A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing -amino acids

Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in reactions catalysed by stereo-selective microbial enzymes: initially a hydantoinase catalyses the cleavage of the hydantoin producing an N-carbamyl amino acid. In certain bacteria where an N-carbamyl amino acid amidohydrolase (NCAAH) is present, the N-carbamyl amino acid intermediate is further converted to amino acid, ammonia and CO₂. In this study we report on a novel Pseudomonas putida strain which exhibits high levels of hydantoin-converting activity, yielding -amino acid products including alanine, valine, and norleucine, with bioconversion yields between 60% and 100%. The preferred substrates are generally aliphatic, but not necessarily short chain, 5-alkylhydantoins. In characterizing the enzymes from this microorganism, we have found that the NCAAH has -selectivity, while the hydantoinase is non-stereoselective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate inhibition, or product inhibition by ammonia. The rate-limiting reaction appears to be the NCAAH-catalysed conversion of the intermediate. Metal-dependence studies suggest that the hydantoinase is dependent on the presence of magnesium and cobalt ions, and is strongly inhibited by the presence of copper ions. The relative paucity of -selective hydantoin-hydrolysing enzyme systems, together with the high level of hydantoinase activity and the unusual substrate selectivity of this P. putida isolate, suggest that is has significant potential in industrial applications.     

Conversion of sodium cyanide to carbon dioxide and ammonia by immobilized cells ofPseudomonas putida

Summary Pseudomonas putida, isolated from contaminated industrial wastewaters and soil sites, was found to utilize sodium cyanide (NaCN) as a sole source of carbon and nitrogen. Cells, immobilized in calcium alginate beads (1–2 mm diameter) were aerated in air-uplift-type fluidized batch bioreactor containing 100–400 ppm of NaCN. Degradation of NaCN was monitored for 168 h by analyzing gaseous and dissolved ammonia (NH₃), CO₂, pH and optical density. The results indicated that the alginate-immobilized cells ofP. putida were able to degrade NaCN into NH₃ and CO₂ in a time-dependent manner.     

微生物胞外长距离电子传递网络研究进展

[目的] 解析一株从黄河三角洲湿地甲烷氧化富集物中分离获得的甲烷氧化菌伴生菌的生理学及电化学特性,并探究该菌株对甲烷氧化过程的影响。[方法] 使用高通量测序技术解析甲烷氧化富集物的菌群结构,采用稀释涂布法、平板划线法分离甲烷氧化菌的伴生菌,通过16S rRNA基因测序技术进行菌株初步鉴定。利用扫描电子显微镜观察菌株形态,并通过气相色谱（gas chromatography,GC）检测伴生菌利用甲烷情况及对甲烷氧化菌氧化甲烷效率的影响。采用双室微生物燃料电池（microbial fuel cells,MFCs）及差分脉冲伏安法（differential pulse voltammetry,DPV）检测菌株的电化学活性。[结果] 黄河三角洲湿地土壤甲烷氧化富集物主要的好氧甲烷氧化菌为甲基杆菌属Methylobacter,同时还发现一些伴生菌。分离得到一株甲醇利用菌P7,其16S rRNA基因序列与恶臭假单胞菌Pseudomonasputida的相似性达99.79%。扫描电镜结果显示该菌株为杆状,长约1.5-2.5μm,宽度约为0.5μm。GC检测结果显示,该菌株不能利用甲烷,但与甲烷氧化菌共培养时,可以促进甲烷氧化（P<0.05）。双室MFCs检测结果显示该菌株具有电活性,最大电流输出密度为28 mA/m²,DPV检测结果显示该菌株主要的氧化峰和还原峰分别位于-0.17 V和-0.25 V。[结论] 本研究从黄河三角洲湿地甲烷氧化富集物中获得一株具有电活性的甲烷氧化菌的伴生菌恶臭假单胞菌Pseudomonas putida P7,该菌株可以促进甲烷氧化。本研究加深了对甲烷氧化过程中伴生菌的生理学特性及功能的认识。     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Biotransformation of isoeugenol to vanillin by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IE27 cells

The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 °C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.     

Microbial degradation of cyanide and thiocyanate

The role played by a bacterial community composed ofPseudomonas putida, strain 21;Pseudomonas stutzeri, strain 18; andPseudomonas sp., strain 5, and by physical and chemical factors in the degradation of CN⁻ and SCN⁻ was studied. It was shown that the degradation of CN⁻ is determined both by the action of bacteria and by abiotic physical and chemical factors (pH, O₂, temperature, the medium agitation rate, etc.). The contribution of chemical degradation was found to increase drastically at pH below 9.0; when air was blown through the medium (irrespective of the pH value); under active agitation of the medium; and when the medium surface interfacing air was increased. Even at elevated pH values (9.0-9.2), suboptimal for bacterial growth, the microbial degradation could account for at most 20–25 mg/1 of CN⁻, regardless of its initial concentration. When CN⁻ and SCN⁻ were concurrently present in the medium, the former compound was the first to be degraded by microorganisms. The rate of bacterial degradation of SCN⁻ under continuous cultivation in a chain of reactors was found to depend on its concentration, the medium flow rate, agitation rate, and the pattern of carbon source supply and could exceed 1 g/(l day). CN⁻ and SCN⁻ are utilized by bacteria solely as nitrogen sources. The mechanism of CN⁻ and SCN⁻ degradation by the microbial community is discussed. Deceased.     

Assessment of a chemostat-coupled modified Robbins device to study biofilms

The combination of a modified Robbins device (MRD) attached to the effluent line of a continuous cultivation vessel was assessed by the adhesion of planktonic bacteria maintained at a controlled growth rate. This combination of a chemostat and an MRD provides a large number of sample surfaces for monitoring both the formation and control of biofilms over extended periods of time. This apparatus was used to monitor the colonization of two soil isolates,Pseudomonas fluorescens (EX101) andPseudomonas putida (EX102) onto silastic rubber surfaces. At a similar _rel, both bacteria attached to the silastic, howeverP. fluorescens formed confluent, dense biofilms in less than 24 h, whereasP. putida adhered as single cells or microcolonies after the same period. The metabolic activity, measured by INT-formazan formation, was similar for both organisms with a peak at 6 h of colonization and a subsequent decrease after 24 h. Long term colonization studies ofP. fluorescens produced a population of greater than 9.5 log cfu cm^–2 at 28 days demonstrating the advantages of the chemostat-MRD association. This technique proved to be successful for studying bacterial adhesion and biofilm formation in tubular devices by bacterial populations at controlled and low growth rates.     

Model description of bacterial 3-methylcatechol production in one- and two-phase systems

Pseudomonas putida MC2 produces 3-methylcatechol from toluene in aqueous medium. A second phase of 1-octanol may improve total product accumulation. To optimise the design of such a biphasic process, a process model was developed, both for one- and two-phase applications. The insights obtained by the model predictions showed the importance of different process parameters (like growth substrate concentration and partition coefficient) on growth of biomass, accumulation of 3-methylcatechol and processing time. For future applications, the process model can be used to ensure enough extraction capacity from aqueous to octanol phase. It is a useful tool to define the optimum process conditions, depending on the desired optimisation parameter: product concentration or processing time.     

A pyoverdine from Pseudomonas putida CFML 90-51 with a Lys ε-amino link in the peptide chain

From Pseudomonas putida CFML 90-51 – a hospital isolate – a pyoverdine was obtained which is characterized by the unusual linkage by the -rather than the -amino group of Lys in the peptide chain. The structure elucidation by spectroscopic methods and degradation reactions is reported.     

Indirect utilization of the phytosiderophore mugineic acid as an iron source to rhizosphere fluorescent Pseudomonas

The phytosiderophore mugineic acid (MA) was studied as a source of iron for rhizosphere fluorescent pseudomonads. ⁵⁵Fe supplied as Fe-MA was taken up by Pseudomonas putida WCS358, B10 and St3 grown under iron deficient conditions. The uptake decreased when the bacteria were grown in the presence of iron. However, no differences in uptake were observed when a siderophore deficient mutant was tested. Since ligand exchange between pseudobactin and MA was shown to occur rapidly with a half-life of 2 h, MA mediated iron uptake probably proceeds through this indirect mechanism. The ecological implications of these findings are discussed.     

Development of a biological mercury removal-recovery system

A mercury removal-recovery system was developed for collection of elemental mercury volatilized by biological mercuric ion reduction. Using the mercury removal-recovery system, removal of mercuric chloride from mercury-containing buffer without nutrients by resting cells of mercury-resistant bacterium, Pseudomonas putida PpY101/pSR134 was tested. Optimum temperature, pH, thiol compounds and cell concentration on removal of mercuric chloride were determined, and 92 to 98% of 40 mg Hg l^–1 was recovered in 24 h. The efficiency of mercuric chloride removal from river water and seawater was as high as that observed when using a buffered solution.     

Isolation and characterization of fluorescent Pseudomonas associated with the roots of rice and banana grown in Sri Lanka

Bacterial populations in different parts of the rhizosphere of rice and banana in Sri lanka were examined. On rice, the number of aerobic bacteria and the population of fluorescent bacteria were higher in the rhizoplane as compared to the exorhizosphere. However, the opposite was observed with banana. Percentage of fluorescent bacteria was significantly higher on banana (10.8%) than on rice from the wet and dry zones of Sri Lanka (4.3% and 2.7%, respectively). In the endorhizosphere fraction of rice, bacterial populations were very low. Fluorescent bacteria were absent.Based on 33 phenotypical tests, 89 fluorescent isolates were grouped into 5 clusters. The three major clusters covered the isolates belonging to the Pseudomonas fluorescens-putida group, whereas the remaining small clusters contained other UV-fluorescent bacteria. SDS-PAGE of total cell proteins enabled classification of the isolates into one of 12 different protein-polymorphic types. Only a partial correlation was found between the latter classification and the phenotypical one. Cyanogenesis was observed with strains of P. fluorescens only. Isolates P. fluorescens RW9S1 and P. cepacia RW5P1 displayed a potent antagonism against several fungi.     

Transfer and expression of a hydrocarbon-degrading plasmid pHCL from Pseudomonas putida to marine bacteria

Transfer of a catabolic plasmid from Pseudomonas putida to indigenous marine bacteria and obligate halophilic bacteria was carried out under both in vitro and in situ conditions. The marine recipients, which could not otherwise grow on hydrocarbon substrates, were able to degrade them after the horizontal transfer of the catabolic plasmid from P. putida. Mating conducted on nutrient plates yielded comparatively more transconjugants than in broth mating under laboratory conditions (10⁶ c.f.u./ml). The transconjugants stably maintained the plasmid when they were maintained in seawater amended with selective pressure (antibiotics/Hg (25 g/l) even after 30 days, whereas under non-selective conditions they progressively lost the plasmid after 24 days. The expression of the plasmid in the marine recipients was investigated by gas chromatographic analysis. The overall objective of this study is to evolve a novel strategy for bioremediation of oil spills and the results of the present study suggest that the present approach would offer a better solution for the removal of harmful substances from the environment by avoiding serious interference with the microbial flora of the ecosystem.     

Kinetics of BTEX biodegradation by a coculture of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> and<Emphasis Type="Italic"> Pseudomonas fluorescens</Emphasis> under hypoxic conditions

Pseudomonas putida and Pseudomonas fluorescens present as a coculture were studied for their abilities to degrade benzene, toluene, ethylbenzene, and xylenes (collectively known as BTEX) under various growth conditions. The coculture effectively degraded various concentrations of BTEX as sole carbon sources. However, all BTEX compounds showed substrate inhibition to the bacteria, in terms of specific growth, degradation rate, and cell net yield. Cell growth was completely inhibited at 500mgl^–1 of benzene, 600mgl^–1 of o-xylene, and 1000mgl^–1 of toluene. Without aeration, aerobic biodegradation of BTEX required additional oxygen provided as hydrogen peroxide in the medium. Under hypoxic conditions, however, nitrate could be used as an alternative electron acceptor for BTEX biodegradation when oxygen was limited and denitrification took place in the culture. The carbon mass balance study confirmed that benzene and toluene were completely mineralized to CO₂ and H₂O without producing any identifiable intermediate metabolites.     

Interactions between populations ofPseudomonas putida leading to the expression of a cryptic dehalogenase gene (dehll)

In appropriate environments containing 2-monochloropropionic acid (2MCPA), mutations in a population of nondehalogenatingPseudomonas putida, strain PP40-040 (parent population), resulted in the formation of 2mcpa⁺ papillae as a result of the decryptification of adehII gene. Increasing the size of the parent population, for example by increasing the availability of a metabolizable substrate such as succinate or lactate, increased the number of 2mcpa⁺ papillae formed because there were more parent cells available for mutation to the 2mcpa⁺ phenotype. The presence of a dehalogenating population, such asP. putida strain PP3, in close proximity to the non-dehalogenating population, also increased the number of 2mcpa⁺ papillae formed. This was due to the excretion of dehalogenases into the growth medium, which caused localized dehalogenation of the available 2MCPA, yielding a metabolizable substrate. This substrate stimulated the growth of the non-dehalogenating population, in turn increasing the number of 2mcpa⁺ papillae formed. Barriers, such as dialysis membranes, which prevented the excretion of the dehalogenases into the growth medium, prevented the stimulation of 2mcpa⁺ papillae formation by preventing release of metabolizable substrates from 2MCPA breakdown. Cell-free extracts (CFE) from dehalogenase-producing populations had a similar effect for the same reason. CFE without dehalogenase activity or in which the dehalogenase activity had been destroyed by heating failed to stimulate parent population growth and 2mcpa⁺ papillae formation. In the case ofPseudomonas putida strain PP3, which carries an easily transposed dehalogenase-encoding transposon, treatment of CFE with DNAase eliminated an additional factor involved in the formation of 2mcpa⁺ papillae.The authors are with the School of Pure and Applied Biology, University of Wales-Cardiff, P.O. Box 915, Cardiff CF1 3TL, UK     

Purification and characterization of dihydroorotase fromPseudomonas putida

Dihydroorotase was purified to homogeneity fromPseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-l-orotate and its methyl ester, and the reactions were reversible. The apparentK _m andV _max values for dihydro-l-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 μmol min⁻¹ mg⁻¹, respectively; and those forN-carbamoyl-dl-aspartate (at pH 6.0) were 2.2 mM and 68 μmol min⁻¹ mg⁻¹, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn²⁺. However, excessive Zn²⁺ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited byN-carbamoylamino acids such asN-carbamoylglycine, with aK _i value of 2.7 mM. The enzyme was also inhibited noncompetitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with aK _i value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.     

Inactivation of type I polyhydroxyalkanoate synthase in <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> resulted in discovery of another potential PHA synthase

Aeromonas hydrophila CGMCC 0911 possessing type I polyhydroxyalkanoate (PHA) synthase (PhaC) produced only PHBHHx from lauric acid but not from glucose. Medium-chain-length (mcl) PHA was produced from lauric acid or glucose only when PhaC of A. hydrophila was inactivated, indicating the existence of another PHA synthase in the wild type. Using PCR cloning strategy, the potential PHA synthase gene (phaC _mcl) was obtained from genomic DNA of the wild type and exhibited strong homology to type II PHA synthase genes of Pseudomonas strains. The phaC _mcl gene was PCR subcloned into plasmid pBBR1MCS2 and expressed in a PHA-negative mutant of Pseudomonas putida. Recombinant P. putida synthesized mcl PHA from gluconate or octanoate. This result proved that wild type A. hydrophila possessed another type II PHA synthase, which was responsible for the synthesis of mcl PHA, besides type I PHA synthase.     

Tolerance of plastic-encapsulated Pseudomonas putida KT2440 to chemical stress

Pseudomonas putida dried in the presence of hydroxyectoine or trehalose can withstand exposure to organic solvents and therefore can be encapsulated inside plastics such as polystyrene. Here we show that P. putida in a plastic-encapsulated dried tablet exhibits remarkable tolerance to chemical stress, comparable to that of spores of Bacillus subtilis.     

Removal of caffeine in sewage by Pseudomonas putida: Implications for water pollution index

A strain of Pseudomonas putida (biotype A) capable of growing on caffeine (1,3,7-trimethylxanthine) was isolated from a domestic wastewater processing operation. It used caffeine as the sole carbon source with a mean growth rate constant (k) of 0.049 h^-1 (approximately 20 h per generation), whereas k for glucose utilization under similar incubation conditions was 0.31 (3.3 h per generation). The isolate contained at least two plasmids, and the increased expression of a 40 kDa protein was attributable to growth on caffeine. Degradation byproducts of caffeine metabolism by the bacterial isolate included other xanthine derivatives. The slow bacterial catabolism of caffeine in sewage has implications for the effectiveness of wastewater purification, re-use and disposal.The author is with the Laboratory for Molecular Ecology, Department of Environmental Analysis and Design, University of California at Irvine, Irvine, CA 92717-5150 U.S.A.