Showing papers on "Pseudomonas putida published in 1996"

Isolation and Characterization of Mutants of the Plant Growth-Promoting Rhizobacterium Pseudomonas putida GR12-2 That Overproduce Indoleacetic Acid

Abstract: Following transposon Tn5 mutagenesis of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2, mutants that were able to grow in the presence of the tryptophan analog 5-fluorotryptophan were selected. Seven of the 50 5-fluorotryptophan-resistant mutants overproduced the phytohormone indoleacetic acid (IAA). Of these seven mutants, the highest level of IAA was observed with strain P. putida GR12-2/aux1, which produced four times the amount of indoleacetic acid synthesized by the wild-type strain. Strain P. putida GR12-2/aux1, in contrast to the wild type, lost the ability to stimulate the elongation of the roots of canola seedlings under gnotobiotic conditions. The growth rate, siderophore production, and 1-aminocyclopropane-1-carboxylate deaminase activity of mutant strain P. putida GR12-2/aux1 were identical to those of the wild-type strain. The role of IAA in the mechanism of plant growth stimulation by P. putida GR12-2 and other plant growth-promoting rhizobacteria is discussed.

Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy.

Abstract: As a representative member of the toluene-degrading population in a biofilter for waste gas treatment, Pseudomonas putida was investigated with a 16S rRNA targeting probe. The three-dimensional distribution of P. putida was visualized in the biofilm matrix by scanning confocal laser microscopy, demonstrating that P. putida was present throughout the biofilm. Acridine orange staining revealed a very heterogeneous structure of the fully hydrated biofilm, with cell-free channels extending from the surface into the biofilm. This indicated that toluene may penetrate to deeper layers of the biofilm, and consequently P. putida may be actively degrading toluene in all regions of the biofilm. Furthermore, measurements of growth rate-related parameters for P. putida showed reduced rRNA content and cell size (relative to that in a batch culture), indicating that the P. putida population was not degrading toluene at a maximal rate in the biofilm environment. Assuming that the rRNA content reflected the cellular activity, a lower toluene degradation rate for P. putida present in the biofilm could be estimated. This calculation indicated that P. putida was responsible for a significant part (65%) of the toluene degraded by the entire community.

Root elongation in various agronomic crops by the plant growth promoting rhizobacterium pseudomonas putida gr12–2

Abstract: Seeds of canola, lettuce, tomato, barley, wheat, and oats were inoculated with either the wild-type plant growth promoting rhizobacterium (PGPR), Pseudomonas putida GR12–2, or the mutant P. putida GR 12–2lacd68 (deficient in the activity of the enzyme 1-aminocyclopropane-1-carboxylate deaminase) alone and in conjunction with either an inhibitor of ethylene biosynthesis, L-α-(aminoethoxyvinyl)-glycine (AVG), or the chemical ethylene generator, (2-chloroethyl) phosphonic acid (ethophon). For the different treatments, variations in root length under gnotobiotic conditions were compared. Canola, lettuce, tomato, and wheat responded to all of the treatments in a similar manner: The root lengths increased when seeds were treated with P. putida GR 12–2 and/or AVG but not with the mutant strain, in comparison with a MgSO4 control treatment, while the ethophon treatment inhibited root elongation. With barley and oat, none of the treatments had any effect on root lengths; however, when the ethophon concent...

Cell Envelope Changes in Solvent-Tolerant and Solvent-Sensitive Pseudomonas putida Strains following Exposure to o-Xylene.

Abstract: Solvent-tolerant and -sensitive Pseudomonas putida strains were studied to determine their cell envelope changes following exposure to o-xylene. Both strains produced trans-unsaturated fatty acids. The tolerant strain showed an increase in total fatty acids, an increase in saturated fatty acids, and modified lipopolysaccharide. It is suggested that these envelope modifications aid in survival at high concentrations of organic solvents.

Effect of Environmental Factors on the trans/cis Ratio of Unsaturated Fatty Acids in Pseudomonas putida S12.

Abstract: The membrane reactions of Pseudomonas putida S12 to environmental stress were investigated. Cells reacted to the addition of six different heavy metals with an increase in the ratio of trans to cis unsaturated fatty acids. A correlation among the increase in the trans/cis ratio, the toxic effects of the heavy metals, and nonspecific permeabilization of the cytoplasmic membrane, as indicated by an efflux of potassium ions, was measured. Cells previously adapted to toxic concentrations of toluene exhibited increased tolerance to all applied concentrations of zinc compared with nonadapted cells. Cells exposed to different temperatures grew optimally at 30(deg)C. The degree of saturation of the membrane fatty acids of these cells decreased with decreasing temperature. An increase in the trans/cis ratio of unsaturated fatty acids took place only at higher temperatures. Osmotic stress, expressed as reduced water activity, was obtained by using different types of solutes. Only in the presence of toxic concentrations of sodium chloride or sucrose did the trans/cis ratio increase. At no applied water activity a significant effect of glycerol on the trans/cis ratio was measured. When cells were exposed to different pHs, a distinct optimum cis/trans isomerase activity was measured at pHs between 4.0 and 5.0, whereas at higher or lower pHs no reaction occurred. This optimum coincided with a loss of viability between pH 4 and 5.

Active efflux of toluene in a solvent resistant bacterium.

Abstract: We investigated the mechanisms behind the organic-solvent resistance of the solvent-tolerant strain Pseudomonas putida S12. By use of 14C-labeled toluene, we obtained evidence that an energy-dependent export system may be responsible for this resistance to toluene.

Molecular cloning of novel genes for polycyclic aromatic hydrocarbon degradation from Comamonas testosteroni GZ39.

Abstract: Three strains of Comamonas testosteroni were isolated from river sediment for the ability to degrade phenanthrene; two of the strains also grew on naphthalene, and one strain also grew on anthracene. The homology of the genes for polycyclic aromatic hydrocarbon degradation in these strains to the classical genes (nah) for naphthalene degradation from Pseudomonas putida NCIB 9816-4 was determined. The three C. testosteroni strains showed no homology to the nah gene probe even under low-stringency conditions. The genes for naphthalene and phenanthrene degradation were cloned from one of the three C. testosteroni strains. Two cosmid clones expressing polycyclic aromatic hydrocarbon dioxygenase activity were identified from a library prepared with genomic DNA from C. testosteroni GZ39. The genes coding for the first two enzymes in the catabolic pathway, phenanthrene dioxygenase and cis-phenanthrene dihydrodiol dehydrogenase, were localized to a 5.4-kb NcoI-PstI fragment by subcloning and gene expression experiments. Further subcloning and analysis revealed a novel organization of the genes, with the gene for cis-phenanthrene dihydrodiol dehydrogenase located between the genes for the individual phenanthrene dioxygenase components. A Southern blot with the cloned genes from C. testosteroni GZ39 confirmed that these genes are distinct from those found in P. putida NCIB 9816-4. Southern blots also demonstrated that C. testosteroni GZ38A possesses genes for phenanthrene degradation that are similar to those cloned from C. testosteroni GZ39. However, C. testosteroni GZ42 possesses genes for phenanthrene degradation that are not homologous to those cloned from C. testosteroni GZ39. This suggests that there are at least two different sets of genes for the degradation of phenanthrene among the three C. testosteroni strains.

Carbon catabolite repression of phenol degradation in Pseudomonas putida is mediated by the inhibition of the activator protein PhlR.

Abstract: Enzymes involved in (methyl)phenol degradation of Pseudomonas putida H are encoded by the catabolic operon (phlA-L) on plasmid pPGH1. Transcription of this operon by the sigma54 (RpoN)-containing RNA polymerase is positively controlled by the gene product of the divergently transcribed phlR in response to the availability of the respective substrate. Additionally, phenol degradation is subject to carbon catabolite repression induced by organic acids (e.g., succinate, lactate, and acetate) or carbohydrates (e.g., glucose and gluconate). Analysis of lacZ fusion to the catabolic promoter and quantified primer extension experiments indicate that carbon catabolite repression also occurs at the transcriptional level of the catabolic operon. In this study, it is furthermore shown that carbon catabolite repression is a negative control. Titration of the postulated negative controlling factor was exclusively observed when extra copies of functional phlR gene were present in the cell. We therefore conclude that PhlR is the target and that carbon catabolite repression of phenol degradation occurs by interfering with the activating function of PhlR.

Catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments.

Abstract: We studied the degradation of toluene for bacteria isolated from hypoxic (i.e., oxygen-limited) petroleum-contaminated aquifers and compared such strains with other toluene degraders. Three Pseudomonas isolates, P. pickettii PKO1, Pseudomonas sp. strain W31, and P. fluorescens CFS215, grew on toluene when nitrate was present as an alternate electron acceptor in hypoxic environments. We examined kinetic parameters (K(m) and Vmax) for catechol 2,3-dioxygenase (C230), a key shared enzyme of the toluene-degradative pathway for these strains, and compared these parameters with those for the analogous enzymes from archetypal toluene-degrading pseudomonads which did not show enhanced, nitrate-dependent toluene degradation. C230 purified from strains W31, PKO1, and CFS215 had a significantly greater affinity for oxygen as well as a significantly greater rate of substrate turnover than found for the analogous enzymes from the TOL plasmid (pWW0) of Pseudomonas putida PaW1, from Pseudomonas cepacia G4, or from P. putida F1. Analysis of the nucleotide and deduced amino acid sequences of C23O from strain PKO1 suggests that this extradiol dioxygenase belongs to a new cluster within the subfamily of C23Os that preferentially cleave monocyclic substrates. Moreover, deletion analysis of the nucleotide sequence upstream of the translational start of the meta-pathway operon that contains tbuE, the gene that encodes the C230 of strain PKO1, allowed identification of sequences critical for regulated expression of tbuE, including a sequence homologous to the ANR-binding site of Pseudomonas aeruginosa PAO. When present in cis, this site enhanced expression of tbuE under oxygen-limited conditions. Taken together, these results suggest the occurrence of a novel group of microorganisms capable of oxygen-requiring but nitrate-enhanced degradation of benzene, toluene, ethylbenzene, and xylenes in hypoxic environments. Strain PKO1, which exemplifies this novel group of microorganisms, compensates for a low-oxygen environment by the development of an oxygen-requiring enzyme with kinetic parameters favorable to function in hypoxic environments, as well as by elevating synthesis of such an enzyme in response to oxygen limitation.

p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and characterization of DNA carrying the cmt operon.

Abstract: Pseudomonas putida F1 utilizes p-cumate (p-isopropylbenzoate) as a growth substrate by means of an eight-step catabolic pathway. A 35.75-kb DNA segment, within which the cmt operon encoding the catabolism of p-cumate is located, was cloned as four separate overlapping restriction fragments and mapped with restriction endonucleases. By examining enzyme activities in recombinant bacteria carrying these fragments and sub-cloned fragments, genes encoding most of the enzymes of the p-cumate pathway were located. Subsequent sequence analysis of 11,260 bp gave precise locations of the 12 genes of the cmt operon. The first three genes, cmtAaAbAc, and the sixth gene, cmtAd, encode the components of p-cumate 2,3-dioxygenase (ferredoxin reductase, large subunit of the terminal dioxygenase, small subunit of the terminal dioxygenase, and ferredoxin, respectively); these genes are separated by cmtC, which encodes 2,3-dihydroxy-p-cumate 3,4-dioxygenase, and cmtB, coding for 2,3-dihydroxy-2,3-dihydro-p-cumate dehydrogenase. The ring cleavage product, 2-hydroxy-3-carboxy-6-oxo-7-methylocta-2,4-dienoate, is acted on by a decarboxylase encoded by the seventh gene, cmtD, which is followed by a large open reading frame, cmtI, of unknown function. The next four genes, cmtEFHG, encode 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase, 2-hydroxypenta-2,4-dienoate hydratase, 4-hydroxy-2-oxovalerate aldolase, and acetaldehyde dehydrogenase, respectively, which transform the decarboxylation product to amphibolic intermediates. The deduced amino acid sequences of all the cmt gene products except CmtD and CmtI have a recognizable but low level of identity with amino acid sequences of enzymes catalyzing analogous reactions in other catabolic pathways. This identity is highest for the last two enzymes of the pathway (4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase [acylating]), which have identities of 66 to 77% with the corresponding enzymes from other aromatic meta-cleavage pathways. Recombinant bacteria carrying certain restriction fragments bordering the cmt operon were found to transform indole to indigo. This reaction, known to be catalyzed by toluene 2,3-dioxygenase, led to the discovery that the tod operon, encoding the catabolism of toluene, is located 2.8 kb downstream from and in the same orientation as the cmt operon in P. putida F1.

Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1.

Abstract: In order to study the toluene and o-xylene catabolic genes of Pseudomonas stutzeri OX1, a genomic library was constructed. A 28-kb EcoRI restriction endonuclease DNA fragment, cloned into the vector plasmid pLAFR1 and designated pFB3401, permitted Pseudomonas putida PaW340 to convert toluene and o-xylene into the corresponding meta-ring fission products. Physical and functional endonuclease restriction maps have been derived from the cloned DNA fragment. Further subcloning into and deletion analysis in the Escherichia coli vector pGEM-3Z allowed the genes for the conversion of toluene or o-xylene into the corresponding catechols to be mapped within a 6-kb region of the pFB3401 insert and their direction of transcription to be determined. Following exposure to toluene, E. coli cells carrying this 6-kb region produce a mixture of o-cresol, m-cresol, and p-cresol, which are further converted to 3-methylcatechol and 4-methylcatechol. Similarly, a mixture of 2,3-dimethylphenol and 3,4-dimethylphenol, further converted into dimethylcatechols, was detected after exposure to o-xylene. The enzyme involved in the first step of toluene and o-xylene degradation exhibited a broad substrate specificity, being able to oxidize also benzene, ethylbenzene, m-xylene, p-xylene, styrene, and naphthalene. Deletions of the 6-kb region which affect the ability to convert toluene or o-xylene into the corresponding methylphenols compromise also their further oxidation to methylcatechols. This suggests that a single enzyme system could be involved in both steps of the early stages of toluene and o-xylene catabolism.

Mannitol, a novel bacterial compatible solute in Pseudomonas putida S12.

Abstract: The aim of this study was to identify the compatible solutes accumulated by Pseudomonas putida S12 subjected to osmotic stress. In response to reduced water activity, P. putida S12 accumulated Nalpha-acetylglutaminylglutamine amide (NAGGN) simultaneously with a novel compatible solute identified as mannitol (using 13C- and 1H-nuclear magnetic resonance, liquid chromatography-mass spectroscopy and high-performance liquid chromatography methods) to maximum concentrations of 74 and 258 micromol g (dry weight) of cells(-1), respectively. The intracellular amounts of each solute varied with both the type and amount of osmolyte applied to induce osmotic stress in the medium. Both solutes were synthesized de novo. Addition of betaine to the medium resulted in accumulation of this compound and depletion of both NAGGN and mannitol. Mannitol and NAGGN were accumulated when sucrose instead of salts was used to reduce the medium water activity. Furthermore, both compatible solutes were accumulated when glucose was substituted by other carbon sources. However, the intracellular quantities of mannitol decreased when fructose, succinate, or lactate were applied as a carbon source. Mannitol was also raised to high intracellular concentrations by other salt-stressed Pseudomonas putida strains. This is the first study demonstrating a principal role for the de novo-synthesized polyol mannitol in osmoadaptation of a heterotrophic eubacterium.

Comparison of factors influencing trichloroethylene degradation by toluene-oxidizing bacteria.

Abstract: The degradation of trichloroethylene (TCE) by toluene-oxidizing bacteria has been extensively studied, and yet the influence of environmental conditions and physiological characteristics of individual strains has received little attention. To consider these effects, the levels of TCE degradation by strains distinguishable on the basis of toluene and nitrate metabolism were compared under aerobic or hypoxic conditions in the presence and absence of nitrate and an exogenous electron donor, lactate. Under aerobic conditions with toluene-induced cells, strains expressing toluene dioxygenases (Pseudomonas putida F1, Pseudomonas sp. strain JS150, Pseudomonas fluorescens CFS215, and Pseudomonas sp. strain W31) degraded TCE at low rates, with less than 12% of the TCE removed in 18 h. In contrast, strains expressing toluene monooxygenases (Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1) degraded 36 to 67% of the TCE over the same period. Under hypoxic conditions (1.7 mg of dissolved oxygen per liter) or when lactate was added as an electron donor, the extent of TCE degradation by toluene-induced cells was generally lower. In the presence of lactate, degradation of TCE by denitrifying strain PKO1 was enhanced by nitrate under conditions in which dissimilatory nitrate reduction was observed. The results of experiments performed with strains F1, G4, PKO1, and KR1 suggested that TCE or an oxidation product induces toluene degradation and that TCE induces its own degradation in the monooxygenase strains. The role of TCE as an inducer of toluene oxygenase activity in PKO1 was confirmed by performing a promoter probe analysis, in which we found that TCE activates transcription from the PKO1 3-monooxygenase operon promoter.

Catabolite repression of the toluene degradation pathway in Pseudomonas putida harboring pWW0 under various conditions of nutrient limitation in chemostat culture.

Abstract: In earlier studies, the pathway of toluene and m- and p-xylene degradation (TOL pathway) in Pseudomonas putida (pWW0) was found to be subject to catabolite repression when the strain was grown at the maximal rate on glucose or succinate in the presence of an inducer. This report describes catabolite repression of the TOL pathway by succinate in chemostat cultures run at a low dilution rate (D = 0.05 h-1) under different conditions of inorganic-nutrient limitation. The activity of benzylalcohol dehydrogenase (BADH) in cell extracts was used as a measure of the expression of the TOL upper pathway. When cells were grown in the presence of 10 to 15 mM succinate under conditions of phosphate or sulfate limitation, the BADH activity in response to the nonmetabolizable inducer o-xylene was less than 2% of that of cells grown under conditions of succinate limitation. Less repression was found under conditions of ammonium or oxygen limitation (2 to 10% and 20 to 35%, respectively, of the BADH levels under succinate limitation). The BADH expression levels determined under the different growth conditions appeared to correlate well with the mRNA transcript levels from the upper pathway promoter (Pu), which indicates that repression was due to a blockage at the transcriptional level. The meta-cleavage pathway was found to be less susceptible to catabolite repression. The results obtained suggest that the occurrence of catabolite repression is related to a high-energy status of the cells rather than to a high growth rate or directly to the presence of growth-saturating concentrations of a primary carbon and energy source.

Biodegradation of Hydrogen Sulfide by a Laboratory-Scale Immobilized Pseudomonas putida CH11 Biofilter

Abstract: A heterotrophic Pseudomonas putida CH11 was isolated from livestock farming wastewater and applied for the treatment of H2S-containing gas. Extensive tests including removal characteristics, metabolic products, and removal efficiencies of H2S by P. putida CH11 were examined in batch and continuous systems. The optimum pH required to remove hydrogen sulfide was found in the range of 6-8. The maximum removal rate and the saturation constant were calculated to be Vm = 1.36 g S/day.kg dry bead and Ks = 45.9 ppm, respectively. The main metabolic product of H2S oxidation was determined to be elemental sulfur. When P. putida CH11 was immobilized within Ca alginate, the cells exhibited high H2S removal efficiency, in excess of 96%, at concentration of hydrogen sulfide from 10 to 150 ppm (flow rates of 36 and 72 L/h). These results suggest that P. putida CH11 immobilized within Ca alginate has the potential to be used as a H2S removal agent.

Involvement of σ54 in exponential silencing of the Pseudomonas putida TOL plasmid Pu promoter

Abstract: Summary The σ54-dependent Pu promoter of the TOL plasmid pWWO of Pseudomonas putida becomes activated by the prokaryotic enhancer-binding XyIR protein when cells encounter m-xylene in the medium. However, even in the presence of the aromatic inducer, Pu activity is silenced in vivo during rapid exponential growth of the cells in rich medium. Various elements known to be involved in the control of the transcriptional activity of the promoter were examined to ascertain the mechanism by which expression of Pu is limited during the exponential phase of growth. A truncated and fully constitutive XyIR derivative deleted of its signal reception N-terminal domain was found to be subjected to the same exponential silencing as the wild-type XyIR when exposed to m-xylene. This indicated that the phenomenon is not due to a late activation of XyIR by the aromatic effector. A Pu variant in which the integration host factor (IHF)-binding site had been functionally replaced by a statically curved DNA segment showed the same induction pattern, thus ruling out variations in the intracellular levels of IHF changes during growth as the element responsible for the inactivity of Pu in rapidly growing cells. On the contrary, overproduction of the σ54 factor allowed Pu expression during exponential phase. As σ54 protein levels remained approximately constant during growth, the exponential silencing of Pu could be caused ultimately by changes in the activity of the factor itself. This effect may not be exclusive to Pu, but could be a general co-regulation mechanism in σ54-dependent promoters that connects transcription of a specific set of genes with the general physiological status of the cells.

Cometabolic degradation of 4-chlorophenol by Alcaligenes eutrophus.

Abstract: Alcaligenes eutrophus was grown in batch cultures using either phenol as a sole substrate or mixtures of phenol and 4-chlorophenol. Phenol was found to be the sole source for carbon and energy while 4-chlorophenol was utilized only as a cometabolite. Maximum growth rates on phenol reached only 0.26 h-1, significantly below the growth rates reported earlier with Pseudomonas putida. The cometabolite was found to decrease biomass yield and increase lag time before logarithmic growth occurred. Both phenol and 4-chlorophenol were found to inhibit the growth rate linearly with maximum concentrations of 1080 ppm and 69 ppm respectively, beyond which no growth occurred. The best-fit parameters are incorporated into a simple, dynamic (i.e. time-varying) model capable of predicting all the batch growth conditions presented here. It is shown that P. putida is capable of faster bioremediation when phenol is the sole carbon source or for mixed substrates with low concentrations of the cometabolite, but for high concentrations of 4-chlorophenol, A. eutrophus becomes superior because of the long lag times that occur in the Pseudomonas species.

Use of an ipb-lux Fusion To Study Regulation of the Isopropylbenzene Catabolism Operon of Pseudomonas putida RE204 and To Detect Hydrophobic Pollutants in the Environment.

Abstract: A DNA segment involved in the regulation of the isopropylbenzene (cumene) catabolism operon (ipb) of plasmid pRE4 from Pseudomonas putida RE204 and the Vibrio fischeri luciferase genes, luxCDABE, were used to create an ipbRo/pA(prm1)-luxCDABE reporter fusion plasmid, pOS25. Escherichia coli HMS174(pOS25) produces light in the presence of inducers of the ipb operon. These inducers were shown to be hydrophobic compounds and to include monoalkylbenzenes, substituted benzenes and toluenes, some alkanes and cycloalkanes, chlorinated solvents, and naphthalenes. Complex hydrocarbon mixtures, such as gasoline, diesel fuel, jet fuels (JP-4 and JP-5), and creosote, were also inducers of ipb-lux. Bacteria carrying the ipb-lux reporter may be useful as bioindicators of hydrocarbon pollution in the environment and may be particularly valuable for examining the bioavailability of inducing pollutants.

Chloroform mineralization by toluene-oxidizing bacteria.

Abstract: Seven toluene-oxidizing bacterial strains (Pseudomonas mendocina KR1, Burkholderia cepacia G4, Pseudomonas putida F1, Pseudomonas pickettii PKO1, and Pseudomonas sp. strains ENVPC5, ENVBF1, and ENV113) were tested for their ability to degrade chloroform (CF). The greatest rate of CF oxidation was achieved with strain ENVBF1 (1.9 nmol/min/mg of cell protein). CF also was oxidized by P. mendocina KR1 (0.48 nmol/min/mg of cell protein), strain ENVPC5 (0.49 nmol/min/mg of cell protein), and Escherichia coli DH510B(pRS202), which contained cloned toluene 4-monooxygenase genes from P. mendocina KR1 (0.16 nmol/min/mg of cell protein). Degradation of [14C]CF and ion analysis of culture extracts revealed that CF was mineralized to CO2 (approximately 30 to 57% of the total products), soluble metabolites (approximately 15%), a total carbon fraction irreversibly bound to particulate cellular constituents (approximately 30%), and chloride ions (approximately 75% of the expected yield). CF oxidation by each strain was inhibited in the presence of trichloroethylene, and acetylene significantly inhibited trichloroethylene oxidation by P. mendocina KR1. Differences in the abilities of the CF-oxidizing strains to degrade other halogenated compounds were also identified. CF was not degraded by B. cepacia G4, P. putida F1, P. pickettii PKO1, Pseudomonas sp. strain ENV113, or P. mendocina KRMT, which contains a tmo mutation.

cis/trans isomerization of unsaturated fatty acids as possible control mechanism of membrane fluidity in Pseudomonas putida P8.

Abstract: Exponentially growing cells ofPseudomonas putida had an increased ratio of saturated to unsaturated fatty acids in response to increased growth temperatures. Resting cells in which fatty acid biosynthesis was stopped reacted to a thermal increase by convertingcis-monounsaturated fatty acids totrans isomers.cis/trans isomerization of up to 60% of the unsaturated fatty acids was also activated by alcohols of different chain length. Their effective concentrations apparently depended on the lipophilic character of the alcohols. Also, a salt shock caused by the addition of NaCl resulted in the production oftrans fatty acids. However, cells that were adapted to growth media of high osmolarity synthesized cyclopropane fatty acids instead oftrans fatty acids. Activity ofcis/trans-isomerase was dependent on the growth phase and was significantly higher during logarithmic growth than during the stationary phase. The results of this study agree with the hypothesis that the isomerization ofcis intotrans unsaturated fatty acids is an emergency action of cells ofP. putida to adapt membrane fluidity to drastic changes of environmental conditions.

Cloning and characterization of styrene catabolism genes from Pseudomonas fluorescens ST

Abstract: A gene bank from Pseudomonas fluorescens ST was constructed in the broad-host-range cosmid pLAFR3 and mobilized into Pseudomonas putida PaW340. Identification of recombinant cosmids containing the styrene catabolism genes was performed by screening transconjugants for growth on styrene and epoxystyrene. Transposon mutagenesis and subcloning of one of the selected genome fragments have led to the identification of three enzymatic activities: a monooxygenase activity encoded by a 3-kb PstI-EcoRI fragment and an epoxystyrene isomerase activity and an epoxystyrene reductase activity encoded by a 2.3-kb BamHI fragment. Escherichia coli clones containing the 3-kb PstI-EcoRI fragment were able to transform styrene into epoxystyrene, and those containing the 2.3-kb BamHI fragment converted epoxystyrene into phenylacetaldehyde or, only in the presence of glucose, into 2-phenylethanol. The three genes appear to be clustered and are probably encoded by the same DNA strand. In E. coli, expression of the epoxystyrene reductase gene was under the control of its own promoter, whereas the expression of the other two genes was dependent on the presence of an external vector promoter.

The Pseudomonas putida peptidoglycan-associated outer membrane lipoprotein is involved in maintenance of the integrity of the cell cell envelope.

Abstract: Pseudomonas putida 14G-3, a derivative of the natural soil inhabitant P. putida KT2440, exhibited a chromosomal insertion of a mini-Tn5/9phoA transposon that resulted in reduced ability to colonize soil. In vitro characterization of P. putida 14G-3 revealed that it exhibited an altered cell morphology and envelope, as revealed by electron microscopy. The derived strain was sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA, produced clumps when it reached high cell densities in the late logarithmic growth phase, and did not grow on low-osmolarity medium. The P. putida DNA surrounding the mini-Tn5/9phoA insertion was cloned and used as a probe to rescue the wild-type gene, which was sequenced. Comparison of the deduced peptide sequence with sequences in the Swiss-Prot database allowed the knocked-out gene to be identified as that encoding the peptidoglycan-associated lipoprotein (Pal or OprL) of P. putida. The protein was identified in coupled transcription and translation assays in vitro.

Gene cloning and characterization of Pseudomonas putida L-methionine-alpha-deamino-gamma-mercaptomethane-lyase.

Abstract: Methionine dependency has been reported in cancer cell lines and primary tumors. Thus, L-methionine deprivation might have potential value for the treatment of human cancers with a methionine requirement. L-Methionine-alpha-deamino-gamma-mercaptomethane-lyase has been reported to decrease plasma methionine levels and to inhibit tumor growth in experimental animals but has not been studied extensively because sufficient homogeneous enzyme was not available. In this study, we cloned the L-methioninase gene from Pseudomonas putida and isolated pure and abundant recombinant enzyme. Both L-methionine and L-cysteine in culture medium were completely degraded by 1 unit/ml purified enzyme. Two hundred and fifty units/kg L-methioninase administered i.v. to mice yielded 0.7 unit/ml of plasma concentration and lowered total plasma sulfur-containing amino acids by more than 75%. Although sensitivity to enzymatic methionine depletion differed among cell lines, leukemia cell lines were generally more sensitive than solid tumor cell lines. The availability of pure recombinant L-methioninase will allow in vivo studies on the antitumor activity and the potential toxicity of enzymatic methionine depletion.

Production of poly(3-hydroxyalkanoates) by Pseudomonas putida KT2442 in continuous cultures

Abstract: The synthesis of poly(3-hydroxyalkanoates) (PHA) by Pseudomonas putida KT2442 growing on long-chain fatty acids was studied in continuous cultures. The effects of the growth rate on the biomass and polymer concentration were determined and it was found that the PHA concentrations decreased with increasing growth rates. The highest volumetric productivity was 0.13 g PHA l-1 h-1 at a specific growth rate (μ) of 0.1 h-1. The molecular mass of the polymer remained constant at all growth rates but changes in the monomeric composition of the PHA synthesized were observed. Variation of the carbon to nitrogen (C/N) ratio of the substrate feed at μ=0.1 h-1 revealed optimal PHA formation at C/N=20 mol/mol. In order to optimize PHA production P. putida KT2442 was cultivated to high cell densities in oxygen-limited continuous cultures. In this way a maximum biomass concentration of 30 g/l containing approximately 23% PHA was achieved. This corresponds to a volumetric productivity of 0.69 g l-1 h-1.

The Pseudomonas aeruginosa tonB gene encodes a novel TonB protein.

Abstract: The Pseudomonas aeruginosa tonB gene was cloned by complementation of the tonB mutation of Pseudomonas putida strain TE516 (W. Bitter, J. Tommassen & P. J. Weisbeek, 1993, Mol Microbiol 7, 117-130). The gene was 1025 bp in length, capable of encoding a protein of 36860 Da. As with previously described TonB proteins, the P. aeruginosa TonB (TonBp.a.) was rich in Pro residues (18.1 %) and contained Glu-Pro/Lys-Pro repeats. Unlike previously described TonB proteins, however, TonBp.a. lacked an N-terminal membrane anchor (signal) sequence and contained, instead, a predicted internal signal/anchor sequence, expected to yield an atypical N-terminal cytoplasmic domain in this protein. TonB proteins are essential components in iron-siderophore uptake in bacteria, apparently functioning as energy transducers in coupling the energized state of the cytoplasmic membrane to outer-membrane receptor function. As expected, tonB derivatives of P. aeruginosa were defective in siderophore-mediated iron acquisition. tonB gene expression was inducible by iron-limitation, consistent with the identification of a Fur consensus binding sequence upstream of the gene. TonBp.a. showed substantially greater similarity to the Escherichia coli TonB protein than the Pseudomonas putida protein (31 % identity vs. 20 % identity) and tonBP.a. was able to complement deficiencies in the acquisition of ferric enterobactin and vitamin B12# and sensitivity to phage o80 of an E. coli tonB strain. The larger size of TonBP.a. and its ability to function in both E. coli and P. putida make it a unique TonB protein whose characterization should enhance our understanding of TonB function in bacteria.

Expression of the TOL plasmid xylS gene in Pseudomonas putida occurs from a alpha 70-dependent promoter or from alpha 70- and alpha 54-dependent tandem promoters according to the compound used for growth.

Abstract: Growth of Pseudomonas putida (pWWO) on alkylbenzoates requires the expression of the meta pathway operon, which is mediated by the XylS protein after binding of a benzoate effector. Alternatively, in cells growing on toluene or its aromatic alcohols, overexpression of xylS mediated by XylR activated by these compounds leads to overproduction of the XylS regulator, which even in the absence of benzoate effectors stimulates transcription from the meta cleavage pathway operon promoter. We show here that in bacteria growing on glycerol or alkylbenzoates, the xylS gene is expressed at a low but constitutive level from a newly found sigma 70-dependent promoter called Ps2. The amount of XylS protein made from the transcript originated from Ps2 was sufficient to allow high levels of expression from the meta cleavage pathway operon promoter when the cells were grown in the presence of 3-methylbenzoate. The transcription initiation point of the transcript generated from Ps2 mapped 9 bp upstream from the proposed ATG of the xylS gene; this transcript contains the ribosome-binding site. The Ps2 promoter was located 110 bp downstream from a previously described sigma54-dependent promoter located upstream from the xylS open reading frame, now called Ps1. In cells growing on toluene or benzyl alcohols, the XylS regulator is overproduced as a consequence of increased expression of the gene through the effect of the two promoters working in tandem: the newly found sigma 70-dependent promoter, whose expression is XylR and toluene independent, and the sigma 54-dependent promoter, whose expression is dependent on XylR activated by its effectors. This expression pathway of the xylS gene explains why sigma 54-deficient P. putida bearing the wild-type TOL plasmid, or the wild-type P. putida strain bearing a TOL plasmid with a knocked-out xylR gene, can grow on alkylbenzoates. Until now this has been one of the unresolved paradoxes in the transcriptional control of the TOL meta cleavage pathway.

3-Hydroxyisobutyrate Dehydrogenase from Pseudomonas putida E23: Purification and Characterization

Abstract: The NAD+-dependent 3-hydroxyisobutyrate dehydrogenase [EC 1.1.1.31] was purified to homogeneity from Pseudomonas putida E23. The enzyme was a tetramer (molecular mass, 120kDa) consisted of identical subunits (molecular mass, 30 kDa). The enzyme was specific for NAD+ (Km, 0.44 mm). The maximal activity was obtained at about pH 10. The enzyme was specific for the l-isomer of 3-hydroxyisobutyrate. In addition to l-3-hydroxyisobutyrate, l-serine, 2-methyl-dl-serine, and 3-hydroxypropionate were substrates. The Km for l-3-hydroxyisobutyrate, l-serine, 2-methyl-dl-serine, and 3-hydroxypropionate were 0.12, 18, 44, and 83 mm, respectively. The enzyme was inhibited by p-chloromercuribenzoate, HgCl2, and AgNO3, but not by EDTA, α,α′-dipyridyl, and o-phenanthroline. The N-terminal 26 amino acid sequence was compared with the sequences deduced from the enzyme genes of rat liver and Pseudomonas aeruginosa.