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PubMedDate not supplied

Effects of temperature on detection of plasmid or chromosomally encoded gfp- and lux-labeled Pseudomonas fluorescens in soil.

Pseudomonas fluorescens is a normal inhabitant of the soil rhizosphere. The use of genetically altered strains of P. fluorescens in bioremediation has led to the need for effective monitoring of such cells released into the environment. In this study, we present data on the persistence in soil of P. fluorescens harboring gfp (green fluorescent protein) or lux (bioluminescence) genes. Comparisons were made between strains marked chromosomally and strains carrying these markers on a plasmid. Overall effects of plasmid carriage on culturability were also examined. Sterile soil microcosms were inoculated with washed cells to a final concentration of ca. 10(6) CFU.g(-1) and placed at 5, 23, and 35-37 degrees C. Samples were taken periodically and examined for culturability and viability, using the substrate responsiveness assay. Our results indicated no significant loss of culturability at 5 and 23 degrees C for a period of over one year. In contrast, cells of P. fluorescens incubated at 35-37 degrees C entered the viable but nonculturable state within 7 days. All cells labeled with gfp retained fluorescence regardless of culturability, suggesting that the green fluorescent protein can be of value in monitoring the presence of cells following their release to the environment. Because fluorescence was maintained regardless of the cells' physiological state, this protein may also be an indicator of cell viability.

Bacteriological Techniques↗

PubMed1989

Cyanase-mediated utilization of cyanate in Pseudomonas fluorescens NCIB 11764.

Pseudomonas fluorescens NCIB 11764 was capable of utilizing cyanate (OCN-) as a sole nitrogen source for growth. Crude cell extracts from cells grown on cyanate, but not on ammonium sulfate, were induced for an enzyme catalyzing cyanate conversion to ammonia. Enzymatic activity was shown to be bicarbonate dependent and specific for cyanate as a substrate, suggesting that cyanate utilization in this organism is facilitated by an enzyme resembling cyanase (cyanate amidohydrolase; EC 3.5.5.3), as described previously in Escherichia coli and Flavobacterium sp.

Aminohydrolases↗

PubMed1999

Disruption of narG, the gene encoding the catalytic subunit of respiratory nitrate reductase, also affects nitrite respiration in Pseudomonas fluorescens YT101.

The Pseudomonas fluorescens YT101 gene narG, which encodes the catalytic alpha subunit of the respiratory nitrate reductase, was disrupted by insertion of a gentamicin resistance cassette. In the Nar(-) mutants, nitrate reductase activity was not detectable under all the conditions tested, suggesting that P. fluorescens YT101 contains only one membrane-bound nitrate reductase and no periplasmic nitrate reductase. Whereas N(2)O respiration was not affected, anaerobic growth with NO(2) as the sole electron acceptor was delayed for all of the Nar(-) mutants following a transfer from oxic to anoxic conditions. These results provide the first demonstration of a regulatory link between nitrate and nitrite respiration in the denitrifying pathway.

Anaerobiosis↗

PubMed1997

Trehalose induces antagonism towards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400.

Pseudomonas fluorescens ATCC 17400 shows in vitro activity against Pythium debaryanum under conditions of iron limitation. A lacZ reporter gene introduced by transposon mutagenesis into the P. fluorescens ATCC 17400 trehalase gene (treA) was induced by a factor released by the phytopathogen Pythium debaryanum. The induction of the lacZ gene was lost upon treatment of the Pythium supernatant with commercial trehalase. A trehalose concentration as low as 1 microM could induce the expression of treA. The mutation did not affect the wild-type potential for fungus antagonism but drastically decreased the osmotolerance of the mutant in liquid culture and suppressed the ability of P. fluorescens ATCC 17400 to utilize trehalose as a carbon source. A subsequent transposon insertion in treP, one of the trehalose phosphotransferase genes upstream of treA, silenced the lacZ gene. This double mutant restricted fungal growth only under conditions of high osmolarity, which probably results in internal trehalose accumulation. These data confirm the role of the disaccharide trehalose in osmotolerance, and they indicate its additional role as an initiator of or a signal for fungal antagonism.

Amino Acid Sequence↗

PubMed2000

Lon protease influences antibiotic production and UV tolerance of Pseudomonas fluorescens Pf-5.

Pseudomonas fluorescens Pf-5 is a soil bacterium that suppresses plant pathogens due in part to its production of the antibiotic pyoluteorin. Previous characterization of Pf-5 revealed three global regulators, including the stationary-phase sigma factor sigma(S) and the two-component regulators GacA and GacS, that influence both antibiotic production and stress response. In this report, we describe the serine protease Lon as a fourth global regulator influencing these phenotypes in Pf-5. lon mutants overproduced pyoluteorin, transcribed pyoluteorin biosynthesis genes at enhanced levels, and were more sensitive to UV exposure than Pf-5. The lon gene was preceded by sequences that resembled promoters recognized by the heat shock sigma factor sigma(32) (sigma(H)) of Escherichia coli, and Lon accumulation by Pf-5 increased after heat shock. Therefore, sigma(H) represents the third sigma factor (with sigma(S) and sigma(70)) implicated in the regulation of antibiotic production by P. fluorescens. Lon protein levels were similar in stationary-phase and exponentially growing cultures of Pf-5 and were not positively affected by the global regulator sigma(S) or GacS. The association of antibiotic production and stress response has practical implications for the success of disease suppression in the soil environment, where biological control organisms such as Pf-5 are likely to encounter environmental stresses.

ATP-Dependent Proteases↗

PubMed1975

Studies on the degradation of pterine and pterine-6-carboxylic acid by Pseudomonas fluorescens UK-1.

Pseudomonas fluorescens UK-1 has been incubated in basal mineral medium which was 0.5mM in pterine or pterine-6-carboxylic acid. During incubation the test organism splits the pteridine ring by liberating carbon dioxide from position 2. Glucose added to the medium greatly enhances both the growth of the organism and the carbon dioxide formation. Despite the structural similarities between pterine and pterine-6-carboxylic acid, only the degradation products derived from pterine are fluorescent in UV-light. Among the degradation products lumazine, pyrazine-2-carboxylic acid, and pyrazine-2-carboxamide have been identified. Also the activities of pterine deaminase and a carbon dioxide-liberating enzyme have been determined.

Aminohydrolases↗

PubMed1996

Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome.

Pseudomonas fluorescens is a saprophytic bacterium commonly isolated from soil, water, and the surfaces and tissues of plants and animals. The species has important applications in biotechnology because it can enhance plant growth and protect crops against disease. A complete physical map of the 6.63 Mbp P. fluorescens SBW25 chromosome was constructed using data obtained from combinations of one- and two-dimensional electrophoresis of completely or partially digested chromosomal DNA with end labelling. In total, 139 restriction sites (15 PacI, 53 SpeI, 71 XbaI) were placed on the physical map and complete maps of the circular chromosome were obtained for both PacI and SpeI; only XbaI fragments linking SpeI fragments were positioned. The average resolution of restriction sites was 48 kbp. A genetic map was derived from the physical map by southern hybridization and 31 genes were positioned including oriC, rDNA operons (rnnA-E), recA, gacA, and pyvD.

Blotting, Southern↗

PubMed2003

The pyoverdine from Pseudomonas chlororaphis D-TR133 showing mutual acceptance with the pyoverdine of Pseudomonas fluorescens CHAO.

From Pseudomonas chlororaphis D-TR 133 a pyoverdine was isolated and its primary structure were elucidated by spectroscopic methods and degradation reactions. Despite some structural differences, its Fe(III) complex and that of the pyoverdine from Pseudomonas fluorescens CHA0 were taken up by either strain with a high rate. This is explained by a structural similarity between the two pyoverdines which were shown to differ in their structures only by the replacement of Lys by Ala in the C-terminal part of the molecules. An unexpected feature is that the main pyoverdine of P. chlororaphis D-TR133 is accompanied by a minor one where specifically one Ala is replaced by Gly. So far amino acid variations in the peptide chain of pyoverdines produced by a given strain had not been observed amongst the producers of the about fifty pyoverdines reported in the literature.

Bacterial Typing Techniques↗

PubMed1979

Thermal inactivation of a heat-resistant lipase produced by the psychotrophic bacterium Pseudomonas fluorescens.

Lipase from Pseudomonas fluorescens was studied for thermostability at temperatures ranging from 100 C to 160 C. The heat treatments were in two media, and heating times necessary to inactivate 90% of the enzyme at constant temperature were extremely long even at high temperatures, e.g. 3.6 min at 140 C in nutrient broth and 2.0 min at 170 C in skim milk. The increments of temperature to reduce these heating times 90% were 37.0 C in nutrient broth and 38.9 C in skim milk. The lipase was inactivated only partly after 20 h at 20 C in 8 M urea, 6 M guanidine hydrochloride, and 1.0% sodium dodecyl sulfate. Four percent 2-mercaptoethanol showed no effect.

Animals↗

PubMed2001

Structural and functional analysis of the phosphonoacetate hydrolase (phnA) gene region in Pseudomonas fluorescens 23F.

The Pseudomonas fluorescens 23F phosphonoacetate hydrolase gene (phnA) encodes a novel carbon-phosphorus bond cleavage enzyme whose expression is independent of the phosphate status of the cell. Analysis of the regions adjacent to the phosphonoacetate hydrolase structural gene (phnA) indicated the presence of five open reading frames (ORFs). These include one (phnR) whose putative product shows high levels of homology to the LysR family of positive transcriptional regulators. Its presence was shown to be necessary for induction of the hydrolase activity. 2-Phosphonopropionate was found to be an inducer (and poor substrate) for phosphonoacetate hydrolase. Unlike phosphonoacetate, which is also an inducer of phosphonoacetate hydrolase, entry of 2-phosphonopropionate into cells appeared to be dependent on the presence of a gene (phnB) that lies immediately downstream of phnA and whose putative product shows homology to the glycerol-3-phosphate transporter. RNA analysis revealed transcripts for the phnAB and phnR operons, which are transcribed divergently; the resulting mRNAs overlapped by 29 nucleotide bases at their 5' ends. Transcripts of phnAB were detected only in cells grown in the presence of phosphonoacetate, whereas transcripts of phnR were observed in cells grown under both induced and uninduced conditions. The expression of three additional genes found in the phnA region did not appear necessary for the degradation of phosphonoacetate and 2-phosphonopropionate by either Pseudomonas putida or Escherichia coli cells.

Alkaline Phosphatase↗

PubMed2003

Two different mechanisms are involved in the extremely high-level benzalkonium chloride resistance of a Pseudomonas fluorescens strain.

A Pseudomonas fluorescens strain, PFRB, which we previously isolated as a contaminant in a batch of benzalkonium chloride (BAC) stock solution, exhibits high-level resistance, not only to BAC, but also to other cationic surfactants belonging to disinfectants classified as quaternary ammonium compounds (QACs). In this study, we analyzed the resistance mechanism of the strain to BAC and other disinfectants. We obtained results suggesting that two different mechanisms, reduced adsorption of BAC to the cell surface and an energy-dependent mechanism which is most probably an efflux system, were implicated in the high-level resistance to BAC. Reduced adsorption of BAC is likely due to the decreased negative cell surface charge of the strain. The putative efflux system seems to be unique in that it excretes only a certain range of cationic membrane-acting disinfectants belonging to QACs.

Adsorption↗

PubMed2005

Purification, refolding, and characterization of recombinant Pseudomonas fluorescens lipase.

Thermostable Pseudomonas fluorescens SIK W1 lipase (PFL), which is responsible for the spoilage of milk, was overexpressed as inclusion bodies in Escherichia coli. Renaturation of solubilized PFL was achieved by using size-exclusion protein refolding chromatography. The renatured enzyme was purified homogeneously using a combination of gel filtration and ion-exchange FPLC. Its specific activity was found to be enhanced in the presence of Ca2+. Secondary structural changes induced by Ca2+ were monitored by circular dichroism, which demonstrated that the activity increase of PFL in the presence of Ca2+ is strongly correlated with significant increases in alpha-helix and beta-sheet content. In the presence of Ca2+, the PFL structure was found resistant to denaturation by guanidine hydrochloride and to enzyme activity loss due to cosolvents like DMSO and trifluoroethanol, suggesting that Ca2+ plays an important role in inducing conformational changes and consequently in maintaining enzyme structural stability.

Calcium↗

PubMed2005

Role of the stress sigma factor RpoS in GacA/RsmA-controlled secondary metabolism and resistance to oxidative stress in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens biocontrol strain CHA0, the two-component system GacS/GacA positively controls the synthesis of extracellular products such as hydrogen cyanide, protease, and 2,4-diacetylphloroglucinol, by upregulating the transcription of small regulatory RNAs which relieve RsmA-mediated translational repression of target genes. The expression of the stress sigma factor sigmaS (RpoS) was controlled positively by GacA and negatively by RsmA. By comparison with the wild-type CHA0, both a gacS and an rpoS null mutant were more sensitive to H2O2 in stationary phase. Overexpression of rpoS or of rsmZ, encoding a small RNA antagonistic to RsmA, restored peroxide resistance to a gacS mutant. By contrast, the rpoS mutant showed a slight increase in the expression of the hcnA (HCN synthase subunit) gene and of the aprA (major exoprotease) gene, whereas overexpression of sigmaS strongly reduced the expression of these genes. These results suggest that in strain CHA0, regulation of exoproduct synthesis does not involve sigmaS as an intermediate in the Gac/Rsm signal transduction pathway whereas sigmaS participates in Gac/Rsm-mediated resistance to oxidative stress.

Bacterial Proteins↗

PubMed1988

Molecular cloning of multiple xylanase genes from Pseudomonas fluorescens subsp. cellulosa.

Pseudomonas fluorescens subsp. cellulosa was shown to express extracellular xylanases. Genes encoding these enzymes were isolated from a gene library of P. fluorescens subsp. cellulosa DNA, constructed in bacteriophage lambda 47.1. One of the phages (PXC) that expressed xylanase also conferred the ability to hydrolyse carboxymethylcellulose. An 11.8 kb HindIII DNA restriction fragment and a 6.2 kb EcoRI DNA fragment were subcloned from two distinct xylanase-expressing phages, into pUC18, to yield recombinant plasmids pGHJ4 and pGHJ5 respectively. Cells of Escherichia coli harbouring either of these two plasmids, or plasmid pJHH1 (comprising the cellulase gene from PXC, previously cloned on a 7.3 kb partial EcoRI DNA fragment in pUC18), expressed xylanase activity. The positions of the xylanase genes in the recombinant plasmids were elucidated by subcloning and transposon mutagenesis. In pJHH1 the xylanase gene was adjacent to the DNA region encoding the endoglucanase. The polysaccharide-degrading genes in pJHH1 were transcribed from different promotors. Hybridization studies revealed that the xylanase genes encoded by pGHJ4 and pGHJ5 showed strong homology. All three cloned enzymes cleaved p-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-cellobioside. Xylan and glucose did not affect expression of xylanase in E. coli strains harbouring pJHH1, pGHJ4 or pGHJ5.

Cloning, Molecular↗

PubMed2004

Positive autoregulation and signaling properties of pyoluteorin, an antibiotic produced by the biological control organism Pseudomonas fluorescens Pf-5.

Pseudomonas fluorescens Pf-5, a rhizosphere bacterium, produces a suite of secondary metabolites that are toxic to seed- and root-rotting plant pathogens. Among these are the polyketide compounds pyoluteorin and 2,4-diacetylphloroglucinol. We provide evidence that pyoluteorin production is influenced by positive autoregulation. Addition of pyoluteorin to liquid cultures of Pf-5 enhanced pyoluteorin production. In addition, pyoluteorin and 2,4-diacetylphloroglucinol mutually inhibit one another's production in Pf-5. For pyoluteorin, both positive autoregulation and negative influences on production by 2,4-diacetylphloroglucinol were demonstrated at the transcriptional level by measuring activity from transcriptional fusions of an ice nucleation reporter gene (inaZ) to three separate pyoluteorin biosynthetic genes. The occurrence of pyoluteorin autoregulation in the rhizosphere was assessed on cucumber seedlings in pasteurized soil with cross-feeding experiments. In the rhizosphere, expression of a pyoluteorin biosynthesis gene by a pyoluteorin-deficient mutant of Pf-5 was enhanced by pyoluteorin produced by coinoculated cells of Pf-5. These data establish that the polyketide pyoluteorin is an autoregulatory compound and functions as a signal molecule influencing the spectrum of secondary metabolites produced by the bacterial cell.

Anti-Bacterial Agents↗

PubMed2001

Detection and analysis of chromosomal arsenic resistance in Pseudomonas fluorescens strain MSP3.

Pseudomonas fluorescens MSP3 isolated from sea water was resistant to arsenate. This bacterium harbored no plasmids, indicating that arsenic resistance was chromosomally encoded. The chromosomal DNA from MSP3 when transformed onto Escherichia coli DH5alpha using pBluescript exhibited resistance to sodium arsenate and sodium arsenite. Three clones MSA1, MSA2, and MSI3 containing the ars genes were obtained and further subcloning resulted in three fragments of size 2.2, 2.6, and 2.1 kb for pMSA11, pMSA12, and pMSI13, respectively, which contained the genes arsRBC of the arsenic operon. An efflux mechanism of detoxification was observed which was ATP dependent. The resistance mechanism was encoded from a single operon which consisted of an arsenite inducible repressor that regulates the expression of arsenate reductase (ars C) and inner membrane associated arsenite export system encoded by ars B. The chromosomal operon was cloned, sequenced, and found to consist of three cistrons, named as ars R, ars B, and ars C. Southern hybridization and mating experiments confirmed the functioning of the ars genes in the operon, thereby conferring increased resistance to sodium arsenate and sodium arsenite.

Adenosine Triphosphatases↗

PubMed2002

Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.

4-Butyrolactone↗

PubMed1998

A seven-gene locus for synthesis of phenazine-1-carboxylic acid by Pseudomonas fluorescens 2-79.

Pseudomonas fluorescens 2-79 produces the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA), which is active against a variety of fungal root pathogens. In this study, seven genes designated phzABCDEFG that are sufficient for synthesis of PCA were localized within a 6.8-kb BglII-XbaI fragment from the phenazine biosynthesis locus of strain 2-79. Polypeptides corresponding to all phz genes were identified by analysis of recombinant plasmids in a T7 promoter/polymerase expression system. Products of the phzC, phzD, and phzE genes have similarities to enzymes of shikimic acid and chorismic acid metabolism and, together with PhzF, are absolutely necessary for PCA production. PhzG is similar to pyridoxamine-5'-phosphate oxidases and probably is a source of cofactor for the PCA-synthesizing enzyme(s). Products of the phzA and phzB genes are highly homologous to each other and may be involved in stabilization of a putative PCA-synthesizing multienzyme complex. Two new genes, phzX and phzY, that are homologous to phzA and phzB, respectively, were cloned and sequenced from P. aureofaciens 30-84, which produces PCA, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine. Based on functional analysis of the phz genes from strains 2-79 and 30-84, we postulate that different species of fluorescent pseudomonads have similar genetic systems that confer the ability to synthesize PCA.

Amino Acid Sequence↗