藤黄绿脓菌素的自诱导及假单胞菌M18抗生物质代谢相关性初步分析

假单胞菌M18的生防功能归功于其分泌吩嗪-1-羧酸和藤黄绿脓菌素。为了研究抗生物质合成代谢相关性及调控机制,分别构建了两种抗生物质合成基因簇插入突变株M18T和M18Z1。用翻译融合表达载体pMEAZ(pltA′-′lacZ)分别转化野生株和突变株M18T、发酵培养并测定β-半乳糖苷酶活性,结果显示,添加藤黄绿脓菌素使突变株M18T(pMEAZ)的β-半乳糖苷酶活性比野生株M18(pMEAZ)增加约6倍,表明藤黄绿脓菌素对自身基因簇具正向自诱导作用。抗生物质的测定结果显示,突变株M18T无藤黄绿脓菌素合成,而吩嗪-1-羧酸的合成量与野生株相同;突变株M18Z1与野生株相比,吩嗪-1-羧酸明显减少,藤黄绿脓菌素却显著提高。过量的吩嗪-1-羧酸又抑制藤黄绿脓菌素的合成。表明,假单胞菌M18中独有的代谢相关方式为:藤黄绿脓菌素不影响吩嗪-1-羧酸,但吩嗪-1-羧酸负调控藤黄绿脓菌素。     

Phytase activity in some groups of bacteria. Search for and cloning of genes for bacterial phytases

A search for phytase genes in 9 Bacillus strains from the collection of IMGAN was implemented. The growth optimum of strains IX-22, IX-12B, K17-2, K18, IMG I, IMG II, M4 and M8 was 50-60 degrees C; the optimal growth temperature for Bacillus sp. 790 was 45-47 degrees C. According to the sequence data of 16S RNA genes, Bacillus sp. 790 belongs to the B. subtilis/amyloliquefaciens group. The other 8 strains were identified as B. licheniformis. Selection of Bacillus strains, potentially containing the phytase genes, was performed via PCR with primers designed on the basis of the conserved sequence regions of the phyA gene from B. amyloliquefaciens FZB45 with chromosomal DNA being used as the template. The nucleotide sequences of all PCR fragments showed a high level of homology to the known Bacillus phytase genes. The gene libraries of B. licheniformis M8 and B. amyloliquefaciens 790 in E. coli were constructed and phytase-containing clones were selected from them. Twenty-four Pseudomonas strains of different species, 5 Xanthomonas maltophilia strains and 1 Xanthomonas malvacearum (all from the mentioned collection) were tested for phytase activity. Such activity was found in 13 Pseudomonas strains and in 6 Xanthomonas strains. The accumulation of phytase in Pseudomonas was shown to take place at later (over 2 days') growth stages. The optimum pH for phytase from 3 Pseudomonas strains were established. The enzymes were found to be most active at pH 5.5.     

Cloning and nucleotide sequence analysis of xylE gene responsible for meta-cleavage of 4-chlorocatechol from Pseudomonas sp. S-47

Pseudomonas sp. S-47 expresses catechol 2,3-dioxygenase (C230) catalyzing the conversion of 4-chlorocatechol (4CC) as well as catechol to 5-chloro-2-hydroxymuconic semialdehyde and 2-hydroxymuconic semialdehyde, respectively, through meta-ring cleavage. The xylE gene encoding C230 for meta-cleavage was cloned from strain S-47 and its nucleotide sequence was analyzed. The pRES101 containing the xylE gene exhibited high C230 activity toward catechol and 4CC without altering the substrate specificity from natural strain. The xylE gene was composed of 924 bp and encoded polypeptide of molecular mass 35 kDa containing 307 amino acids. A deduced amino acid sequence of the C230 from strain S-47 exhibited over 80% identity with those of Pseudomonas putida mt-2, Pseudomonas putida G7, and Pseudomonas sp. CF600. However, it shows below 45% identity with those of Pseudomonas cepacia LB400 and Pseudomonas sp. KKS102. The C230 of strain S-47 was conserved in the amino acids (His150, His214, Glu261) for metal binding ligands and those (His199, His242, and Tyr251) for catalytic sites. Therefore, Pseudomonas sp. S-47 can be explained as acting by degrading catechol as well as 4CC by xylE-encoding C230 which was fused by N domain of nahH and C domain of dmpB from other Pseudomonas strains.     

Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain SU.

Strains of Arthrobacter catalyze a hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) to p-hydroxybenzoate. The reaction requires ATP and coenzyme A (CoA), indicating activation of the substrate via a thioester, like that reported for Pseudomonas sp. strain CBS3 (J. D. Scholten, K.-H. Chang, P. C. Babbit, H. Charest, M. Sylvestre, and D. Dunaway-Mariano, Science 253:182-185, 1991). The dehalogenase genes of Arthrobacter sp. strain SU were cloned and expressed in Escherichia coli. Analyses of deletions indicate that dehalogenation depends on three open reading frames (ORFs) which are organized in an operon. There is extensive sequence homology to corresponding gene products in Pseudomonas sp. strain CBS3, suggesting that ORF1 and ORF2 encode a 4-CBA-CoA-ligase and a 4-CBA-CoA dehalogenase, respectively. ORF3 possibly represents a thioesterase, although no homology to the enzyme from Pseudomonas sp. strain CBS3 exists.     

Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain SU.

Strains of Arthrobacter catalyze a hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) to p-hydroxybenzoate. The reaction requires ATP and coenzyme A (CoA), indicating activation of the substrate via a thioester, like that reported for Pseudomonas sp. strain CBS3 (J. D. Scholten, K.-H. Chang, P. C. Babbit, H. Charest, M. Sylvestre, and D. Dunaway-Mariano, Science 253:182-185, 1991). The dehalogenase genes of Arthrobacter sp. strain SU were cloned and expressed in Escherichia coli. Analyses of deletions indicate that dehalogenation depends on three open reading frames (ORFs) which are organized in an operon. There is extensive sequence homology to corresponding gene products in Pseudomonas sp. strain CBS3, suggesting that ORF1 and ORF2 encode a 4-CBA-CoA-ligase and a 4-CBA-CoA dehalogenase, respectively. ORF3 possibly represents a thioesterase, although no homology to the enzyme from Pseudomonas sp. strain CBS3 exists.     

RpoS as an intermediate in RsmA-dependent regulation of secondary antifungal metabolites biosynthesis in Pseudomonas sp. M18

To investigate the regulatory mechanism governing antifungal metabolite biosynthesis, two kinds of global regulator genes in Pseudomonas sp. M18, an rpoS and an rsmA gene, were cloned and mutated by inserting with an aacC1 cassette, respectively. Two mutants showed the same regulatory mode with repression of phenazine-1-carboxylic acid and remarkable enhancement of pyoluteorin. In the rpoS-mutant, a translational rsmA'-'lacZ fusion was expressed at the same level corresponding to that in the wild-type strain. In the rsmA-mutant, however, expression of the translational rpoS'-'lacZ fusion was only about 30% of that in the wild-type strain. The results indicated that the absence of RsmA leads to repression of the rpoS gene expression, which has further been confirmed with construction of chromosomal rpoS-lacZ fusion in the rsmA-mutant and the wild-type strain, respectively. The findings showed a new regulatory cascade controlling antifungal metabolite production in Pseudomonas sp. M18, suggesting that RpoS may serve as a mediator in RsmA-dependent regulation of secondary metabolite biosynthesis.     

Draft genome sequence of Serratia sp. strain M24T3, isolated from pinewood disease nematode Bursaphelenchus xylophilus

Here we report the draft genome sequence of Serratia sp. strain M24T3, which is associated with pinewood nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease. Serratia sp. strain M24T3 has been identified as a bionematocide for B. xylophilus in vitro, and multiple genes potentially involved in virulence and nematotoxity were identified.     

Biochemical and genetic analyses of ferulic acid catabolism in Pseudomonas sp. Strain HR199.

The gene loci fcs, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase, ech, encoding enoyl-CoA hydratase/aldolase, and aat, encoding beta-ketothiolase, which are involved in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199 (DSM7063), were localized on a DNA region covered by two EcoRI fragments (E230 and E94), which were recently cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The nucleotide sequences of parts of fragments E230 and E94 were determined, revealing the arrangement of the aforementioned genes. To confirm the function of the structural genes fcs and ech, they were cloned and expressed in Escherichia coli. Recombinant strains harboring both genes were able to transform ferulic acid to vanillin. The feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities of the fcs and ech gene products, respectively, were confirmed by photometric assays and by high-pressure liquid chromatography analysis. To prove the essential involvement of the fcs, ech, and aat genes in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199, these genes were inactivated separately by the insertion of omega elements. The corresponding mutants Pseudomonas sp. strain HRfcsOmegaGm and Pseudomonas sp. strain HRechOmegaKm were not able to grow on ferulic acid or on eugenol, whereas the mutant Pseudomonas sp. strain HRaatOmegaKm exhibited a ferulic acid- and eugenol-positive phenotype like the wild type. In conclusion, the degradation pathway of eugenol via ferulic acid and the necessity of the activation of ferulic acid to the corresponding CoA ester was confirmed. The aat gene product was shown not to be involved in this catabolism, thus excluding a beta-oxidation analogous degradation pathway for ferulic acid. Moreover, the function of the ech gene product as an enoyl-CoA hydratase/aldolase suggests that ferulic acid degradation in Pseudomonas sp. strain HR199 proceeds via a similar pathway to that recently described for Pseudomonas fluorescens AN103.     

Taxonomic characterisation of Pseudomonas strain L48 and formal proposal of Pseudomonas entomophila sp. nov

An entomopathogenic, Gram-negative bacterium isolated from a female specimen of the fruit fly Drosophila melanogaster was taxonomically characterised. Strain L48(T) was strictly aerobic, non-fermentative, oxidase and catalase positive, rod-shaped, and motile due to a polar inserted flagellum. Phylogenetic analysis of the 16S rRNA gene and three other housekeeping genes placed strain L48 (T) in the Pseudomonas putida phylogenetic group. DNA-DNA hybridisation studies together with phenotypic metabolic tests and MALDI-TOF MS analysis justified the proposal of strain L48(T) as a representative of a novel species, for which the name Pseudomonas entomophila sp. nov. is proposed. The type strain is deposited in culture collections under accession numbers CCUG 61470(T) and CECT 7985(T).     

Isolation and Characterization of ACC Deaminase Gene from Two Plant Growth-Promoting Rhizobacteria

Lowering of plant ethylene by deamination of its immediate precursor 1-aminocyclopropane-1-carboxylate (ACC) is a key trait found in many rhizobacteria. We isolated and screened bacteria from the rhizosphere of wheat for their ACC-degrading ability. The ACC deaminase gene (acdS) isolated from two bacterial isolates through PCR amplification was cloned and sequenced. Nucleotide sequence alignment of these genes with previously reported genes of Pseudomonas sp. strain ACP and Enterobacter cloacae strain UW4 showed variation in their sequences. In the phylogenetic analysis, distinctness of these two genes was observed as a separate cluster. 16S rDNA sequencing of two isolates identified them to be Achromobacter sp. and Pseudomonas stutzeri.     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素 (Pyoluteorin ,Plt )和吩嗪 1 羧酸 (Phenazine 1 carboxylicacid ,PCA)并抑制多种植物病菌的生长。从M18中克隆双基因调控系统gacS gacA的组成基因gacA ,并构建了该基因抗性插入突变株M18G。在KMB培养基中 ,M18G合成Plt的能力受到完全抑制 ,而PCA的积累约比野生型提高 31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明 ,在M18G菌株中 ,Plt合成的抑制并不引起PCA的过量积累 ,PCA的过量积累也不引起Plt合成的抑制。由此推测 ,gacA在基因表达的水平上全局性地执行着调控功能     

Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.     

Plasmid pCBI carries genes for anaerobic benzoate catabolism in Alcaligenes xylosoxidans subsp. denitrificans PN-1

Pseudomonas sp. strain PN-1 is reclassified as Alcaligenes xylosoxidans subsp. denitrificans PN-1. Strain PN-1 is a gram-negative, rod-shaped organism, is motile by means of lateral flagella, is oxidase positive, and does not ferment sugars. Plasmid pCBI, carrying genes for the anaerobic degradation of benzoate in strain PN-1, is 17.4 kilobase pairs in length and is transmissible to a number of denitrifying Pseudomonas aeruginosa and Pseudomonas stutzeri strains. A restriction endonuclease map was constructed.     

Pseudomonas japonica sp. nov., a novel species that assimilates straight chain alkylphenols

A bacterial strain, WL(T), which was isolated from an activated sludge, was able to degrade alkylphenols. 16S rDNA sequence analysis indicated that strain WL(T) belonged to the genus Pseudomonas (sensu stricto) and formed a monophyletic clade with the type strain of Pseudomonas graminis and other members in the Pseudomonas putida subcluster with sequence similarity values higher than 97%. Genomic relatedness based on DNA-DNA hybridization of strain WL(T) to these strains is 2-41%. Strain WL(T) contained ubiquinone-9 as the main respiratory quinone, and the G+C content of DNA was 66 mol%. The organism contained hexadecanoic acid (16:0), hexadecenoic acid (16:1) and octadecenoic acid (18:1) as major cellular fatty acids. The hydroxy fatty acids detected were 3-hydroxydecanoic acid (3-OH 10:0), 3-hydroxydodecanoic acid (3-OH 12:0) and 2-hydroxydodecanoic acid (2-OH 12:0). These results, as well as physiological and biochemical characteristics clearly indicate that the strain WL(T) represents a new Pseudomonas species, for which the name Pseudomonas japonica is proposed. The type strain is strain WL(T) (=IAM 15071T=TISTR 1526T).     

Production of the siderophore aerobactin by a halophilic pseudomonad.

A bacterial strain, isolated from a cyanobacterial culture, was identified as Pseudomonas sp. strain X40. Under iron-limiting conditions, the Pseudomonas sp. produced aerobactin, a dihydroxamate siderophore previously found only in the family Enterobacteriaceae. Aerobactin was identified by electrophoretic mobility, spectrophotometric titration, proton nuclear magnetic resonance spectroscopy, mass spectrometry, acid hydrolysis, and biological activity. Aerobactin was used as a siderophore in the Pseudomonas sp. and Escherichia coli. Two iron-repressed outer membrane proteins were observed in the Pseudomonas sp., neither of which had electrophoretic mobility identical to that of the aerobactin outer membrane receptor protein from E. coli. DNA hybridization assays showed no hybridization to the aerobactin genes from the E. coli plasmid pColV, indicating that the genetic determinants for aerobactin production by Pseudomonas strain X40 differ substantially from those found in the archetypic enteric plasmid pColV-K30.     

Production of the siderophore aerobactin by a halophilic pseudomonad.

A bacterial strain, isolated from a cyanobacterial culture, was identified as Pseudomonas sp. strain X40. Under iron-limiting conditions, the Pseudomonas sp. produced aerobactin, a dihydroxamate siderophore previously found only in the family Enterobacteriaceae. Aerobactin was identified by electrophoretic mobility, spectrophotometric titration, proton nuclear magnetic resonance spectroscopy, mass spectrometry, acid hydrolysis, and biological activity. Aerobactin was used as a siderophore in the Pseudomonas sp. and Escherichia coli. Two iron-repressed outer membrane proteins were observed in the Pseudomonas sp., neither of which had electrophoretic mobility identical to that of the aerobactin outer membrane receptor protein from E. coli. DNA hybridization assays showed no hybridization to the aerobactin genes from the E. coli plasmid pColV, indicating that the genetic determinants for aerobactin production by Pseudomonas strain X40 differ substantially from those found in the archetypic enteric plasmid pColV-K30.     

Mechanistic insights into the global response to phenol in the phenol-biodegrading strain Pseudomonas sp. M1 revealed by quantitative proteomics

Quantitative proteomics was used to gain insights into the global adaptive response to phenol in the phenol-biodegrading strain Pseudomonas sp. M1 when an alternative carbon source (pyruvate or succinate) is present. A phylogenetic analysis indicated Pseudomonas citronellolis as the closest species to the environmental strain M1, while P. aeruginosa is the closest species with the genome sequence available. After two-dimensional gel electrophoresis (2-DE) separation, protein identification by MS/MS ion search allowed the assignment of 87 out of 136 selected protein spots, 56 of which matched P. aeruginosa proteins present in databases. Coordinate induction of six enzymes of the phenol catabolic pathway in cells grown in pyruvate and phenol was revealed by expression proteomics. When succinate was the alternative carbon source (C-source), these catabolic proteins were not expressed. The global response of Pseudomonas sp. M1 to phenol-induced stress involved, among others, proteins of the energy metabolism, stress response proteins, and transport proteins. Quantitative and/or qualitative differences were registered in M1 response to different phenol concentrations or to identical phenol concentrations when cells were grown in pyruvate or succinate medium. They were attributed to differences observed in the specific growth rate, in the expression of phenol catabolism, and in resistance to phenol of Pseudomonas sp. M1 grown under different conditions.     

RNA聚合酶σ^38亚基缺失对假单胞菌抗生物质合成代谢的影响

设计引物从假单胞菌M18基因组DNA中扩增并获得rpoS基因的378bp保守区段。以此为探针,从假单胞菌基因组文库中克隆了包括rpoS基因全序列及其相邻序列的3·1kbEcoRⅠ-XhoⅠ片段。通过抗性基因(抗庆大霉素基因)的定点插入构建了σ38亚基缺失突变株M18S。HPLC检测结果显示,σ38亚基缺失引起该菌株的抗生物质合成代谢的显著变化。与野生株相比,缺失突变株的吩嗪-1-羧酸在PPM和KMB中2种培养基中合成量由58μg/mL和10·2μg/mL分别减少到20·4μg/mL和0μg/mL;而缺失突变株的藤黄绿脓菌素则相反,在PPM和KMB两种培养基中合成量由0·5μg/mL和20·5μg/mL分别提高到75·4μg/mL和185·6μg/mL。表明σ38亚基可区别性调控假单胞菌M18的抗生物质合成代谢。rpoS基因的互补实验和两种抗生素基因与β-半乳糖苷酶基因的翻译融合表达实验进一步验证了上述的结果:σ38亚基正调控吩嗪-1-羧酸的表达,而负调控藤黄绿脓菌素的表达。