Degradation of 2-bromo-, 2-chloro- and 2-fluorobenzoate by Pseudomonas putida CLB 250

Pseudomonas putida strain CLB 250 (DSM 5232) utilized 2-bromo-, 2-chloro- and 2-fluorobenzoate as sole source of carbon and energy. Degradation is suggested to be initiated by a dioxygenase liberating halide in the first catabolic step. After decarboxylation and rearomatization catechol is produced as a central metabolite which is degraded via the ortho-pathway. After inhibition of ring cleavage activities with 3-chlorocatechol, 2-chlorobenzoate was transformed to catechol in nearly stoichiometric amounts. Other ortho-substituted benzoates like anthranilate and 2-methoxybenzoate seem to be metabolized via the same route.     

Degradation of high concentrations of cresols by Pseudomonas sp. CP4

Pseudomonas sp. CP₄, a potent phenol-degrading laboratory isolate could mineralize all three isomers of cresol. This strain readily utilized up to 1.4, 1.1 and 2.2 g/l of o- m- and p-cresol, respectively as the sole sources of carbon and energy. These are the highest concentrations of cresols reported to be degraded by a bacterial strain. The rates of degradation of the three isomers were in the order: o- > p- > m-cresol. All the isomers of cresol were catabolized through a meta-cleavage pathway. Fairly high catechol 2,3-dioxygenase (C230) activity against catechol was observed in the cell-free extracts of the culture grown on these compounds and were in the order: m- > o- > p-cresol.     

Biodegradation of chlorophenol mixtures by Pseudomonas putida

The dynamic growth behavior of Pseudomonas putida has been studied when resting calls were inoculated into a growth medium containing inhibitory concentrations of mixtures of phenol and monochlorophenols. Resting cells inoculated into single carbon substrate media did not demonstrate enhanced cell lysis by any of the phenol substrates. The apprarent death rate was reduced as the concentrations of phenol or chlorophenols were increased. This behavior was modeled by employing a constant specific death rate (k(d) = 0.0075 h(-1)) and assuming all organic species result in a lag-phase, specific growth rate which may be larger or smaller than k(d).Logarithmic biomass growth on pure monochlorophenols did not occur within 2 weeks after inoculation. Logarithmic growth phases were only observed when the monochlorophenols were cometabolized with phenol. The delay time over which the lag phase exists increased exponentially with phenol concentration and linearly with monochlorophenol concentration. The log growth yield coefficient decreased linearly with monochlorophenol concentration.The lag-phase, specific growth rate was found to decrease exponentially with the concentration of monochlorophenols. This resulted in a 50% lag growth rate inhibition for both 3- and 4-chlorophenol of 9 ppm and for 2-chlorophenol of only 2 ppm. The new, empirical correlations are shown to closely model the complete lag and log growth behavior ot P. putida on phenol and chlorophenol mixtures. (c) 1992 John Wiley & Sons, Inc.     

Factors affecting 2-hydroxypropiophenone formation by benzoylformate decarboxylase from Pseudomonas putida

Benzoylformate (100 mM) was quantitatively converted to the acyloin compound, 2-hydroxypropiophenone (61.76 mM) and benzaldehyde (38.2 mM) by an enzyme extract from Pseudomonas putida ATCC 12633 in the presence of 1.6M acetaldehyde. Biotransformations were carried out at pH 6.0 and 30 degrees C with an incubation time of 60 min. Activity of the acyloin forming enzyme, benzoylformate decarboxylase, was 1.23 units/mL in the biotransformation mixture. Acyloin formation increased dramatically with pH in the range 4-5 and had a broad activity plateau in the pH range 5-8. A broad temperature optimum for acyloin formation was also observed in the range 20-40 degrees C.     

Dynamics of phenol degradation by Pseudomonas putida

Pure cultures of Pseudomonas putida (ATCC 17484) were grown in continuous culture on phenol at dilution rates of 0.074-0.085 h(-1) and subjected to step increases in phenol feed concentration. Three distinct patterns of dynamic response were obtained depending on the size of the step change used: low level, moderate level, or high level. During low level responses no accumulations of phenol or non-phenol, non-glucose-dissolved organic carbon, DOC(NGP), were observed. Moderate level responses were characterized by the transient accumulation of DOC(NGP) with a significant delay prior to phenol leakage. High level responses demonstrated a rapid onset of phenol leakage and no apparent accumulations of DOC(NGP). The addition of phenol to a continuous culture of the same organism on glucose did not result in transient DOC(NGP) accumulations, although transient phenol levels exceeded 90 mg l(-1). These results were consistent with intermediate metabolite production during phenol step tests coupled with substrate-inhibited phenol uptake and suggested that traditional kinetic models based on the Haldane equation may be inadequate for describing the dynamics of phenol degrading systems. (c) 1993 John Wiley & Sons, Inc.     

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

The intermediate and terminal products of cyanide and thiocyanate decomposition by individual strains of the genus Pseudomonas, P. putida strain 21 and P. stutzeri strain 18, and by their association were analyzed. The activity of the enzymes of nitrogen and sulfur metabolism in these strains was compared with that of the collection strains P. putida VKM B-2187^T and P. stutzeri VKM B-975^T. Upon the introduction of CN⁻ and SCN⁻ into cell suspensions of strains 18 and 21 in phosphate buffer (pH 8.8), the production of NH ₄ ⁺ was observed. Due to the high rate of their utilization, NH₃, NH ₄ ⁺ , and CNO⁻ were absent from the culture liquids of P. putida strain 21 and P. stutzeri strain 18 grown with CN⁻ or SCN⁻. Both Pseudomonas strains decomposed SCN⁻ via cyanate production. The cyanase activity was 0.75 μmol/(min mg protein) for P. putida strain 21 and 1.26 μmol/(min mg protein) for P. stutzeri strain 18. The cyanase activity was present in the cells grown with SCN⁻ but absent in cells grown with NH ₄ ⁺ . Strain 21 of P. putida was a more active CN⁻ decomposer than strain 18 of P. stutzeri. Ammonium and CO₂ were the terminal nitrogen and carbon products of CN⁻ and SCN⁻ decomposition. The terminal sulfur products of SCN⁻ decomposition by P. stutzeri strain 18 and P. putida strain 21 were thiosulfate and tetrathionate, respectively. The strains utilized the toxic compounds in the anabolism only, as sources of nitrogen (CN⁻ and SCN⁻) and sulfur (SCN⁻). The pathway of thiocyanate decomposition by the association of bacteria of the genus Pseudomonas is proposed based on the results obtained. Original Russian Text ? N.V. Grigor’eva, T.F. Kondrat’eva, E.N. Krasil’nikova, G.I. Karavaiko, 2006, published in Mikrobiologiya, 2006, Vol. 75, No. 3, pp. 320–328.     

The Degradation of Dimethoate and the Mineral Immobilizing Function for Cd2+ by Pseudomonas putida

Organophosphorus pollution and heavy metal pollution are prominent in China and have caused increasingly severe environmental pollution. This research used Pseudomonas putida to degrade dimethoate so as to induce the formation of calcium carbonate (CaCO₃) and calcium phosphate (Ca₃(PO₄)₂) in beef extract peptone medium. In addition, the mineral immobilizing function of the generated Ca₃(PO₄)₂ and CaCO₃ for Cd²⁺ was studied by adding different concentrations of Cd²⁺ to the culture solution. Meanwhile, transmission electron microscopy (TEM), scanning electronic microscopy (SEM), X-ray diffraction, gas chromatography and atomic absorption spectrophotometry were used to investigate the biodegradation of dimethoate, the concentration variation of Ca²⁺ and Cd²⁺, the mineral and chemical compositions of the precipitates. The results showed that the growth of P. Putida could increase the pH value of the culture solution and effectively degrade the organophosphorus pesticide dimethoate. Besides, the concentration of Ca²⁺ in the culture solution decreased significantly in the first four days and then tended to be stable. Moreover, the TEM and SEM results presented that there were large amounts of biogenic sedimentary CaCO₃ and a little Ca₃(PO₄)₂ in the precipitates. Furthermore, in the employed culture system, the removal rates of Cd²⁺, when added at two different concentrations (6 ppm and 15 ppm), reached 100%. Therefore, this study provided a new idea for treating wastewater polluted with organophosphorus pesticide and heavy metals by using microorganisms.     

Degradation of trichloroethylene by a growth-arrestedPseudomonas putida

A toluene-oxidizing strain ofPseudomonas mendocina KR1 containing toluene-4-mono-oxygenase (TMO) completely degrades TCE with the addition of toluene as a co-substrate in aerobic condition. In order to constructin situ bioremediation system for TCE degradation without any growth-stimulating nutrients or toxic inducers such as toluene, we used the carbon-starvation promoter ofPseudomonas putida MK1 (Kim, Y.et al., J. bacteriol., 1995). Upon entry into the stationary phase due to the deprivation of nutrients, this promoter is strongly induced without further cell growth. The TMO gene cluster (4.5 kb) was spliced downstream of the carbon starvation promoter ofPseudomona putida MK1, already cloned in pUC19. TMO under the carbon starvation promoter was not expressed inE. coli cells either in stationary phase or exponential phase. For TMO expression inPseudomonas strains,tmo and carbon starvation promoter region were recloned into a modified broad-host range vector pMMB67HES which was made from pMMB67HE (8.9 kb) by deletion oftac promoter andlacI ^q (about 1.5 kb). Indigo was produced by TMO under the carbon starvation promoter in aPseudomonas strain of post-exponential phase on M9 (0.2% glucose and 1mM indole) or LB. 18% of TCE was degraded in 14 hours after entering the stationary phase at the initial concentration of 6.6μ M in liquid phase.     

Stabilization of creatinase from Pseudomonas putida by random mutagenesis.

Creatinase (creatine amidinohydrolase, EC 3.5.3.3) from Pseudomonas putida is a homodimer of 45 kDa subunit molecular mass, the three-dimensional structure of which is known at 1.9 A resolution. Three point mutants, A109V, V355M, and V182I, as well as one double mutant combining A109V and V355M, and the triple mutant with all three replacements, were compared with wild-type creatinase regarding their physical and enzymological properties. High-resolution crystal data for wild-type creatinase and the first two mutants suggest isomorphism at least for these three proteins (R. Huber, pers. comm.). Physicochemical measurements confirm this prediction, showing that the mutations have no effect either on the quaternary structure and gross conformation or the catalytic properties as compared to wild-type creatinase. The replacement of V182 (at the solvent-exposed end of the first helix of the C-terminal domain) does not cause significant differences in comparison with the wild-type enzyme. The other point mutations stabilize the first step in the biphasic denaturation transition without affecting the second one. In sum, the enhanced stability seems to reflect slight improvements in the local packing without creating new well-defined bonds. The increase in hydrophobicity generated by the introduction of additional methyl groups (A109V, V182I) must be compensated by minor readjustments of the global structure. Secondary or quaternary interactions are not affected. In going from single to double and triple mutants, to a first approximation, the increments of stabilization are additive.     

Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes

Eighteen 4-t-octylphenol-degrading bacteria were isolated and screened for the presence of degradative genes by polymerase chain reaction method using four designed primer sets. The primer sets were designed to amplify specific fragments from multicomponent phenol hydroxylase, single component monooxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase genes. Seventeen of the 18 isolates exhibited the presence of a 232 bp amplicon that shared 61-92% identity to known multicomponent phenol hydroxylase gene sequences from short and/or medium-chain alkylphenol-degrading strains. Twelve of the 18 isolates were positive for a 324 bp region that exhibited 78-95% identity to the closest published catechol 1,2-dioxygenase gene sequences. The two strains, Pseudomonas putida TX2 and Pseudomonas sp. TX1, contained catechol 1,2-dioxygenase genes also have catechol 2,3-dioxygenase genes. Our result revealed that most of the isolated bacteria are able to degrade long-chain alkylphenols via multicomponent phenol hydroxylase and the ortho-cleavage pathway.     

Characterization of 2-nitrophenol uptake system of Pseudomonas putida B2

Uptake of substituted nitrophenols from the bulk solution into the cytoplasm limited reaction rates by Pseudomonas putida B2. Initial enzymatic conversion of 2-nitrophenol (ONP) to catechol is by an intracellular soluble enzyme, nitrophenol oxygenase [Zeyer J and Kearney PC. 1984. J Agric Food Chem 32: 238–242]. Addition of N-ethylmaleimide (NEM) to cell suspensions led to a decrease in specific reaction rates for ONP, dependent on the ratio of NEM to cellular protein. Maximal NEM inhibition resulted in an 80–90% decrease in the ONP reaction rate which could not be reversed following dilution. Cell-free enzyme extract isolated from NEM-inactivated cells demonstrated less than 20% loss of the specific ONP reaction rates. NEM apparently acted by inhibiting a protein which facilitated uptake of nitrophenol into the cytoplasm, prior to the first catabolic enzyme. Both intact organisms and protoplasts exhibited the same 80–90% decrease in reaction rate which established that NEM inhibition was localized in the plasma membrane. NEM elicited variable effects on reaction rates for a series of ring substituted 2-nitrophenols. The data indicated that uptake of substituted 2-nitrophenols involved at least two transport systems, one sensitive to NEM inactivation and a second insensitive uptake process. Received 05 November 1996/ Accepted in revised form 29 May 1997     

蚯蚓对细菌降解土壤中菲的作用

通过30d室内培养试验,分别研究了接种蚯蚓(E)、细菌(B)以及同时接种细菌和蚯蚓(BE)对土壤中菲降解的影响.结果表明: 在土壤中菲的初始污染浓度为50 mg*kg-1的条件下,各处理间菲的降解率差异显著,其降解率的大小顺序依次为:BE》B》E》CK(对照); 在150 mg*kg-1菲的初始污染浓度下,BE处理中菲的降解率高达98.86%,显著高于CK和E处理.B处理中细菌的双加氧酶活性在3种菲初始污染浓度下没有显著差异,而BE处理中双加氧酶的活性随着土壤中菲的初始污染浓度的升高而增加.在相同菲污染浓度下BE处理中蚯蚓体内的菲含量明显高于E处理.表明蚯蚓能够通过生物富集作用降低土壤中菲的浓度,而蚯蚓与细菌的相互作用能够进一步促进土壤中菲的降解.     

恶臭假单胞菌丙氨酸消旋酶基因的克隆、序列分析及表达

从恶臭假单胞菌(Pseudomonas putida)200的基因组出发,用PCR方法克隆到两个独立作用的丙氨酸消旋酶基因,称之为dadX和alr。DadX编码357个氨基酸长的多肽,计算分子量为38.82kDa,alr编码409个氨基酸长的多肽,计算分子量为44.182kDa。序列分析显示,DadX的氨基酸序列与Pseudomonas putidaKT2440,铜绿假单胞菌(Pseudomonas aeruginosa),鼠伤寒沙门氏菌(Salmonella typhimurium)和大肠杆菌(Escherichia coli)的DadX比较,相似性分别为96.64%、71.99%、44.88%和47.37%。Alr的氨基酸序列与Pseudomonas putidaKT2440比较,同源性为94.38%,而与铜绿假单胞菌(P.aeruginosa)、鼠伤寒沙门氏菌(S.typhimurium)和大肠杆菌(E.coli)的Alr比较,同源性均较低,分别为22.89%、25.72%和26.44%。在P.putida200的DadX和Alr氨基酸序列中部发现有对于酶活性至关重要的保守区域,如磷酸吡哆醛(PLP)结合位点。DadX和alr在大肠杆菌中得到表达,DadX丙氨酸消旋酶只对丙氨酸有消旋作用,而Alr丙氨酸消旋酶可以作用于丙氨酸和丝氨酸两种底物,且对丝氨酸特异性更高。Alr的表达不依赖于外源启动子,说明在其结构基因上游存在启动子结构。     

Degradation of tetradecyltrimethylammonium by Pseudomonas putida A ATCC 12633 restricted by accumulation of trimethylamine is alleviated by addition of Al 3+ ions

Aims: To establish if tetradecyltrimethylammonium (TDTMA) might be degraded by pure culture of Pseudomonas strains, and how the presence of a Lewis’ acid in the medium influences its biodegradability. Methods and Results: From different strains of Pseudomonas screened, only Pseudomonas putida A ATCC 12633 grows with 50 mg l^?1 of TDTMA as the sole carbon and nitrogen source. A monooxygenase activity catalyzed the initial step of the biodegradation. The trimethylamine (TMA) produced was used as nitrogen source or accumulated inside the cell. To decrease the intracellular TMA, the culture was divided, and 0·1 mmol l^?1 AlCl₃ added. In this way, the growth and TDTMA consumption increased. The internal concentration of TMA, determined using the fluorochrome Morin, decreased by the formation of Al³⁺ : TMA complex. Conclusions: Pseudomonas putida utilized TDTMA as its sole carbon and nitrogen source. The TMA produced in the initial step of the biodegradation by a monooxygenase activity was used as nitrogen source or accumulated inside the cell, affecting the bacterial growth. This effect was alleviated by the addition of AlCl₃. Significance and Impact of the Study: The use of Lewis’ acids to sequester intracellular amines offers an alternative to achieve an efficient utilization of TDTMA by Ps. putida.     

Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267

Aims: Plant growth‐promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially identified, and their role in control of Phytophthora damping‐off of cucumber evaluated. Methods and Results: The biosurfactants were shown to lyse zoospores of Phy. capsici and inhibit growth of the fungal pathogens Botrytis cinerea and Rhizoctonia solani. In vitro assays further showed that the biosurfactants of strain 267 are essential in swarming motility and biofilm formation. In spite of the zoosporicidal activity, the biosurfactants did not play a significant role in control of Phytophthora damping‐off of cucumber, since both wild type strain 267 and its biosurfactant‐deficient mutant were equally effective, and addition of the biosurfactants did not provide control. Genetic characterization revealed that surfactant biosynthesis in strain 267 is governed by homologues of PsoA and PsoB, two nonribosomal peptide synthetases involved in production of the cyclic lipopeptides (CLPs) putisolvin I and II. The structural relatedness of the biosurfactants of strain 267 to putisolvins I and II was supported by LC‐MS and MS‐MS analyses. Conclusions: The biosurfactants produced by Ps. putida 267 were identified as putisolvin‐like CLPs; they are essential in swarming motility and biofilm formation, and have zoosporicidal and antifungal activities. Strain 267 provides excellent biocontrol activity against Phytophthora damping‐off of cucumber, but the lipopeptide surfactants are not involved in disease suppression. Significance and Impact of the Study: Pseudomonas putida 267 suppresses Phy. capsici damping‐off of cucumber and provides a potential supplementary strategy to control this economically important oomycete pathogen. The putisolvin‐like biosurfactants exhibit zoosporicidal and antifungal activities, yet they do not contribute to biocontrol of Phy. capsici and colonization of cucumber roots by Ps. putida 267. These results suggest that Ps. putida 267 employs other, yet uncharacterized, mechanisms to suppress Phy. capsici.     

Broad spectrum and narrow spectrum mercury resistance encoded by different plasmids of a Pseudomonas putida strain

Abstract Pseudomonas putida strain H harbours two plasmids of different sizes. It was demonstrated that the large plasmid pPGH1 confers a broad spectrum resistance to mercurials, whereas the small plasmid pPGH2 confers a narrow spectrum one. Under the influence of the small plasmid the resistance of cells against poisoning with 2,4-di-chlorophenol or o -cresol increases in comparison to cells without this plasmid. Both plasmids proved to be not self-transmissible, but pPGH1 is transferable by mobilisation by means of the IncP-1 vectors R68.45 or RP1.     

Production and characterization of a new bioemulsifier from Pseudomonas putida ML2

AIMS: To characterize the bioemulsifier produced by a nonfluorescent strain of Pseudomonas putida isolated from a polluted sediment and to determine the influence of pH, temperature, media composition, and carbon and nitrogen source on growth and emulsifying activity. METHODS AND RESULTS: Different indexes were employed to determine the emulsifying properties of culture supernatants of P. putida ML2 in defined and complex media. Surface tension of cell-free supernatants was measured. Purification and chemical analysis of the emulsifier was performed. Confirmed results indicate that a polysaccharide with hexasaccharide repeating units is responsible for the emulsifying activity in a mineral medium with glucose as sole carbon source. Moreover, an emulsifier is produced when growing on naphthalene. CONCLUSIONS: Culture media composition influences the amount and the properties of the emulsifier produced by this P. putida strain. Under nitrogen limiting conditions, a polysaccharide is responsible for the emulsifying activity in defined mineral media. In complex nitrogen rich medium, a different kind of emulsifier is produced. The exopolymer may contribute to hydrocarbons solubilization. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first exopolysaccharide with emulsifying properties produced by a Pseudomonas strain reported to the present. Also chemical composition is significantly different from previous reports. This strain has potential use in bioremediation and the purified polysaccharide may be used in food and cosmetic industry. Moreover, the production of the exopolymer may play a role on biofilm formation.     

Water stress effects on toluene biodegradation by Pseudomonas putida

We quantified the effects of matric and solute waterpotential on toluene biodegradation by Pseudomonasputida mt-2, a bacterial strain originally isolated fromsoil. Across the matric potential range of 0 to – 1.5 MPa,growth rates were maximal for P. putida at – 0.25MPa and further reductions in the matric potentialresulted in concomitant reductions in growth rates.Growth rates were constant over the solute potential range0 to – 1.0 MPa and lower at – 1.5 MPa. First ordertoluene depletion rate coefficients were highest at0.0 MPa as compared to other matric water potentialsdown to – 1.5 MPa. Solute potentials down to – 1.5 MPadid not affect first order toluene depletion ratecoefficients. Total yield (protein) and carbon utilizationefficiency were not affected by water potential, indicatingthat water potentials common to temperate soils were notsufficiently stressful to change cellular energyrequirements. We conclude that for P. putida: (1)slightly negative matric potentials facilitate faster growthrates on toluene but more negative water potentials resultin slower growth, (2) toluene utilization rate per cell massis highest without matric water stress and is unaffected bysolute potential, (3) growth efficiency did not differ acrossthe range of matric water potentials 0.0 to – 1.5 MPa.     

Modification of phospholipid composition in Pseudomonas putida A ATCC 12633 induced by contact with tetradecyltrimethylammonium

AIMS: The aim of this work was to establish if the response to tetradecyltrimethylammonium (TDTMA), a representative quaternary ammonium compound (QAC), involves changes in the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633. METHODS AND RESULTS: Pseudomonas putida was exposed to 50 mg l(-1) of TDTMA for 15 min, and PL composition was analysed. With respect to control values, phosphatidic acid and phosphatidylglycerol increased by 140% and 120%, respectively; cardiolipin decreased about 60%. In TDTMA-adapted bacteria, the most significant change was a 380% increase in phosphatidic acid. Accompanying this change was a 130% increase in phosphatidylglycerol and a 70% decrease in cardiolipin. The changes in adapted cells were reverted after two subcultures without biocide. CONCLUSIONS: Pseudomonas putida responded to TDTMA through quantitative changes in PLs with specific variations in the content of phosphatidic acid, phosphatidylglycerol and cardiolipin. These modifications indicated that these PLs are involved in cellular responses to QACs, utilizing phosphatidic acid principally to neutralize the high positive charge density given for the ammonium quaternary moiety from TDTMA. SIGNIFICANCE AND IMPACT OF THE STUDY: The changes in PL composition give a new insight about the response inflicted by Ps. putida when these bacteria are exposed to QACs.