Showing papers on "Pseudomonas putida published in 2006"
TL;DR: The view that AHL molecules play a role in the biocontrol activity of rhizobacteria through the induction of systemic resistance to pathogens is supported.
Abstract: N-acyl-L-homoserine lactone (AHL) signal molecules are utilized by Gram-negative bacteria to monitor their population density (quorum sensing) and to regulate gene expression in a density-dependent manner. We show that Serratia liquefaciens MG1 and Pseudomonas putida IsoF colonize tomato roots, produce AHL in the rhizosphere and increase systemic resistance of tomato plants against the fungal leaf pathogen, Alternaria alternata. The AHL-negative mutant S. liquefaciens MG44 was less effective in reducing symptoms and A. alternata growth as compared to the wild type. Salicylic acid (SA) levels were increased in leaves when AHL-producing bacteria colonized the rhizosphere. No effects were observed when isogenic AHL-negative mutant derivatives were used in these experiments. Furthermore, macroarray and Northern blot analysis revealed that AHL molecules systemically induce SA- and ethylene-dependent defence genes (i.e. PR1a, 26 kDa acidic and 30 kDa basic chitinase). Together, these data support the view that AHL molecules play a role in the biocontrol activity of rhizobacteria through the induction of systemic resistance to pathogens.
418 citations
TL;DR: Although the bacterial inoculation was found to result in significant increment in plant biomass, it stimulated bacterial and suppressed fungal counts in the rhizosphere and is important with respect to enumerating microbial diversity of the colder regions as well as understanding the potential biotechnological applications of native microbes.
Abstract: The morphological, biochemical, and physiological characteristics of a phosphate solubilizing and antagonistic bacterial strain, designated as B0, isolated from a sub-alpine Himalayan forest site have been described. The isolate is gram negative, rod shaped, 0.8 × 1.6 μm in size, and psychrotrophic in nature that could grow from 0 to 35°C (optimum temp. 25°C). It exhibited tolerance to a wide pH range (3–12; optimum 8.0) and salt concentration up to 4% (w/v). Although it was sensitive to kanamycin, gentamicin, and streptomycin ( 1000 μg mL−1). The isolate showed maximum similarity with Pseudomonas
putida based on 16S rRNA analysis. It solubilized tricalcium phosphate under in vitro conditions. The phosphate solubilization was estimated along a temperature range (4–28°C), and maximum activity (247 μg mL−1) was recorded at 21°C after 15 days of incubation. The phosphate solubilizing activity coincided with a concomitant decrease in pH of the medium. The isolate also exhibited antifungal activity against phytopathogenic fungi in Petri dish assays and produced chitinase, s-l,3-glucanase, salicylic acid, siderophore, and hydrogen cyanide. The plant growth promotion and antifungal properties were demonstrated through a maize-based bioassay under greenhouse conditions. Although the bacterial inoculation was found to result in significant increment in plant biomass, it stimulated bacterial and suppressed fungal counts in the rhizosphere. The present study is important with respect to enumerating microbial diversity of the colder regions as well as understanding the potential biotechnological applications of native microbes.
232 citations
TL;DR: In this paper, a survey of 185 plant-associated, phytopathogenic, soil and river Pseudomonas isolates resulted in 76% producing biofilms at the air-liquid interface after selection in static microcosms.
Abstract: The ability to form biofilms is seen as an increasingly important colonization strategy among both pathogenic and environmental bacteria. A survey of 185 plant-associated, phytopathogenic, soil and river Pseudomonas isolates resulted in 76% producing biofilms at the air–liquid (A–L) interface after selection in static microcosms. Considerable variation in biofilm phenotype was observed, including waxy aggregations, viscous and floccular masses, and physically cohesive biofilms with continuously varying strengths over 1500-fold. Calcofluor epifluorescent microscopy identified cellulose as the matrix component in biofilms produced by Pseudomonas asplenii, Pseudomonas corrugata, Pseudomonas fluorescens, Pseudomonas marginalis, Pseudomonas putida, Pseudomonas savastanoi and Pseudomonas syringae isolates. Cellulose expression and biofilm formation could be induced by the constitutively active WspR19 mutant of the cyclic-di-GMP-associated, GGDEF domain-containing response regulator involved in the P. fluorescens SBW25 wrinkly spreader phenotype and cellular aggregation in Pseudomonas aeruginosa PA01. WspR19 could also induce P. putida KT2440, which otherwise did not produce a biofilm or express cellulose, as well as Escherichia coli K12 and Salmonella typhimurium LT2, both of which express cellulose yet lack WspR homologues. Statistical analysis of biofilm parameters suggest that biofilm development is a more complex process than that simply described by the production of attachment and matrix components and bacterial growth. This complexity was also seen in multivariate analysis as a species-ecological habitat effect, underscoring the fact that in vitro biofilms are abstractions of those surface and volume colonization processes used by bacteria in their natural environments.
231 citations
TL;DR: Corn rhizosphere isolates, Gluconacetobacter azotocaptans DS1, Pseudomonas putida CQ179, and Azospirillum lipoferum N7, provided significant plant growth promotion expressed as increased root/shoot weight when compared to uninoculated plants, in sand and/or soil.
Abstract: Alcohol production from corn is gaining importance in Ontario, Canada, and elsewhere. A major cost of corn production is the cost of chemical fertilizers and these continue to increase in price. The competitiveness of alcohol with fossil fuels depends on access to low-cost corn that allows growers to earn a sustainable income. In this study we set out to determine if we can identify root-associated microorganisms from Ontario-grown corn that can enhance the nutrient flow to corn roots, directly or indirectly, and help minimize the use of extraneous fertilizer. Bacteria were isolated from corn rhizosphere and screened for their capacity to enhance corn growth. The bacteria were examined for their ability to fix nitrogen, solubilize phosphate, and produce indole acetic acid (IAA) and antifungal substances on potato dextrose agar. Bacterial suspensions were applied to pregerminated seed of four corn varieties (39D82, 39H84, 39M27, and 39T68) planted in sterilized sand and unsterilized cornfield soil. The plants were grown under greenhouse conditions for 30 days. Three isolates were identified as having growth-promoting effect. These bacteria were identified as to species by biochemical tests, fatty acid profiles, and 16S rDNA sequence analysis. Corn rhizosphere isolates, Gluconacetobacter azotocaptans DS1, Pseudomonas putida CQ179, and Azospirillum lipoferum N7, provided significant plant growth promotion expressed as increased root/shoot weight when compared to uninoculated plants, in sand and/or soil. All strains except P. putida CQ179 were capable of nitrogen fixation and IAA production. Azospirillum brasilense, however, produced significantly more IAA than the other isolates. Although several of the strains were also able to solubilize phosphate and produce metabolites inhibitory to various fungal pathogens, these properties are not considered as contributing to growth promotion under the conditions used in this study. These bacteria will undergo field tests for their effect on corn growth.
224 citations
TL;DR: The authors showed that toluene is sensed by Pseudomonas putida KT2440 cells as a stressor rather than as a nutrient and that the inhibition by the aromatic compounds of many functions was the tradeoff for activating stress tolerance genes at a minimal cost in terms of energy.
208 citations
TL;DR: This study enabled us to select an ideal combination of efficient Rhizobium strain and PGPR for pigeonpea grown in the semiarid tropics.
Abstract: Plant-growth promoting rhizobacteria (PGPR), in conjuction with efficient Rhizobium, can affect the growth and nitrogen fixation in pigeonpea by inducing the occupancy of introduced Rhizobium in the nodules of the legume. This study assessed the effect of different plant-growth promoting rhizobacteria (Azotobacter chroococcum, Azospirillum brasilense, Pseudomonas fluorescens, Pseudomonas putida and Bacillus cereus) on pigeonpea (Cajanus cajan (L) Milsp.) cv. P-921 inoculated with Rhizobium sp. (AR-2-2 k). A glasshouse experiment was carried out with a sandy-loam soil in which the seeds were treated with Rhizobium alone or in combination with several PGPR isolates. It was monitored on the basis of nodulation, N 2 fixation, shoot biomass, total N content in shoot and legume grain yield. The competitive ability of the introduced Rhizobium strain was assessed by calculating nodule occupancy. The PGPR isolates used did not antagonize the introduced Rhizobium strain and the dual inoculation with either Pseudomonas putida, P. fluorescens or Bacillus cereus resulted in a significant increase in plant growth, nodulation and enzyme activity over Rhizobium-inoculated and uninoculated control plants. The nodule occupancy of the introduced Rhizobium strain increased from 50% (with Rhizobium alone) to 85% in the presence of Pseudomonas putida. This study enabled us to select an ideal combination of efficient Rhizobium strain and PGPR for pigeonpea grown in the semiarid tropics.
208 citations
01 Jan 2006
TL;DR: It is argued that toluene is sensed by P. putida KT2440 as a stressor rather than as a nutrient and that the inhibition by the aromatic compounds of many functions the authors tested is the tradeoff for activating stress tolerance genes at a minimal cost in terms of energy.
202 citations
TL;DR: The results of proteomic analysis of Pseudomonas putida cultured in monocyclic aromatic compounds suggest that proteome analysis complements and supports predictive information obtained by genomic sequence analysis.
Abstract: Proteomic analysis of Pseudomonas putida KT2440 cultured in monocyclic aromatic compounds was performed using 2-DE/MS and cleavable isotope-coded affinity tag (ICAT) to determine whether proteins involved in aromatic compound degradation pathways were altered as predicted by genomic analysis (Jimenez et al., Environ Microbiol. 2002, 4, 824-841). Eighty unique proteins were identified by 2-DE/MS or MS/MS analysis from P. putida KT2440 cultured in the presence of six different organic compounds. Benzoate dioxygenase (BenA, BenD) and catechol 1,2-dioxygenase (CatA) were induced by benzoate. Protocatechuate 3,4-dixoygenase (PcaGH) was induced by p-hydroxybenzoate and vanilline. beta-Ketoadipyl CoA thiolase (PcaF) and 3-oxoadipate enol-lactone hydrolase (PcaD) were induced by benzoate, p-hydroxybenzoate and vanilline, suggesting that benzoate, p-hydroxybenzoate and vanilline were degraded by different dioxygenases and then converged in the same beta-ketoadipate degradation pathway. An additional 110 proteins, including 19 proteins from 2-DE analysis, were identified by cleavable ICAT analysis for benzoate-induced proteomes, which complemented the 2-DE results. Phenylethylamine exposure induced beta-ketoacyl CoA thiolase (PhaD) and ring-opening enzyme (PhaL), both enzymes of the phenylacetate (pha) biodegradation pathway. Phenylalanine induced 4-hydroxyphenyl-pyruvate dioxygenase (Hpd) and homogentisate 1,2-dioxygenase (HmgA), key enzymes in the homogentisate degradation pathway. Alkyl hydroperoxide reductase (AphC) was induced under all aromatic compounds conditions. These results suggest that proteome analysis complements and supports predictive information obtained by genomic sequence analysis.
157 citations
TL;DR: The UV-B responses and recovery abilities of the different bacteria were consistent with the irradiation levels in their native environment and the recovery strategies through dark and light repair were different in all strains.
Abstract: Acinetobacter johnsonii A2 isolated from the natural community of Laguna Azul (Andean Mountains at 4,560 m above sea level), Serratia marcescens MF42, Pseudomonas sp. strain MF8 isolated from the planktonic community, and Cytophaga sp. strain MF7 isolated from the benthic community from Laguna Pozuelos (Andean Puna at 3,600 m above sea level) were subjected to UV-B (3,931 J m−2) irradiation. In addition, a marine Pseudomonas putida strain, 2IDINH, and a second Acinetobacter johnsonii strain, ATCC 17909, were used as external controls. Resistance to UV-B and kinetic rates of light-dependent (UV-A [315 to 400 nm] and cool white light [400 to 700 nm]) and -independent reactivation following exposure were determined by measuring the survival (expressed as CFU) and accumulation of cyclobutane pyrimidine dimers (CPD). Significant differences in survival after UV-B irradiation were observed: Acinetobacter johnsonii A2, 48%; Acinetobacter johnsonii ATCC 17909, 20%; Pseudomonas sp. strain MF8, 40%; marine Pseudomonas putida strain 2IDINH, 12%; Cytophaga sp. strain MF7, 20%; and Serratia marcescens, 21%. Most bacteria exhibited little DNA damage (between 40 and 80 CPD/Mb), except for the benthic isolate Cytophaga sp. strain MF7 (400 CPD/Mb) and Acinetobacter johnsonii ATCC 17909 (160 CPD/Mb). The recovery strategies through dark and light repair were different in all strains. The most efficient in recovering were both Acinetobacter johnsonii A2 and Cytophaga sp. strain MF7; Serratia marcescens MF42 showed intermediate recovery, and in both Pseudomonas strains, recovery was essentially zero. The UV-B responses and recovery abilities of the different bacteria were consistent with the irradiation levels in their native environment.
151 citations
TL;DR: A strain of Pseudomonas putida was found capable of metabolizing p-nitrophenol (PNP) as a sole source of carbon, nitrogen and energy, and its degradation potential at 300 and 500 ppm was examined in a medium devoid of carbon and nitrogen source.
146 citations
TL;DR: This is the first report of a Pseudomonas biocontrol strain that produces HPR in which the production of this compound correlates with its biocOntrol activity.
Abstract: A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and phenazine-1-carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F. oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol a...
TL;DR: The available evidence suggest that the biofilm forming organisms do not possess comprehensive genetic programs for biofilm development, but instead the bacteria appear to have evolved a number of different mechanisms to optimize surface colonization, of which they express a subset in response to the prevailing environmental conditions.
Abstract: Surface-associated microbial communities in many cases display dynamic developmental patterns. Model biofilms formed by Pseudomonas aeruginosa and Pseudomonas putida in laboratory flow-chamber setups represent examples of such behaviour. Dependent on the experimental conditions the bacteria in these model biofilms develop characteristic multicellular structures through a series of distinct steps where cellular migration plays an important role. Despite the appearance of these characteristic developmental patterns in the model biofilms the available evidence suggest that the biofilm forming organisms do not possess comprehensive genetic programs for biofilm development. Instead the bacteria appear to have evolved a number of different mechanisms to optimize surface colonization, of which they express a subset in response to the prevailing environmental conditions. These mechanisms include the ability to regulate cellular adhesiveness and migration in response to micro-environmental signals including those secreted by the bacteria themselves.
TL;DR: Adaptation of the membrane barrier, uptake of phosphate, maintenance of the intracellular pH and redox status, and translational control of metabolism are key mechanisms of the response of P. putida to abiotic stresses.
Abstract: The metabolically versatile soil bacterium Pseudomonas putida has to cope with numerous abiotic stresses in its habitats. The stress responses of P. putida KT2440 to 4°C, pH 4.5, 0.8 M urea, and 45 mM sodium benzoate were analyzed by determining the global mRNA expression profiles and screening for stress-intolerant nonauxotrophic Tn5 transposon mutants. In 392 regulated genes or operons, 36 gene regions were differentially expressed by more than 2.5-fold, and 32 genes in 23 operons were found to be indispensable for growth during exposure to one of the abiotic stresses. The transcriptomes of the responses to urea, benzoate, and 4°C correlated positively with each other but negatively with the transcriptome of the mineral acid response. The CbrAB sensor kinase, the cysteine synthase CysM, PcnB and VacB, which control mRNA stability, and BipA, which exerts transcript-specific translational control, were essential to cope with cold stress. The cyo operon was required to cope with acid stress. A functional PhoP, PtsP, RelA/SpoT modulon, and adhesion protein LapA were necessary for growth in the presence of urea, and the outer membrane proteins OmlA and FepA and the phosphate transporter PstBACS were indispensable for growth in the presence of benzoate. A lipid A acyltransferase (PP0063) was a mandatory component of the stress responses to cold, mineral acid, and benzoate. Adaptation of the membrane barrier, uptake of phosphate, maintenance of the intracellular pH and redox status, and translational control of metabolism are key mechanisms of the response of P. putida to abiotic stresses.
TL;DR: The results strongly suggest that co-adhesion and synergistic interaction with biofilm-forming species might represent an important mechanism, and a possible alternative strategy to production of adhesion determinants, for persistence and propagation of E. coli in the environment.
TL;DR: A methodology was developed for the extraction of medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) from Pseudomonas putida and it was determined that almost all of the PHA could be recovered with no detectable loss of molecular weight.
TL;DR: Modification of shoot and root system dry weights occured in soybean but not in alfalfa in presence of Pseudomonas strains.
Abstract: Allfalfa and soybean are the most important leguminous plants in the agricultural system of the semiarid pampas of Argentina. The possible action of phosphate solubilizing bacteria on the leguminous-rhizobia symbiosis was studied since in this region the available phosphorus distribution is not uniform. The strains used were Sinorhizobium meliloti 3DOh13, a good solubilizer of iron and phosphorus for alfalfa, Bradyrhizobium japonicum TIIIB for soybean and two phosphorus-solubilizing strains of Pseudomonas putida (SP21 and SP22) for growth promotion treatments. Modification of shoot and root system dry weights occured in soybean but not in alfalfa in presence of Pseudomonas strains.
TL;DR: Development of biodecaffeination techniques using these enzymes or using whole cells offers an attractive alternative to the present existing chemical and physical methods removal of caffeine, which are costly, toxic and non-specific to caffeine.
Abstract: Catabolism of caffeine (1,3,7-trimethylxanthine) in microorganisms commences via two possible mechanisms: demethylation and oxidation. Through the demethylation route, the major metabolite formed in fungi is theophylline (1,3-dimethylxanthine), whereas theobromine (3,7-dimethylxanthine) is the major metabolite in bacteria. In certain bacterial species, caffeine has also been oxidized directly to trimethyl uric acid in a single step. The conversion of caffeine to its metabolites is primarily brought about by N-demethylases (such as caffeine demethylase, theobromine demethylase and heteroxanthinedemethylase), caffeine oxidase and xanthine oxidase that are produced by several caffeine-degrading bacterial species such as Pseudomonas
putida and species within the genera Alcaligenes, Rhodococcus and Klebsiella. Development of biodecaffeination techniques using these enzymes or using whole cells offers an attractive alternative to the present existing chemical and physical methods removal of caffeine, which are costly, toxic and non-specific to caffeine. This review mainly focuses on the biochemistry of microbial caffeine degradation, presenting recent advances and the potential biotechnological application of caffeine-degrading enzymes.
TL;DR: In this article, the new various metal complexes of 5-benzoyl-4-hydroxy-2-methyl-6-phenyl-2H-pyridazin-3-one were synthesized.
TL;DR: The most sensitive species among the bacteria was P. putida which was inhibited by the allelochemicals even at 1 mм concentration, and catechol has also been found to have an antifungal effect on the fungi used in the study, whereas no antIfungal effects of pyrogallol were observed.
Abstract: Catechol and pyrogallol are allelochemicals which belong to phenolic compounds synthesized in plants. Their antimicrobial activities were investigated on three bacteria (Pseudomonas putida, Pseudomonas pyocyanea, Corynebacterium xerosis) and two fungi (Fusarium oxysporum, Penicillium italicum) phytopathogenic species as test organisms using the disc diffusion method. Both catechol and pyrogallol were found to have antibacterial effects on all the bacteria used in the study at 5 and 10 mM concentrations. Catechol has also been found to have an antifungal effect on the fungi used in the study, whereas no antifungal effects of pyrogallol were observed. The most sensitive species among the bacteria was P. putida which was inhibited by the allelochemicals even at 1 mM concentration.
TL;DR: Evidence is presented that proteins with GGDEF and EAL domains are involved in the regulation of biofilm formation and biofilm dispersion in Pseudomonas putida, which supports the emerging theme that GGDEF-domain and Eal-domain proteins areinvolved in regulating the transition of bacteria between a roaming lifestyle and a sessile biofilm lifestyle.
Abstract: Microbial biofilm formation often causes problems in medical and industrial settings, and knowledge about the factors that are involved in biofilm development and dispersion is useful for creating strategies to control the processes. In this report, we present evidence that proteins with GGDEF and EAL domains are involved in the regulation of biofilm formation and biofilm dispersion in Pseudomonas putida. Overexpression in P. putida of the Escherichia coli YedQ protein, which contains a GGDEF domain, resulted in increased biofilm formation. Overexpression in P. putida of the E. coli YhjH protein, which contains an EAL domain, strongly inhibited biofilm formation. Induction of YhjH expression in P. putida cells situated in established biofilms led to rapid dispersion of the biofilms. These results support the emerging theme that GGDEF-domain and EAL-domain proteins are involved in regulating the transition of bacteria between a roaming lifestyle and a sessile biofilm lifestyle.
TL;DR: A more detailed view of the effect of toluene on the intracellular energy management of P. putida S12 is yielded and several novel leads have been obtained for further targeted investigations.
Abstract: The aim of this study was to assess the cellular response of the solvent-tolerant Pseudomonas putida S12 to toluene as the single effector. Proteomic analysis (two-dimensional difference-in-gel-electrophoresis) was used to assess the response of P. putida S12 cultured in chemostats. This approach ensures constant growth conditions, both in the presence and absence of toluene. A considerable negative effect of toluene on the cell yield was found. The need for energy in the defence against toluene was reflected by differentially expressed proteins for cell energy management. In toluene-stressed cells the balance between proton motive force (PMF) enforcing and dissipating systems was shifted. NAD(P)H generating systems were upregulated whereas the major proton-driven system, ATP synthase, was downregulated. Other differentially expressed proteins were identified: outer membrane proteins, transport proteins, stress-related proteins and translation-related proteins. In addition, a protein with no assigned function was found. This study yielded a more detailed view of the effect of toluene on the intracellular energy management of P. putida S12 and several novel leads have been obtained for further targeted investigations. © 2006 The Authors.
TL;DR: Although plate counts decreased for both gfp-tagged antagonists, PRD16 showed a better survival in the rhizosphere of tomato roots independent of the inoculation method, as evidenced by all three methods.
TL;DR: Phenol had larger degradation rate than SA, whereas the inhibition of P. putida by phenol was less significant than by SA, and the cells were more favored to degrade phenol than SA under comparable conditions.
TL;DR: It is demonstrated that quorum-sensing signals induce the production of cyclic lipopeptides putisolvin I and II and consequently control biofilm formation by Pseudomonas putida.
Abstract: Pseudomonas putida strain PCL1445 produces two cyclic lipopeptides, putisolvin I and putisolvin II, which possess surface tension-reducing abilities and are able to inhibit biofilm formation and to break down existing biofilms of several Pseudomonas spp., including P. aeruginosa. Putisolvins are secreted in the culture medium during growth at late exponential phase, indicating that production is possibly regulated by quorum sensing. In the present study, we identified a quorum-sensing system in PCL1445 that is composed of ppuI, rsaL, and ppuR and shows very high similarity with gene clusters of P. putida strains IsoF and WCS358. Strains with mutations in ppuI and ppuR showed a severe reduction of putisolvin production. Expression analysis of the putisolvin biosynthetic gene in a ppuI background showed decreased expression, which could be complemented by the addition of synthetic 3-oxo-C10-N-acyl homoserine lactone (3-oxo-C10-AHL) or 3-oxo-C12-AHL to the medium. An rsaL mutant overproduces AHLs, and production of putisolvins is induced early during growth. Analysis of biofilm formation on polyvinylchloride showed that ppuI and ppuR mutants produce a denser biofilm than PCL1445, which correlates with decreased production of putisolvins, whereas an rsaL mutant shows a delay in biofilm production, which correlates with early production of putisolvins. The results demonstrate that quorum-sensing signals induce the production of cyclic lipopeptides putisolvin I and II and consequently control biofilm formation by Pseudomonas putida.
TL;DR: This article cloned the ligAB and ligC genes of SYK-6, which respectively encode protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase, into a broad host range plasmid vector, pKT230MC.
Abstract: Sphingomonas paucimobilis SYK-6, which can degrade various low molecular weight compounds derived from plant polyphenols such as lignin, lignan, and tannin, metabolizes these substances via 2-pyrone-4,6-dicarboxylic acid (PDC). We focused on this metabolic intermediate as a potential raw material for novel, bio-based polymers. We cloned the ligAB and ligC genes of SYK-6, which respectively encode protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase, into a broad host range plasmid vector, pKT230MC. The resulting plasmid, pDVABC, was introduced into the PpY1100 strain of Pseudomonas putida, and we found that PDC could be stably produced from protocatechuate and accumulated. In addition, we examined the efficiency of production of PDC from protocatechuate on a 5-L scale in a Luria-Bertani medium containing 100 mM glucose and determined that PDC was stably produced from protocatechuate to yield 10 g/L or more.
TL;DR: This study reports the first P. putida infection in fish outside of Japan, and shows that in the advanced form of the disease, almost all layers of the skin down to the epidermis were lost.
TL;DR: Pseudomonas putida KT2440, a saprophytic soil bacterium that colonizes the plant root, is a suitable microorganism for the removal of pollutants and a stable host for foreign genes used in biotransformation processes and the conditions for the optimal preservation of the strain and its derivatives for long-term storage are investigated.
Abstract: Pseudomonas putida KT2440, a saprophytic soil bacterium that colonizes the plant root, is a suitable microorganism for the removal of pollutants and a stable host for foreign genes used in biotransformation processes. Because of its potential use in agriculture and industry, we investigated the conditions for the optimal preservation of the strain and its derivatives for long-term storage. The highest survival rates were achieved with cells that had reached the stationary phase and which had been subjected to freeze-drying in the presence of disaccharides (trehalose, maltose, and lactose) as lyoprotectants. Using fluorescence polarization techniques, we show that cell membranes of KT2440 were more rigid in the stationary phase than in the exponential phase of growth. This is consistent with the fact that cells grown in the stationary phase exhibited a higher proportion of C17:cyclopropane as a fatty acid than cells in the exponential phase. Mutants for the cfaB gene, which encodes the main C17:cyclopropane synthase, and for the cfaA gene, which encodes a minor C17:cyclopropane synthase, were constructed. These mutants were more sensitive to freeze-drying than wild-type cells, particularly the mutant with a knockout in the cfaB gene that produced less than 2% of the amount of C17:cyclopropane produced by the parental strain.
TL;DR: A simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center is reported.
Abstract: Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450.
TL;DR: Pseudomonas putida CSV86 exhibited diauxic growth on aromatic compounds plus glucose, with utilization of aromatics in the first log phase and of glucose in the second log phase, suggesting that de novo protein synthesis was essential.
Abstract: Pseudomonas putida CSV86, a naphthalene-degrading organism, exhibited diauxic growth on aromatic compounds plus glucose, with utilization of aromatics in the first log phase and of glucose in the second log phase. Glucose supplementation did not suppress the activity of degrading enzymes, which were induced upon addition of aromatic compounds. The induction was inhibited by chloramphenicol, suggesting that de novo protein synthesis was essential. Cells showed cometabolism of aromatic compounds and organic acids; however, organic acids suppressed glucose utilization.