ISSN:0962-1083    

DOI:10.1111/j.1365-294X.1995.tb00253.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

ISSN:0962-1083    

DOI:10.1111/j.1365-294X.1995.tb00254.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

ISSN:0962-1083    

DOI:10.1111/j.1365-294X.1995.tb00255.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

ISSN:0962-1083    

DOI:10.1111/j.1365-294X.1995.tb00256.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractOver the last decade, major advancements have occurred in the application of nucleic‐acid‐based methods to detect and determine the levels of catabolic genes in environmental samples. Studies have focused on validating methods in microcosms, studying changes in the structure and expression of microbial communities in response to contaminants, and improving the sensitivity of the methods. Only in the last few years have these methods transitioned from development and validation to efforts to apply these methods for monitoring in situ bioremediation. Methods that analyse nucleic acids extracted from environmental samples are of value to bioremediation because they allow analysis independent of the artefacts that can arise from laboratory biodegradative potential assays and laboratory culture‐based enumerations and from the inability to culture a large proportion of the micro‐organisms in the environment In theory, these methods enable a more comprehensive perspective, and a more defensible interpretation, of the microbial community response to intrinsic and engineered bioremediation processes. Results from the first studies applying nucleic‐acid‐based methods to intrinsic or engineered bioremediation indicate that these methods have both potential and limitations. The rapidly increasing number of cloned and sequenced catabolic genes, methodological advancements such as the ability to track specific micro‐organisms without prior sequence data, and the potential use of bioaugmentation in the field suggest that the utility of these methods forin situbioremediation will increase in the

ISSN:0962-1083    

DOI:10.1111/j.1365-294X.1995.tb00257.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractWe investigated the use of multiplex polymerase chain reaction (FCR) techniques coupled with Southern analysis to detect xenobiotic‐degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions ofPseudomonas putidamt‐2 (pWWO),P. oleovorans(OCT), andAlcaligenes eutrophusJMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions ofxylM(PCR product = 631 bp),alkB(546 bp) andtfdA(710 bp), which are found on pWWO, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide‐stained gels of the initial PCR reaction indicated detectable amplification of between 10* to 10* cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 μL) of the first reaction increased the ethidium bromide‐based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradarJve cells, at a lower detection limit of =>10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added targ

ISSN:0962-1083    

DOI:10.1111/j.1365-294X.1995.tb00258.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractHorizontal gene transfer in the Bacteria has been demonstrated to occur under natural conditions. The ecological impact of gene transfer events depends on the new genetic material being expressed in recipient organisms, and on natural selection processes operating on these recipients. The phylogenetic distribution ofcbaABgenes for chlorobenzoate 3,4‐(4,5)‐dioxygenase, which are carried withinTn5271 on the IncPβ plas‐mid pBRC60, was investigated using isolates from freshwater microcosms and from the Niagara River watershed. The latter included isolates from surface water, groundwater and bioremediation reactor samples. ThecbaABgenes have become integrated, through interspecific transfer, primarily into species of the β Proteobacteria (44/48 isolates). Only four isolates, identified asPseudomonas fluorescens(3/48) andXanthomonas maltophilia(1/48), belonged to the Γ Proteobacteria, despite the observation that pBRC60 was capable of mobilizing these genes into a wide range of β and Γ Proteobacteria in the laboratory. The natural host range correlated with the distribution of themeta‐ring‐fission pathway for metabolism of protocatechuates formed when thecbaABgenes were expressed (45/48 isolates). We proposed the hypothesis that natural selection has favoured recipients that successfully integrate the activity of the transferred dioxygenase with the conservedmetaring‐fission pathway. The hypothesis was tested by transferring a plasmid construct containing thecbaABgenes into type strains representative of the β and γ Proteobacteria. The concept of applying mobile catabolic genes to probe the phylogenetic distribution of compatible degradative pat

ISSN:0962-1083    

DOI:10.1111/j.1365-294X.1995.tb00259.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractIndividualmerRTΔPregions were amplified from DNA directly isolated from soil and sediment samples using consensus primers derived from the conservedmersequences ofTn501,Tn21and pMER419. Soil and sediment samples were taken from four sites in the British Isles; one ‘pristine’ (SB) and three polluted (SO, SE, T2) with respect to mercury. The sizes of the PCR products amplified (= 1 kb) were consistent with their generation frommerdeterminants related to the archetypal elements found in Gram negative bacteria. Forty‐five individual clones of sequences obtained from these four sites were isolated which hybridized (>70% homology) to amerRTΔPprobe from Tn501. The diversity of these amplified mer genes was analysed using Restriction Fragment Length Polymorphism (RFLP) profiling. Fourteen RFLP classes were distinguished, 12 of which proved to be novel and only two of which had been identified in an earlier study of 40 Gram negative mercury resistant bacteria cultured from the same four sites. UPGMA analysis was used to examine the relationships between the 22 classes of determinant identified. The T2 site, which has the longest history of mercury exposure, was found to have the greatest level of diversity in terms of numbers of classes of determinant, while the SO site, which had the highest mercury levels showed relatively low variation. Variation ofmergenes within and between the sequences from cultivated bacteria and from total bacterial DNA shows clearly that analysing only sequences from cultivated organisms results in a gross underestimation of genetic va

ISSN:0962-1083    

DOI:10.1111/j.1365-294X.1995.tb00260.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractTwo novel 3‐chlorobenzoate‐degrading bacteria were previously isolated from an aquifer in which no such bacteria could be enriched prior to the introduction of the 3‐chlorobenzoate‐degrading strain,Pseudomonassp. B13. To understand the origin of 3‐chlorobenzoate‐degrading genes in the two novel isolates, the 16S ribosomal RNA,clcD (dienelactone hydrolase) andclcA (chlorocatechol oxygenase) genes from these bacteria were amplified and sequenced. The partial 16S rRNA gene sequences and REP‐PCR patterns showed that these two novel isolates were identical but differed from strain B13. Phylogenetic analyses revealed that the novel isolates were closely related toAlcaligenes eutrophusin the beta subclass of theProteobacteria, whereas strain B13 was related toPseudomonas aeruginosaandP. mendocinain the gamma subclass of theProteobacteria.In contrast, theclcD andclcA gene sequences were identical on strain B13 and these two isolates, indicating that the 3‐chlorobenzoate‐degrading genes were transferred from strain B13 to these isolates. What cannot be established is when this

ISSN:0962-1083    

DOI:10.1111/j.1365-294X.1995.tb00261.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

AbstractThirty closely spaced cores were obtained from Miocene‐aged fluvial, lacustrine and palaeosol subsurface sediments ranging in depth from 173 to 197 m at a site in south‐central Washington to investigate the size and composition of the microbial community in relation to sediment geochemical and geophysical properties. Total phospholipid fatty acid (PLFA) analysis indicated that the greatest concentrations of microbial bio‐mass were in low‐permeability lacustrine sediments that also contained high concentrations of organic carbon. Community structure, based on lipid analyses and on in situ hybridization of bacterial cells with 16S RNA‐directed DNA probes, also revealed the presence of metabolically active bacteria that respire sulphate and/or Fe(III) in the lacustrine sediments. Concentrations of pore water sulphate were low (4–8 mg/L) and HCI‐extractable Fe was predominantly Fe(II) in the same samples where total biomass and organic carbon were highest. The low hydraulic conductivity (10‐6to<10‐9cm/s) of these sediments has likely contributed to the long term maintenance of both bacteria and organic carbon by limiting the supply of soluble electron acceptors for microbial respiration. These results suggest that the current subsurface microbial population was derived from organisms that were present during lake sedimentation = 6–

ISSN:0962-1083    

DOI:10.1111/j.1365-294X.1995.tb00262.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY