Biomedical subjects

L J Forney

Publications and source records attributed to L J Forney.

At least 19 recordsLinked to original sources

PubMed2006

Microbial community structure in three deep-sea carbonate crusts.

Carbonate crusts in marine environments can act as sinks for carbon dioxide. Therefore, understanding carbonate crust formation could be important for understanding global warming. In the present study, the microbial communities of three carbonate crust samples from deep-sea mud volcanoes in the eastern Mediterranean were characterized by sequencing 16S ribosomal RNA (rRNA) genes amplified from DNA directly retrieved from the samples. In combination with the mineralogical composition of the crusts and lipid analyses, sequence data were used to assess the possible role of prokaryotes in crust formation. Collectively, the obtained data showed the presence of highly diverse communities, which were distinct in each of the carbonate crusts studied. Bacterial 16S rRNA gene sequences were found in all crusts and the majority was classified as alpha-, gamma-, and delta- Proteobacteria. Interestingly, sequences of Proteobacteria related to Halomonas and Halovibrio sp., which can play an active role in carbonate mineral formation, were present in all crusts. Archaeal 16S rRNA gene sequences were retrieved from two of the crusts studied. Several of those were closely related to archaeal sequences of organisms that have previously been linked to the anaerobic oxidation of methane (AOM). However, the majority of archaeal sequences were not related to sequences of organisms known to be involved in AOM. In combination with the strongly negative delta 13C values of archaeal lipids, these results open the possibility that organisms with a role in AOM may be more diverse within the Archaea than previously suggested. Different communities found in the crusts could carry out similar processes that might play a role in carbonate crust formation.

Anaerobiosis↗

PubMed2004

Analysis of diversity among 3-chlorobenzoate-degrading strains of Rhodopseudomonas palustris.

The phenotypic and genetic characteristics of 14 strains of the purple nonsulfur bacterium Rhodopseudomonas palustris were studied to assess diversity within this species. While all strains had certain phenotypic characteristics in common, including the ability to metabolize benzoate and degrade 2- and 3-chlorobenzoate, there were also significant differences among the strains such as the rate of growth in media containing benzoate as a carbon source. Genetic characterization of the strains revealed there were three divergent lineages in the species. Based on 16S rRNA gene sequences, the 14 strains could be grouped into three distinct clusters (A, B, and C), and this clustering was congruent with that based on gene sequences of form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Although BOX-PCR genomic DNA fingerprints of all 14 strains exhibited differences, analysis of the fingerprint images and UPGMA/product-moment analysis of similarities showed there were three groupings that were entirely consistent with clusters based on other characteristics of the strains. Thus, regardless of the method of analysis used, strains in groups A and B consistently clustered together and were separate from those of group C. These results suggest that strains in groups A-B and C represent phylogenetically related clones that have diverged from one another. This indicates that at least three lineages of Rhodopseudomonas palustris exist among the strains included in this study, and that each may be particularly well adapted to a distinct ecological niche.

Base Sequence↗

PubMed2004

Juice irradiation with Taylor-Couette flow: UV inactivation of Escherichia coli.

A novel reactor is described with flow characteristics that approach that of ideal plug flow but with a residence time that is uncoupled from the hydrodynamics or boundary layer characteristics. The design described consists of an inner cylinder that rotates within a stationary but larger outer cylinder. At low rotation rates, a laminar, hydrodynamic configuration called Taylor-Couette flow is established, which consists of a system of circumferential vortices within the annular fluid gap. The latter constitutes a spatially periodic flow that is the hydrodynamic equivalent to cross flow over a tube bank or lamp array. These vortices provide radial mixing, reduce the boundary layer thickness, and are independent of the axial flow rate and thus the fluid residence time. An additional feature of the rotating design is the repetitive exposure of the fluid parcels to a minimum number of lamps, which substantially reduces the maintenance requirements. Inactivation data for Escherichia coli (ATCC 15597) were recorded in commercial apple and grape juice that are relatively opaque to UV radiation. With initial E. coli concentrations of approximately 10(6) CFU/ml, Taylor-Couette flow was found to provide a 3- to 5-log improvement in the inactivation efficiency compared with simple channel flow between concentric cylinders.

Beverages↗

PubMed2003

Ultraviolet disinfection: similitude in Taylor-Couette and channel flow.

The inactivation data for Escherichia coli are recorded for the three reactor geometries of Taylor-Couette flow and flow between either concentric cylinders or a square channel. All of the data are shown to be correlated with the assumption of plug flow. In particular, the effects of nonuniform radiation levels are accounted for by integration across the fluid channel as done previously. However, a new correction factor is introduced that is shown to be inversely proportional to the laminar, velocity boundary thickness to account for the effects of a concentration boundary layer of surviving pathogen. It has also been demonstrated that the common problems of nonuniform radiation levels and concentration boundary layer effects in UV reactors are largely eliminated with the use of Taylor-Couette flow. Moreover, the repetitive exposure of fluid parcels to a small number of lamps in the rotating Taylor-Couette flow decreases maintainance requirements compared to the hydrodynamic equivalent of cross-flow over a tube bank or lamp array. Over a 3-log reduction in the inactivation of E. coli was demonstrated compared to a conventional channel with the same radiation dosage. Moreover, greater than a 2-log reduction was evident compared to flow through concentric cylinders.

Disinfection↗

PubMed2003

The 64 508 bp IncP-1beta antibiotic multiresistance plasmid pB10 isolated from a waste-water treatment plant provides evidence for recombination between members of different branches of the IncP-1beta group.

The complete 64508 bp nucleotide sequence of the IncP-1beta antibiotic-resistance plasmid pB10, which was isolated from a waste-water treatment plant in Germany and mediates resistance against the antimicrobial agents amoxicillin, streptomycin, sulfonamides and tetracycline and against mercury ions, was determined and analysed. A typical class 1 integron with completely conserved 5' and 3' segments is inserted between the tra and trb regions. The two mobile gene cassettes of this integron encode a beta-lactamase of the oxacillin-hydrolysing type (Oxa-2) and a gene product of unknown function (OrfE-like), respectively. The pB10-specific gene load present between the replication module (trfA1) and the origin of vegetative replication (oriV) is composed of four class II (Tn3 family) transposable elements: (i). a Tn501-like mercury-resistance (mer) transposon downstream of the trfA1 gene, (ii). a truncated derivative of the widespread streptomycin-resistance transposon Tn5393c, (iii). the insertion sequence element IS1071 and (iv). a Tn1721-like transposon that contains the tetracycline-resistance genes tetA and tetR. A very similar Tn501-like mer transposon is present in the same target site of the IncP-1beta degradative plasmid pJP4 and the IncP-1beta resistance plasmid R906, suggesting that pB10, R906 and pJP4 are derivatives of a common ancestor. Interestingly, large parts of the predicted pB10 restriction map, except for the tetracycline-resistance determinant, are identical to that of R906. It thus appears that plasmid pB10 acquired as many as five resistance genes via three transposons and one integron, which it may rapidly spread among bacterial populations given its high promiscuity. Comparison of the pB10 backbone DNA sequences with those of other sequenced IncP-1beta plasmids reveals a mosaic structure. While the conjugative transfer modules (trb and tra regions) and the replication module are very closely related to the corresponding segments of the IncP-1beta resistance plasmid R751 and even more similar to the IncP-1beta degradative plasmids pTSA and pADP-1, the stable inheritance operons klcAB-korC and kleAEF are most similar to those of the IncP-1beta resistance plasmid pB4, and clearly less similar to the other IncP-1beta plasmids. This suggests that IncP-1beta plasmids can undergo recombination in the environment, which may enhance plasmid diversity and bacterial adaptability.

Anti-Bacterial Agents↗

PubMed2003

Spatial distribution of Rhodopseudomonas palustris ecotypes on a local scale.

The number, spatial distribution, and significance of genetically distinguishable ecotypes of prokaryotes in the environment are poorly understood. Oda et al. (Y. Oda, B. Star, L. A. Huisman, J. C. Gottschal, and L. J. Forney, Appl. Environ. Microbiol. 69:xxx-xxx, 2003) have shown that Rhodopseudomonas palustris ecotypes were lognormally distributed along a 10-m transect and that multiple strains of the species could coexist in 0.5-g sediment samples. To extend these observations, we investigated the clonal diversity of R. palustris in 0.5-g samples taken from the corners and center of a 1-m square. A total of 35 or 36 clones were recovered by direct plating from each sample and were characterized by BOX A1R repetitive element-PCR genomic DNA fingerprinting. Isolates with fingerprint images that were >/=80% similar to each other were defined as the same genotype. Among the 178 isolates studied, 32 genotypes were identified, and each genotype contained between 1 and 40 isolates. These clusters were consistent with minor variations found in 16S rRNA gene sequences. The Shannon indices of the genotypic diversity within each location ranged from 1.08 (5 genotypes) to 2.18 (13 genotypes). Comparison of the rank abundance of genotypes found in pairs of locations showed that strains from three locations were similar to each other, with Morisita-Horn similarity coefficients ranging from 0.59 to 0.71. All comparisons involving the remaining two locations resulted in coefficients between 0 and 0.12. From these results we inferred that the patterns of ecotype diversity at the sampling site are patchy at a 1-m scale and postulated that factors such as mixing, competitive interactions, and microhabitat variability are likely to be responsible for the maintenance of the similarities between some locations and the differences between others.

DNA Fingerprinting↗

PubMed2001

Structure of microbial communities in activated sludge: potential implications for assessing the biodegradability of chemicals.

Various methods used to assess the biodegradability of chemicals often employ activated sludge as an inoculum since chemicals that ultimately enter the environment are often discharged through wastewater. Differences in the structure and function of activated sludge microbial communities that may complicate interpretation of biodegradation tests could arise from differences in wastewater composition, wastewater treatment plant (WWTP) operation, or manipulations done after collection of the activated sludge. In this study, various methods were used to characterize the structure of microbial communities found in freshly collected activated sludge from WWTPs in Japan, Europe, and the United States, as well as sludge that had been continuously fed either sewage or a glucose-peptone mixture for several weeks after collection. Comparisons of biomass levels, whole-community substrate utilization (determined using Biolog GN and GP plates), and phospholipid fatty acid (PLFA) profiles indicated there were both geographical and temporal differences among freshly collected activated sludge samples. Moreover, marked shifts in the structure of activated sludge microbial communities occurred upon continuous cultivation in the laboratory for 5 weeks using a glucose-peptone feed. These shifts were evident from whole-community substrate utilization and PLFA profiles as well as differences in the profiles of 16S rDNA genes from numerically dominant populations obtained by denaturing gradient gel electrophoresis and terminal restriction fragment analyses. Further studies are needed to better define the variability within and between activated sludge from wastewater treatment plants and laboratory reactors and to assess the impact of such differences on the outcome of biodegradability tests.

Bacteria↗

PubMed2001

Characterization and distribution of esterase activity in activated sludge.

The location and activity of esterase enzymes in activated sludge from three municipal wastewater treatment plants were characterized using model substrates and denaturing and non-denaturing polyacrylamide gel electrophoresis (PAGE) of particulate, freeze-thaw (primarily periplasmic enzymes and those associated with outer cell surfaces) and extracellular fractions of activated sludge bacteria. Particulate and freeze-thaw fractions had a similar spectrum of substrate specificity and contained significant levels of protein and esterase activity against model substrates, C2-C18 monoesters of p-nitrophenol and C2-C8 diesters of fluorescein. Esterase activity was highest with substrates that had short alkyl chains (C4) and decreased as the chain lengths increased beyond C8. Extracellular fractions contained very low levels of protein (<0.1 mg/l) and showed no esterase activity against any of the model substrates tested. Multiple bands were observed upon analysis of particulate and freeze-thaw fractions by non-denaturing PAGE in combination with activity staining using various alpha-naphthol ester substrates (C2-C8). Our results indicate that esterase enzymes in activated sludge are fairly diverse from a structural standpoint but exhibit a high level of functional redundancy, with different enzymes catalyzing the same reactions in different sludges. Extracellular esterase activity was totally absent for the substrates we tested and the esterase activity that we observed was closely linked to a particulate floc or cellular material.

Electrophoresis, Polyacrylamide Gel↗

PubMed1999

Detection of methanogenic archaea in seawater particles and the digestive tract of a marine fish species.

A methanogen-specific nested PCR approach was used to detect methanogenic archaea in seawater particles of the North Sea and the feces and the digestive tract of flounder (Platichthys flesus), a fish found in the North Sea. A number of 16S rDNA sequences with 97.6-99.5% similarity to Methanococcoides methylutens were found in the seawater particles as well as the digestive tract and fecal samples.

Animals↗

PubMed1999

Degradation of 3-chlorobenzoate under low-oxygen conditions in pure and mixed cultures of the anoxygenic photoheterotroph Rhodopseudomonas palustris DCP3 and an aerobic Alcaligenes species.

The presence or absence of molecular oxygen has been shown to play a crucial role in the degradability of haloaromatic compounds. In the present study, it was shown that anaerobic phototrophic 3-chlorobenzoate (3CBA) metabolism by Rhodopseudomonas palustris DCP3 is oxygen tolerant up to a concentration of 3 microM O2. Simultaneous oxidation of an additional carbon source permitted light-dependent anaerobic 3CBA degradation at oxygen input levels which, in the absence of such an additional compound, would result in inhibition of light-dependent dehalogenation. Experiments under the same experimental conditions with strain DCP3 in coculture with an aerobic 3CBA-utilizing heterotroph, Alcaligenes sp. strain L6, revealed that light-dependent dehalogenation of 3CBA did not occur. Under both oxygen limitation (O2 < 0.1 microM) and low oxygen concentrations (3 microM O2), all the 3CBA was metabolized by the aerobic heterotroph. These data suggest that biodegradation of (halo)aromatics by photoheterotrophic bacteria such as R. palustris DCP3 may be restricted to anoxic photic environments.

Aerobiosis↗

PubMed1998

Enhancement of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation in soil by dissemination of catabolic plasmids.

Few studies have been done to evaluate the transfer of catabolic plasmids from an introduced donor strain to indigenous microbial populations as a means to remediate contaminated soils. In this work we determined the effect of the conjugative transfer of two 2,4-D degradative plasmids to indigenous soil bacterial populations on the rate of 2,4-D degradation in soil. We also assessed the influence of the presence of 2,4-D on the number of transconjugants formed. The two plasmids used, pEMT1k and pEMT3k, encode 2,4-D degradative genes (tfd) that differ in DNA sequence as well as gene organisation, and confer different growth rates to Ralstonia eutropha JMP228 when grown with 2,4-D as a sole carbon source. In an agricultural soil (Ardoyen) treated with 2,4-D (100 ppm) there were ca. 10(7) CFU of transconjugants per gram bearing pEMT1k as well as a high number of pEMT3k bearing transconjugants (ca 10(6) CFU/g). In this soil the formation of a high number of 2,4-D degrading transconjugants resulted in faster degradation of 2,4-D as compared to the uninoculated control soil. In contrast, only transconjugants with pEMT1k were detected (at a level of ca. 10(3) CFU/g soil) in the untreated Ardoyen soil. High numbers of transconjugants that carried pEMT1k were also found in a second experiment done using forest soil (Lembeke) treated with 100 ppm 2,4-D. However, unlike in the Ardoyen soil, no transconjugants with pEMT3k were detected and the transfer of plasmid pEMT1k to indigenous bacteria did not result in a higher rate of decrease of 2,4-D. This may be because 2,4-D was readily metabolised by indigenous bacteria in this soil. The results indicate that bioaugmentation with catabolic plasmids may be a viable means to enhance the bioremediation of soils which lack an adequate intrinsic ability to degrade a given xenobiotic.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1998

Association of marine archaea with the digestive tracts of two marine fish species.

Recent studies have shown that archaea which were always thought to live under strict anoxic or extreme environmental conditions are also present in cold, oxygenated seawater, soils, the digestive tract of a holothurian deep-sea-deposit feeder, and a marine sponge. In this study, we show, by using PCR-mediated screening in other marine eukaryotes, that marine archaea are also present in the digestive tracts of flounder and grey mullet, two fish species common in the North Sea, in fecal samples of flounder, and in suspended particulate matter of the North Sea water column. No marine archaea could be detected in the digestive tracts of mussels or the fecal pellets of a copepod species. The archaeal 16S ribosomal DNA clone libraries of feces of flounder and the contents of the digestive tracts of grey mullet and flounder were dominated by group II marine archaea. The marine archaeal clones derived from flounder and grey mullet digestive tracts and feces formed a distinct cluster within the group II marine archaea, with 76.7 to 89. 8% similarity to previously described group II clones. Fingerprinting of the archaeal community of flounder digestive tract contents and feces by terminal restriction fragment length polymorphism of archaeal 16S rRNA genes after restriction with HhaI showed a dominant fragment at 249 bp, which is likely to be derived from group II marine archaea. Clones of marine archaea that were closely related to the fish-associated marine archaea clones were obtained from suspended particulate matter of the water column at two stations in the North Sea. Terminal restriction fragment length polymorphism fingerprinting of the archaeal community present in suspended particulate matter showed the same fragment pattern as was found for the archaeal community of the flounder digestive tract contents and feces. These data demonstrate that marine archaea are present in the digestive tracts and feces of very common marine fish. It is possible that the marine archaea associated with the digestive tracts of marine fish are liberated into the water column through the feces and subsequently contribute to the marine archaeal community of suspended particulate matter.

Animals↗

PubMed1998

Parallel and divergent genotypic evolution in experimental populations of Ralstonia sp.

Genetic rearrangements within a population of bacteria were analyzed to understand the degree of divergence occurring after experimental evolution. We used 18 replicate populations founded from Ralstonia sp. strain TFD41 that had been propagated for 1,000 generations with 2,4-dichlorophenoxyacetic acid (2,4-D) as the carbon source. Genetic divergence was examined by restriction fragment length polymorphism analysis of the incumbent plasmid that carries the 2,4-D catabolic genes and by amplification of random regions of the genome via PCR. In 18 evolved clones examined, we observed duplication within the plasmid, including the tfdA gene, which encodes a 2,4-D dioxygenase that catalyzes the first step in the 2,4-D catabolic pathway. In 71 of 72 evolved clones, a common 2.4-kb PCR product was lost when genomic fingerprints produced by PCR amplification using degenerate primers based on repetitive extragenic palindromic (REP) sequences (REP-PCR) were compared. The nucleotide sequence of the 2.4-kb PCR product has homology to the TRAP (tripartite ATP-independent periplasmic) solute transporter gene family. Hybridization of the 2. 4-kb REP-PCR product from the ancestor to genomic DNA from the evolved populations showed that the loss of the PCR product resulted from deletions in the genome. Deletions in the plasmid and presence and/or absence of other REP-PCR products were also found in these clones but at much lower frequencies. The common and uncommon genetic changes observed show that both parallel and divergent genotypic evolution occurred in replicate populations of this bacterium.

Chlorophenols↗

PubMed1997

Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA.

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.

DNA, Bacterial↗

PubMed1997

Microbial Succession during a Field Evaluation of Phenol and Toluene as the Primary Substrates for Trichloroethene Cometabolism.

Microbial community composition and succession were studied in an aquifer that was amended with phenol, toluene, and chlorinated aliphatic hydrocarbons to evaluate the effectiveness of these aromatic substrates for stimulating trichloroethene (TCE) bioremediation. Samples were taken after the previous year's field studies, which used phenol as the primary substrate, and after three successive monthly treatments of phenol plus 1,1-dichloroethene (1,1-DCE) plus TCE, phenol plus TCE, and toluene plus TCE. Dominant eubacteria in the community were assessed after each of the four treatments by characterizing isolates from the most dilute most-probable-number tubes and by extracting DNA from aquifer samples. The succession of dominant phenol- and toluene-degrading strains was evaluated by genomic fingerprinting, cellular fatty acid methyl ester (FAME) analysis, and amplified ribosomal DNA restriction analysis (ARDRA). 1,1-DCE was found to drastically reduce microbial growth and species richness, which corresponded to the reduction in bioremediation effectiveness noted previously for this treatment (G. D. Hopkins and P. L. McCarty, Environ. Sci. Technol. 29:1628-1637, 1995). Only a few gram-positive isolates could be obtained after treatment with 1,1-DCE, and these were not seen after any other treatments. Microbial densities returned to their original levels following the subsequent phenol-TCE treatment, but the original species richness was not restored until after the subsequent toluene-TCE treatment. Genomic fingerprinting and FAME analysis indicated that six of the seven originally dominant microbial groups were still dominant after the last treatment, indicating that the community is quite resilient to toxic disturbance by 1,1-DCE. FAME analysis indicated that six microbial taxa were dominant: three members of the (beta) subclass of the class Proteobacteria (Comamonas-Variovorax, Azoarcus, and Burkholderia) and three gram-positive groups (Bacillus, Nocardia, and an unidentified group). ARDRA revealed that the dominant community members were stable during the three nontoxic treatments and that virtually all of the bands could be accounted for by isolates from five of the dominant taxa, indicating that the isolation protocol used likely recovered most of the dominant members of this community.

Journal Article↗

PubMed1997

Phenol- and toluene-degrading microbial populations from an aquifer in which successful trichloroethene cometabolism occurred.

We characterized the bacterial populations that grew in a Moffett Field, Calif., aquifer following three sequential field tests of phenol- or toluene-driven cometabolism of trichloroethene (TCE). Reducing the toluene and phenol concentrations in most-probable-number (MPN) tubes from 50 to 5 ppm increased the population density measured for these degraders by 1.5 and 1 log units, respectively, suggesting that natural populations might be quite sensitive to these substrates. Phenol and toluene degraders were isolated from the terminal MPN dilution tubes; 63 genetically distinct strains were identified among the 273 phenol- and toluene-degrading isolates obtained. TCE was cometabolized by 60% of the genetically distinct strains. Most strains (57%) grew on both phenol and toluene, and 78% of these strains hybridized to the toluene ortho-monooxygenase (TOM) probe. None of the strains hybridized to probes from the four other toluene oxygenase pathways. Gram-positive strains comprised 30% of the collection; all of these grew on phenol, and 47% of them also grew on toluene, but none hybridized to the TOM probe. Among the gram-negative strains, 86% of those that grew on both toluene and phenol hybridized to the TOM probe, while only 5% of those that were TOM-positive grew on toluene alone. A larger proportion of TCE degraders was found among gram-negative than gram-positive strains and among organisms that grew on phenol than those that grew on toluene. Hybridization of strains to the TOM probe was somewhat predictive of their TCE-cometabolizing ability, especially for strains isolated on toluene, but there was also a significant number (20%) of strains that hybridized to the TOM probe but were poor TCE cooxidizers. No Moffett Field isolates were as effective as Burkholderia cepacia G4 in cooxidizing TCE. Most of the aquifer strains ranged from moderately effective to ineffective in TCE cooxidation. Such populations, however, apparently accounted for the successful phenol- and toluene-stimulated TCE removal that occurred during the field assessment of this remediation process. This suggests that naturally occurring communities of only moderate TCE-cooxidizing ability may support successful TCE bioremediation as long as the phenol or toluene present is not limiting. This activity, however, may not be sustainable for the long term, because TCE-inactive populations that consumed toluene at rates equal to that of the best TCE degraders were present and hence would be expected to eventually dominate the community.

Journal Article↗

PubMed1997

Pristine environments harbor a new group of oligotrophic 2,4-dichlorophenoxyacetic acid-degrading bacteria.

2,4-Dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated from pristine environments which had no history of 2,4-D exposure. By using 2,4-D dye indicator medium or 14C-labeled 2,4-D medium, six strains were isolated from eight enrichment cultures capable of degrading 2,4-D. Phylogenetic analyses based on 16S ribosomal DNA (rDNA) sequencing and physiological properties revealed that one isolate from Hawaiian volcanic soil could be classified in the genus Variovorax (a member of the beta subdivision of the class Proteobacteria) and that the other five isolates from Hawaiian volcanic soils, Saskatchewan forest soil, and Chilean forest soil have 16S rDNAs with high degrees of similarity to those of the Bradyrhizobium group (a member of the alpha subdivision of the class Proteobacteria). All the isolates grow slowly on either nutrient media (0.1 x Bacto Peptone-tryptone-yeast extract-glucose [PTYG] or 0.1 x Luria broth [LB] medium) or 2,4-D medium, with mean generation times of 16 to 30 h, which are significantly slower than previously known 2,4-D degraders. Nutrient-rich media such as full-strength PTYG and LB medium did not allow their growth. PCR amplification using internal consensus sequences of tfdA (a gene encoding an enzyme for the first step of 2,4-D mineralization, found in pJP4 of Alcaligenes eutrophus JMP134 and some other 2,4-D-degrading bacteria) as primers and Southern hybridization with pJP4-tfdA as a probe revealed that the isolate belonging to the genus Variovorax carried the tfdA gene. This gene was transmissible to A. eutrophus JMP228 carrying a plasmid with a mutant tfdA gene. The other five isolates did not appear to carry tfdA, and 2,4-D-specific alpha-ketoglutarate-dependent dioxygenase activity could not be detected in cell lysates. These results indicate that 2,4-D-degrading bacteria in pristine environments are slow-growing bacteria and that most of their phylogenies and catabolic genes differ from those of 2,4-D degraders typically isolated from agricultural soils or contaminated environments.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1997

A novel means to develop strain-specific DNA probes for detecting bacteria in the environment.

A simple means to develop strain-specific DNA probes for use in monitoring the movement and survival of bacteria in natural and laboratory ecosystems was developed. The method employed amplification of genomic DNA via repetitive sequence-based PCR (rep-PCR) using primers specific for repetitive extragenic palindromic (REP) elements, followed by cloning of the amplified fragments. The cloned fragments were screened to identify those which were strain specific, and these were used as probes for total genomic DNA isolated from microbial communities and subjected to rep-PCR. To evaluate the utility of the approach, we developed probes specific for Burkholderia cepacia G4 and used them to determine the persistence of the strain in aquifer sediment microcosms following bioaugmentation. Two of four probes tested were found to specifically hybridize to DNA fragments of the expected sizes in the rep-PCR fingerprint of B. cepacia G4 but not to 64 genetically distinct bacteria previously isolated from the aquifer. One of these probes, a 650-bp fragment, produced a hybridization signal when as few as 10 CFU of B. cepacia G4 were present in a mixture with 10(6) CFU nontarget strains, indicating that the sensitivity of these probes was comparable to those of other PCR-based detection methods. The probes were used to discriminate groundwater and microcosm samples that contained B. cepacia G4 from those which did not. False-positive results were obtained with a few samples, but these were readily identified by using hybridization to the second probe as a confirmation step. The general applicability of the method was demonstrated by constructing probes specific to three other environmental isolates.

Bacteria↗