恶臭假单胞菌丙氨酸消旋酶基因的克隆、序列分析及表达

从恶臭假单胞菌(Pseudomonas putida)200的基因组出发,用PCR方法克隆到两个独立作用的丙氨酸消旋酶基因,称之为dadX和alr。DadX编码357个氨基酸长的多肽,计算分子量为38.82kDa,alr编码409个氨基酸长的多肽,计算分子量为44.182kDa。序列分析显示,DadX的氨基酸序列与Pseudomonas putidaKT2440,铜绿假单胞菌(Pseudomonas aeruginosa),鼠伤寒沙门氏菌(Salmonella typhimurium)和大肠杆菌(Escherichia coli)的DadX比较,相似性分别为96.64%、71.99%、44.88%和47.37%。Alr的氨基酸序列与Pseudomonas putidaKT2440比较,同源性为94.38%,而与铜绿假单胞菌(P.aeruginosa)、鼠伤寒沙门氏菌(S.typhimurium)和大肠杆菌(E.coli)的Alr比较,同源性均较低,分别为22.89%、25.72%和26.44%。在P.putida200的DadX和Alr氨基酸序列中部发现有对于酶活性至关重要的保守区域,如磷酸吡哆醛(PLP)结合位点。DadX和alr在大肠杆菌中得到表达,DadX丙氨酸消旋酶只对丙氨酸有消旋作用,而Alr丙氨酸消旋酶可以作用于丙氨酸和丝氨酸两种底物,且对丝氨酸特异性更高。Alr的表达不依赖于外源启动子,说明在其结构基因上游存在启动子结构。     

Cloning, DNA sequence analysis, and expression in Escherichia coli of the gene for mandelate racemase from Pseudomonas putida

The gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) was cloned in Pseudomonas aeruginosa (ATCC 15692). The selection for the cloned gene was based upon the inability of P. aeruginosa to grow on (R)-mandelate as sole carbon source by virtue of the absence of mandelate racemase in its mandelate pathway. Fragments of P. putida DNA obtained by digestion of chromosomal DNA with Sau3A were ligated into the BamHI site of the Gram-negative vector pKT230 and transformed into the P. aeruginosa host. A transformant able to utilize (R)-mandelate as sole carbon source was characterized, and the plasmid was found to contain approximately five kilobase pairs of P. putida DNA. Subcloning of this DNA revealed the position of the gene for the racemase within the cloned DNA from P. putida. The dideoxy-DNA sequencing procedure was used to determine the sequence of the gene and its translated sequence. The amino acid sequence and molecular weight for mandelate racemase deduced from the gene sequence (38 570) are in excellent agreement with amino acid composition and molecular weight data for the polypeptide recently determined with enzyme isolated from P. putida; these recent determinations of the polypeptide molecular weight differ significantly from the originally reported value of 69,500 [Fee, Judith A., Hegeman, G.D., & Kenyon, G.L. (1974) Biochemistry 13,2528], which was used to demonstrate that alpha-phenylglycidate, an active site directed irreversible inhibitor, binds to the enzyme with a stoichiometry of 1:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa.

Pseudomonas aeruginosa K437 lacks the ferripyoverdine receptor and, as a result, grows poorly on an iron-deficient minimal medium supplemented with ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and pyroverdine. By using a phagemid-based in vivo cloning system, attempts were made to clone the receptor gene by complementing this growth defect. Several recombinant phagemids carrying P. aeruginosa chromosomal DNA which provided for good growth on EDDHA-pyoverdine-containing medium and which concomitantly restored production of the ferripyroverdine receptor in strain K437 were isolated. These phagemids contained a common 4.6-kb SphI fragment which similarly restored production of the receptor in K437. Nucleotide sequencing of the SphI fragment revealed a single large open reading frame, designated fpvA (ferripyoverdine uptake), of 2439 bp. The predicted translation product of fpvA has a molecular mass of 89,395 Da. N-terminal amino acid sequence analysis of the purified ferripyoverdine receptor confirmed fpvA as the receptor gene. Moreover, it indicated that the receptor is initially synthesized as a precursor with a signal sequence of 27 amino acids which is cleaved to yield the mature protein. The deduced FpvA polypeptide exhibited homology to regions shown to be conserved in TonB-dependent receptor proteins. FpvA also shared strong homology (41.3% identity) with the PupA protein of Pseudomonas putida WCS358. This protein is the receptor for the iron-bound form of pseudobactin, a compound structurally very similar to pyoverdine.     

Cloning, nucleotide sequence and structural analysis of the Clostridium acetobutylicum dnaJ gene

Abstract The complete dnaJ gene of Clostridium acetobutylicum was isolated by chromosome walking using the previously cloned 5' end of the gene as a probe. Nucleotide sequencing of a positively reacting 2.2-kb Hin cII fragment, contained in the recombinant plasmid pKG4, revealed that the reading frame of the dnaJ gene of C. acetobutylicum consists of 1125 bp, encoding a protein of 374 amino acids with a calculated M _r of 40376 and an isoelectric points of 9.54. The deduced amino acid sequence showed high similarity to the DnaJ proteins of other bacteria (e.g. Escherichia coli, Bacillus subtilis ) as well as of an archaeon ( Methanosarcina mazei ) and to the corresponding proteins of eukaryotes ( Saccharomyces cerevisiae, Homo sapiens ). The areas of similarity included a conserved N-terminal domain of about 70 amino acids, a glycine-rich region of about 30 residues, and a central domain containing four repeats of a CXXCXGXG motif, whereas the C-terminal domain was less conserved. Northern (RNA) blot analysis indicated that dnaJ is induced by heat shock and that it is part of the dnaK operon of C. acetobutylicum . The 5' end (901 bp) of another gene ( orfB ), downstream of dnaJ and not heat-inducible, showed no significant similarity to other sequences available in EMBL and GenBank databases.     

Cloning of genes specifying carbohydrate catabolism in Pseudomonas aeruginosa and Pseudomonas putida.

A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.     

Cloning and nucleotide sequence of the aspartase gene of Pseudomonas fluorescens

The aspartase gene (aspA) of Pseudomonas fluorescens was cloned and the nucleotide sequence of the 2,066-base-pair DNA fragment containing the aspA gene was determined. The amino acid sequence of the protein deduced from the nucleotide sequence was confirmed by N- and C-terminal sequence analysis of the purified enzyme protein. The deduced amino acid composition also fitted the previous amino acid analysis results well (Takagi et al. (1984) J. Biochem. 96, 545-552). These results indicate that aspartase of P. fluorescens consists of four identical subunits with a molecular weight of 50,859, composed of 472 amino acid residues. The coding sequence of the gene was preceded by a potential Shine-Dalgarno sequence and by a few promoter-like structures. Following the stop codon there was a structure which is reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 62.3%. Inspection of the codon usage for the aspA gene revealed as high as 80.0% preference for G or C at the third codon position. The deduced amino acid sequence was 56.3% homologous with that of the enzyme of E. coli W (Takagi et al. (1985) Nucl. Acids Res. 13, 2063-2074). Cys-140 and Cys-430 of the E. coli enzyme, which had been assigned as functionally essential (Ida & Tokushige (1985) J. Biochem. 98, 793-797), were substituted by Ala-140 and Ala-431, respectively, in the P. fluorescens enzyme.     

Sequence analysis of the hutH gene encoding histidine ammonia-lyase in Pseudomonas putida.

The complete nucleotide sequence of the hutH gene, encoding histidine ammonia-lyase (histidase), in Pseudomonas putida ATCC 12633 has been determined from the appropriate portions of the hut region that had been cloned into Escherichia coli. The resulting DNA sequence revealed an open reading frame of 1,530 base pairs, corresponding to a protein subunit of approximate molecular weight 53,600, in the location previously identified for the histidase gene by Tn1000 mutagenesis. Translation began at a GTG codon, but direct protein sequencing revealed that the initiating amino acid was removed posttranslationally to provide an N-terminal threonine; 11 additional residues completely agreed with the predicted amino acid sequence. This sequence excluded the possibility that a dehydroalanine unit, the postulated coenzyme for histidase, is attached at the N terminus of histidase subunits. Comparison of the P. putida histidase gene sequence with that of a Bacillus subtilis region encoding histidase revealed 42% identity at the protein level. Although the hutU (urocanase) and hutH (histidase) genes are induced by urocanate and normally are transcribed as a unit beginning with hutU, analysis of the region immediately upstream of the histidase gene revealed a potential weak promoter that may possibly be used to maintain a basal level of histidase for the generation of inducer (urocanate) when histidine levels are elevated.     

Cloning and expression of the polyhydroxyalkanote depolymerase gene from Pseudomonas putida, and characterization of the gene product

A polyhydroxyalkanote (PHA) depolymerase gene ( pha Z) was cloned by PCR from Pseudomonas putida and over-expressed in Escherichia coli as inclusion bodies. Nucleotide sequence analysis predicted an 852 bp open reading frame encoding a protein of 283 amino acids with a predicted molecular weight of 31283 Da. The deduced amino acid sequence had at least 80% homology to the PHA depolymerase from other Pseudomonas strains and consisted a conserved lipase box-like sequence (G-X-S(102)-X-G). The inclusion bodies were refolded and biochemically characterized. The depolymerase activity was optimal at 40 degrees C and pH 8.     

Cloning, nucleotide sequence, and overexpression of the gene coding for delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.

The structural gene coding for the delta 5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. putida biotype B genomic library in lambda gt11. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bp upstream of the ATG start codon. Escherichia coli BL21(DE3) transformed with the pKK-KSI plasmid containing the ksi gene expressed a high level of isomerase activity when induced by isopropyl-beta-D-thiogalactopyranoside. KSI was purified to homogeneity by a simple and rapid procedure utilizing fractional precipitation and an affinity column of deoxycholate-ethylenediamine-agarose as a major chromatographic step. The molecular weight of KSI was 14,535 (calculated, 14,536) as determined by electrospray mass spectrometry. The purified KSI showed a specific activity (39,807 mumol min-1 mg-1) and a Km (60 microM) which are close to those of KSI originally obtained from P. putida biotype B.     

Cloning of genes for naphthalene metabolism in Pseudomonas putida.

Plasmid pIG7 DNA cloned in Pseudomonas putida with the broad-host-range vectors pRK290 and pKT240 expresses the genes encoding nephthalene oxidation in the presence of the intermediate substrate, salicylate, or the gratuitous inducer, anthranilate. Two operons, nahAF and nahGK, cloned from the EcoRI fragment A (25 kilobases) are under wild-type regulation by the nahR locus. Deletion plasmids provide a restriction map of both operons. Double transformants containing structural and regulatory cistron nahR in trans are used to demonstrate positive control of expression.     

Cloning of pectate lyase gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida.

Pectate lyase (PL) depolymerizes pectin and other polygalacturonates (PGAs) and is thought to play a role in bacterial invasion of plants. Production of PL by the soft-rotting pathogen Pseudomonas fluorescens CY091 is regulated by Ca2+. In the presence of Ca2+, this bacterium constitutively synthesizes PL in media containing glucose, glycerol, or PGA and excretes over 87% of total PL into culture fluids. In the absence of Ca2+, the organism fails to use PGA as a carbon source and produces very low levels of PL in media containing glucose or glycerol. Of the small amount of PL produced by the bacterium in Ca(2+)-deficient media, over 78% was detected within the cells, indicating that Ca2+ is critical not only for the production but also for the secretion of PL. The pel gene, encoding an alkaline PL (pI 10.0, Mr 41,000) was cloned and located on the overlapping region of a 4.3-kb SalI and a 7.1-kb EcoRI fragment. The 7.1-kb EcoRI fragment appears to contain a promoter for pel gene expression. A 1.7-kb SalI-XhoI subfragment of the 4.3-kb SalI fragment was cloned into pUC18 to give pROTM2. Escherichia coli cells carrying pROTM2 produce 50 to 100 times more PL than do cells carrying other pectolytic constructs. Production of PL by E. coli (pROTM2) was not affected by carbon sources or by Ca2+. The pI and Mr of PL from E. coli corresponded to values for its counterpart from P. fluorescens. A 0.7-kb BglII-ClaI fragment encoding the pel structural sequence was used to detect pel homologs in various species of fluorescent pseudomonads. Homologous sequences were observed in 10 of 11 strains of P. fluorescens, P. viridiflava, and P. putida. The pel gene in fluorescent pseudomonads is well conserved and may exist and remain repressed in certain strains or species which exhibit nonpectolytic phenotypes under laboratory conditions.     

Cloning, sequencing and analysis of the structural gene and regulatory region of the Pseudomonas aeruginosa chromosomal ampC beta-lactamase.

The chromosomal gene from Pseudomonas aeruginosa encoding beta-lactamase has been cloned, and the sequence determined and compared with corresponding sequences of beta-lactamases from members of the enterobacteriaceae. Upstream of the beta-lactamase gene is an open reading frame which we postulate encodes a regulatory protein, AmpR. We identified a helix-turn-helix region in AmpR and a putative AmpR-binding site.