Genetic exchange in soil between introduced chlorobenzoate degraders and indigenous biphenyl degraders.

Pseudomonas aeruginosa JB2, a chlorobenzoate degrader, was inoculated into soil having indigenous biphenyl degraders but no identifiable 2-chlorobenzoate (2CBa) or 2,5-dichlorobenzoate (2,5DCBa) degraders. The absence of any indigenous chlorobenzoate degraders was noted by the failure to obtain enrichment cultures with the addition of 2CBa, 3CBa, or 2,5DCBa and by the failure of soil DNA to hybridize to the tfdC gene, which encodes ortho fission of chlorocatechols. In contrast, DNA extracted from inoculated soils hybridized to this probe. Bacteria able to utilize both biphenyl and 2CBa as growth substrates were absent in uninoculated soil, but their presence increased with time in the inoculated soils. This increase was related kinetically to the growth of biphenyl degraders. Pseudomonas sp. strain AW, a dominant biphenyl degrader, was selected as a possible parental strain. Eight of nine recombinant strains, chosen at random, had high phenotypic similarity (90% or more) to the inoculant; the other, strain JB2-M, had 78% similarity. Two hybrid strains, P. aeruginosa JB2-3 and Pseudomonas sp. JB2-M, were the most effective of all strains, including strain AW, in metabolizing polychlorinated biphenyls (Aroclor 1242). Repetitive extragenic palindromic-PCR analysis of putative parental strains JB2 and AW and the two recombinant strains JB2-3 and JB2-M showed similar fragments among the recombinants and JB2 but not AW. These results indicate that the bph genes were transferred to the chlorobenzoate-degrading inoculant from indigenous biphenyl degraders.     

A survey of the cellular responses in Pseudomonas putida KT2440 growing in sterilized soil by microarray analysis

Genome-wide scanning of gene expression by microarray techniques was successfully performed on RNA extracted from sterilized soil inoculated with Pseudomonas putida KT2440/pSL1, which contains a chloroaromatic degrading plasmid, in the presence or absence of 3-chlorobenzoic acid (3CB). The genes showing significant changes in their expression in both the triplicate-microarray analysis using amplified RNA and the single-microarray analysis using unamplified RNA were investigated. Pathway analysis revealed that the benzoate degradation pathway underwent the most significant changes following treatment with 3CB. Analysis based on categorization of differentially expressed genes against 3CB revealed new findings about the cellular responses of the bacteria to 3CB. Genes specifically involved in the transport of 3CB were upregulated, including a K(+)/H(+) antiporter complex, a universal stress protein, two cytochrome P450 proteins and an efflux transporter. The downregulated expression of several genes involved in carbon metabolism and the genes belonging to a prophage in the presence of 3CB was observed. This study demonstrated the applicability of the method of soil RNA extraction for microarray analysis of gene expression in bacteria growing in sterilized soil.     

Matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry- and MALDI biotyper-based identification of cultured biphenyl-metabolizing bacteria from contaminated horseradish rhizosphere soil

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.     

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.     

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.     

Cometabolic oxidation of polychlorinated biphenyls in soil with a surfactant-based field application vector.

Polychlorinated biphenyl (PCB)-degradative genes, under the control of a constitutive promoter, were cloned into a broad-host-range plasmid and a transposon. These constructs were inserted into a surfactant-utilizing strain, Pseudomonas putida IPL5, to create a field application vector (FAV) in which a surfactant-degrading organism cometabolizes PCB. By utilizing a surfactant not readily available to indigenous populations and a constitutive promoter, selective growth and PCB-degradative gene expression are decoupled from biphenyl. Since PCB degradation via the biphenyl degradation pathway is nonadaptive in the absence of biphenyl, there is no selective pressure for PCB gene maintenance. The recombinant strains exhibited degradative activity against 25 of 33 PCB congeners in Aroclor 1248 in the absence of biphenyl. Whole-cell enzyme assays indicated that PCB-degradative activity of a recombinant strain carrying the PCB genes on a plasmid was approximately twice that of the same strain carrying the PCB genes on a transposon. Plasmid loss rates in the absence of antibiotic selection averaged 7.4% per cell division and were highly variable between experiments. Surfactant-amended slurries of PCB-contaminated electric power plant substation soil were inoculated with approximately 10(5) recombinant cells per ml. The populations of the added strains increased to greater than 10(9) cells per ml in 2 days, and cell growth coincided with PCB degradation. By 15 days, 50 to 60% of the indicator congener 2,3,2',5'-tetrachlorobiphenyl was degraded. The effectiveness of PCB degradation by the plasmid-containing strain depended on plasmid stability. The transposon-encoded PCB genes were much more stable, and in surfactant-amended soil slurries, PCB degradation was more consistent between experiments.     

Isolation of lightning-competent soil bacteria

Artificial transformation is typically performed in the laboratory by using either a chemical (CaCl(2)) or an electrical (electroporation) method. However, laboratory-scale lightning has been shown recently to electrotransform Escherichia coli strain DH10B in soil. In this paper, we report on the isolation of two "lightning-competent" soil bacteria after direct electroporation of the Nycodenz bacterial ring extracted from prairie soil in the presence of the pBHCRec plasmid (Tc(r), Sp(r), Sm(r)). The electrotransformability of the isolated bacteria was measured both in vitro (by electroporation cuvette) and in situ (by lightning in soil microcosm) and then compared to those of E. coli DH10B and Pseudomonas fluorescens C7R12. The electrotransformation frequencies measured reached 10(-3) to 10(-4) by electroporation and 10(-4) to 10(-5) by simulated lightning, while no transformation was observed in the absence of electrical current. Two of the isolated lightning-competent soil bacteria were identified as Pseudomonas sp. strains.     

Development and Application of a New Method To Extract Bacterial DNA from Soil Based on Separation of Bacteria from Soil with Cation-Exchange Resin

A new method for the extraction of bacterial DNA from soil has been developed. Soil samples of 50 g were dispersed, and bacteria were released by use of a cation-exchange resin; subsequently, bacteria were separated from soil particles by low-speed centrifugation and lysed with lysozyme and ionic detergent, and the DNA was then purified by CsCl-ethidium bromide equilibrium density centrifugation. The extracted DNA was of high molecular weight and sufficiently pure for restriction enzyme digestion, DNA-DNA hybridization, and amplification by the polymerase chain reaction. The advantages of the new method are that the separation of bacteria from soil is considerably faster than by repeated blending, more samples can be handled, and furthermore no aerosols are formed during separation. Also, we investigated whether the CsCl-ethidium bromide equilibrium density centrifugation could be replaced by purification using Gene-Clean. However, this method produced DNAs which were insufficiently pure for several types of analysis. The new method was used to study survival of a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading Pseudomonas cepacia DBO1 (pRO101) in unamended soil and in soil amended with 2,4-D. We found that the degrading strain, irrespective of inoculation level, was able to grow to the same high numbers in soil amended with 2,4-D, while the strain in nonamended soil were maintained at the inoculation level. Detection based on DNA extraction and subsequent dot blot DNA-DNA hybridization was in accordance with detection by plating on selective medium.     

Changes in bacterial populations and in biphenyl dioxygenase gene diversity in a polychlorinated biphenyl-polluted soil after introduction of willow trees for rhizoremediation

The aim of this study was to analyze the structural and functional changes occurring in a polychlorinated-biphenyl (PCB)-contaminated soil ecosystem after the introduction of a suitable host plant for rhizoremediation (Salix viminalis). We have studied the populations and phylogenetic distribution of key bacterial groups (Alpha- and Betaproteobacteria, Acidobacteria, and Actinobacteria) and the genes encoding iron-sulfur protein alpha (ISPalpha) subunits of the toluene/biphenyl dioxygenases in soil and rhizosphere by screening gene libraries using temperature gradient gel electrophoresis. The results, based on the analysis of 415 clones grouped into 133 operational taxonomic units that were sequence analyzed (>128 kbp), show that the rhizospheric bacterial community which evolved from the native soil community during the development of the root system was distinct from the soil community for all groups studied except for the Actinobacteria. Proteobacteria were enriched in the rhizosphere and dominated both in rhizosphere and soil. There was a higher than expected abundance of Betaproteobacteria in the native and in the planted PCB-polluted soil. The ISPalpha sequences retrieved indicate a high degree of catabolic and phylogenetic diversity. Many sequences clustered with biphenyl dioxygenase sequences from gram-negative bacteria. A distinct cluster that was composed of sequences from this study, some previously described environmental sequences, and a putative ISPalpha from Sphingomonas wittichii RW1 seems to contain greater diversity than the presently recognized toluene/biphenyl dioxygenase subfamily. Moreover, the rhizosphere selected for two ISPalpha sequences that accounted for almost 60% of the gene library and were very similar to sequences harbored by Pseudomonas species.     

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.     

榕树根际土壤广谱拮抗菌株的筛选、鉴定及特性

于江西省吉安市内一株明万历年间植下的古榕树下,采集根际土样并使用23种指示菌通过管碟法和菌丝生长速率法对土壤内放线菌进行拮抗筛选,最终得到一株传代稳定的广谱拮抗菌株AHF-20.根据对菌株的形态观察、生理生化特性以及分子生物学鉴定,将该拮抗菌株鉴定为链霉菌,并对该菌株的抑菌活性物质进行了研究.结果表明:链霉菌AHF-20的发酵产物对23种测试指示菌全部具有拮抗效果,且抑菌能力稳定性较好,对温度、光照、紫外线、酸碱都有一定的耐受性,于121 ℃下加热20 min后依然存在抑菌活性.根据活性物质的极性使用正丁醇萃取发酵产物,获得的正丁醇粗提物稀释至1 μg·mL^-1后,对大肠杆菌仍有抑制效果,可见具有较好的生防利用潜力和开发成新型微生物药物的可能性.     

Enhanced mineralization of polychlorinated biphenyls in soil inoculated with chlorobenzoate-degrading bacteria.

An Altamont soil containing no measurable population of chlorobenzoate utilizers was examined for the potential to enhance polychlorinated biphenyl (PCB) mineralization by inoculation with chlorobenzoate utilizers, a biphenyl utilizer, combinations of the two physiological types, and chlorobiphenyl-mineralizing transconjugants. Biphenyl was added to all soils, and biodegradation of 14C-Aroclor 1242 was assessed by disappearance of that substance and by production of 14CO2. Mineralization of PCBs was consistently greatest (up to 25.5%) in soils inoculated with chlorobenzoate degraders alone. Mineralization was significantly lower in soils receiving all other treatments: PCB cometabolizer (10.7%); chlorobiphenyl mineralizers (8.7 and 14.9%); and mixed inocula of PCB cometabolizers and chlorobenzoate utilizers (11.4 and 18.0%). However, all inoculated soils had higher mineralization than did the uninoculated control (3.1%). PCB disappearance followed trends similar to that observed with the mineralization data, with the greatest degradation occurring in soils inoculated with the chlorobenzoate-degrading strains Pseudomonas aeruginosa JB2 and Pseudomonas putida P111 alone. While the mechanism by which the introduction of chlorobenzoate degraders alone enhanced biodegradation of PCBs could not be elucidated, the possibility that chlorobenzoate inoculants acquired the ability to metabolize biphenyl and possibly PCBs was explored. When strain JB2, which does not utilize biphenyl, was inoculated into soil containing biphenyl and Aroclor 1242, the frequency of isolates able to utilize biphenyl and 2,5-dichlorobenzoate increased progressively with time from 3.3 to 44.4% between 15 and 48 days, respectively. Since this soil contained no measurable level of chlorobenzoate utilizers yet did contain a population of biphenyl utilizers, the possibility of genetic transfer between the latter group and strain JB2 cannot be excluded.     

Use of exogenous specialised bacteria in the biological detoxification of a dump site‐polychlorobiphenyl‐contaminated soil in slurry phase conditions

The possibility of biologically detoxifying a contaminated soil from an Italian dump site containing about 1500 mg/kg (in dry soil) of polychlorinated biphenyls was studied in the laboratory in this work. The soil, which contained indigenous aerobic bacteria capable of growing on biphenyl or on monochlorobenzoic acids at concentration of about 300 CFU per g of air‐dried soil, was amended with inorganic nutrients, saturated with water and treated in aerobic 3‐L batch slurry reactors (soil suspension at 20% w/v). Either Pseudomonas sp. CPE1 strain, capable of cometabolising low‐chlorinated biphenyls into chlorobenzoic acids, or a bacterial co‐culture capable of aerobically dechlorinating polychlorobiphenyls constituted by this bacterium and the two chlorobenzoic acid degrading bacteria Pseudomonas sp. CPE2 strain and Alcaligenes sp. CPE3 strain, were used as inocula (final concentration of about 10⁸ CFU/mL for each bacterium), in the absence and in the presence of biphenyl (4 g/kg of air dried soil). Significant soil polychlorobiphenyl depletions were observed in all the reactors after 119 days of treatment. The soil inoculation with the sole CPE1 was found to slightly enhance the polychlorobiphenyl depletions (about 20%) and the soil detoxification; the effect was higher in the presence of biphenyl. The use of the polychlorobiphenyl mineralising bacterial co‐culture as inoculum resulted in a strong enhancement of the depletions of both the soil polychlorobiphenyls (from 50 to 65%) and of the original soil ecotoxicity. The bacterial biomass inoculated was found to implant into the soil; the higher specialised biomass availability thus reached in the inoculated soil was probably responsible of a more extensive biodegradation of polychlorobiphenyls and therefore of the higher detoxification yields observed in the inoculated reactors. The soil ecotoxicity, measured through two different soil contact assays, i.e., the Lepidium sativum germination test and the Collembola mortality test, was often found to decrease proportionally with the soil polychlorobiphenyl concentration. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 240–249, 1999.     

Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes

Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif⁺-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10⁴ cells.     

Identification of a toluene-degrading bacterium from a soil sample through H(2)(18)O DNA stable isotope probing

DNA stable isotope probing (DNA-SIP) with H(2)(18)O was used to identify a toluene-degrading bacterium in soil amended with 48 ppm toluene. After quantification of toluene degradation rates in soil, DNA was extracted from soil incubated with H(2)(18)O, H(2)(16)O, H(2)(16)O and 48 ppm toluene, or H(2)(18)O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H(2)(16)O. With extracts from soils to which only H(2)(18)O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H(2)(18)O and toluene was analyzed. We suggest that this third band formed because toluene does not contain any oxygen atoms and toluene-degrading organisms had to transfer oxygen atoms from H(2)(18)O into metabolic intermediates to form nucleic acids de novo. We extracted the third DNA band and amplified a large fraction of the bacterial 16S rRNA gene. Direct sequencing of the PCR product obtained from the labeled DNA, as well as cloned 16S rRNA amplicons, identified a known toluene degrader, Rhodococcus jostii RHA1. A toluene-degrading bacterial strain was subsequently isolated from soil and shown to be Rhodococcus jostii RHA1. Finally, quantitative real-time PCR analysis showed that the abundance of the 16S rRNA gene of Rhodococcus jostii RHA1 increased in soil after toluene exposure but not in soils from which toluene was withheld. This study indicates that H(2)(18)O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.