Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments.

Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.     

Evaluation of strains isolated by growth on naphthalene and biphenyl for hybridization of genes to dioxygenase probes and polychlorinated biphenyl-degrading ability.

Approximately equal numbers of bacteria were isolated from primarily tropical soils by growth on biphenyl and naphthalene to compare their competence in polychlorinated biphenyl (PCB) degradation. The strains isolated by growth on biphenyl catalyzed more extensive PCB degradation than the strains isolated by growth on naphthalene, suggesting that naphthalene cocontamination may be only partially effective in stimulating the cometabolism of lower chlorinated PCBs. Probes were made from the bph, nah, and tod genes encoding the large iron iron sulfur protein of the dioxygenase complex and hybridized to 19 different strains. The hybridization patterns did not correlate well with the substrates of isolation, suggesting that there is considerable diversity in these genes in nature and that probe hybridization is not a reliable indication of catabolic capacity. The strains with the most extensive PCB degradation capacity did strongly hybridize to the bph probe, but a few strains that exhibited strong hybridization had poor PCB-degrading ability. Of the 19 strains studied, 5 hybridized to more than one probe and 2, including one strong PCB degrader, hybridized to all three probes. Southern blots showed that the bph and nah probes hybridized to separate bands, suggesting that multiple dioxygenases were present. Multiple dioxygenases may be an important feature of competitive decomposers in nature and hence may not be rare. Most of the isolates identified were members of the beta subgroup of the Proteobacteria, a few were gram positive, and none were true Pseudomonas species.     

Molecular diagnostics and chemical analysis for assessing biodegradation of polychlorinated biphenyls in contaminated soils

Summary The microbial populations in PCB-contaminated electric power substation capacitor bank soil (TVA soil) and from another PCB-contaminated site (New England soil) were compared to determine their potential to degrade PCB. Known biphenyl operon genes were used as gene probes in colony hybridizations and in dot blots of DNA extracted from the soil to monitor the presence of PCB-degrading organisms in the soils. The microbial populations in the two soils differed in that the population in New England soil was enriched by the addition of 1000 p.p.m. 2-chlorobiphenyl (2-CB) whereas the population in the TVA capacitor bank soil was not affected. PCB degradative activity in the New England soil was indicated by a 50% PCB disappearance (gas chromatography), accumulation of chlorobenzoates (HPLC), and¹⁴CO₂ evolution from¹⁴C-2CB. The PCB-degrading bacteria in the New England soil could be identified by their positive hybridization to thebph gene probes, their ability to produce the yellowmeta-cleavage product from 2,3-dihydroxybiphenyl (2,3-DHB), and the degradation of specific PCB congeners by individual isolates in resting cell assays. Although the TVA capacitor bank soil lacked effective PCB-degrading populations, addition of a PCB-degrading organism and 10 000 p.p.m. biphenyl resulted in a >50% reduction of PCB levels. Molecular characterization of soil microbial populations in laboratory scale treatments is expected to be valuable in the design of process monitoring and performance verification approaches for full scale bioremediation.     

Diversity of the C-terminal portion of the biphenyl dioxygenase large subunit

The biphenyl dioxygenase (BPDO) catalyses a stereospecific dioxygenation of biphenyl and analogs of it. Aside from being involved in the destruction and detoxification of toxic pollutants in soil, in the context of the green chemistry concept, this enzyme is a promising biocatalyst to design new more selective and more environmentally friendly approaches to manufacture fine chemicals. At this time, most of our knowledge about the variability of key residues determining the substrate specificity and regiospecificity of the enzyme oxygenase component (BphAE) toward biphenyl analogs and about the effect of altering these residues on catalytic properties is based on investigations made with BphAEs from cultured organisms and engineered enzymes derived from them. The purpose of this work was to examine the diversity of the amino acid sequence patterns of the alpha subunit (BphA) C-terminal domain deduced from PCR products amplified from DNA extracted from cultured bacteria of various phylogenetic lines and from the soil microflora of PCB-contaminated soils. Of special interest were segments of the C-terminal portion called regions I, III and IV. Altogether, the phylogenetic tree obtained from aligning the deduced amino acid sequences of BphAs C-terminal domain from cultured bacteria belonging to various ecological niches and from uncultured soil bacteria reveals that most of the BphAs were linked to the three clusters of BphAs previously reported. However, few belong to new branches that diverge from the previously known branches showing a high diversity of BphAs in natural environment. Furthermore, data show a wide distribution of BphAs with family linkages that not only crosses bacterial taxonomic frontiers but also ecological niches. Nevertheless, in spite of this divergence, the sequence patterns of regions III and IV amino acids that are known to influence substrate specificity and regiospecificity are rather conserved among BphAs and the pattern was independent of the family cluster to which they belong. In most cases, regions III and IV amino acid patterns are closer to those of Pseudomonas pseudoalcaligenes KF707 BphA1 than to the most versatile Burkholderia xenovorans LB400 BphA. This might suggest that the PCB-degrading potency of soil bacteria is closer to the one observed for KF707 BphAE than from LB400 BphAE. However, the fact that among less than 20 PCR products amplified from soil DNA that we have sequenced, one of them was very homologous to that of LB400 BphA and in addition, residues 335 and 336 of LB400 were replaced by residues that previous enzyme engineering had shown to extend the range of PCB substrate used by the enzyme strongly suggest that PCB-degrading bacteria are evolving in soil to optimize their PCB-degrading capacity.     

一种用分子杂交技术检测谷氨酸生产菌溶原性的方法

谷氨酸生产菌用于发酵生产谷氨酸和其他氨基酸,由于外源噬菌体污染或者由溶原菌内诱导出的噬菌体污染,往往给工业发酵带来严重的危害,于是测定谷氨酸生产菌是否为溶原菌便成了当务之急。本文以溶原菌E.coli K_(12)为研究模型,首先用~(32)P标记的λDNA作探针,建立了检测溶原菌株的分子诊断方法,继而通过寄主菌株从生产环境样品中分离筛选到7_6和7_(?)两大类共20多株噬菌体。由该两类噬菌体制得的~(32)P-7_(?)和7_(?)探针,可分别与以生产谷氨酸的棒杆菌B_9、7338和T_(?)三个菌株为寄主,从环境样品中分离的18个和6个噬菌体分离物杂交。而采用缺口翻译系统制备的7_(?)和7_(?)混合探针,或混合使用7_(?)和(?)探针,使得检测方法更为简便易行。通过Southern Blot技术和菌落杂交对菌DNA的分析,结果表明三个寄主菌株均不是所分离的噬菌体的溶原菌。最后,本文还就烈性噬菌体和温和噬菌体之间的同源性与分子诊断的可行性作了初步探讨。     

Bioremediation of polychlorinated biphenyl-contaminated soil using carvone and surfactant-grown bacteria

Partial bioremediation of polychlorinated biphenyl (PCB)-contaminated soil was achieved by repeated applications of PCB-degrading bacteria and a surfactant applied 34 times over an 18-week period. Two bacterial species, Arthrobacter sp. strain B1B and Ralstonia eutrophus H850, were induced for PCB degradation by carvone and salicylic acid, respectively, and were complementary for the removal of different PCB congeners. A variety of application strategies was examined utilizing a surfactant, sorbitan trioleate, which served both as a carbon substrate for the inoculum and as a detergent for the mobilization of PCBs. In soil containing 100 μg Aroclor 1242 g⁻¹ soil, bioaugmentation resulted in 55–59% PCB removal after 34 applications. However, most PCB removal occurred within the first 9 weeks. In contrast, repeated addition of surfactant and carvone to non-inoculated soil resulted in 30–36% PCB removal by the indigenous soil bacteria. The results suggest that bioaugmentation with surfactant-grown, carvone-induced, PCB-degrading bacteria may provide an effective treatment for partial decontamination of PCB-contaminated soils. Received: 9 March 2000 / Received revision: 27 June 2000 / Accepted: 16 July 2000     

A sulfone group-labeled TEM-DNA probe: comparison with a 32P-labeled probe in dot-hybridization

A non-radioactive DNA probe for the TEM-type beta-lactamase gene was obtained by using the 'Chemiprobe' system. It was used along with a 32P-labeled TEM probe to screen for TEM beta-lactamase gene in 107 bacterial isolates representing 7 Gram-negative genera and previously classified as TEM-positive or negative. The DNA to be tested was extracted from these bacterial isolates by the Birnboim-Doly method and, after blotting into charged nylon membranes, it was submitted to hybridization with either the TEM 'Chemiprobe' or the 32P-TEM probe. The TEM 'Chemiprobe' could detect as few as 25 pg specific DNA if it was used at a concentration of 5 ng per cm2 of membrane. The results obtained by both probes were concordant in 93.5% of the entire sample. The TEM 'Chemiprobe' was specific since only one false positive was observed. Furthermore, it appeared at least as sensitive as the 32P-labeled TEM probe. As the dot-hybridization with the sulfone-labeled probe was sensitive, simple and easy to perform, it will be useful for large-scale screening in clinical laboratory.     

Degradation of polychlorinated biphenyl (PCB) by a consortium obtained from a contaminated soil composed of Brevibacterium,Pandoraea and Ochrobactrum

An indigenous polychlorinated biphenyl (PCB)-degrading bacterial consortium was obtained from soils contaminated by transformer oil with a high content of PCBs. The PCB degrader strains were isolated and identified as Brevibacterium antarcticum, Pandoraea pnomenusa, and Ochrobactrum intermedium by 16S rRNA gene sequence phylogenetic analysis. The PCB-degrading ability of the consortium and of individual strains was determined by using GC/MS. The PCB-degrading capacities of the consortium were evaluated for three concentrations of transfomer oil ranging from 55 to 152 μM supplemented with 0.001% biphenyl and 0.1% of Tween 80 surfactant. PCB biodegradation by the consortium was favored in the presence of both additives and the greatest extent of biodegradation (67.5%) was obtained at a PCB concentration of 55 μM. Each bacterial species exhibited a particular pattern of degradation relating to specific PCB congeners. Isolated strains showed a moderate degradation capability towards tetra-, hepta-, and octa-chlorobiphenyls; although no effect on penta-, hexa-, and nona-chlorobiphenyls was observed. Recently, PCB degradation capacity was recognized in a Pandorea member; however, this is the first study that describes the ability of Brevibacterium and Ochrobactrum species to degrade PCBs.     

Identification and Analysis of Polychlorinated Biphenyls (PCBs)-Biodegrading Bacterial Strains in Shanghai

As one of China’s great metropolises, Shanghai is vulnerable to various forms of industrial and agricultural contamination associated with its development. Polychlorinated biphenyls (PCBs) are man-made chemicals that never existed in nature until the 1900s when they started to be released into the environment. PCBs are hazardous environmental contaminants that bind strongly to soil. In this study, four soil samples were screened for the presence of PCB-degrading bacteria. The 16 S rDNAs were amplified from those genomes and the products (~1.5 kb) were purified and sequenced for the isolation and identification of bacterial species. Four Pseudomonas strains (strain 1-212 from sample 1; strain 2-241 from sample 2; strain 3-318 from sample 3; and strain 4-150 from sample 4) were selected for analysis by HPLC. Setting the content of the biphenyl in CK as 100%, the biphenyl contents was 2.32% in 1-212, 73.11% in 2-241, 69.83% in 3-318, and 86.16% in 4-150. The results of this study suggest directions for future research, including genetic screening, cloning and restructuring, and provide guidance for the cultivation of PCBs-degrading bacteria.     

Two approaches to biological decontamination of groundwater and soil polluted by aromatics-characterization of microbial populations.

As part of the EU project MULTIBARRIERS, six new endogenous aerobic bacterial isolates able to grow in the presence of BTmX (benzene, toluene, m-xylene) were characterized with respect to their growth specificities. Preliminary analysis included restriction fragment length polymorphism profiles and 16S rDNA sequencing. The diversity of these strains was confirmed by denaturing gradient gel electrophoresis. Additional aerobic bacterial strains were isolated from the rhizospheres of plants grown in polychlorinated biphenyl (PCB)-contaminated soils. Pot experiments were designed to show the beneficial effect of plants on the bacterial degradation of PCBs. The effect of PCB removal from soil was evaluated and bacteria isolated from three different plant species were examined for the presence of the bph operon.     

DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community

We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded, ³²P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 × 10⁴ cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1-μg DNA sample.     

Psychrotolerant Bacteria Isolated from Arctic Soil That Degrade Polychlorinated Biphenyls at Low Temperatures

Psychrotolerant polychlorinated biphenyl (PCB)-degrading bacteria were isolated at 7°C from PCB-contaminated Arctic soil by using biphenyl as the sole organic carbon source. These isolates were distinguished from each other by differences in substrates that supported growth and substrates that were oxidized. 16S ribosomal DNA sequences suggest that these isolates are most closely related to the genus Pseudomonas. Total removal of Aroclor 1242, and rates of removal of selected PCB congeners, by cell suspensions of Arctic soil isolates and the mesophile Burkholderia cepacia LB400 were determined at 7, 37, and 50°C. Total removal values of Aroclor 1242 at 7°C by LB400 and most Arctic soil isolates were similar (between 2 and 3.5 μg of PCBs per mg of cell protein). However the rates of removal of some individual PCB congeners by Arctic isolates were up to 10 times higher than corresponding rates of removal by LB400. Total removal of Aroclor 1242 and the rates of removal of individual congeners by the Arctic soil bacteria were higher at 37°C than at 7°C but as much as 90% lower at 50°C than at 37°C. In contrast, rates of PCB removal by LB400 were higher at 50°C than at 37°C. In all cases, temperature did not affect the congener specificity of the bacteria. These observations suggest that the PCB-degrading enzyme systems of the bacteria isolated from Arctic soil are cold adapted.     

Microbial Diversity and Community Structure in Two Different Agricultural Soil Communities

Abstract In this study, two different agricultural soils were investigated: one organic soil and one sandy soil, from Stend (south of Bergen), Norway. The sandy soil was a field frequently tilled and subjected to crop rotations. The organic soil was permanent grazing land, infrequently tilled. Our objective was to compare the diversity of the cultivable bacteria with the diversity of the total bacterial population in soil. About 200 bacteria, randomly isolated by standard procedures, were investigated. The diversity of the cultivable bacteria was described at phenotypic, phylogenetic, and genetic levels by applying phenotypical testing (Biolog) and molecular methods, such as amplified rDNA restriction analysis (ARDRA); hybridization to oligonucleotide probes; and REP-PCR. The total bacterial diversity was determined by reassociation analysis of DNA isolated from the bacterial fraction of environmental samples, combined with ARDRA and DGGE analysis. The relationship between the diversity of cultivated bacteria and the total bacteria was elucidated. Organic soil exhibited a higher diversity for all analyses performed than the sandy soil. Analysis of cultivable bacteria resulted in different resolution levels and revealed a high biodiversity within the population of cultured isolates. The difference between the two agricultural soils was significantly higher when the total bacterial population was analyzed than when the cultivable population was. Thus, analysis of microbial diversity must ultimately embrace the entire microbial community DNA, rather than DNA from cultivable bacteria.     

The Anopheles punctulatus complex: DNA probes for identifying the Australian species using isotopic, chromogenic, and chemiluminescence detection systems

Isotopic and enzyme-labeled species-specific DNA probes were made for the three known members of the Anopheles punctulatus complex of mosquitoes in Australia (Anopheles farauti Nos. 1, 2, and 3). Species-specific probes were selected by screening total genomic libraries made from the DNA of individual species with 32P-labeled DNA of homologous and heterologous mosquito species. The 32P-labeled probes for A. farauti Nos. 1 and 2 can detect less than 0.2 ng of DNA while the 32P-labeled probe for A. farauti No. 3 has a sensitivity of 1.25 ng of DNA. Probes were then enzyme labeled for chromogenic and chemiluminescence detection and compared to isotopic detection using 32P-labeled probes. Sequences of the probe repeat regions are presented. Species identifications can be made from dot blots or squashes of freshly killed mosquitoes or mosquitoes stored frozen, dried, and held at room temperature or fixed in isopropanol or ethanol with isotopic, chromogenic, or chemiluminescence detection systems. The use of nonisotopic detection systems will enable laboratories with minimal facilities to identify important regional vectors.     

Sequence similarities in the genes encoding polychlorinated biphenyl degradation by Pseudomonas strain LB400 and Alcaligenes eutrophus H850

DNA-DNA hybridization was used to compare the Pseudomonas strain LB400 genes for polychlorinated biphenyl (PCB) degradation with those from seven other PCB-degrading strains. Significant hybridization was detected to the genome of Alcaligenes eutrophus H850, a strain similar to LB400 in PCB-degrading capability. These two organisms showed a strong conservation of restriction sites in the region of DNA encoding PCB metabolism. No other sequence similarities were detected in the two genomes. DNA from the other PCB-degrading strains showed no hybridization to the probe, which demonstrated the existence of at least two distinct classes of genes encoding PCB degradation.     

Comparative effect of microwaves and boiling on the denaturation of DNA

The effect of heat and microwave denaturation of small volumes of double-stranded plasmid DNA has been compared. Samples of intact plasmid DNA had plasmid DNA linearized by digestion with EcoRI were conventionally denatured in a boiling water bath or denatured by 2450 MHz of microwave energy for 0-300 s. Heat denaturation for periods longer than 120 s caused breakdown of linearized plasmid DNA; however, microwave denaturation for 10-300 s caused no apparent degradation of linearized DNA. Breakdown of DNA forms II and III was noted in plasmid DNA subjected to 300 s of either heat or microwave denaturation but breakdown of forms II and III occurred more quickly with heat than with microwave treatment. Microwave treatment was also found to be better than heat to denature 32P-labeled DNA probes subsequently used to detect homologous DNA samples immobilized on nitrocellulose filters. A microwave-treated 32P-labeled DNA probe was able to hybridize to DNA samples 20 times more dilute than a heat-treated 32P-labeled DNA probe. Depending on the form of DNA to be analyzed, these results indicate that small volumes of DNA solutions and radiolabeled DNA probes can be effectively denatured in a conventional microwave oven.     

Adaptation mechanisms of bacteria during the degradation of polychlorinated biphenyls in the presence of natural and synthetic terpenes as potential degradation inducers

In this study, we examined the effect of polychlorinated biphenyls (PCBs) in the presence of natural and synthetic terpenes and biphenyl on biomass production, lipid accumulation, and membrane adaptation mechanisms of two PCB-degrading bacterial strains Pseudomonas stutzeri and Burkholderia xenovorans LB400. According to the results obtained, it could be concluded that natural terpenes, mainly those contained in ivy leaves and pine needles, decreased adaptation responses induced by PCBs in these strains. The adaptation processes under investigation included growth inhibition, lipid accumulation, composition of fatty acids, cis/trans isomerization, and membrane saturation. Growth inhibition effect decreased upon addition of these natural compounds to the medium. The amount of unsaturated fatty acids that can lead to elevated membrane fluidity increased in both strains after the addition of the two natural terpene sources. The cells adaptation changes were more prominent in the presence of carvone, limonene, and biphenyl than in the presence of natural terpenes, as indicated by growth inhibition, lipid accumulation, and cis/trans isomerization. Addition of biphenyl and carvone simultaneously with PCBs increased the trans/cis ratio of fatty acids in membrane fractions probably as a result of fluidizing effects of PCBs. This stimulation is more pronounced in the presence of PCBs as a sole carbon source. This suggests that PCBs alone have a stronger effect on bacterial membrane adaptation mechanisms than when added together with biphenyl or natural or synthetic terpenes.     

Analyses of polycyclic aromatic hydrocarbon-degrading bacteria isolated from contaminated soils

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria isolated from PAH-contaminated soils were analyzed genotypically and phenotypically for their capacity for metabolism of naphthalene and other PAH substrates. The methods used for the analyses were DNA hybridization using NAH7-derived gene probes, PAH spray plate assays, ¹⁴C-PAH mineralization assays, and dioxygenase activity assays. The results of the analyses showed a dominant number of PAH-degrading bacteria with a NAH7-like genotype. The results support the continued use of the nahA probe for contaminated soils to monitor the genetic potential of indigenous microorganisms to degrade PAHs. However, the finding of non-it nahA-hybridizing PAH-degrading bacteria show the limitation of NAH7-derived gene probes. Fifteen percent (13/89) of PAH-degrading bacteria isolated were not detected with the nahA gene probe. Four isolates (designated A5PH1, A8AN3, B1PH2, and B10AN1) did not hybridize with any of the NAH7-derived gene probes ( nahA, nahG, nahH, and nahR) used in this study. Considering the numerous unculturable microorganisms in nature and their potential genotypes, NAH7-derived gene probes may underestimate the microbial potential to catabolize PAHs. This necessitates development of new gene probes for enumeration and isolation of PAH-degrading bacteria to better understand the in situ microbial potential to degrade PAHs.     

Comparison of three nonradioactive and a radioactive DNA probe for the detection of target DNA by DNA hybridization

The specificity and sensitivity of three methods for the preparation and detection of nonradioactive probe DNA (biotin-nick translation, biotin-photolabel, and antigen-chemical linkage) were evaluated and compared with a nick-translated³²P-labeled DNA probe in DNA hybridization studies. The DNA probes were prepared from a restriction fragment (HindIII-3) from bacteriophage P1 DNA, and target DNA consisted of purified phage P1 DNA or P1 prophage DNA in lysogens ofEscherichia coli. A probe concentration of 50 ng/ml resulted in clear detection with the three nonradioactiveHindIII-3 DNA probes, whereas the specificity of the³²P-HindIII-3 DNA probe was satisfactory at a concentration of 25 ng/ml. However, the detection of false positives was greater with the³²P-labeled probe. The sensitivity of the radiolabeled DNA probe was marginally greater than that of the nonradioactive probes in dot blot hybridizations with purified phage P1 DNA. However, when the preparation time, ease of use, safety, duration of storage, and expense were compared for the four methods of labeling, the nonradiolabeled probes were generally superior to the radiolabeled probe.