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 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.     

Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate.

Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis commonly produce a capsule-like exopolysaccharide called alginate. The alginate-producing (Alg+) phenotype results in a mucoid colony morphology and is an unstable trait. A mutant of P. aeruginosa FRD (a cystic fibrosis isolate) was obtained which was temperature sensitive for alginate production ( Algts ). At elevated growth temperatures (41 degrees C), no alginate was detected in culture supernatants of the Algts mutant, but yields of alginate increased as the temperature of incubation was reduced. The mutation responsible for the Algts phenotype, alg-50(Ts), has been mapped to a region of the FRD chromosome closely linked to trp-2. The alg-50(Ts) marker did not map near the met-l-linked chromosomal mutations responsible for the instability of the Alg+ phenotype. A broad host range cosmid cloning system based upon derivatives of plasmid RK2 was used to construct a P. aeruginosa clone bank. After transfer of the clone bank to the Algts mutant, hybrid plasmids were obtained which complemented the Algts defect. Deletion mapping of the original 20.3 kilobases of P. aeruginosa DNA cloned showed that a 4.7-kilobase fragment would complement the alg-50(Ts) mutation.     

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 of the Pseudomonas aeruginosa gene encoding CDP-diglyceride synthetase

The CDP-diglyceride synthetase (CDS)-encoding gene (cds) from Pseudomonas aeruginosa PAO1 was cloned and sequenced. The gene possessed an open reading frame of 813 bp capable of encoding a putative polypeptide of 271 amino acids (aa) (28 699 Da). The deduced aa sequence of CDS revealed a 67% similarity (45% identity) to Escherichia coli CDS.     

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 sequencing of the Pseudomonas aeruginosa PAK pilin gene

A 1.2-kilobase (kb) HindIII restriction fragment containing the pilin gene from Pseudomonas aeruginosa PAK has been cloned and sequenced. The pilin protein is 144 amino acids in length with a positively charged leader sequence of 6 amino acids. There is probably only one copy of the gene per chromosome.     

Molecular characterization of imipenem-resistant Pseudomonas aeruginosa in Hiroshima, Japan

Pseudomonas aeruginosa showing resistance to imipenem were found in 100 of 1,058 strains (9.5%) from six hospitals (a-f) in Hiroshima City, Japan. Of the 100 strains, 14 (14%) were double disk synergy test positive using sodium mercaptoacetic acid disks, and 18 (18%) were bla(IMP-1) or bla(VIM-2) allele positive by polymerase chain reaction (PCR). Among 100 imipenem-resistant strains, 32 were categorized into multi-drug resistant strains, in which 13 were positive for the metallo-beta-lactamase gene. Fifty-one strains (51%) among the 100 imipenem-resistant strains had elevated RND efflux pump activity against levofloxacin. But only 6 of 51 strains were classified as multi-drug resistant strains. The pulsed field gel electrophoresis analysis of the Spe I-digested DNA from the 100 isolates suggested not only clonal spread but spread of heterogeneous clones started to contribute to the prevalence of metallo-beta-lactamase producing P. aeruginosa strains in Japanese hospitals.     

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, sequencing, and expression of the gene for NADH-sensitive citrate synthase of Pseudomonas aeruginosa

The structural gene for the allosteric citrate synthase of Pseudomonas aeruginosa has been cloned from a genomic library by using the Escherichia coli citrate synthase gene as a hybridization probe under conditions of reduced stringency. Subcloning of portions of the original 10-kilobase-pair (kbp) clone led to isolation of the structural gene, with its promoter, within a 2,083-bp length of DNA flanked by sites for KpnI and BamHI. The nucleotide sequence of this fragment is presented; the inferred amino acid sequence was 70 and 76% identical, respectively, with the citrate synthase sequences from E. coli and Acinetobacter anitratum, two other gram-negative bacteria. DEAE-cellulose chromatography of P. aeruginosa citrate synthase from an E. coli host harboring the cloned P. aeruginosa gene gave three peaks of activity. All three enzyme peaks had subunit molecular weights of 48,000; the proteins were identical by immunological criteria and very similar in kinetics of substrate saturation and NADH inhibition. Because the cloned gene contained only one open reading frame large enough to encode a polypeptide of such a size, the three peaks must represent different forms of the same protein. A portion of the cloned P. aeruginosa gene was used as a hybridization probe under stringent conditions to identify highly homologous sequences in genomic DNA of a second strain classified as P. aeruginosa and isolates of P. putida, P. stutzeri, and P. alcaligenes. When crude extracts of each of these four isolates were mixed with antiserum raised against purified P. aeruginosa citrate synthase, however, only the P. alcaligenes extract cross-reacted.     

The sequence and function of the recA gene and its protein in Pseudomonas aeruginosa PAO

Summary The recA gene of Pseudomonas aeruginosa has been isolated and its nucleotide sequence has been determined. The coding region of the recA gene has 1038 bp specifying 346 amino acids. The recA protein of P. aeruginosa showed a striking homology with that of Escherichia coli except for the carboxy-terminal region both at the nucleotide and amino acid level. The recA ⁺-carrying plasmids restored the UV sensitivity and recombination ability of several rec mutants of P. aeruginosa. The precise location of the recA gene on the chromosome was deduced from the analysis of R plasmids.     

群体感应淬灭酶克隆表达及其对铜绿假单胞菌的影响

【背景】由于抗生素的大量使用,导致细菌耐药性越来越强,寻找新的抗细菌感染药物成为研究热点。【目的】克隆表达群体感应淬灭酶,探究其对铜绿假单胞菌毒力及致病性的影响。【方法】利用PCR技术从产群体感应淬灭酶的芽孢杆菌QSI-1基因组DNA中克隆出aiiA基因,将其克隆到表达载体pET30a并导入大肠杆菌E.coliBL21(DE3)中进行诱导表达,通过镍柱亲和层析获得纯化的N-酰基高丝氨酸内酯酶。用不同浓度的淬灭酶作用于铜绿假单胞菌,检测其对铜绿假单胞菌毒力因子产生以及生物膜形成能力的影响;以秀丽隐杆线虫为模型,考察其对线虫感染铜绿假单胞菌存活率的影响。【结果】克隆表达出群体感应淬灭酶,该酶能显著抑制铜绿假单胞菌毒力因子产生和生物膜的形成,并能降低铜绿假单胞菌对感染线虫的致死率。【结论】群体感应淬灭酶可作为一种能高效抑制细菌致病性的物质,为临床治疗细菌性感染提供新的策略。     

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.