficient colicin V. The presence of the export signal at the N-terminus of CvaC contrasts with the observed C-terminal location of the export signal for hemolysin, which also utilizes an MDR-like protein for its secretion. It was also found that the CvaA component of the colicin V export system shows amino acid sequence similarities with another component involved in hemolysin export, HlyD. The role of the second component in these systems and the possibility that other members of the MDR-like subfamily will also have corresponding second components are discussed. A third component used in both colicin V and hemolysin extracellular secretion is the E. coli host outer membrane protein, TolC.     

Plasmid expression vectors with elements of the E. coli alkaline phosphatase gene

A family of plasmid expression vectors, containing fragments of the E. coli alkaline phosphatase gene (phoA), has been constructed for the lambda PR promoter-directed thermoinducible superproduction of fusions of heterologous polypeptides to the N- or C-terminus of E. coli alkaline phosphatase and its leader peptide. Effective expression and export to periplasm of resulting fusion proteins, which may retain enzymatic activity of the phosphatase, has been shown.     

Use of signature-tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization

The pathogenesis of cholera begins with colonization of the host intestine by Vibrio cholerae . The toxin co-regulated pilus (TCP), a fimbrial structure produced by V . cholerae , is absolutely required for colonization (i.e. the persistence, survival and growth of V . cholerae in the upper intestinal milieu), but many other aspects of the colonization process are not well understood. In this study, we use signature-tagged transposon mutagenesis (STM) to conduct a screen for random insertion mutations that affect colonization in the suckling mouse model for cholera. Of approximately 1100 mutants screened, five mutants (approximately 0.5%) with transposon insertions in TCP biogenesis genes were isolated, validating the use of STM to identify attenuated mutants. Insertions in lipopolysaccharide, biotin and purine biosynthetic genes were also found to cause colonization defects. Similar results were observed for mutations in homologues of pta and ptfA , two genes involved in phosphate transfer. Finally, our screen identified several novel genes, disruption of which also caused colonization defects in the mouse model. These results demonstrate that STM is a powerful method for isolating colonization-defective mutants of V . cholerae .