Diverse responses to UV-B radiation and repair mechanisms of bacteria isolated from high-altitude aquatic environments

Acinetobacter johnsonii A2 isolated from the natural community of Laguna Azul (Andean Mountains at 4,560 m above sea level), Serratia marcescens MF42, Pseudomonas sp. strain MF8 isolated from the planktonic community, and Cytophaga sp. strain MF7 isolated from the benthic community from Laguna Pozuelos (Andean Puna at 3,600 m above sea level) were subjected to UV-B (3,931 J m-2) irradiation. In addition, a marine Pseudomonas putida strain, 2IDINH, and a second Acinetobacter johnsonii strain, ATCC 17909, were used as external controls. Resistance to UV-B and kinetic rates of light-dependent (UV-A [315 to 400 nm] and cool white light [400 to 700 nm]) and -independent reactivation following exposure were determined by measuring the survival (expressed as CFU) and accumulation of cyclobutane pyrimidine dimers (CPD). Significant differences in survival after UV-B irradiation were observed: Acinetobacter johnsonii A2, 48%; Acinetobacter johnsonii ATCC 17909, 20%; Pseudomonas sp. strain MF8, 40%; marine Pseudomonas putida strain 2IDINH, 12%; Cytophaga sp. strain MF7, 20%; and Serratia marcescens, 21%. Most bacteria exhibited little DNA damage (between 40 and 80 CPD/Mb), except for the benthic isolate Cytophaga sp. strain MF7 (400 CPD/Mb) and Acinetobacter johnsonii ATCC 17909 (160 CPD/Mb). The recovery strategies through dark and light repair were different in all strains. The most efficient in recovering were both Acinetobacter johnsonii A2 and Cytophaga sp. strain MF7; Serratia marcescens MF42 showed intermediate recovery, and in both Pseudomonas strains, recovery was essentially zero. The UV-B responses and recovery abilities of the different bacteria were consistent with the irradiation levels in their native environment.     

Inability of muconate cycloisomerases to cause dehalogenation during conversion of 2-chloro-cis,cis-muconate.

The conversion of 2-chloro-cis,cis-muconate by muconate cycloisomerase from Pseudomonas putida PRS2000 yielded two products which by nuclear magnetic resonance spectroscopy were identified as 2-chloro- and 5-chloromuconolactone. High-pressure liquid chromatography analyses showed the same compounds to be formed also by muconate cycloisomerases from Acinetobacter calcoaceticus ADP1 and Pseudomonas sp. strain B13. During 2-chloro-cis,cis-muconate turnover by the enzyme from P. putida, 2-chloromuconolactone initially was the major product. After prolonged incubation, however, 5-chloromuconolactone dominated in the resulting equilibrium. In contrast to previous assumptions, both chloromuconolactones were found to be stable at physiological pH. Since the chloromuconate cycloisomerases of Pseudomonas sp. strain B13 and Alcaligenes eutrophus JMP134 have been shown previously to produce the trans-dienelactone (trans-4-carboxymethylene-but-2-en-4-olide) from 2-chloro-cis,cis-muconate, they must have evolved the capability to cleave the carbon-chlorine bond during their divergence from normal muconate cycloisomerases.     

Isolation and study of Acinetobacter sp. mutant strains defective in production of exopolysaccharides

Nitrosoguanidine-induced mutants of Acinetobacter sp. defective in exopolysaccharide biosynthesis did not differ from the parent strain in distinguishing physiological and biochemical properties, such as requirements for growth factors, utilization of mono- and disaccharides, and resistance to antibiotics. The genetic relation of parent and mutant strains was shown by 16S rRNA PCR analysis. The comparative study of parent and mutant strains with respect to resistance to unfavorable environmental factors confirmed our hypothesis that Acinetobacter sp. exopolysaccharides perform protective functions. Hybridization experiments revealed the conjugal transfer of plasmid R68.45 from Pseudomonas putida BS228 (R68.45) to mutant but not to the parent Acinetobacter sp. strains. The role of the Acinetobacter sp. exopolysaccharides in providing the genetic stability of this bacterium is discussed.     

Characterization of a Pseudomonas putida rough variant evolved in a mixed-species biofilm with Acinetobacter sp. strain C6

Genetic differentiation by natural selection is readily observed among microbial populations, but a more comprehensive understanding of evolutionary forces, genetic causes, and resulting phenotypic advantages is not often sought. Recently, a surface population of Pseudomonas putida bacteria was shown to evolve rapidly by natural selection of better-adapted variants in a mixed-species biofilm consortium (S. K. Hansen, P. B. Rainey, J. A. Haagensen, and S. Molin, Nature 445:533-536, 2007). Adaptation was caused by mutations in a wapH homolog (PP4943) involved in core lipopolysaccharide biosynthesis. Here we investigate further the biofilm physiology and the phenotypic characteristics of the selected P. putida rough colony variants. The coexistence of the P. putida population in a mixed-species biofilm with Acinetobacter sp. strain C6 is dependent on the benzoate excreted from Acinetobacter during the catabolism of benzyl alcohol, the sole carbon source. Examination of biofilm development and the dynamics of the wild-type consortium revealed that the biofilm environment became oxygen limited, possibly with low oxygen concentrations around Acinetobacter microcolonies. In contrast to P. putida wild-type cells, which readily dispersed from the mixed-species biofilm in response to oxygen starvation, the rough variant cells displayed a nondispersal phenotype. However, in monospecies biofilms proliferating on benzoate, the rough variant (like the wild-type population) dispersed in response to oxygen starvation. A key factor explaining this conditional, nondispersal phenotype is likely to be the acquired ability of the rough variant to coaggregate specifically with Acinetobacter cells. We further show that the P. putida rough variant displayed enhanced production of a cellulose-like polymer as a consequence of the mutation in wapH. The resulting phenotypic characteristics of the P. putida rough variant explain its enhanced fitness and ability to form tight structural associations with Acinetobacter microcolonies.     

Characterization of hydrocarbon-degrading microbial populations in contaminated and pristine Alpine soils

Biodegradation of petroleum hydrocarbons in cold environments, including Alpine soils, is a result of indigenous cold-adapted microorganisms able to degrade these contaminants. In the present study, the prevalence of seven genotypes involved in the degradation of n-alkanes (Pseudomonas putida GPo1 alkB; Acinetobacter spp. alkM; Rhodococcus spp. alkB1, and Rhodococcus spp. alkB2), aromatic hydrocarbons (P. putida xylE), and polycyclic aromatic hydrocarbons (P. putida ndoB and Mycobacterium sp. strain PYR-1 nidA) was determined in 12 oil-contaminated (428 to 30,644 mg of total petroleum hydrocarbons [TPH]/kg of soil) and 8 pristine Alpine soils from Tyrol (Austria) by PCR hybridization analyses of total soil community DNA, using oligonucleotide primers and DNA probes specific for each genotype. The soils investigated were also analyzed for various physical, chemical, and microbiological parameters, and statistical correlations between all parameters were determined. Genotypes containing genes from gram-negative bacteria (P. putida alkB, xylE, and ndoB and Acinetobacter alkM) were detected to a significantly higher percentage in the contaminated (50 to 75%) than in the pristine (0 to 12.5%) soils, indicating that these organisms had been enriched in soils following contamination. There was a highly significant positive correlation (P < 0.001) between the level of contamination and the number of genotypes containing genes from P. putida and Acinetobacter sp. but no significant correlation between the TPH content and the number of genotypes containing genes from gram-positive bacteria (Rhodococcus alkB1 and alkB2 and Mycobacterium nidA). These genotypes were detected at a high frequency in both contaminated (41.7 to 75%) and pristine (37.5 to 50%) soils, indicating that they are already present in substantial numbers before a contamination event. No correlation was found between the prevalence of hydrocarbon-degradative genotypes and biological activities (respiration, fluorescein diacetate hydrolysis, lipase activity) or numbers of culturable hydrocarbon-degrading soil microorganisms; there also was no correlation between the numbers of hydrocarbon degraders and the contamination level. The measured biological activities showed significant positive correlation with each other, with the organic matter content, and partially with the TPH content and a significant negative correlation with the soil dry-mass content (P < 0.05 to 0.001).     

松材线虫对其携带的细菌繁殖的影响

1.引言松材线虫病（Bursaphelenchus xylophilus）是松树的一种毁灭性病害,在日本、中国、韩国和北美、尼日利亚和葡萄牙等国家蔓延,造成了巨大经济损失,其中以日本和中国受害最重.一直认为松材线虫是引起该病的唯一病原,但近十几年来的研究发现,细菌在致病过程中可能起着重要作用,相继从病木和松材线虫体上分离到能对黑松苗有致萎活性的细菌.赵博光等首次根据实验提出松材线虫病是线虫和细菌共同侵染引起的复合侵染病害的假说,并在以后的试验中得到了验证.关于松材线虫对其细菌繁殖的影响研究鲜有报道.本试验采用从感病松树上分离并鉴定了的细菌菌株中选取假单胞属7株、其它属的细菌菌株3株,     

Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment

Aniline-degraders were isolated from activated sludge and environmental samples and classified into eight phylogenetic groups. Seven groups were classified into Gram-negative bacteria, such as Acidovorax sp., Acinetobacter sp., Delftia sp., Comamonas sp., and Pseudomonas sp., suggesting the possible dominance of Gram-negative aniline-degraders in the environment. Aniline degradative genes were cloned from D. acidovorans strain 7N, and the nucleotide sequence of the 8,039-bp fragment containing eight open reading frames was determined. Their deduced amino acid sequences showed homologies to glutamine synthetase (GS)-like protein, glutamine amidotransferase (GA)-like protein, large and small subunits of aniline dioxygenase, reductase, LysR-type regulator, small ferredoxin-like protein, and catechol 2,3-dioxygenase, suggesting a high similarity of this gene cluster to those in P. putida strain UCC22 and Acinetobacter sp. strain YAA. Polymerase chain reaction (PCR) and sequencing analyses of GS-like protein gene segments of other Gram-negative bacteria suggested that Gram-negative bacteria have aniline degradative gene that can be divided into two distinctive groups.     

Construction of artificially structured microbial consortia (ASMC) using dielectrophoresis: examining bacterial interactions via metabolic intermediates within environmental biofilms

The construction of artificial biofilms with defined internal architectures is described. Bacterial cells are suspended in a low conductivity medium, guided to specific areas in a microelectrode array by dielectrophoresis (DEP), and then immobilised using the flocculating agent poly(ethylenimine). Multispecies biofilms can be constructed by introducing different species at different times. The rapid construction of such biofilms with defined internal architectures provides, when combined with visual reporters of gene activity, a powerful new method for the investigation of the effects of the spatial organisation on interactions between bacterial species in biofilms. To demonstrate the utility of the technique as a method for investigating metabolic interactions in biofilms, aggregates were constructed from Acinetobacter sp. C6 and Pseudomonas putida::gfp. The Acinetobacter degrades benzyl alcohol, overproducing benzoate, which in turn is consumed by the Pseudomonas strain. The P. putida has a chromosomally expressed cassette encoding a gfp downstream of the promoter which controls degradation of benzoate, making the interaction between the two strains in the metabolism of benzyl alcohol visible by the production of green fluorescent protein (GFP). Microscopic observation of the biofilms, including the use of confocal laser scanning microscopy (CLSM), confirmed that metabolic exchange occurred. In addition, it was observed that the bacteria appear to have a preferred biofilm architecture, with P. putida in the bottom layer, and Acinetobacter at the top.     

Metabolic commensalism and competition in a two-species microbial consortium

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

Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus

Biophysical and genetic properties of six independently isolated plasmids encoding the degradation of the herbicides 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid are described. Four of the plasmids, pJP3, pJP4, pJP5, and pJP7, had molecular masses of 51 megadaltons, belonged to the IncP1 incompatibility group, and transferred freely to strains of Escherichia coli, Rhodopseudomonas sphaeroides, Rhizobium sp., Agrobacterium tumefaciens, Pseudomonas putida, Pseudomonas fluorescens, and Acinetobacter calcoaceticus. In addition, these four plasmids conferred resistance to merbromin, phenylmercury acetate, and mercuric ions, had almost identical restriction endonuclease cleavage patterns, and encoded degradation of m-chlorobenzoate. The two other plasmids, pJP2 and pJP9, did not belong to the IncP1 incompatibility group, had molecular masses of 37 megadaltons, encoded the degradation of phenoxyacetic acid, and possessed identical restriction endonuclease cleavage patterns.     

A survey of indigenous microbial hydrocarbon degradation genes in soils from Antarctica and Brazil

Total community DNA from 29 noncontaminated soils and soils impacted by petroleum hydrocarbons and chloro-organics from Antarctica and Brazil were screened for the presence of nine catabolic genes, encoding alkane monooxygenase or aromatic dioxygenases, from known bacterial biodegradation pathways. Specific primers and probes targeting alkane monooxygenase genes were derived from Pseudomonas putida ATCC 29347 (Pp alkB), Rhodococcus sp. strain Q15 (Rh alkB1, Rh alkB2), and Acinetobacter sp. ADP-1 (Ac alkM). In addition, primers and probes detecting aromatic dioxygenase genes were derived from P. putida ATCC 17484 (ndoB), P. putida F1 (todC1), P. putida ATCC 33015 (xylE and cat23), and P. pseudoalcaligenes KF707 (bphA). The primers and probes were used to analyze total community DNA extracts by using PCR and hybridization analysis. All the catabolic genes, except the Ac alkM, were detected in contaminated and control soils from both geographic regions, with a higher frequency in the Antarctic soils. The alkane monooxygenase genes, Rh alkB1 and Rh alkB2, were the most frequently detected alk genes in both regions, while Pp alkB was not detected in Brazil soils. Genes encoding the aromatic dioxygenases toluene dioxygenase (todC1) and biphenyl dioxygenase (bphA) were the most frequently detected in Antarctica, and todC1 and catechol-2,3-dioxygenase (cat23) were the most frequent in Brazil soils. Hybridization analysis confirmed the PCR results, indicating that the probes used had a high degree of homology to the genes detected in the soil extracts and were effective in detecting biodegradative potential in the indigenous microbial population.     

Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4'-dichlorobiphenyl

Acinetobacter sp. strain 4CB1 was isolated from a polychlorobiphenyl-contaminated soil sample by using 4-chlorobenzoate as a sole source of carbon and energy. Resting cells of Acinetobacter sp. strain 4CB1 hydrolytically dehalogenated 4-chlorobenzoate under aerobic and anaerobic conditions, but 4-hydroxybenzoate accumulated only under anaerobic conditions. Cell extracts of Acinetobacter sp. strain 4CB1 oxidized 4-hydroxybenzoate by an NADH-dependent monooxygenase to form protocatechuate, which was subsequently oxidized by both ortho- and meta-protocatechuate dioxygenase reactions. When grown on biphenyl, Acinetobacter sp. strain P6 cometabolized 4,4'-dichlorobiphenyl primarily to 4-chlorobenzoate; however, when this strain was grown in a coculture with Acinetobacter sp. strain 4CB1, 4-chlorobenzoate did not accumulate but was converted to inorganic chloride. When resting cells of Acinetobacter sp. strain 4CB1 were incubated anaerobically with 3,4-dichlorobenzoate, they accumulated 4-carboxy-1,2-benzoquinone as a final product. Since 3,4-dichlorobenzoate is a product that is formed from the cometabolism of 3,4-dichloro-substituted tetrachlorobiphenyls by Acinetobacter sp. strain P6, the coculture has a potential application for dehalogenation and mineralization of specific polychlorobiphenyl congeners.     

Oxidation of dimethyl sulfide by various aromatic compound oxygenases from bacteria

As a result of the determination of dimethyl sulfide (DMS) oxidizing activity of bacterial aromatic compound oxygenases, multicomponent monooxygenases (DmpKLMNOP from Pseudomonas sp. CF600, AphKLMNOP from Comamonas testosteroni TA441, and TodABCDEF from Pseudomonas sp. JS150), single component monooxygenases (TfdB from Pseudomonas putida EST4011 and XylMA from Pseudomonas putida mt-2), and dioxygenases (CumA1A2A3A4 from Pseudomonas fluorescens IP01 and PahAaAbAcAd from Pseudomonas putida OUS82) showed DMS-oxidizing activity, while CarAaAcAd from Pseudomonas sp. CA10 and SoxC from Rhodococcus sp. IGTS8 did not. These results indicate the possibilities that these oxygenases might oxidize DMS to DMSO under the natural condition in the environment.Present address: Laboratory of Microbiology, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan     

Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4'-dichlorobiphenyl.

Acinetobacter sp. strain 4CB1 was isolated from a polychlorobiphenyl-contaminated soil sample by using 4-chlorobenzoate as a sole source of carbon and energy. Resting cells of Acinetobacter sp. strain 4CB1 hydrolytically dehalogenated 4-chlorobenzoate under aerobic and anaerobic conditions, but 4-hydroxybenzoate accumulated only under anaerobic conditions. Cell extracts of Acinetobacter sp. strain 4CB1 oxidized 4-hydroxybenzoate by an NADH-dependent monooxygenase to form protocatechuate, which was subsequently oxidized by both ortho- and meta-protocatechuate dioxygenase reactions. When grown on biphenyl, Acinetobacter sp. strain P6 cometabolized 4,4'-dichlorobiphenyl primarily to 4-chlorobenzoate; however, when this strain was grown in a coculture with Acinetobacter sp. strain 4CB1, 4-chlorobenzoate did not accumulate but was converted to inorganic chloride. When resting cells of Acinetobacter sp. strain 4CB1 were incubated anaerobically with 3,4-dichlorobenzoate, they accumulated 4-carboxy-1,2-benzoquinone as a final product. Since 3,4-dichlorobenzoate is a product that is formed from the cometabolism of 3,4-dichloro-substituted tetrachlorobiphenyls by Acinetobacter sp. strain P6, the coculture has a potential application for dehalogenation and mineralization of specific polychlorobiphenyl congeners.     

Cloning of a DNA fragment encoding part of a 70-kDa heat shock protein of Candida albicans

Abstract Muropeptide composition of peptidoglycan from the Gram-negative bacteria Aeromonas sp., Acinetobacter acetoaceticus, Agrobacterium tumefaciens, Enterobacter cloacae, Proteus morganii, Pseudomonas aeruginosa, Pseudomonas putida, Vibrio parahaemolyticus Yersinia enterocolitica and Escherichia coli , was analyzed by HPLC In all instances peptidoglycan was built up from the same subunits. A wide disparity in the relative abundance of muropeptides and all structural parameters was observed. The contribution of LD-A2pm-A2pm cross-linked muropeptides was extremely variable; from 1 to 45% of cross-linked muropeptides. Muropeptides with the dipeptides Lys-Lys or Arg-Lys, indicative of murein-bound (lipo)proteins, were detected in all instances although abundance was very variable.     

Comparison of enzymatic and immunochemical properties of 2,3-dihydroxybiphenyl-1,2-dioxygenases from four Pseudomonas strains

Abstract A 2,3-dihydroxybiphenyl-1,2-dioxygenase gene has been cloned from chromosomal DNA of Pseudomonas sp. DJ-12 which can grow on biphenyl or 4-chlorobiphenyl as the sole carbon and energy source. Enzymatic and immunochemical properties of the cloned 2,3-dihydroxybiphenyl-1,2-dioxygenase were characterized, and compared with those of P. pseudoalcaligenes KF707, Pseudomonas sp. KKS102, and P. putida OU83. The dioxygenase of Pseudomonas sp. DJ-12 was similar to those of P. pseudoalcaligenes KF707, and Pseudomonas sp. KKS102, but significantly different from that of P. putida OU83 in electrophoretic mobilities on native PAGE and SDS-PAGE. The dioxygenases of Pseudomonas sp. DJ-12 and P. putida OU83 exhibited the highest ring-fission activity to 3-methylcatechol, and those of P. pseudoalcaligenes KF707 and Pseudomonas sp. KKS102 to 2,3-dihydroxybiphenyl among 2,3-dihydroxybiphenyl, catechol, 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol as substrates. 2,3-dihydroxybiphenyl-1,2-dioxygenase of P. pseudoalcaligenes KF707 was immunochemically related to that of Pseudomonas sp. KKS102, but was different from those of Pseudomonas sp. DJ-12 and P. putida OU83.     

Cloning of a carbofuran hydrolase gene from Achromobacter sp. strain WM111 and its expression in gram-negative bacteria.

A 14-kilobase-pair (kbp) EcoRI DNA fragment that encodes an enzyme capable of rapid hydrolysis of N-methylcarbamate insecticides (carbofuran hydrolase) was cloned from carbofuran-degrading Achromobacter sp. strain WM111. When used to probe Southern blots containing plasmid and total DNAs from WM111, this 14-kbp fragment hybridized strongly to a 14-kbp EcoRI fragment from the greater than 100-kbp plasmid harbored by this strain but weakly to EcoRI-digested total DNA from Achromobacter sp. strain WM111, indicating that the gene for N-methylcarbamate degradation (mcd) is plasmid encoded. Further subcloning localized the mcd gene on a 3-kbp ScaI-ClaI fragment. There was little or no expression of this gene in the alternative gram-negative hosts Pseudomonas putida, Alcaligenes eutrophus, Acinetobacter calcoaceticus, and Achromobacter pestifer. Western blotting (immunoblotting) of the protein products produced by low-level expression in P. putida confirmed that this 3-kbp fragment encodes the two 70+-kilodalton protein products seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified carbofuran hydrolase.     

Metabolism of Chlorotoluenes by Burkholderia sp. Strain PS12 and Toluene Dioxygenase of Pseudomonas putida F1: Evidence for Monooxygenation by Toluene and Chlorobenzene Dioxygenases

The degradation of toluene by Pseudomonas putida F1 and of chlorobenzenes by Burkholderia sp. strain PS12 is initiated by incorporation of dioxygen into the aromatic nucleus to form cis-dihydrodihydroxybenzenes. Toluene-grown cells of P. putida F1 and 3-chlorobenzoate-grown cells of Burkholderia sp. strain PS12 were found to monooxygenate the side chain of 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols. Further metabolism of these products was slow, and the corresponding chlorobenzoates were usually observed as end products, whereas the 3-chlorobenzoate produced from 3-chlorotoluene in Burkholderia sp. strain PS12 was metabolized further. Escherichia coli cells containing the toluene dioxygenase genes from P. putida F1 oxidized 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols as major products, demonstrating that this enzyme is responsible for the observed side chain monooxygenation. Two methyl- and chloro-substituted 1,2-dihydroxycyclohexadienes were formed as minor products from 2- and 3-chlorotoluene, whereas a chloro- and methyl-substituted cyclohexadiene was the only product formed from 4-chlorotoluene. The toluene dioxygenase of P. putida F1 and chlorobenzene dioxygenase from Burkholderia sp. strain PS12 are the first enzymes described that efficiently catalyze the oxidation of 2-chlorotoluene.     

高效柴油降解菌Acinetobacter sp.W3分离鉴定及降解酶基因扩增分析

从柴油污染的海水样品中分离高效柴油降解细菌,分析菌株对柴油的降解能力及降解酶基因,为海洋柴油污染的生物修复奠定基础。选取浙江定海港柴油污染的海水样品,进行降解菌的富集培养;采用常规方法分离筛选高效柴油降解菌。利用革兰氏染色、形态学观察、生理生化鉴定及16S rDNA分析等方法对降解菌株进行种属鉴定。采用紫外吸收法测定菌株对柴油的降解率。采用PCR方法、核酸序列测定和比对,对其降解酶基因进行扩增分析。筛选出一株高效降解菌,形态学观察及生理生化鉴定初步确定为不动杆菌。16S rDNA序列分析及比对结果表明,其16S rDNA序列与威尼斯不动杆菌(Acinetobacter venetianus)属的序列同源性达到99.7%,命名为不动杆菌W3(Acinetobactersp.W3),该菌对柴油的7 d降解率达到84.7%。PCR方法从Acinetobactersp.W3菌株中的基因组DNA和质粒DNA上扩增到了大小为540 bp的烷烃羟化酶基因alkB和864 bp的CYP153A部分DNA片段,分别与Acinetobacter venetianus1-D-2的alkB和Acinetobactersp.OC4、Acinetobactersp.EB104的CYP153具有99%和98%的同源性。从定海港口柴油污染海水分离得到一株高效柴油降解菌Acinetobactersp.W3,该菌属于不动杆菌属,含有烷烃降解酶基因,能高效降解柴油污染物,有望应用于海水柴油污染的生物修复。     

Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.