Phospholipid biosynthesis and solvent tolerance in Pseudomonas putida strains.

The role of the cell envelope in the solvent tolerance mechanisms of Pseudomonas putida was investigated. The responses of a solvent-tolerant strain, P. putida Idaho, and a solvent-sensitive strain, P. putida MW1200, were examined in terms of phospholipid content and composition and of phospholipid biosynthetic rate following exposure to a nonmetabolizable solvent, o-xylene. Following o-xylene exposure, P. putida MW1200 exhibited a decrease in total phospholipid content. In contrast, P. putida Idaho demonstrated an increase in phospholipid content 1 to 6 h after exposure. Analysis of phospholipid biosynthesis showed P. putida Idaho to have a higher basal rate of phospholipid synthesis than MW1200. This rate increased significantly following exposure to xylene. Both strains showed little significant turnover of phospholipid in the absence of xylene. In the presence of xylene, both strains showed increased phospholipid turnover. The rate of turnover was significantly greater in P. putida Idaho than in P. putida MW1200. These results suggest that P. putida Idaho has a greater ability than the solvent-sensitive strain MW1200 to repair damaged membranes through efficient turnover and increased phospholipid biosynthesis.     

Indigo formation by microorganisms expressing styrene monooxygenase activity.

The transformation of indole to indigo by microorganisms expressing styrene monooxygenase (SMO) has been studied. Styrene and indole are structurally very similar, and thus we looked at a variety of styrene-degrading strains for indole transformation to indigo. Two strains, Pseudomonas putida S12 and CA-3, gave a blue color on solid media when grown in the presence of indole. Indole induces its own transformation on solid media but is a poor inducer in liquid media. Styrene is the best inducer of indole transformation in both strains. Arginine represses styrene consumption and indigo formation rates in P. putida S12 compared to phenylacetic acid-grown cells, while the opposite effect is seen for P. putida CA-3. Characterization of an SMO- and styrene oxide isomerase (SOI)-negative transposon mutant of P. putida CA-3 and an SOI-negative N-methyl-N'-nitro-N-nitrosoguanidine mutant of P. putida S12 reveals the involvement of both SMO and SOI in indole transformation to indigo. Both strains stoichiometrically produce high-purity indigo from indole.     

The metabolism of caffeine by a Pseudomonas putida strain

1) A bacterium capable of growing aerobically with caffeine (1,3,7-trimethylxanthine) as sole source of carbon and nitrogen was isolated from soil. The morphological and physiological characteristics of the bacterium were examined. The organism was identified as a strain of Pseudomonas putida and is referred to as Pseudomonas putida C1. 15 additional caffeine-degrading bacteria were isolated, and all of them were also identified as Pseudomonas putida strains. The properties of the isolates are discussed in comparison with 6 Pseudomonas putida strains of the American Type Culture Collection. 2) The degradation of caffeine by Pseudomonas putida C1 was investigated; the following 14 metabolites were identified: 3,7-dimethylxanthine (theobromine), 1,7-dimethylxanthine, 7-methylxanthine, xanthine, 3,7-dimethyluric acid, 1,7-dimethyluric acid, 7-methyluric acid, uric acid, allantoin, allantoic acid, ureidoglycolic acid, glyoxylic acid, urea, and formaldehyde. Formaldehyde has been demonstrated to be the product of oxidative N-demethylation mediated by an inducible demethylase. A pathway of caffeine degradation is proposed.     

Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.     

Molecular analysis of the Pseudomonas aeruginosa regulatory genes ptxR and ptxS.

We have previously described two Pseudomonas aeruginosa genes, ptxR, which enhances toxA and pvc (the pyoverdine chromophore operon) expression, and ptxS, the first gene of the kgu operon for the utilization of 2-ketogluconate by P. aeruginosa. ptxS interferes with the effect of ptxR on toxA expression. In this study, we have utilized DNA hybridization experiments to determine the presence of ptxR and ptxS homologous sequences in several gram-negative bacteria. ptxR homologous sequences were detected in P. aeruginosa strains only, while ptxS homologous sequences were detected in P. aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens. Using Northern blot hybridization experiments and a ptxS-lacZ fusion plasmid, we have shown that P. aeruginosa ptxR and ptxS are expressed in P. putida and P. fluorescens. Additional Northern blot hybridization experiments confirmed that ptxS is transcribed in P. putida and P. fluorescens strains that carried no plasmid. The presence of a PtxS homologue in these strains was examined by DNA-gel shift experiments. Specific gel shift bands were detected when the lysates of P. aeruginosa, P. putida, and P. fluorescens were incubated with the ptxS operator site as probe. kgu-hybridizing sequences were detected in P. putida and P. fluorescens. These results suggest that (i) ptxR is present in P. aeruginosa, while ptxS is present in P. aeruginosa, P. putida, and P. fluorescens; (ii) both ptxR and ptxS are expressed in P. putida and P fluorescens; and (iii) a PtxS homologue may exist in P. putida and P. fluorescens.     

Effect of trichloroethylene on the competitive behavior of toluene-degrading bacteria

The influence of trichloroethylene (TCE) on a mixed culture of four different toluene-degrading bacterial strains (Pseudomonas putida mt-2, P. putida F1, P. putida GJ31, and Burkholderia cepacia G4) was studied with a fed-batch culture. The strains were competing for toluene, which was added at a very low rate (31 nmol mg of cells [dry weight] h). All four strains were maintained in the mixed culture at comparable numbers when TCE was absent. After the start of the addition of TCE, the viabilities of B. cepacia G4 and P. putida F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE, although at considerably different rates. P. putida mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE occurred. While P. putida mt-2 took over the culture, mutants of this strain which could no longer grow on p-xylene arose. Most of them had less or no meta-cleavage activity and were able to grow on toluene with a higher growth rate. The results indicate that cometabolic degradation of TCE has a negative effect on the maintenance and competitive behavior of toluene-utilizing organisms that transform TCE.     

Mechanism of cyanide and thiocyanate decomposition by an association of Pseudomonas putida and Pseudomonas stutzeri strains

The intermediate and terminal products of cyanide and thiocyanate decomposition by individual strains of the genus Pseudomonas, P. putida strain 21 and P. stutzeri strain 18, and by their association were analyzed. The activity of the enzymes of nitrogen and sulfur metabolism in these strains was compared with that of the collection strains P. putida VKM B-2187T and P. stutzeri VKM B-975T. Upon the introduction of CN- and SCN- into cell suspensions of strains 18 and 21 in phosphate buffer (pH 8.8), the production of NH4+ was observed. Due to the high rate of their utilization, NH3, NH4+, and CNO- were absent from the culture liquids of P. putida strain 21 and P. stutzeri strain 18 grown with CN- or SCN-. Both Pseudomonas strains decomposed SCN- via cyanate production. The cyanase activity was 0.75 micromol/(min mg protein) for P. putida strain 21 and 1.26 micromol/(min mg protein) for P. stutzeri strain 18. The cyanase activity was present in the cells grown with SCN- but absent in cells grown with NH4+. Strain 21 of P. putida was a more active CN- decomposer than strain 18 of P. stutzeri. Ammonium and CO2 were the terminal nitrogen and carbon products of CN- and SCN- decomposition. The terminal sulfur products of SCN- decomposition by P. stutzeri strain 18 and P. putida strain 21 were thiosulfate and tetrathionate, respectively. The strains utilized the toxic compounds in the anabolism only, as sources of nitrogen (CN- and SCN-) and sulfur (SCN-). The pathway of thiocyanate decomposition by the association of bacteria of the genus Pseudomonas is proposed based on the results obtained.     

Metabolic engineering of strains of Ralstonia eutropha and Pseudomonas putida for biotechnological production of 2-methylcitric acid

In this study strains of Ralstonia eutropha H16 and Pseudomonas putida KT2440 were engineered which are suitable for biotechnological production of 2-methylcitric acid (2MC). Analysis of a previous mutant of R. eutropha able to accumulate 2MC recommended this strain as a candidate for fermentative production of 2MC. This knowledge was used for construction of strains of R. eutropha H16 and P. putida KT2440 capable of enhanced production of 2MC. In both bacteria the chromosomal genes encoding the 2-methyl-cis-aconitate hydratase (acnM) were disrupted by directed insertion of a copy of an additional 2-methylcitrate synthase gene (prpC) yielding strains R. eutropha DeltaacnM(Re)OmegaKmprpC(Pp) and P. putida DeltaacnM(Pp)OmegaKmprpC(Re). In both strains 2-methylcitrate synthase was expressed under control of the constitutive kanamycin-resistance gene (OmegaKm) resulting in up to 20-fold higher specific 2-methylcitrate synthase activities in comparison to the wild type. The disruption of the acnM gene by insertion of prpC led to a propionate- and levulinate-negative phenotype of the engineered strains, and analysis of supernatant of these strains revealed overproduction and accumulation of 2MC in the medium. A two stage cultivation regime comprising an exponential growth phase and a 2MC production phase was developed and applied to both engineered strains for optimum production of 2MC. Whereas gluconate, fructose or succinate were provided as carbon source for the exponential growth phase, a combination of propionate or levulinate as precursor substrate for provision of propionyl-CoA and succinate or fumarate as precursor substrate for provision of oxaloacetate were used in the production phase to make sure that the 2-methylcitrate synthase was provided with their substrates. Employing the optimised feeding regime P. putida DeltaacnM(Pp)OmegaKmprpC(Re) and R. eutropha DeltaacnM(Re)OmegaKmprpC(Pp) produced 2MC up to maximal concentrations of 7.2 g/L or 26.5 mM and 19.2 g/L or 70.5 mM, respectively, during 144 h of cultivation.     

Pseudomonas fluorescens biovar V: its resolution into distinct component groups and the relationship of these groups to other P. fluorescens biovars, to P. putida, and to psychrotrophic pseudomonads associated with food spoilage

A numerical taxonomic analysis was performed to evaluate the appropriateness of a single biovar designation (biovar V) for all Pseudomonas fluorescens isolates negative for denitrification, levan production and phenazine pigmentation and to determine the relationship of biovar V strains to other taxa within the same Pseudomonas RNA homology group. Seventy-two strains assigned to P. fluorescens biovar V and four strains of P. fragi were characterized and the data subjected to a numerical taxonomic analysis along with comparable data for 17 previously characterized strains of this biovar and 89 P. putida strains. Seven distinct biovar V clusters containing three or more strains were revealed, and the carbon sources useful for their differentiation were identified. Cluster 1 (38 strains) closely resembled two atypical P. fluorescens I strains. It was also related to P. fluorescens biovar IV and to P. fragi. Cluster 2 (5 strains) was related to cluster 1. Cluster 3 (7 strains) was identical to a major group of meat spoilage psychrotrophic pseudomonads (P. lundensis). Cluster 4 (3 strains) was not related to any other group examined. Cluster 5 consisted of six isolates initially designated P. putida A along with four P. fluorescens biovar V strains all of which resembled P. putida more than they resembled the other P. fluorescens groups. Cluster 6 (16 strains) was distinct from the other biovar V clusters, but was closely related to P. fluorescens biovars I and II. Cluster 7 (3 strains) shared many characteristics with cluster 5. Separate P. fluorescens biovar designations are proposed for cluster 6 and for the combined clusters 1 and 2. A new P. putida biovar is proposed for the combined clusters 5 and 7.     

Identification of the Anabaena sp. strain PCC7120 cyanophycin synthetase as suitable enzyme for production of cyanophycin in gram-negative bacteria like Pseudomonas putida and Ralstonia eutropha

The cyanophycin synthetase gene cphA1 encoding the major cyanophycin synthetase (CphA) of Anabaena sp. strain PCC7120 was expressed in Escherichia coli conferring so far the highest specific CphA activity to E. coli (6.7 nmol arginine per min and mg protein). CphA1 and cphA genes of Synechocystis sp. strains PCC6803 and PCC6308 and Synechococcus strain MA19 were also expressed in wild types and polyhydroxyalkanoate-negative (PHA) mutants of Pseudomonas putida and Ralstonia eutropha. Recombinant strains of these bacteria expressing cphA1 accumulated generally more cyanophycin (23.0 and 20.0% of cellular dry matter, CDM, respectively) than recombinants expressing any other cphA (6.8, 9.0, or 15.8% of CDM for P. putida strains and 7.3, 12.6, or 14.1% of CDM for R. eutropha). Furthermore, PHA-negative mutants of P. putida (9.7, 10.0, 17.5, or 24.0% of CDM) and R. eutropha (8.9, 13.8, 16.0, or 22.0% of CDM) accumulated generally more cyanophycin than the corresponding PHA-positive parent strains (6.8, 9.0, 15.8, and 23.0% of CDM for P. putida strains and 7.3, 12.6, 14.1, or 20.0% of CDM for R. eutropha strains). Recombinant strains of Gram-positive bacteria (Bacillus megaterium, Corynebacterium glutamicum) were not suitable for cyanophycin production due to accumulation of less cyanophycin and retarded release of cyanophycin. PHA-negative mutants of P. putida and R. eutropha expressing cphA1 of Anabaena sp. strain PCC7120 are therefore preferred candidates for industrial production of cyanophycin.     

Biofilm formation of Pseudomonas putida IsoF: the role of quorum sensing as assessed by proteomics

Pseudomonas putida strains are frequently isolated from the rhizosphere of plants and many strains promote plant-growth, exhibit antagonistic activities against plant pathogens and have the capacity to degrade pollutants. Factors that appear to contribute to the rhizosphere fitness are the ability of the organism to form biofilms and the utilization of cell-to-cell-communication systems (quorum sensing, QS) to co-ordinate the expression of certain phenotypes in a cell density dependent manner. Recently, the ppu QS locus of the tomato rhizosphere isolate P. putida Iso F was characterized and an isogenic QS-negative ppuI mutant P. putida F117 was generated. In the present study we investigated the impact of QS and biofilm formation on the protein profile of surface-associated proteins of P. putida IsoF. This was accomplished by comparative proteome analyses of the P. putida wild type IsoF and the QS-deficient mutant F117 grown either in planktonic cultures or in 60 h old mature biofilms. Differentially expressed proteins were identified by peptide mass fingerprinting and database search in the completed P. putida KT2440 genome sequence. The sessile life style affected 129 out of 496 surface proteins, suggesting that a significant fraction of the bacterial genome is involved in biofilm physiology. In surface-attached cells 53 out of 484 protein spots were controlled by the QS system, emphasizing its importance as global regulator of gene expression in P. putida IsoF. Most interestingly, the impact of QS was dependent on whether cells were grown on a surface or in suspension; about 50% of the QS-controlled proteins identified in planktonic cultures were found to be oppositely regulated when the cells were grown as biofilms. Fifty-seven percent of all identified surface-controlled proteins were also regulated by the ppu QS system. In conclusion, our data provide strong evidence that the set of QS-regulated proteins overlaps substantially with the set of proteins differentially expressed in sessile cells.     

Phenotypic characteristics of Pseudomonas putida isolated from different environments

Various typing methods have been suggested to differentiate isolates of P. putida species with the aim of developing epidemiological tools. 34 P putida strains were isolated from the samples of crude oil and oil derivates contaminated soil (n=27), biopreparates used for biodegradation of soil contamination (n=3) and hospital materials (n=4). The biochemical typing was assessed using ID32GN tests (bio-Merieux). The strains were grouped into 11 biotypes. Antibiotic-containing discs were used for routine antibiogramsby disc diffusion assay. The strains were most resistant to cefoperazone and ticarcillin (29.4% and 26.5% of strains, respectively). The intracellular esterases of P. putida were separated by polyacrylamide gel electrophoresis and stained with Fast Blue using alpha-naphthyl acetate, beta-naphthyl propionate and indoxyl acetate as substrates. On the basis of the indicators dye migration the electrophoresis process was observed. By calculating the RF (retention factor) the distribution of stripes was obtained with great accuracy. The use of biochemical tests, sensitivity tests and zymotyping provides the possibility to inter- and intraspecies differentiation of P. putida.     

Plasmid control of the Pseudomonas aeruginosa and Pseudomonas putida phenotypes and of linalool and p-cymene oxidation.

Two Pseudomonas strains (PpG777 and PaG158) were derived from the parent isolate Pseudomonas incognita (putida). Strain PpG777 resembles the parental culture in growth on linalool as a source of carbon and slight growth on p-cymene, whereas PaG158 grows well on p-cymene, but not on linalool or other terpenes tested, and has a P. aeruginosa phenotype. Curing studies indicate that linalool metabolism is controlled by an extrachromosomal element whose loss forms a stable strain PaG158 with the p-cymene growth and P. aeruginosa phenotype characters. The plasmid can be transferred by PpG777 to both P. putida and P. aeruginosa strains. Surprisingly, the latter assume the P. putida phenotype. We conclude that the genetic potential to oxidize p-cymene is inherent in PpG777 but expression is repressed. Similarly, this observation implies that support of linalool oxidation effectively conceals the P. aeruginosa character.     

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.     

Established and abandoned tea (Camillia sinensis L.) rhizosphere: dominant bacteria and their antagonism

Some parts of the Indian Himalayan region are covered by established and abandoned tea bushes. Rhizospheric soils of these plants were studied for bacterial dominance and antagonism. Representatives of Bacillus and Pseudomonas genera were found to dominate the rhizosphere of established and abandoned tea bushes, respectively. Amongst the isolated species Bacillus subtilis and Bacillus mycoides appeared to be closely associated with roots of established tea bushes while the rhizosphere of abandoned tea bushes was dominated by Pseudomonas putida. Four isolates of both B. subtilis and P. putida were selected on the basis of maximum antibacterial activity. The bacteriocin-like activity of B. subtilis and P putida strains was detected to be active over a range of temperature 0-50 degrees C and was sensitive to proteolytic enzymes. Incubation of indicator strains with different concentrations of bacteriocin-like substances confirmed their bactericidal activity. Various species of Bacillus and Pseudomonas behaved antagonistically amongst themselves due to the production of bacteriocins under in vitro conditions.     

松材线虫对其携带的细菌繁殖的影响

1.引言松材线虫病（Bursaphelenchus xylophilus）是松树的一种毁灭性病害,在日本、中国、韩国和北美、尼日利亚和葡萄牙等国家蔓延,造成了巨大经济损失,其中以日本和中国受害最重.一直认为松材线虫是引起该病的唯一病原,但近十几年来的研究发现,细菌在致病过程中可能起着重要作用,相继从病木和松材线虫体上分离到能对黑松苗有致萎活性的细菌.赵博光等首次根据实验提出松材线虫病是线虫和细菌共同侵染引起的复合侵染病害的假说,并在以后的试验中得到了验证.关于松材线虫对其细菌繁殖的影响研究鲜有报道.本试验采用从感病松树上分离并鉴定了的细菌菌株中选取假单胞属7株、其它属的细菌菌株3株,     

PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains.

Degenerate PCR primers, UP-1 and UP-2r, for the amplification of DNA gyrase subunit B genes (gyrB) were designed by using consensus amino acid sequences of gyrases from Escherichia coli, Pseudomonas putida, and Bacillus subtilis. In addition to the degenerate sequences, these primers have sequences at the 5' end which allow direct sequencing of amplified PCR products. With these primers, DNA segments of the predicted size were amplified from a variety of gram-negative and gram-positive genera. The nucleotide sequences of the amplified gyrB DNA from three P. putida strains were determined directly from the amplified fragments. The base substitution frequency of gyrB between the strains of P. putida was much higher than that of the 16S rRNA gene. With a specific set of PCR primers, it was possible to amplify gyrB fragments selectively from P. putida or its subgroups. The direct sequencing method of gyrB developed in this study provides a rapid and convenient system for bacterial identification, taxonomic analysis, and monitoring of bacteria in the natural environment.     

Recruitment and expression of toluene/trichloroethylene biodegradation genes in bacteria native to deep-subsurface sediments.

Four plasmids, each encoding a combination of either an Escherichia coli or Pseudomonas putida promoter and either toluene dioxygenase or toluene monooxygenase, were electroporated into five bacterial strains isolated from sediments found at depths of 91 to 295 m. Four of these engineered bacterial strains demonstrated both toluene and trichloroethylene degradation activities.     

Species of Pseudomonas obtained at 7 degrees C and 30 degrees C during aerobic storage of lamb carcasses.

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30 degrees C were Ps. fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7 degrees C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% SSM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group (Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (> 74% SSM) and Ps. lundensis (> 80%). The recovery of pseudomonads seemed to be affected by the sampling day.