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Showing papers on "Pseudomonas putida published in 2002"


Journal ArticleDOI
TL;DR: It is suggested that bacterial IAA plays a major role in the development of the host plant root system.
Abstract: Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system.

1,737 citations


Journal ArticleDOI
TL;DR: Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes.
Abstract: Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.

1,308 citations


Journal ArticleDOI
TL;DR: The number of efflux pump operons has been found to correlate with the degree of solvent tolerance in different P. putida strains, and the operation of these efflux pumps seems to be coupled to the proton motive force via the TonB system, although the intimate mechanism of energy transfer remains elusive.
Abstract: Organic solvents can be toxic to microorganisms, depending on the inherent toxicity of the solvent and the intrinsic tolerance of the bacterial species and strains. The toxicity of a given solvent correlates with the logarithm of its partition coefficient in n-octanol and water (log Pow). Organic solvents with a log Pow between 1.5 and 4.0 are extremely toxic for microorganisms and other living cells because they partition preferentially in the cytoplasmic membrane, disorganizing its structure and impairing vital functions. Several possible mechanisms leading to solvent-tolerance in gram-negative bacteria have been proposed: (a) adaptive alterations of the membrane fatty acids and phospholipid headgroup composition, (b) formation of vesicles loaded with toxic compounds, and (c) energy-dependent active efflux pumps belonging to the resistance-nodulation-cell division (RND) family, which export toxic organic solvents to the external medium. In these mechanisms, changes in the phospholipid profile and extrusion of the solvents seem to be shared by different strains. The most significant changes in phospholipids are an increase in the melting temperature of the membranes by rapid cis-to-trans isomerization of unsaturated fatty acids and modifications in the phospholipid headgroups. Toluene efflux pumps are involved in solvent tolerance in several gram-negative strains, e.g., Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa. The AcrAB-TolC and AcrEF-TolC efflux pumps are important for n-hexane tolerance in E. coli. A number of P. putida strains have been isolated that tolerate toxic hydrocarbons such as toluene, styrene, and p-xylene. At least three efflux pumps (TtgABC, TtgDEF, and TtgGHI) are present in the most extensively characterized solvent-tolerant strain, P. putida DOT-T1E, and the number of efflux pumps has been found to correlate with the degree of solvent tolerance in different P. putida strains. The operation of these efflux pumps seems to be coupled to the proton motive force via the TonB system, although the intimate mechanism of energy transfer remains elusive. Specific and global regulators control the expression of the efflux pump operons of E. coli and P. putida at the transcriptional level.

763 citations


Journal ArticleDOI
TL;DR: The global view on the mineralization of aromatic compounds by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegradation/biotransformation processes, and reveals this bacterium as a useful model system for studying biochemical, genetic, evolutionary and ecological aspects of the catabolism of aromatic compound.
Abstract: Summary Analysis of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central aromatic intermediates, that is, the protocatechuate (pca genes) and catechol (cat genes) branches of the β-ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes). Two additional gene clusters that might be involved in the catabolism of N-heterocyclic aromatic compounds (nic cluster) and in a central meta-cleavage pathway (pcm genes) were also identified. Furthermore, the genes encoding the peripheral pathways for the catabolism of p-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n-phenylalkanoic acids (fad) were mapped in the chromosome of P. putida KT2440. Although a repetitive extragenic palindromic (REP) element is usually associated with the gene clusters, a supraoperonic clustering of catabolic genes that channel different aromatic compounds into a common central pathway (catabolic island) was not observed in P. putida KT2440. The global view on the mineralization of aromatic compounds by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegradation/biotransformation processes, and reveals this bacterium as a useful model system for studying biochemical, genetic, evolutionary and ecological aspects of the catabolism of aromatic compounds.

465 citations



Journal ArticleDOI
TL;DR: Three recombinant host strains are developed for the functional analysis of the novel alkane hydroxylase genes and sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane Hydroxylases are as distantly related to each other as to the remaining alkanes.
Abstract: We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47 Delta B, which encodes all proteins necessary for growth on medium-chain-length alkanes (C(6) to C(12)), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C(12) to C(16) alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.

272 citations


Journal ArticleDOI
TL;DR: The presence of multiple alkane hydroxylases in the two rhodococcal strains is reminiscent of other multiple-degradative-enzyme systems reported in Rhodococcus.
Abstract: The alkane hydroxylase systems of two Rhodococcus strains (NRRL B-16531 and Q15, isolated from different geographical locations) were characterized. Both organisms contained at least four alkane monooxygenase gene homologs (alkB1, alkB2, alkB3, and alkB4). In both strains, the alkB1 and alkB2 homologs were part of alk gene clusters, each encoding two rubredoxins (rubA1 and rubA2; rubA3 and rubA4), a putative TetR transcriptional regulatory protein (alkU1; alkU2), and, in the alkB1 cluster, a rubredoxin reductase (rubB). The alkB3 and alkB4 homologs were found as separate genes which were not part of alk gene clusters. Functional heterologous expression of some of the rhodococcal alk genes (alkB2, rubA2, and rubA4 [NRRL B-16531]; alkB2 and rubB [Q15]) was achieved in Escherichia coli and Pseudomonas expression systems. Pseudomonas recombinants containing rhodococcal alkB2 were able to mineralize and grow on C12 to C16 n-alkanes. All rhodococcal alkane monooxygenases possessed the highly conserved eight-histidine motif, including two apparent alkane monooxygenase signature motifs (LQRH[S/A]DHH and NYXEHYG[L/M]), and the six hydrophobic membrane-spanning regions found in all alkane monooxygenases related to the Pseudomonas putida GPo1 alkane monooxygenase. The presence of multiple alkane hydroxylases in the two rhodococcal strains is reminiscent of other multiple-degradative-enzyme systems reported in Rhodococcus.

233 citations


Journal ArticleDOI
TL;DR: In chemostats, where the organisms cannot establish in fixed positions, the two strains will compete for the primary carbon source, benzyl alcohol, which apparently gives Acinetobacter strain C6 a growth advantage, probably because it converts Benzyl alcohol to benzoate with a higher yield per time unit than P. putida R1.
Abstract: We analyzed metabolic interactions and the importance of specific structural relationships in a benzyl alcohol-degrading microbial consortium comprising two species, Pseudomonas putida strain R1 and Acinetobacter strain C6, both of which are able to utilize benzyl alcohol as their sole carbon and energy source. The organisms were grown either as surface-attached organisms (biofilms) in flow chambers or as suspended cultures in chemostats. The numbers of CFU of P. putida R1 and Acinetobacter strain C6 were determined in chemostats and from the effluents of the flow chambers. When the two species were grown together in chemostats with limiting concentrations of benzyl alcohol, Acinetobacter strain C6 outnumbered P. putida R1 (500:1), whereas under similar growth conditions in biofilms, P. putida R1 was present in higher numbers than Acinetobacter strain C6 (5:1). In order to explain this difference, investigations of microbial activities and structural relationships were carried out in the biofilms. Insertion into P. putida R1 of a fusion between the growth rate-regulated rRNA promoter rrnBP1 and a gfp gene encoding an unstable variant of the green fluorescent protein made it possible to monitor the physiological activity of P. putida R1 cells at different positions in the biofilms. Combining this with fluorescent in situ hybridization and scanning confocal laser microscopy showed that the two organisms compete or display commensal interactions depending on their relative physical positioning in the biofilm. In the initial phase of biofilm development, the growth activity of P. putida R1 was shown to be higher near microcolonies of Acinetobacter strain C6. High-pressure liquid chromatography analysis showed that in the effluent of the Acinetobacter strain C6 monoculture biofilm the metabolic intermediate benzoate accumulated, whereas in the biculture biofilms this was not the case, suggesting that in these biofilms the excess benzoate produced by Acinetobacter strain C6 leaks into the surrounding environment, from where it is metabolized by P. putida R1. After a few days, Acinetobacter strain C6 colonies were overgrown by P. putida R1 cells and new structures developed, in which microcolonies of Acinetobacter strain C6 cells were established in the upper layer of the biofilm. In this way the two organisms developed structural relationships allowing Acinetobacter strain C6 to be close to the bulk liquid with high concentrations of benzyl alcohol and allowing P. putida R1 to benefit from the benzoate leaking from Acinetobacter strain C6. We conclude that in chemostats, where the organisms cannot establish in fixed positions, the two strains will compete for the primary carbon source, benzyl alcohol, which apparently gives Acinetobacter strain C6 a growth advantage, probably because it converts benzyl alcohol to benzoate with a higher yield per time unit than P. putida R1. In biofilms, however, the organisms establish structured, surface-attached consortia, in which heterogeneous ecological niches develop, and under these conditions competition for the primary carbon source is not the only determinant of biomass and population structure.

214 citations


Journal ArticleDOI
TL;DR: Surprisingly, it was observed that the most abundant compounds in these fractions were cyclic dipeptides (diketopiperazines, DKPs), a rather novel finding in Gram-negative bacteria.
Abstract: The most universal cell-cell signaling mechanism in Gram-negative bacteria occurs via the production and response to a class of small diffusible molecules called N-acylhomoserine lactones (AHLs). This communication is called quorum sensing and is responsible for the regulation of several physiological processes and many virulence factors in pathogenic bacteria. The detection of these molecules has been rendered possible by the utilization of genetically engineered bacterial biosensors which respond to the presence of exogenously supplied AHLs. In this study, using diverse bacterial biosensors, several biosensor activating fractions were purified by organic extraction, HPLC and TLC of cell-free culture supernatants of plant growth-promoting Pseudomonas putida WCS358. Surprisingly, it was observed that the most abundant compounds in these fractions were cyclic dipeptides (diketopiperazines, DKPs), a rather novel finding in Gram-negative bacteria. The purification, characterization, chemical synthesis of four DKPs are reported and their possible role in cell-cell signaling is discussed.

184 citations


Journal ArticleDOI
TL;DR: Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by the newly isolated and promising strain Pseudomonas putida 21BN, which was identified as rhamnolipids, the amphiphilic surface-active glycolipids usually secreted by pseudomonas spp.
Abstract: Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by the newly isolated and promising strain Pseudomonas putida 21BN. The biosurfactants were identified as rhamnolipids, the amphiphilic surface-active glycolipids usually secreted by Pseudomonas spp. Their production was observed when the strain was grown on soluble substrates, such as glucose or on poorly soluble substrates, such as hexadecane, reaching values of 1.2 g l(-1). When grown on hexadecane as the sole carbon source the biosurfactant lowered the surface tension of the medium to 29 mN m(-1) and formed stable and compact emulsions with emulsifying activity of 69%.

173 citations



Journal ArticleDOI
TL;DR: In this paper, peak assignments in 31 P NMR spectra of alkaline soil extracts were analyzed and it was shown that the resonance at 0.5-1.9ppm was caused by DNA-P and the other diesters were represented by teichoic acids.
Abstract: This study involved a critical assessment of peak assignments in 31 P NMR spectra of alkaline soil extracts and included 31 P NMR spectroscopy of (1) 500 mM NaOH solutions of RNA, DNA, and lecithin; (2) 50 mM H 2 SO 4 , 500 mM NaHCO 3 (pH 8.5), and 100 mM NaOH extracts from Pseudomonas putida (Gram-negative bacterium), Bacillus subtilis (Gram-positive bacterium), Penicillium citrinum (a fungus), Aspergillus niger (a fungus), and leaves of Betula pubescens , Picea abies , and Pinus cembra ; (3) 100 mM NaOH solutions of evaporated methanol–chloroform extracts from B. subtilis , P. putida , B. pubescens leaves, and from organic and mineral soil horizons; (4) 100 mM NaOH and 50 mM H 2 SO 4 extracts from delipidized bacterial cells, and 100 mM NaOH extracts from delipidized birch leaves and soil samples. Results showed that the resonance at 0 ppm, previously assigned to phospholipids and nucleic acids, was caused by DNA-P. Resonances of phospholipids of plant and microbial origin were observed at about 1.5–1.7 and 0.6–0.7 ppm, in regions previously assigned to teichoic acid-P by various authors. Non-lipidic compounds extracted from B. subtilis and resonating at 1.9 ppm probably were represented by teichoic acids. 31 P NMR spectroscopy cannot differentiate signals derived from P of phospholipids and non-lipidic compounds in the low field of the diester region of spectra of alkaline extracts from soils, while quantitative differentiation of DNA-P and the other diesters seems quite a simple task. An unknown resonance at −1.5 ppm remained unidentified. The real concentrations of diester-P in soils can be considerably underestimated and those of monoester-P overestimated, when analyzed in alkaline extracts, because of almost complete hydrolysis of RNA and partial hydrolysis of phospholipids to monoesters.

Journal ArticleDOI
TL;DR: Cloned a genomic region of the plant growth-promoting P. putida strain IsoF that provoked induction of a bioluminescent AHL reporter plasmid is cloned, suggesting that the quorum-sensing system influences biofilm structural development.
Abstract: Recent reports have shown that several strains of Pseudomonas putida produce N-acylhomoserine lactones (AHLs). These signal molecules enable bacteria to coordinately express certain phenotypic traits in a density-dependent manner in a process referred to as quorum sensing. In this study we have cloned a genomic region of the plant growth-promoting P. putida strain IsoF that, when present in trans, provoked induction of a bioluminescent AHL reporter plasmid. Sequence analysis identified a gene cluster consisting of four genes: ppuI and ppuR, whose predicted amino acid sequences are highly similar to proteins of the LuxI-LuxR family, an open reading frame (ORF) located in the intergenic region between ppuI and ppuR with significant homology to rsaL from Pseudomonas aeruginosa, and a gene, designated ppuA, present upstream of ppuR, the deduced amino acid sequence of which shows similarity to long-chain fatty acid coenzyme A ligases from various organisms. Using a transcriptional ppuA::luxAB fusion we demonstrate that expression of ppuA is AHL dependent. Furthermore, transcription of the AHL synthase ppuI is shown to be subject to quorum-sensing regulation, creating a positive feedback loop. Sequencing of the DNA regions flanking the ppu gene cluster indicated that the four genes form an island in the suhB-PA3819 intergenic region of the currently sequenced P. putida strain KT2440. Moreover, we provide evidence that the ppu genes are not present in other AHL-producing P. putida strains, indicating that this gene cluster is so far unique for strain IsoF. While the wild-type strain formed very homogenous biofilms, both a ppuI and a ppuA mutant formed structured biofilms with characteristic microcolonies and water-filled channels. These results suggest that the quorum-sensing system influences biofilm structural development.

Journal ArticleDOI
TL;DR: Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolate showed multiple phenotypes in terms of utilization and degradation pathways, suggesting that PAH-degrading bacteria are diverse.
Abstract: Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source. Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection. Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways. The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp. strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates. Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates. Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp. strain U2. However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains. These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria.

Journal ArticleDOI
TL;DR: Different storage conditions appear to govern the selection of pseudomonads in gilt-head sea bream fish, while proteolytic and less lipolytic strains dominated under modified-atmosphere packaging.
Abstract: The population dynamics of pseudomonads in gilt-head sea bream Mediterranean fish (Sparus aurata) stored under different conditions were studied. Phenotypic analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins were performed to identify a total of 106 Pseudomonas strains isolated from S. aurata stored under different temperatures (at 0, 10, and 20°C) and packaging conditions (air and a modified atmosphere of 40% CO2-30% N2-30% O2). Pseudomonas lundensis was the predominant species, followed by Pseudomonas fluorescens, while Pseudomonas fragi and Pseudomonas putida were detected less frequently. Fluorescent Pseudomonas strains dominated under air conditions, while proteolytic and less lipolytic strains dominated under modified-atmosphere packaging. Different storage conditions appear to govern the selection of pseudomonads in gilt-head sea bream fish.

Journal ArticleDOI
TL;DR: It is shown that most Rhodococcus isolates contain three to five quite divergent homologues of the Pseudomonas putida GPo1 alkB gene, and two Mycobacterium isolates each contain one homologue, however there is no evidence for the presence of alkB homologue in the remaining strains.
Abstract: We isolated Gram-positive alkane-degraders from soil and a tricking-bed reactor, and show using polymerase chain reaction (PCR) with degenerate alkane hydroxylase primers and Southern blots that most Rhodococcus isolates contain three to five quite divergent homologues of the Pseudomonas putida GPo1 alkB gene. Two Mycobacterium isolates each contain one homologue, however there is no evidence for the presence of alkB homologues in the remaining strains.

Journal ArticleDOI
TL;DR: Activity of the ipdc promoter, measured by quantifying light production, increased fivefold in the presence of L-tryptophan, confirming that ipdc expression is induced by tryptophan.
Abstract: The phytohormone indole-3-acetic acid (IAA) accumulates in the culture medium of the plant growth-promoting bacterium Pseudomonas putida GR12-2 only when grown in the presence of exogenous tryptoph...

Journal ArticleDOI
TL;DR: Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.
Abstract: 13C/12C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.

Journal ArticleDOI
TL;DR: Most of us feel, from time to time, that other authors have not acknowledged the work of the authors' own or other groups or have omitted to interpret important aspects of their own data.
Abstract: Most of us feel, from time to time, that other authors have not acknowledged the work of our own or other groups or have omitted to interpret important aspects of their own data. Perhaps we have observations that, although not sufficient to merit a full paper, add a further dimension to one published by others. In other instances we may have a useful piece of methodology that we would like to share.

Journal ArticleDOI
TL;DR: Heterogeneity in biopolymer properties on an individual bacterium and within a population of bacterial cells may be much greater than previously believed and should be incorporated into models of bacterial adhesion.


Journal ArticleDOI
TL;DR: PCRs showed that the blaVIM-1-containing cassette was identical to that previously found in strains of different species from other Italian hospitals and that the cassette array of In110 was not identical but clearly related to that of In70, pointing to a common origin of this cassette and to a related evolutionary history of their cognate integrons.
Abstract: Successful carbapenem-based chemotherapy for the treatment of Pseudomonas infections has been seriously hindered by the recent appearance of IMP- and VIM-type metallo-β-lactamases, which confer high-level resistance to carbapenems and most other β-lactams. Recently, multidrug-resistant Pseudomonas putida isolates for which carbapenem MICs were ≥32 μg/ml were recovered from cultures of urine from three inpatients in the general intensive care unit of the Ospedale di Circolo, Varese, Italy. Enzyme assays revealed production of a metallo-β-lactamase activity, while molecular analysis detected in each isolate a blaVIM-1 determinant carried by an apparently identical medium-sized plasmid. Conjugation experiments were unsuccessful in transferring the β-lactamase determinant to Escherichia coli or Pseudomonas aeruginosa. Macrorestriction analysis by pulsed-field gel electrophoresis demonstrated that the isolates were of clonal origin. PCR mapping and sequencing of the variable region of the plasmid-borne class 1 integron carrying the blaVIM-1 determinant (named In110) showed that the blaVIM-1-containing cassette was identical to that previously found in strains of different species from other Italian hospitals and that the cassette array of In110 was not identical but clearly related to that of In70 (a blaVIM-1-containing plasmid-borne integron from an Achromobacter xylosoxidans isolate), pointing to a common origin of this cassette and to a related evolutionary history of their cognate integrons.

Journal ArticleDOI
TL;DR: P Phenotypic examination of the recovered bacteria revealed that they belong mainly to the genus Pseudomonas, Enterobacter and Acinetobacter.
Abstract: Potential hydrocarbon degrader bacteria were isolated from soil samples that have been exposed to crude petroleum oil spills. Bacterial population of these polluted soils showed counts ranging between 9.5 x 10(5) and 237.5 x 10(5) CFU/g soil with 2 different colony types of bacterial strains which have been recovered on the agar plates. Results indicated that longer aged contamination exhibited a greater number of microorganisms. Phenotypic examination of the recovered bacteria revealed that they belong mainly to the genus Pseudomonas, Enterobacter and Acinetobacter. Turbidity, dry weight and physical appearance were used as an indication for the ability of these bacteria to grow on diesel. Action of three different Pseudomonas species, Acinetobacter lowffi, Enterobacter cloacae and Rhodococcus erythropolis on 0.1 % (v/v) diesel was followed at 1, 2, 6 and 12 h. Pseudomonas putida and P. mallei and Enterobacter cloacae indicated a positive reaction; however, Pseudomonas maltophilia and Acinetobacter lowffi showed no effect.

Journal ArticleDOI
TL;DR: The results obtained show that the growth-promoting effects of ACC-utilizing rhizobacteria depend significantly on the nutrient status of the plant.
Abstract: Responses of rape (Brassica napus var. oleifera L.) to inoculation with plant growth promoting rhizobacteria, Pseudomonas putida Am2, Pseudomonas putida Bm3, Alcaligenes xylosoxidans Cm4, and Pseud...

Journal ArticleDOI
TL;DR: The results show that the protein engineering of P450(cam) for high selectivity of substrate oxidation is more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of enzyme-substrate interactions.
Abstract: Oxygenated derivatives of the monoterpene (+)-α-pinene are found in plant essential oils and used as fragrances and flavorings. (+)-α-Pinene is structurally related to (+)-camphor, the natural substrate of the heme monooxygenase cytochrome P450cam from Pseudomonas putida. The aim of the present work was to apply the current understanding of P450 substrate binding and catalysis to engineer P450cam for the selective oxidation of (+)-α-pinene. Consideration of the structures of (+)-camphor and (+)-α-pinene lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations. All mutants showed greatly enhanced binding and rate of oxidation of (+)-α-pinene. Some mutants had tighter (+)-α-pinene binding than camphor binding by the wild-type. The most active was the Y96F/V247L mutant, with a (+)-α-pinene oxidation rate of 270 nmol (nmol of P450cam)-1 min-1, which was 70% of the rate of camphor oxidation by wild-type P450cam. Camphor is oxidized by wild-type P450cam exclusively ...

Journal ArticleDOI
TL;DR: This work investigated the biodegradation potential of phenol using mixed liquors of Pseudomonas putida and activated sludge and found that a second-order polynomial regression model could properly interpret the experimental data with an R2-value and F-value of 0.9997.

Journal ArticleDOI
TL;DR: Results show an excellent correlation between successful naphthalene rhizoremediation by the Barmultra-P.
Abstract: Previously, we have described the selection of a plant-bacterium pair that is efficient in rhizoremediating naphthalene pollution in microcosm studies. After repeated selection for efficient root tip colonization upon inoculation of seeds of grass cv. Barmultra and for stable and efficient growth on naphthalene, Pseudomonas putida PCL1444 was selected as the most efficient colonizer of Barmultra roots. Here, we report the analysis of Barmultra root exudate composition and our subsequent tests of the growth rate of the bacterium and of the expression of the naphthalene degradation genes on individual exudate components. High performance liquid chromatography analysis of the organic acid and sugar root-exudate components revealed that glucose and fructose are the most abundant sugars, whereas succinic acid and citric acid are the most abundant organic acids. Tn5luxAB mutants of PCL1444 impaired in naphthalene degradation appeared to be impaired in genes homologous to genes of the upper naphthalene degradation pathway present in various Pseudomonas strains and to genes of the lower pathway genes for naphthalene degradation in P. stutzeri. Highest expression for both pathways involved in naphthalene degradation during growth in minimal medium with the carbon source to be tested was observed at the start of the logarithmic phase. Naphthalene did not induce the upper pathway, but a different pattern of expression was observed in the lower pathway reporter, probably due to the conversion of naphthalene to salicylic acid. Salicylic acid, which is described as an intermediate of the naphthalene degradation pathway in many Pseudomonas strains, did induce both pathways, resulting in an up to sixfold higher expression level at the start of the logarithmic phase. When expression levels during growth on the different carbon sources present in root exudate were compared, highest expression was observed on the two major root exudate components, glucose and succinic acid. These results show an excellent correlation between successful naphthalene rhizoremediation by the Barmultra-P. putida PCL1444 pair and both efficient utilization of the major exudate components for growth and high transcription of the naphthalene catabolic genes on the major exudate components. Therefore, we hypothesize that efficient root colonizing and naphthalene degradation is the result of the applied colonization enrichment procedure.

Journal ArticleDOI
TL;DR: Bacterial adherence to hydrocarbons (BATH) assays showed cells grown on the higher concentrations of mono-chlorophenol to be more hydrophobic than those grown on phenol and lower concentrations of Mono- chlorophenol, suggesting that increased hydrophobicity and autoaggregation of P. putida CP1 were a response to toxicity of the added substrates.
Abstract: A bacterium, CP1, identified as Pseudomonas putida strain, was investigated for its ability to grow on and degrade mono-chlorophenols and phenols as sole carbon sources in aerobic shaking batch culture. The organism degraded up to 1.56 mM 2- and 3-chlorophenol, 2.34 mM 4-chlorophenol and 8.5 mM phenol using an ortho-cleavage pathway. P. putida CP1, acclimated to degrade 2-chlorophenol, was capable of 3-chlorocatechol degradation, while P. putida, acclimated to 4-chlorophenol degradation, degraded 4-chlorocatechol. Growth of P. putida CP1 on higher concentrations of the mono-chlorophenols, >or=1.56 mM 4-chlorophenol and >or=0.78 mM 2- and 3-chlorophenol, resulted in decreases in cell biomass despite metabolism of the substrates, and the formation of large aggregates of cells in the culture medium. Increases in cell biomass with no clumping of the cells resulted from growth of P. putida CP1 on phenol or on lower concentrations of mono-chlorophenol. Bacterial adherence to hydrocarbons (BATH) assays showed cells grown on the higher concentrations of mono-chlorophenol to be more hydrophobic than those grown on phenol and lower concentrations of mono-chlorophenol. The results suggested that increased hydrophobicity and autoaggregation of P. putida CP1 were a response to toxicity of the added substrates.

Journal ArticleDOI
TL;DR: Among the housekeeping genes, only genes of the translational apparatus were located in segments with an atypical signature, suggesting that the synthesis of ribosomal proteins is uncoupled from the rapidly changing translational demands of the cell by the separate utilization of tRNA pools.
Abstract: Summary The compositional bias of the G+C, di- and tetranucleotide contents in the 6 181 862 bp Pseudomonas putida KT2440genome was analysed in sliding windows of 4000 bp in steps of 1000 bp. The genome has a low GC skew (mean 0.066) between the leading and lagging strand. The values of GC contents (mean 61.6%) and of dinucleotide relative abundance exhibit skewed Gaussian distributions. The variance of tetranucleotide frequencies, which increases linearly with increasing GC content, shows two overlapping Gaussian distributions of genome sections with low (minor fraction) or high variance (major fraction). Eighty per cent of the chromosome shares similar GC contents and oligonucleotide bias, but 105 islands of 4000 bp or more show atypical GC contents and/or oligonucleotide signature. Almost all islands provide added value to the metabolic proficiency of P. putida as a saprophytic omnivore. Major features are the uptake and degradation of organic chemicals, ion transport and the synthesis and secretion of secondary metabolites. Other islands endow P. putida with determinants of resistance and defenceor with constituents and appendages of the cell wall. A total of 29 islands carry the signature of mobile elements such as phage, transposons, insertion sequence (IS) elements and group II introns, indicating recent acquisition by horizontal gene transfer. The largest gene carries the most unusual sequence that encodes a multirepeat threonine-rich surface adhesion protein. Among the housekeeping genes, only genes of the translational apparatus were located in segments with an atypical signature, suggesting that the synthesis of ribosomal proteins is uncoupled from the rapidly changing translational demands of the cell by the separate utilization of tRNA pools.

Journal ArticleDOI
TL;DR: The repertoire of sigma factors in P. putida KT2440 was analysed and 19 of which corresponded to the subfamily of extracytoplasmic function (ECF) sigma Factors, which showed similarity to the Escherichia coli FecI sigma factor, which is involved in iron acquisition.
Abstract: Pseudomonas putida KT2440 is highly successful in colonizing a variety habitats, including aquatic and edaphic niches. In accordance with this ability and with the need to adapt to changing environmental conditions, P. putida has developed sophisticated mechanisms of transcriptional regulation. We analysed, at the genome level, the repertoire of sigma factors in P. putida KT2440 and identified 24 sigma factors, 19 of which corresponded to the subfamily of extracytoplasmic function (ECF) sigma factors. We detected 13 ECF sigma factors that showed similarity to the Escherichia coli FecI sigma factor, which is involved in iron acquisition. In 11 cases, a fecR-like gene was found adjacent to the fecI-like gene and, in 10 cases, a gene encoding an iron receptor lies in the vicinity of the fecI/fecR cluster. This may explain the ability of P. putida KT2440 to grow under low iron availability conditions. Five fecI/fecR/iron receptor gene clusters from P. putida were also identified in the human pathogen Pseudomonas aeruginosa.