Nucleotide sequence and expression of the algE gene involved in alginate biosynthesis by Pseudomonas aeruginosa.

Alginate (Alg), a random polymer of mannuronic acid and glucuronic acid residues, is synthesized and secreted by Pseudomonas aeruginosa primarily during its infection of the lungs of cystic fibrosis patients. The molecular biology and biochemistry of the enzymatic steps leading to the production of the Alg precursor GDP-mannuronic acid have been elucidated, but the mechanism of polymer formation and export of Alg are not understood. We report the nucleotide sequence of a 2.4-kb DNA fragment containing the algE gene, previously designated alg76, encoding the AlgE protein (Mr 54,361) that is believed to be involved in these late steps of Alg biosynthesis. Expression of algE appears to occur from its own promoter. The promoter region contains several direct and inverted repeat sequences and shares structural similarity with promoters of several other alg genes from P. aeruginosa. In addition, the AlgE protein was overproduced from the tac promoter in P. aeruginosa. N-terminal amino acid sequence analysis showed that the polypeptide contains a signal peptide which is cleaved to form the mature protein during AlgE export from the cell cytoplasm.     

Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa.

Reporter gene technology was employed to detect the activity of an alginate promoter of Pseudomonas aeruginosa when the organism was grown as a biofilm on a Teflon mesh substratum and as planktonic cells in liquid medium. Alginate biosynthetic activity was determined with a mucoid cell line derived from a cystic fibrosis isolate and containing an alginate algC promoter fused to a lacZ reporter gene. Reporter activity was demonstrated with chromogenic and fluorogenic substrates for beta-galactosidase. Expression of algC was shown to be upregulated in biofilm cells compared with planktonic cells in liquid medium. Gene up-expression correlated with alginate biosynthesis as measured by Fourier transform infrared spectroscopy, uronic acid accumulation, and alginate-specific enzyme-linked immunosorbent assay. The algC promoter was shown to have maximum activity in planktonic cultures during the late lag and early log phases of the cell growth cycle. During a time course experiment, biofilm algC activity exceeded planktonic activity except during the period immediately following inoculation into fresh medium. In continuous-culture experiments, conversion of lacZ substrate was demonstrated microscopically in individual cells by epifluorescence microscopy.     

Cloning and characterization of a Pseudomonas aeruginosa gene involved in the negative regulation of phosphate taxis.

Pseudomonas aeruginosa PAO1 exhibited a positive chemotactic response to P(i). The chemotactic response was induced by P(i) limitation. An alkaline phosphatase (AP) constitutive mutant showed a chemotactic response to P(i), regardless of whether the cells were starved for P(i). Sequence analysis and complementation studies showed that the P. aeruginosa phoU gene was involved both in the regulation of AP expression and in the induction of P(i) taxis. However, unlike AP expression, P(i) taxis was not regulated by the phoB gene product.     

Cloning and heterologous expression of a gene encoding an alkane-induced extracellular protein involved in alkane assimilation from Pseudomonas aeruginosa.

Pseudomonas aeruginosa PG201 produces a 16-kDa extracellular protein in media containing n-hexadecane as a carbon source but not in media containing glycerol or glucose. This protein was purified, and the N-terminal amino acid sequence was determined. The amino acid composition of the protein was found to be very similar to that of the so-called protein-like activator for n-alkane oxidation (PA) from P. aeruginosa S7B1. This extracellular protein was previously characterized (K. Hisatsuka, T. Nakahara, Y. Minoda, and K. Yamada, Agric. Biol. Chem. 41:445-450, 1977) and found to stimulate the growth of P. aeruginosa on n-hexadecane and to possess emulsifying activity. To study the role(s) of the PA protein and to make it accessible for possible future applications, we have cloned the PA-encoding (pra) gene and determined its nucleotide sequence. This analysis revealed a protein-coding region of 162 amino acids, with the first 25 residues being reminiscent of those of a typical bacterial signal sequence. The pra gene was inactivated by insertional mutagenesis, and the resulting strain was found to lack extracellular PA protein and to be retarded in its growth in n-hexadecane-containing media. These results are consistent with the growth stimulatory role of the PA protein. The pra gene was expressed in Escherichia coli, and substantial amounts of the recombinant protein were found in the extracellular growth medium. The recombinant protein was purified by metal chelate affinity chromatography. The ability to produce secreted PA protein by E. coli provides a simple and safe means to analyze its function(s) in alkane assimilation in the future.     

Cloning of Pseudomonas aeruginosa algG, which controls alginate structure.

The biochemical mechanism by which alpha-L-guluronate (G) residues are incorporated into alginate by Pseudomonas aeruginosa is not understood. P. aeruginosa first synthesizes GDP-mannuronate, which is used to incorporate beta-D-mannuronate residues into the polymer. It is likely that the conversion of some beta-D-mannuronate residues to G occurs by the action of a C-5 epimerase at either the monomer (e.g., sugar-nucleotide) or the polymer level. This study describes the results of a molecular genetic approach to identify a gene involved in the formation or incorporation of G residues into alginate by P. aeruginosa. Mucoid P. aeruginosa FRD1 was chemically mutagenized, and mutants FRD462 and FRD465, which were incapable of incorporating G residues into alginate, were independently isolated. Assays using a G-specific alginate lyase from Klebsiella aerogenes and 1H-nuclear magnetic resonance analyses showed that G residues were absent in the alginates secreted by these mutants. 1H-nuclear magnetic resonance analyses also showed that alginate from wild-type P. aeruginosa contained no detectable blocks of G. The mutations responsible for defective incorporation of G residues into alginate in the mutants FRD462 and FRD465 were designated algG4 and algG7, respectively. Genetic mapping experiments revealed that algG was closely linked (greater than 90%) to argF, which lies at 34 min on the P. aeruginosa chromosome and is adjacent to a cluster of genes required for alginate biosynthesis. The clone pALG2, which contained 35 kilobases of P. aeruginosa DNA that included the algG and argF wild-type alleles, was identified from a P. aeruginosa gene bank by a screening method that involved gene replacement. A DNA fragment carrying algG was shown to complement algG4 and algG7 in trans. The algG gene was physically mapped on the alginate gene cluster by subcloning and Tn501 mutagenesis.     

Cloning of genes from mucoid Pseudomonas aeruginosa which control spontaneous conversion to the alginate production phenotype.

Strains of Pseudomonas aeruginosa causing chronic pulmonary infections in patients with cystic fibrosis are known to convert to a mucoid form in vivo characterized by the production of the exopolysaccharide alginate. The alginate production trait is not stable, and mucoid strains frequently convert back to the nonmucoid form in vitro. The DNA involved in these spontaneous alginate conversions, referred to as algS, was shown here to map near hisI and pru markers on the chromosome of strain FRD, an isolate from a cystic fibrosis patient. Although cloning algS+ by trans-complementation was not possible, a clone (pJF5) was isolated that caused algS mutants to convert to the Alg+ phenotype at detectable frequencies (approximately 0.1%) in vitro. Gene replacement with transposon-marked pJF5 followed by mapping studies showed that pJF5 contained DNA transducibly close to algS in the chromosome. Another clone was identified called pJF15 which did contain algS+ from mucoid P. aeruginosa. The plasmid-borne algS+ locus could not complement spontaneous algS mutations in trans, but its cis-acting activity was readily observed after gene replacement with the algS mutant chromosome by using an adjacent transposon as the selectable marker. pJF15 also contained a trans-active gene called algT+ in addition to the cis-active gene algS+. The algT gene was localized on pJF15 by using deletion mapping and transposon mutagenesis. By using gene replacement, algT::Tn501 mutants of P. aeruginosa were constructed which were shown to be complemented in trans by pJF15. Both algS and algT were located on a DNA fragment approximately 3 kilobases in size. The algS gene may be a genetic switch which regulates the process of alginate conversion.     

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.     

Cloning and expression of the alkaline proteinase gene from Pseudomonas aeruginosa IFO 3455.

The alkaline proteinase gene from Pseudomonas aeruginosa IFO 3455 was cloned and expressed in Escherichia coli.     

Nucleotide sequence and expression of the Pseudomonas aeruginosa algF gene controlling acetylation of alginate

Colonization of the cystic fibrosis lung by Pseudomonas aeruginosa is greatly facilitated by the production of an exopolysaccharide called alginate. In this study we determined the nucleotide sequence of an alginate modification gene, algF, which controls the addition of acetyl groups to alginate. Expression of algF using a T7 promoter-expression system showed that algF codes for a 24.5 kDa polypeptide (predicted size 22 832 Da) that is processed to 19.5 kDa. The N-terminus of the processed polypeptide matched the predicted amino acid sequence of AlgF starting at Asp-29. An algF mutant failed to produce alginate owing to a polar effect on the downstream algA gene. Although the algA gene, provided in trans, restored synthesis of alginate, the alginate was non-acetylated. We show that a plasmid containing both the algF and algA gene complements the alginate acetylation defect of the algF mutant strain.     

Cloning and expression of the catA and catBC gene clusters from Pseudomonas aeruginosa PAO.

A 9.9-kilobase (kb) BamHI restriction endonuclease fragment encoding the catA and catBC gene clusters was selected from a gene bank of the Pseudomonas aeruginosa PAO1c chromosome. The catA, catB, and catC genes encode enzymes that catalyze consecutive reactions in the catechol branch of the beta-ketoadipate pathway: catA, catechol-1,2-dioxygenase (EC 1.13.11.1); catB, muconate lactonizing enzyme (EC 5.5.1.1); and catC, muconolactone isomerase (EC 5.3.3.4). A recombinant plasmid, pRO1783, which contains the 9.9-kb BamHI restriction fragment complemented P. aeruginosa mutants with lesions in the catA, catB, or catC gene; however, this fragment of chromosomal DNA did not contain any other catabolic genes which had been placed near the catA or catBC cluster based on cotransducibility of the loci. Restriction mapping, deletion subcloning, and complementation analysis showed that the order of the genes on the cloned chromosomal DNA fragment is catA, catB, catC. The catBC genes are tightly linked and are transcribed from a single promoter that is on the 5' side of the catB gene. The catA gene is approximately 3 kb from the catBC genes. The cloned P. aeruginosa catA, catB, and catC genes were expressed at basal levels in blocked mutants of Pseudomonas putida and did not exhibit an inducible response. These observations suggest positive regulation of the P. aeruginosa catA and catBC cluster, the absence of a positive regulatory element from pRO1783, and the inability of the P. putida regulatory gene product to induce expression of the P. aeruginosa catA, catB, and catC genes.     

Cloning and expression of a Bacillus licheniformisα-amylase gene in Pseudomonas aeruginosa

Abstract The gene for B. licheniformis α-amylase has been cloned in P. aeruginosa . Synthesis of the enzyme occurs in late log phase and goes on during stationary phase. Although P. aeruginosa is a secretory bacterium, α-amylase is not efficiently secreted into the extracellular medium; 85% of the enzyme is retained in the periplasm.     

Cloning and expression of a gene segment encoding the enzymatic moiety of Pseudomonas aeruginosa exotoxin A.

Using the broad-host-range plasmid vector pRO1614, we cloned a segment of the gene from Pseudomonas aeruginosa PA103 encoding the enzymatically active part of the exotoxin A protein. Expression of the cloned gene segment has been achieved both in Escherichia coli and in a nontoxigenic P. aeruginosa host, as assayed by the production of exotoxin A-related antigen and by the ability of the gene product to ADP-ribosylate elongation factor 2. Western blot hybridization analysis revealed a series of polypeptides antigenically related to exotoxin A, the largest of which had a molecular weight of ca. 50,000.     

Cloning of a catabolite repression control (crc) gene from Pseudomonas aeruginosa, expression of the gene in Escherichia coli, and identification of the gene product in Pseudomonas aeruginosa.

Mutants which are defective in catabolite repression control (CRC) of multiple independently regulated catabolic pathways have been previously described. The mutations were mapped at 11 min on the Pseudomonas aeruginosa chromosome and designated crc. This report describes the cloning of a gene which restores normal CRC to these Crc- mutants in trans. The gene expressing this CRC activity was subcloned on a 2-kb piece of DNA. When this 2-kb fragment was placed in a plasmid behind a phage T7 promoter and transcribed by T7 RNA polymerase, a soluble protein with a molecular weight (MW) of about 30,000 was produced in Escherichia coli. A soluble protein of identical size was overproduced in a Crc- mutant when it contained the 2-kb fragment on a multicopy plasmid. This protein could not be detected in the mutant containing the vector without the 2-kb insert or with no plasmid. When a 0.3-kb AccI fragment was removed from the crc gene and replaced with a kanamycin resistance cassette, the interrupted crc gene no longer restored CRC to the mutant, and the mutant containing the interrupted gene no longer overproduced the 30,000-MW protein. Pools of intracellular cyclic AMP and the activities of adenylate cyclase and phosphodiesterase were measured in mutant and wild-type strains with and without a plasmid containing the crc gene. No consistent differences between any strains were found in any case. These results provide original evidence for a 30,000-MW protein encoded by crc+ that is required for wild-type CRC in P. aeruginosa and confirms earlier reports that the mode of CRC is cyclic AMP independent in this bacterium.     

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.     

Cloning and characteristics of recA gene in Pseudomonas aeruginosa

A recA-like gene from Pseudomonas aeruginosa was cloned and identified by means of interspecific complementation of gene recA repair defect in Escherichia coli. The gene was mapped in the PvuII-HindIII Ps. aeruginosa chromosome fragment of 1.5 kbp in length. Having been recloned in pUC18 or 19 plasmids in either of possible orientations, this fragment was shown to complement three different defects of E. coli recA mutants: in repair, recombination and SOS functions.     

Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the sputum of cystic fibrosis patients produce copious quantities of an exopolysaccharide known as alginic acid. Since clinical isolates of the mucoid variants are unstable with respect to alginate synthesis and revert spontaneously to the more typical nonmucoid phenotype, it has been difficult to isolate individual structural gene mutants defective in alginate synthesis. The cloning of the genes controlling alginate synthesis has been facilitated by the isolation of a stable alginate-producing strain, 8830. The stable mucoid strain was mutagenized with ethyl methanesulfonate to obtain various mutants defective in alginate biosynthesis. Several nonmucoid (Alg-) mutants were isolated. A mucoid P. aeruginosa gene library was then constructed, using a cosmid cloning vector. DNA isolated from the stable mucoid strain 8830 was partially digested with the restriction endonuclease HindIII and ligated to the HindIII site of the broad host range cosmid vector, pCP13. After packaging in lambda particles, the recombinant DNA was introduced via transfection into Escherichia coli AC80. The clone bank was mated (en masse) from E. coli into various P. aeruginosa 8830 nonmucoid mutants with the help of pRK2013, which provided donor functions in trans, and tetracycline-resistant exconjugants were screened for the ability to form mucoid colonies. Three recombinant plasmids, pAD1, pAD2, and pAD3, containing DNA inserts of 20, 9.5, and 6.2 kilobases, respectively, were isolated based on their ability to restore alginate synthesis in various strain 8830 nonmucoid (Alg-) mutants. Mutants have been assigned to at least four complementation groups, based on complementation by pAD1, pAD2, or pAD3 or by none of them. Introduction of pAD1 into the spontaneous nonmucoid strain 8822, as well as into other nonmucoid laboratory strains of P. aeruginosa such as PAO and SB1, was found to slowly induce alginate synthesis. This alginate-inducing ability was found to reside on a 7.5-kilobase EcoRI fragment that complemented the alg-22 mutation of strain 8852. The pAD1 chromosomal insert which complements the alg-22 mutation was subsequently mapped at ca. 19 min of the P. aeruginosa PAO chromosome.