Nucleotide sequence and expression in Escherichia coli of the Pseudomonas aeruginosa lasA gene.

Pseudomonas aeruginosa PAO-E64 is a mutant which produces parental levels of elastase antigen but has no elastolytic activity at 37 degrees C. The lesion (lasA1) in PAO-E64 is not a mutation in the structural gene for P. aeruginosa elastase (P.A. Schad, R.A. Bever, T.I. Nicas, F. Leduce, L.F. Hanne, and B.H. Iglewski, J. Bacteriol. 169: 2691-2696, 1987). A 1.7-kilobase segment of DNA that complements the lasA1 lesion was sequenced. Computer analysis of the DNA sequence showed that it contained an open reading frame which encoded a 41,111-dalton protein. The lasA gene was expressed under an inducible PT-7 promoter, and a 40,000-dalton protein was detected in Escherichia coli lysates. The lasA protein was localized in the outer membrane fraction of E. coli. This lasA protein produced in E. coli activated the extracellular elastase produced by the P. aeruginosa mutant, PAO-E64.     

Cloning of Escherichia coli and Pseudomonas aeruginosa phosphomannose isomerase genes and their expression in alginate-negative mutants of Pseudomonas aeruginosa.

The phosphomannose isomerase (pmi) gene of Escherichia coli was cloned on a broad-host-range cosmid vector and expressed in Pseudomonas aeruginosa at a low level. Plasmid pAD3, which harbors the E. coli pmi gene, contains a 6.2-kilobase-pair HindIII fragment derived from the chromosome of E. coli. Subcloning produced plasmids carrying the 1.5-kilobase-pair HindIII-HpaI subfragment of pAD3 that restored alginic acid production in a nonmucoid, alginate-negative mutant of P. aeruginosa. This fragment also complemented mannose-negative, phosphomannose isomerase-negative mutants of E. coli and showed no homology by DNA-DNA hybridization to P. aeruginosa chromosomal DNA. By using a BamHI constructed cosmid clone bank of the stable alginate producing strain 8830, we have been able to isolate a recombinant plasmid of P. aeruginosa origin that also restores alginate production in the alginate-negative mutant. This new recombinant plasmid, designated pAD4, contained a 9.9-kilobase-pair EcoRI-BamHI fragment with the ability to restore alginate synthesis in the alginate-negative P. aeruginosa. This fragment showed no homology to E. coli chromosomal DNA or to plasmid pAD3. Both mucoid and nonmucoid strains of P. aeruginosa had no detectable levels of phosphomannose isomerase activity as measured by mannose 6-phosphate-to-fructose 6-phosphate conversion. However, P. aeruginosa strains harboring the cloned pmi gene of E. coli contained measurable levels of phosphomannose isomerase activity as evidenced by examining the conversion of mannose 6-phosphate to fructose 6-phosphate.     

Heterologous expression and regulation of the lysA genes of Pseudomonas aeruginosa and Escherichia coli

Summary Chlorsulfuron-resistant mutants of Arabidopsis thaliana were isolated by screening for growth of seedlings in the presence of the herbicide. Both whole plants and derived tissue cultures were resistant to concentrations of the herbicide approximately 300-fold higher than that required to prevent growth of the wild-type. The resistance is due to a single dominant nuclear mutation at a locus designated csr which has been genetically mapped to chromosome-3. Acetohydroxy acid synthase activity in extracts from chlorsulfuron-resistant plants was much less-susceptible to inhibition by chlorsulfuron and a structurally related inhibitor than the activity in wild-type extracts. This suggests that the csr locus is the structural gene for acetohydroxy acid synthase.     

Cloning and expression of Pseudomonas aeruginosa flagellin in Escherichia coli.

The flagellin gene was isolated from a Pseudomonas aeruginosa PAO1 genomic bank by conjugation into a PA103 Fla- strain. Flagellin DNA was transferred from motile recipient PA103 Fla+ cells by transformation into Escherichia coli. We show that transformed E. coli expresses flagellin protein. Export of flagellin to the E. coli cell surface was suggested by positive colony blots of unlysed cells and by isolation of flagellin protein from E. coli supernatants.     

Molecular cloning and expression of perhydrolase genes from Pseudomonas aeruginosa and Burkholderia cepacia in Escherichia coli

Two bacterial perhydrolase genes, perPA and perBC, were cloned from Pseudomonas aeruginosa and Burkholderia cepacia, respectively, using PCR amplification with primers designed to be specific for conserved amino acid sequences of the already-known perhydrolases. The amino acid sequence of PerPA was identical to a putative perhydrolase of P. aeruginosa PAO1 genome sequences, whereas PerBC of B. cepacia was a novel bacterial perhydrolase showing similarity of less than 80% with all other existing perhydrolases. Most importantly, the perPA gene was expressed as a soluble intracellular form to an extent of more than 50% of the total protein content in Escherichia coli. Two perhydrolase enzymes were confirmed to exhibit the halogenation activity towards Phenol Red and monochlorodimedone. These results suggested that we successfully obtained the newly identified members of the bacterial perhydrolase family, expanding the pool of available perhydrolases.     

Ordering tryptophan synthase genes of Pseudomonas aeruginosa by cloning in Escherichia coli.

The Pseudomonas aeruginosa tryptophan synthase genes, trpA and trpB, which are induced by their substrate indoleglycerol phosphate, were cloned along with their controlling region into the BamHI site of pBR322 to produce the 10.7-megadalton plasmid pZAZ5. SalI partial digestion and ligation yielded a smaller plasmid, pZAZ167, with the chromosomal insert reduced in size from 8.1 to 3.4 megadaltons. Both pZAZ5 and pZAZ167 display Pseudomonas-like regulation of the trpA and trpB genes. Deletion of an EcoRI fragment or a BglII fragment from pZAZ167 yielded plasmids pZAZ168 and pZAZ169; the former expresses trpB but not trpA, and the latter has lost both activities. A deleted form of pZAZ5 designated pZAZ101 was obtained by excising a BglII-BamHI segment and religating the trip gene segment in the opposite orientation. This plasmid expresses trpA and trpB constitutively. The physical maps of these plasmids establish the gene order: promoter-trpB-trpA.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: II. Cloning and expression in Escherichia coli

The genes for the large and small subunits of anthranilate synthase (trpE and trpG, respectively) have been cloned from Pseudomonas aeruginosa PAC174 into E. coli by R-prime formation with the broad-host- range plasmid R68.44. Sequential subcloning into plasmid vectors reduced the active Pseudomonas DNA fragment to a length of 3.1 kb. We obtained evidence that this region contains the promoter for its own expression and retains a vestigial regulatory response to tryptophan scarcity or excess.      

Cloning and expression of the pilin gene of Pseudomonas aeruginosa PAK in Escherichia coli.

Many strains of Pseudomonas aeruginosa possess pili which have been implicated in the pathogenesis of the organism. This report presents the cloning and expression in Escherichia coli of the gene encoding the structural subunit of the pili of P. aeruginosa PAK. Total DNA from this strain was partially digested with Sau3A and inserted into the cloning vector pUC18. Recombinant E. coli clones were screened with oligonucleotide probes prepared from the constant region of the previously published amino acid sequence of the mature pilin subunit. Several positive clones were identified, and restriction maps were generated. Each clone contained an identical 1.1-kilobase HindIII fragment which hybridized to the oligonucleotide probes. Western blot analysis showed that all of the clones expressed small amounts of the P. aeruginosa pilin subunit, which has a molecular mass of ca. 18,000. This expression occurred independently of the orientation of the inserted DNA fragments in the cloning vector, indicating that synthesis was directed from an internal promoter. However, subclones containing the 1.1-kilobase HindIII fragment in a specific orientation produced an order of magnitude more of the pilin subunit. While the expressed pilin antigen was located in both the cytoplasmic and outer membrane fractions of E. coli, none appeared to be polymerized into a pilus structure.     

Nucleotide sequence and expression in Escherichia coli of the cephalosporin acylase gene of a Pseudomonas strain.

The gene encoding cephalosporin acylase, which hydrolyzes 7-beta-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) to 7-aminocephalosporanic acid (7ACA) and glutaric acid, was cloned from a Pseudomonas sp. strain V22 and expressed in Escherichia coli, in a two-cistron system, and the enzyme was purified and characterized. The purified enzyme was composed of two non-identical subunits, their molecular weights were estimated by SDS-PAGE to be 40,000 and 22,000, and had a pI of 4.6. The amino acid sequence of the enzyme, deduced from the nucleotide sequence, showed high similarity (97%) with that of a previously reported acyI-encoded cephalosporin acylase. Cephalosporin acylase also resembles the bacterial gamma-glutamyl transpeptidases (GGTs) with respect to their molecular organization and amino acid sequence, but differs from them with respect to catalytic and immunological properties. Purified enzyme exhibited not only cephalosporin acylase activity, but also GGT activity. The Km values of the enzyme for GL-7ACA and L-gamma-glutamyl-p-nitroanilide were 6.1 and 3.8 mM, respectively. Cephalosporin acylase was not recognized by antibodies prepared against bacterial GGTs.     

The expression of Pseudomonas aeruginosa PAK pilin gene mutants in Escherichia coli

Previous work has demonstrated the expression of the cloned pilin gene of Pseudomonas aeruginosa PAK within Escherichia coli and has pinpointed this protein's localization exclusively to the cytoplasmic membrane (Finlay et al., 1986). To define regions of the pilin subunit necessary for its stability and transport within E. coli, we constructed six mutants of the pilin gene and studied their expression and localization using a T7 promoter system. Two of the mutants have either a 4- or 8-amino-acid deletion at the N-terminus and both were stably expressed and transported primarily to the cytoplasmic membrane of E. coli. The other four mutants are C-terminal truncations having between 36 and 56 amino acids of the N-terminal region of the unprocessed pilin. Studies with these truncated mutants revealed that only the first 36 residues of the unprocessed pilin subunit were required for insertion into the E. coli membrane.     

Nucleotide sequence of the genes for tryptophan synthase in Pseudomonas aeruginosa

We have determined the DNA sequence of the two adjacent genes for the alpha and beta chains of tryptophan synthase in Pseudomonas aeruginosa, along with 34 5'-flanking and 799 3'-flanking base pairs. The gene order is trpBA as predicted from earlier genetic studies, and the two cistrons overlap by 4 bp; a ribosome binding site for the second gene is evident in the coding sequence of the first gene. We have also determined the location of three large deletions eliminating portions of each gene. A detailed comparison of the deduced P. aeruginosa amino acid sequence with those published for E. coli, Bacillus subtilis, and Saccharomyces cerevisiae shows much similarity throughout the beta and most of the alpha subunit. Most of the residues implicated by chemical modification or mutation as being critical for enzymatic activity are conserved, along with many others, suggesting that three-dimensional structure has remained largely constant during evolution. We also report the construction of a recombinant plasmid that overproduces a slightly modified alpha subunit from P. aeruginosa that can form a functionally effective multimer with normal E. coli beta 2 subunit in vivo.      

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene.

Unlike enteric bacteria, Pseudomonas spp. generally lack thymidine phosphorylase and thymidine kinase activities, thus preventing their utilization of exogenous thymine or thymidine and precluding specific radioactive labeling of their DNA in vivo. To overcome this limitation, a DNA fragment encoding thymidine kinase (EC 2.7.1.21) from Escherichia coli was cloned into pKT230, a small, broad-host-range plasmid derived from plasmid RSF1010. From transformed E. coli colonies, the recombinant plasmid bearing the thymidine kinase gene was conjugally transferred to Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes. Thymidine kinase activity was expressed in all of these species, and all gained the ability to incorporate exogenous [2-14C]thymidine into their DNA. Thymidine incorporation into P. stutzeri was enhanced 12-fold more in mutants lacking thymidylate synthetase activity. These mutants produced higher levels of thymidine kinase and were thymidine auxotrophs; thymineless death resulted from removal of thymidine from a growing culture.     

Cloning and expression of pca genes from Pseudomonas putida in Escherichia coli

Beta-Ketoadipate elicits expression of five structural pca genes encoding enzymes that catalyse consecutive reactions in the utilization of protocatechuate by Pseudomonas putida. Three derivatives of P. putida PRS2000 were obtained, each carrying a single copy of Tn5 DNA inserted into a separate region of the genome and preventing expression of different sets of pca genes. Selection of Tn5 in or near the pca genes in these derivatives was used to clone four structural pca genes and to enable their expression as inserts in pUC19 carried in Escherichia coli. Three of the genes were clustered as components of an apparent operon in the order pcaBDC. This observation indicates that rearrangement of the closely linked genes accompanied divergence of their evolutionary homologues, which are known to appear in the order pcaDBC in the Acinetobacter calcoaceticus pcaEFDBCA gene cluster. Additional evidence for genetic reorganization during evolutionary divergence emerged from the demonstration that the P. putida pcaE gene lies more than 15 kilobase pairs (kbp) away from the pcaBDC operon. An additional P. putida gene, pcaR, was shown to be required for expression of the pca structural genes in response to beta-ketoadipate. The regulatory pcaR gene is located about 15 kbp upstream from the pcaBDC operon.