phoA Gene Fusions Research Articles

Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.

The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria. Introduction of the Mycobacterium fortuitum beta-lactamase gene (blaF*) into this vector led to the production in M. smegmatis of protein fusions with PhoA activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Sequences of the M. tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined. They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M. leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences. This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.

Membrane topology of multidrug resistance protein expressed in Escherichia coli. N-terminal domain.

Expression of eukaryotic polytopic membrane proteins in Escherichia coli could provide an invaluable system for structure-function studies. Recently, the functional expression of a mouse multidrug resistance protein (Mdr1) in E. coli was described (Bibi, E., Gros, P., and Kaback, H. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9209-9213). In the present study, the phoA gene fusion approach has been utilized to determine the membrane topology of the N-terminal domain of Mdr. The results support the idea that the N-terminal half of Mdr contains six transmembrane helices (TMs). However, our observations suggest that the previously proposed TM4 (amino acid residues Thr214-Ala232) is located at the periplasmic face of the membrane. Alternatively, we propose that another stretch of amino acid residues (Leu243 (out) to Ile260 (in)) traverses the membrane. This assignment is indicated also by the following observations: 1) deletion of a segment containing the originally predicted TM4 (delta T214-K241) does not reverse the orientation of the Mdr-alkaline phosphatase hybrid that is located in the following cytoplasmic loop; 2) a "sandwich" chimera, in which alkaline phosphatase is inserted in-frame between amino acid residues Leu226 and Ser227, exhibits high alkaline phosphatase activity. The existence of an externally exposed hydrophobic domain in Mdr may have special structural and functional implications, and these may also be relevant to other members of the ABC superfamily.

Cloning of a galacturonic acid uptake gene fromErwinia chrysanthemiEC16

A 3.4 kb fragment of Erwinia chrysanthemi EC16 DNA capable of complementing galacturonic acid uptake mutants (exuT) was identified and cloned into a multicopy vector. In E. chrysanthemi B374 exuT mutants, the cloned DNA provided for growth of the mutant strains on galacturonic acid by complementing the galacturonic acid uptake defect. Alkaline phosphatase (phoA) gene fusions with the cloned DNA suggested that most of the cloned DNA was necessary for complementation of exuT mutant strains. Using anti-alkaline phosphatase antibody, a hybrid ExuT-PhoA protein was localized to the membrane fraction of the bacterium.

An "all-purpose" cellulase reporter for gene fusion studies and application to the paracrystalline surface (S)-layer protein of Caulobacter crescentus.

The secreted endoglucanase (CenA) from the Gram-positive bacterium Cellulomonas fimi and a deletion derivative (delta CenA) lacking the N-terminal leader peptide of native CenA were used to explore the potential of delta CenA as a reporter molecule in Caulobacter crescentus. Expression of cenA in C. crescentus yielded extracellular endoglucanase activity, suggesting that the N-terminal leader peptide of CenA could direct the enzyme to the periplasm where it subsequently leaked into the medium. In contrast, expression of delta cenA yielded only cell-associated endoglucanase activity; this suggested that the enzyme retained activity in the C. crescentus cytoplasm. Using the putative cytoplasmic and periplasmic forms of delta CenA as markers, a simple assay for periplasmic delta CenA hybrids was developed. This assay indicated that delta CenA activity was largely independent of cellular location. To facilitate the use of delta CenA as a reporter, a broad host range translational fusion vector (pEC215) incorporating delta cenA was constructed. This vector was used to investigate factors important to the expression of the gene (rsaA) encoding the paracrystalline surface protein (S-layer) of the bacterium. It was found that altering the 5' untranslated region of the rsaA mRNA reduced gene expression by 70%. One rsaA:delta cenA gene fusion resulting from these experiments that incorporated only rsaA translation initiation information was further modified to serve as a general reporter for creating transcriptional gene fusions with other promoters. Gene fusions between alkaline phosphatase (phoA) and either cenA or lacZ were used to supplement information about RsaA secretion derived from rsaA:phoA gene fusions. It was found that linkage of the N-terminal leader peptide of CenA to PhoA yielded 50-100 times more cell-associated PhoA activity in C. crescentus than linkage of the RsaA N terminus. Taken together, these experiments indicated that delta CenA was useful for tagging proteins localized to the cytoplasm, exported to the periplasm, or secreted from the cell, as well as for monitoring events in the cytoplasm such as examining factors important to the level of gene expression. Further, because delta CenA was active in all cell compartments, it could be used to estimate the efficiency of hybrid protein export-secretion from enzyme activity measurements alone. In short, delta CenA possessed many of the attributes of an "all-purpose" reporter.

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.

A simple and efficient system for the construction of phoA gene fusions in Gram-negative bacteria

We have developed a two-plasmid system for generating gene fusions between phoA and cloned genes encoding envelope proteins. The vector plasmid carries a temperature-sensitive replication system and can be rescued at high temperature by insertion of an IS 1-based transposon carrying the ori region of pBR322 and a phoA gene lacking transcription and translation initiation signals. The vector plasmid also carries the transfer origin of the conjugative plasmid, F, permitting transfer into a suitable recipient strain. We have used this system in the analysis of the bla gene cloned from pBR322.

The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides

The topology of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides has been examined by generating gene fusions with alkaline phosphatase. Gene fusions were generated at random locations within the fbcB gene encoding the cytochrome b subunit. These fusion products were expressed in Escherichia coli and were screened for alkaline phosphatase activity on chromogenic plates. 33 in-frame fusions which showed activity were further characterized. The fusion junctions of all those fusions which had a high specific activity were clustered in three regions of the cytochrome b polypeptide, and thus these regions were tentatively assigned as being near the periplasmic surface. The data are consistent with a model containing eight transmembrane helices. In order to explore the validity of the gene fusion approach for a protein not normally expressed in E. coli, the topology of the L-subunit of the photosynthetic reaction center from R. sphaeroides was also explored using phoA gene fusions. A similar protocol was used as with the cytochrome b subunit. The gene fusions with high specific activity were shown to be in regions of the L-subunit polypeptide known to be at or near the periplasmic surface, as defined by the high resolution structure determined by X-ray crystallography. These data demonstrate the utility of this approach for determining membrane protein topology and extend potential applications to include at least some proteins not normally expressed in E. coli.

Isolation and characterization of extragenic suppressor mutants of thetolA-876 periplasmic-leaky allele inEscherichia coliK-12

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.

Isolation and characterization of extragenic suppressor mutants of the tolA-876 periplasmic-leaky allele in Escherichia coli K-12

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.

Transcriptional regulation by iron of a Vibrio cholerae virulence gene and homology of the gene to the Escherichia coli fur system

We have previously described an iron-regulated virulence determinant in Vibrio cholerae. Strain MBG40, which contains a TnphoA insertion mutation in the iron-regulated gene irgA, has reduced virulence in a newborn mouse model and has lost the major 77-kDa iron-regulated outer membrane protein. We report here the cloning of the irgA'-'phoA gene fusion, the sequencing of the 5'-proximal portion of irgA, and the definition of its promoter region by primer extension. The deduced amino acid sequence of the amino-terminal portion of IrgA is homologous to the ferrienterochelin receptor of Escherichia coli (FepA), suggesting that IrgA may be the iron-vibriobactin outer membrane receptor. Iron regulation of irgA in an E. coli background and that of the E. coli gene slt-IA in a V. cholerae background are reciprocal, suggesting a common mechanism of iron regulation. Regulation of irgA by iron in V. cholerae occurs at the transcriptional level, and there is an interrupted dyad symmetric sequence in the vicinity of the promoter that is homologous to Fur binding sites of E. coli. Unlike iron-regulated genes in E. coli, however, transcription of irgA requires an additional 900 bp of upstream DNA that contains an open reading frame in inverse orientation to irgA.

Export of FepA::PhoA fusion proteins to the outer membrane of Escherichia coli K-12

A library of fepA::phoA gene fusions was generated in order to study the structure and secretion of the Escherichia coli K-12 ferric enterobactin receptor, FepA. All of the fusion proteins contained various lengths of the amino-terminal portion of FepA fused in frame to the catalytic portion of bacterial alkaline phosphatase. Localization of FepA::PhoA fusion proteins in the cell envelope was dependent on the number of residues of mature FepA present at the amino terminus. Hybrids containing up to one-third of the amino-terminal portion of FepA fractionated with their periplasm, while those containing longer sequences of mature FepA were exported to the outer membrane. Outer membrane-localized fusion proteins expressed FepA sequences on the external face of the outer membrane and alkaline phosphatase moieties in the periplasmic space. From sequence determinations of the fepA::phoA fusion joints, residues within FepA which may be exposed on the periplasmic side of the outer membrane were identified.

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.

Analysis of expression and thermoregulation of the Yersinia pseudotuberculosis inv gene with hybrid proteins.

A series of translational fusions between the Yersinia pseudotuberculosis inv locus and lacZ was constructed. Each Lac+ fusion strain expressed a hybrid protein containing invasin, the product of the inv locus, at its amino-terminal end. Analysis of these gene fusions allowed determination of the direction of translation of the inv gene. Previous studies of Y. pseudotuberculosis invasion have shown that entry into animal cells is temperature regulated. It is shown here that control of expression of the inv gene is also temperature regulated. phoA gene fusions to inv, when present in Y. pseudotuberculosis, were expressed at lower levels when bacteria were grown at 37 degrees C rather than at 28 degrees C. Similar fusions, in contrast, were regulated in a temperature-independent fashion in Escherichia coli, as was the wild-type inv gene. This implies that Y. pseudotuberculosis has chromosomally encoded trans-acting functions that normally thermoregulate expression of inv.

Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions.

Gene iucD of the aerobactin operon of the Escherichia coli plasmid ColV-K30 encodes a membrane-bound enzyme synthesizing N6-hydroxylysine, the first product of the aerobactin biosynthesis pathway. The entire nucleotide sequence of the cloned iucD gene was determined, from which the primary and some aspects of the secondary structure of the encoded peptide were deduced. E. coli cells harboring multicopy plasmid pVLN12 (iucD+) hyperproduced an approximately 50-kilodalton peptide which was purified and identified as the product of the gene by examination of its amino-terminal sequence. Two iucD'-'lacZ gene fusions were constructed in vitro and four iucD'-'phoA gene fusions were generated in vivo by mutagenesis of iucD with transposon TnphoA (Tn5 IS50L::phoA). Analysis of the corresponding fusion proteins suggested at least two domains of attachment of the IucD protein to the inner side of the cytoplasmic membrane. The first apparent membrane-bound domain was found within the first 25 amino acids of the protein and showed a sequence which resembled that of the signal peptides.

Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin.

The transposon TnphoA was used to generate fusions between phoA, the gene for alkaline phosphatase (PhoA), and genes encoding proteins that are secreted by Vibrio cholerae. One of the PhoA+ mutants isolated showed a dramatic reduction in its ability to colonize the intestines of suckling mice. This mutant no longer produced a 20.5-kDa protein (TcpA) that we show is the major subunit of a V. cholerae pilus. Amino-terminal sequence analysis of the TcpA pilus subunit showed that it shares amino acid homology with the pilins produced by several other pathogenic bacteria. The TcpA pilus was coordinately expressed with cholera toxin under various culture conditions, and this effect appeared to be dependent on the transcriptional activator encoded by the toxR gene. We conclude that the toxR gene plays a central role in the transcriptional regulation of multiple virulence genes of V. cholerae.