Modeling of phenoxy acid herbicide mineralization and growth of microbial degraders in 15 soils monitored by quantitative real-time PCR of the functional tfdA gene

Mineralization potentials, rates, and kinetics of the three phenoxy acid (PA) herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP), were investigated and compared in 15 soils collected from five continents. The mineralization patterns were fitted by zero/linear or exponential growth forms of the three-half-order models and by logarithmic (log), first-order, or zero-order kinetic models. Prior and subsequent to the mineralization event, tfdA genes were quantified using real-time PCR to estimate the genetic potential for degrading PA in the soils. In 25 of the 45 mineralization scenarios, ～60% mineralization was observed within 118 days. Elevated concentrations of tfdA in the range 1 × 10(5) to 5 × 10(7) gene copies g(-1) of soil were observed in soils where mineralization could be described by using growth-linked kinetic models. A clear trend was observed that the mineralization rates of the three PAs occurred in the order 2,4-D > MCPA > MCPP, and a correlation was observed between rapid mineralization and soils exposed to PA previously. Finally, for 2,4-D mineralization, all seven mineralization patterns which were best fitted by the exponential model yielded a higher tfdA gene potential after mineralization had occurred than the three mineralization patterns best fitted by the Lin model.     

Transcription dynamics of the functional tfdA gene during MCPA herbicide degradation by Cupriavidus necator AEO106 (pRO101) in agricultural soil

A modified protocol for simultaneous extraction of RNA and DNA, followed by real-time polymerase chain reaction quantification, was used to investigate tfdA gene expression during in situ degradation of the herbicide MCPA (4-chloro-2-methylphenoxy-acetic acid) in soil. tfdA encodes an alpha-ketoglutarate-dependent dioxygenase catalysing the first step in the degradation pathway of MCPA and 2,4-D (2,4-dichlorophenoxy-acetic acid). A linear recovery of tfdA mRNA over three orders of magnitude was shown, and the tfdA mRNA level was normalized using the tfdA mRNA/DNA ratio. The density of active cells required for tfdA mRNA detection was 10(5) cells g(-1) soil. Natural soil microcosms inoculated with Cupriavidus necator (formerly Ralstonia eutropha) AEO106 (pRO101) cells were amended with four different MCPA concentrations (2, 20, 50 and 150 mg kg(-1)). Mineralization rates were estimated by quantification of 14CO2 emission from degradation of 14C-MCPA. tfdA mRNA was detected 1 h after amendment at all four concentrations. In soils amended with 2 and 20 mg kg(-1), the mRNA/DNA ratio for tfdA demonstrated a sharp transient maximum of tfdA expression from no to full expression within 3 and 6 h respectively, followed by a decline and complete loss of expression after 19 and 43 h. A more complex pattern of tfdA expression was observed for the higher 50 and 150 mg kg(-1) amendments; this coincided with growth of C. necator AEO106 (pRO101) in the system. Repeated amendment with MCPA after 2 weeks in the 20 mg kg(-1) scenario revealed a sharp increase of tfdA mRNA, and absence of a mineralization lag phase. For all amendments, tfdA mRNA was detectable only during active mineralization, and thus revealed a direct correlation between tfdA mRNA presence and microbial degrader activity. The present study demonstrates that direct analysis of functional gene expression dynamics by quantification of mRNA can indeed be made in natural soil.     

Comparison of 16S rRNA gene phylogeny and functional tfdA gene distribution in thirty-one different 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid degraders

31 different bacterial strains isolated using the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, were investigated for their ability to mineralize 2,4-D and the related herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA). Most of the strains mineralize 2,4-D considerably faster than MCPA. Three novel primer sets were developed enabling amplification of full-length coding sequences (CDS) of the three known tfdA gene classes known to be involved in phenoxy acid degradation. 16S rRNA genes were also sequenced; and in order to investigate possible linkage between tfdA gene classes and bacterial species, tfdA and 16S rRNA gene phylogeny was compared. Three distinctly different classes of tfdA genes were observed, with class I tfdA sequences further partitioned into the two sub-classes I-a and I-b based on more subtle differences. Comparison of phylogenies derived from 16S rRNA gene sequences and tfdA gene sequences revealed that most class II tfdA genes were encoded by Burkholderia sp., while class I-a, I-b and III genes were found in a more diverse array of bacteria.     

In situ exposure to low herbicide concentrations affects microbial population composition and catabolic gene frequency in an aerobic shallow aquifer

The aim of this study was to evaluate how the in situ exposure of a Danish subsurface aquifer to phenoxy acid herbicides at low concentrations (<40 micro g l(-1)) changes the microbial community composition. Sediment and groundwater samples were collected inside and outside the herbicide-exposed area and were analyzed for the presence of general microbial populations, Pseudomonas bacteria, and specific phenoxy acid degraders. Both culture-dependent and culture-independent methods were applied. The abundance of microbial phenoxy acid degraders (10(0) to 10(4) g(-1) sediment) was determined by most probable number assays, and their presence was only detected in herbicide-exposed sediments. Similarly, PCR analysis showed that the 2,4-dichlorophenoxyacetic acid degradation pathway genes tfdA and tfdB (10(2) to 10(3) gene copies g(-1) sediment) were only detected in sediments from contaminated areas of the aquifer. PCR-restriction fragment length polymorphism measurements demonstrated the presence of different populations of tfd genes, suggesting that the in situ herbicide degradation was caused by the activity of a heterogeneous population of phenoxy acid degraders. The number of Pseudomonas bacteria measured by either PCR or plating on selective agar media was higher in sediments subjected to high levels of phenoxy acid. Furthermore, high numbers of CFU compared to direct counting of 4',6-diamidino-2-phenylindole-stained cells in the microscope suggested an increased culturability of the indigenous microbial communities from acclimated sediments. The findings of this study demonstrate that continuous exposure to low herbicide concentrations can markedly change the bacterial community composition of a subsurface aquifer.     

Impact of soil matric potential on the fine-scale spatial distribution and activity of specific microbial degrader communities

The impact of the soil matric potential on the relationship between the relative abundance of degraders and their activity and on the spatial distribution of both at fine scales was determined to understand the role of environmental conditions in the degradation of organic substrates. The mineralization of (13) C-glucose and (13) C-2,4-dichlorophenoxyacetic acid (2,4-D) was measured at different matric potentials (-0.001, -0.01 and -0.316?MPa) in 6?×?6?×?6?mm(3) cubes excised from soil cores. At the end of the incubation, total bacterial and 2,4-D degrader abundances were determined by quantifying the 16S rRNA and the tfdA genes, respectively. The mineralization of 2,4-D was more sensitive to changes in matric potential than was that of glucose. The amount and spatial structure of 2,4-D mineralization decreased with matric potential, whilst the spatial variability increased. On the other hand, the spatial variation of glucose mineralization was less affected by changes in matric potential. The relationship between the relative abundance of 2,4-D degraders and 2,4-D mineralization was significantly affected by matric potential: the relative abundance of tfdA needed to be higher to reach a given level of 2,4-D mineralization in dryer than in moister conditions. The data show how microbial interactions with their microhabitat can have an impact on soil processes at larger scales.     

Degradation of 4-chloro-2-methylphenoxyacetic acid in top- and subsoil is quantitatively linked to the class III tfdA gene

The tfdA gene is known to be involved in the first step of the degradation of the phenoxy acid herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in several soil bacteria, but bacteria containing other tfdA-like genes have been isolated as well. A quantitative real-time PCR method was used to monitor the increase in the concentration of tfdA genes during degradation of MCPA in sandy topsoil and subsoil over a period of 115 days. Quantitative PCR revealed growth in the tfdA-containing bacterial community, from 500 genes g(-1) soil to approximately 3 x 10(4) genes g(-1) soil and to 7 x 10(5) genes g(-1) soil for topsoil initially added to 2.3 mg MCPA kg(-1) (dry weight) soil and 20 mg MCPA kg(-1) (dry weight) soil, respectively. We analyzed the diversity of the tfdA gene during the degradation experiment. Analyses of melting curves of real-time PCR amplification products showed that a shift in the dominant tfdA population structure occurred during the degradation period. Further denaturing gradient gel electrophoresis and sequence analysis revealed that the tfdA genes responsible for the degradation of MCPA belonged to the class III tfdA genes, while the tfdA genes present in the soil before the occurrence of degradation belonged to the class I tfdA genes. The implications of these results is that the initial assessment of functional genes in soils does not necessarily reflect the organisms or genes that would carry out the degradation of the compounds in question.     

Microbial degradation of 2,4-dichlorophenoxyacetic acid on the Greenland ice sheet

The Greenland ice sheet (GrIS) receives organic carbon (OC) of anthropogenic origin, including pesticides, from the atmosphere and/or local sources, and the fate of these compounds in the ice is currently unknown. The ability of supraglacial heterotrophic microbes to mineralize different types of OC is likely a significant factor determining the fate of anthropogenic OC on the ice sheet. Here we determine the potential of the microbial community from the surface of the GrIS to mineralize the widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Surface ice cores were collected and incubated for up to 529 days in microcosms simulating in situ conditions. Mineralization of side chain- and ring-labeled [(14)C]2,4-D was measured in the samples, and quantitative PCR targeting the tfdA genes in total DNA extracted from the ice after the experiment was performed. We show that the supraglacial microbial community on the GrIS contains microbes that are capable of degrading 2,4-D and that they are likely present in very low numbers. They can mineralize 2,4-D at a rate of up to 1 nmol per m(2) per day, equivalent to ～26 ng C m(-2) day(-1). Thus, the GrIS should not be considered a mere reservoir of all atmospheric contaminants, as it is likely that some deposited compounds will be removed from the system via biodegradation processes before their potential release due to the accelerated melting of the ice sheet.     

Enhanced mineralization of [U-(14)C]2,4-dichlorophenoxyacetic acid in soil from the rhizosphere of Trifolium pratense

Enhanced biodegradation in the rhizosphere has been reported for many organic xenobiotic compounds, although the mechanisms are not fully understood. The purpose of this study was to discover whether rhizosphere-enhanced biodegradation is due to selective enrichment of degraders through growth on compounds produced by rhizodeposition. We monitored the mineralization of [U-(14)C]2,4-dichlorophenoxyacetic acid (2,4-D) in rhizosphere soil with no history of herbicide application collected over a period of 0 to 116 days after sowing of Lolium perenne and Trifolium pratense. The relationships between the mineralization kinetics, the number of 2,4-D degraders, and the diversity of genes encoding 2,4-D/alpha-ketoglutarate dioxygenase (tfdA) were investigated. The rhizosphere effect on [(14)C]2,4-D mineralization (50 microg g(-1)) was shown to be plant species and plant age specific. In comparison with nonplanted soil, there were significant (P < 0.05) reductions in the lag phase and enhancements of the maximum mineralization rate for 25- and 60-day T. pratense soil but not for 116-day T. pratense rhizosphere soil or for L. perenne rhizosphere soil of any age. Numbers of 2,4-D degraders in planted and nonplanted soil were low (most probable number, <100 g(-1)) and were not related to plant species or age. Single-strand conformational polymorphism analysis showed that plant species had no impact on the diversity of alpha-Proteobacteria tfdA-like genes, although an impact of 2,4-D application was recorded. Our results indicate that enhanced mineralization in T. pratense rhizosphere soil is not due to enrichment of 2,4-D-degrading microorganisms by rhizodeposits. We suggest an alternative mechanism in which one or more components of the rhizodeposits induce the 2,4-D pathway.     

2,4-D impact on bacterial communities, and the activity and genetic potential of 2,4-D degrading communities in soil

The key role of telluric microorganisms in pesticide degradation is well recognized but the possible relationships between the biodiversity of soil microbial communities and their functions still remain poorly documented. If microorganisms influence the fate of pesticides, pesticide application may reciprocally affect soil microorganisms. The objective of our work was to estimate the impact of 2,4-D application on the genetic structure of bacterial communities and the 2,4-D-degrading genetic potential in relation to 2,4-D mineralization. Experiments combined isotope measurements with molecular analyses. The impact of 2,4-D on soil bacterial populations was followed with ribosomal intergenic spacer analysis. The 2,4-D degrading genetic potential was estimated by real-time PCR targeted on tfdA sequences coding an enzyme specifically involved in 2,4-D mineralization. The genetic structure of bacterial communities was significantly modified in response to 2,4-D application, but only during the intense phase of 2,4-D biodegradation. This effect disappeared 7 days after the treatment. The 2,4-D degrading genetic potential increased rapidly following 2,4-D application. There was a concomitant increase between the tfdA copy number and the 14C microbial biomass. The maximum of tfdA sequences corresponded to the maximum rate of 2,4-D mineralization. In this soil, 2,4-D degrading microbial communities seem preferentially to use the tfd pathway to degrade 2,4-D.     

Isolation and characterization of a stable 2,4-dichlorophenoxyacetic acid degrading bacterium, Variovorax paradoxus, using chemostat culture

A strain of Variovorax paradoxus degrading 2,4-dichlorophenoxyacetic acid (2,4-D) was isolated from the Dijon area (France) using continuous chemostat culture. This strain, designated TV1, grew on up to 5 mM 2,4-D and efficiently degraded the herbicide as sole carbon source as well as in presence of soil extracts. It also degraded phenol and 2-methyl, 4-chlorophenoxyacetic acid at 3 mM and 2,4-dichlorophenol at 1 mM. This organism contained a stable 200 kb plasmid, designated pTV1, which showed no similarity in its restriction pattern with the archetypal 2,4-D catabolic plasmid pJP4. However, pTV1 contained an 11 kb BamHI fragment which hybridized at low stringency with the 2,4-D degradative genes tfdA, tfdB and tfdR from pJP4. PTV1 partial tfdA sequence showed 77 % similarity with the archetypal tfdA gene sequence from Ralstonia eutropha JMP134. Tn5 mutagenesis confirmed the involvement of this gene in the 2,4-D catabolic pathway. © Rapid Science Ltd. 1998     

Mineralization and co-metabolic degradation of phenoxyalkanoic acid herbicides by a pure bacterial culture isolated from an aquifer

A mecoprop [(+/-)-2-(4-chloro-2-methylphenoxy)propionic acid; MCPP]-degrading bacterium identified as Stenotrophomonas maltophilia PM was isolated from a Danish aquifer. Besides mecoprop, the bacterium was also able to degrade MCPA [(4-chloro-2-methylphenoxy)acetic acid)], MCPB [(4-chloro-2-methylphenoxy)butyric acid], 4-CPA [(4-chlorophenoxy)acetic acid], 2, 4-D [(2, 4-dichlorophenoxy)acetic acid], 2, 4-DP [(+/-)-2-(2, 4-dichlorophenoxy)propionic acid] and 2, 4-DB [(2, 4-dichlorophenoxy)butyric acid]. The bacterium was able to grow using these individual phenoxyalkanoic acids as the sole source of carbon and energy. In addition, it was able to co-metabolically degrade the phenoxyalkanoic acid 2, 4, 5-T [(2, 4, 5-trichlorophenoxy)acetic acid)] in the presence of mecoprop. At high 2, 4, 5-T concentrations (100 and 52 mg/l), however, only partial degradation of both mecoprop and 2, 4, 5-T was obtained, thus indicating the production of toxic metabolites. Bacterial yields were highest when grown on the monochlorinated phenoxyalkanoic acids as compared to the dichlorinated analogues, an exception being growth on 4CPA, which resulted in the lowest yield at all. Using [ring-U-14C]-labeled herbicides it was shown that the lower yield on 2, 4-D than on mecoprop was accompanied by greater CO2 generation, thus indicating that less energy is available from the complete oxidation of the dichlorinated phenoxyalkanoic acids than the monochlorinated analogues.     

Characterization of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid]-degrading Alcaligenes sp.CS1 and Ralstonia sp. CS2 isolated from agricultural soils

The herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] is widely applied to corn fields in order to control broad-leaved weeds. However, it is often detected in groundwater where it can be a persistent contaminant. Two mecoprop-degrading bacterial strains were isolated from agricultural soils through their capability to degrade ( R/S )-mecoprop rapidly. 16S rDNA sequencing of the isolates demonstrated that one was closely related to the genera Alcaligenes sp. (designated CS1) and the other to Ralstonia sp. (designated CS2). Additionally, these isolates demonstrated ability to grow on other related herbicides, including 2,4- D (2,4-dichlorophenoxyacetic acid), MCPA [4-chloro-2-methyl phenoxy acetic acid] and ( R/S )-2,4-DP [2-(2,4-dichlorophenoxy)propionic acid] as sole carbon sources. tfdABC gene-specific probes derived from the 2,4- D -degrading Variovorax paradoxus TV1 were used in hybridization analyses to establish whether tfd -like genes are present in mecoprop-degrading bacteria. Hybridization analysis demonstrated that both Alcaligenes sp. CS1 and Ralstonia sp. CS2 harboured tfdA , tfdB and tfdC genes on plasmids that have approximately > 60% sequence similarity to the tfdA , tfdB and tfdC genes of V. paradoxus . It is therefore likely that tfd -like genes may be involved in the degradation of mecoprop, and we are currently investigating this further.     

tfdA-like genes in 2,4-dichlorophenoxyacetic acid-degrading bacteria belonging to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in alpha-Proteobacteria

The 2,4-dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate dioxygenase gene (tfdA) homolog designated tfdAalpha was cloned and characterized from 2,4-D-degrading bacterial strain RD5-C2. This Japanese upland soil isolate belongs to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in the alpha subdivision of the class Proteobacteria on the basis of its 16S ribosomal DNA sequence. Sequence analysis showed 56 to 60% identity of tfdAalpha to representative tfdA genes. A MalE-TfdAalpha fusion protein expressed in Escherichia coli exhibited about 10 times greater activity for phenoxyacetate than 2,4-D in an alpha-ketoglutarate- and Fe(II)-dependent reaction. The deduced amino acid sequence of TfdAalpha revealed a conserved His-X-Asp-X(146)-His-X(14)-Arg motif characteristic of the active site of group II alpha-ketoglutarate-dependent dioxygenases. The tfdAalpha genes were also detected in 2,4-D-degrading alpha-Proteobacteria previously isolated from pristine environments in Hawaii and in Saskatchewan, Canada (Y. Kamagata, R. R. Fulthorpe, K. Tamura, H. Takami, L. J. Forney, and J. M. Tiedje, Appl. Environ. Microbiol. 63:2266-2272, 1997). These findings indicate that the tfdA genes in beta- and gamma-Proteobacteria and the tfdAalpha genes in alpha-Proteobacteria arose by divergent evolution from a common ancestor.     

Evidence for Acquisition in Nature of a Chromosomal 2,4-Dichlorophenoxyacetic Acid/(alpha)-Ketoglutarate Dioxygenase Gene by Different Burkholderia spp

We characterized the gene required to initiate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by the soil bacterium Burkholderia sp. strain TFD6, which hybridized to the tfdA gene of the canonical 2,4-D catabolic plasmid pJP4 under low-stringency conditions. Cleavage of the ether bond of 2,4-D by cell extracts of TFD6 proceeded by an (alpha)-ketoglutarate-dependent reaction, characteristic of TfdA (F. Fukumori and R. P. Hausinger, J. Bacteriol. 175:2083-2086, 1993). The TFD6 tfdA gene was identified in a recombinant plasmid which complemented a tfdA transposon mutant of TFD6 created by chromosomal insertion of Tn5. The plasmid also expressed TfdA activity in Escherichia coli DH5(alpha), as evidenced by enzyme assays with cell extracts. Sequence analysis of the tfdA gene and flanking regions from strain TFD6 showed 99.5% similarity to a tfdA gene cloned from the chromosome of a different Burkholderia species (strain RASC) isolated from a widely separated geographical area. This chromosomal gene has 77.2% sequence identity to tfdA from plasmid pJP4 (Y. Suwa, W. E. Holben, and L. J. Forney, abstr. Q-403, in Abstracts of the 94th General Meeting of the American Society for Microbiology 1994.). The tfdA homologs cloned from strains TFD6 and RASC are the first chromosomally encoded 2,4-D catabolic genes to be reported. The occurrence of highly similar tfdA genes in different bacterial species suggests that this chromosomal gene can be horizontally transferred.     

Microbiological and Geochemical Heterogeneity in an In Situ Uranium Bioremediation Field Site

The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.     

Analysis, cloning, and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134.

Plasmid pJP4 of Alcaligenes eutrophus JMP134 contains all genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D). Five of these genes, tfdB, tfdC, tfdD, tfdE, and tfdF, have recently been localized and cloned (R. H. Don, A. J. Weightman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). Gene tfdA, which codes for the 2,4-D monooxygenase, has now been found by mutagenesis with transposon Tn5. A 3-kilobase fragment of pJP4 cloned in a broad-host-range vector could complement the 2,4-D-negative phenotype of two mutants which lacked 2,4-D monooxygenase activity. The cloned tfdA gene was also transferred to A. eutrophus JMP222, which is a cured derivative of JMP134. The recombinant strain could utilize phenoxyacetic acid as a sole source of carbon and energy. Pseudomonas sp. strain B13, containing the cloned tfdA, was able to degrade phenoxyacetic acid and 4-chlorophenoxyacetic acid. Gene tfdA was subcloned and analyzed by deletions. Expression of 2,4-D monooxygenase in Escherichia coli containing a 1.4-kilobase subfragment was demonstrated by radioisotopic enzyme assay, and a protein of 32,000-dalton molecular mass was detected by labeling experiments. A 2-kilobase subfragment containing tfdA has been sequenced. Sequence analysis revealed an open reading frame of 861 bases which was identified as the coding region of tfdA by insertion mutagenesis.     

In Situ Exposure to Low Herbicide Concentrations Affects Microbial Population Composition and Catabolic Gene Frequency in an Aerobic Shallow Aquifer

The aim of this study was to evaluate how the in situ exposure of a Danish subsurface aquifer to phenoxy acid herbicides at low concentrations (<40 μg l⁻¹) changes the microbial community composition. Sediment and groundwater samples were collected inside and outside the herbicide-exposed area and were analyzed for the presence of general microbial populations, Pseudomonas bacteria, and specific phenoxy acid degraders. Both culture-dependent and culture-independent methods were applied. The abundance of microbial phenoxy acid degraders (10⁰ to 10⁴ g⁻¹ sediment) was determined by most probable number assays, and their presence was only detected in herbicide-exposed sediments. Similarly, PCR analysis showed that the 2,4-dichlorophenoxyacetic acid degradation pathway genes tfdA and tfdB (10² to 10³ gene copies g⁻¹ sediment) were only detected in sediments from contaminated areas of the aquifer. PCR-restriction fragment length polymorphism measurements demonstrated the presence of different populations of tfd genes, suggesting that the in situ herbicide degradation was caused by the activity of a heterogeneous population of phenoxy acid degraders. The number of Pseudomonas bacteria measured by either PCR or plating on selective agar media was higher in sediments subjected to high levels of phenoxy acid. Furthermore, high numbers of CFU compared to direct counting of 4′,6-diamidino-2-phenylindole-stained cells in the microscope suggested an increased culturability of the indigenous microbial communities from acclimated sediments. The findings of this study demonstrate that continuous exposure to low herbicide concentrations can markedly change the bacterial community composition of a subsurface aquifer.     

The metabolic pathway of 2,4-dichlorophenoxyacetic acid degradation involves different families of tfdA and tfdB genes according to PCR-RFLP analysis

Abstract: Twenty-five 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacteria from geographically diverse locations and presenting various degrees of similarity or no similarity to the tfdA and tfdB genes from Alcaligenes eutrophus JMP134 were analysed by PCR-RFLP (restriction length fragment polymorphism). Primers for the 2,4-D etherase gene were derived by sequence alignment of the tfdA genes from A. eutrophus JMP134 and Burkholderia sp. RASC. Primers for the 2,4-dichlorophenolhydroxylase gene were based on the tfdB gene sequence from A. eutrophus JMP134 by taking codon degeneration and variations in amino acid residue sequences into consideration. PCR amplification using the tfdA primer set produced fragments of 0.3 kb from 17 strains which showed varying degrees of similarity to the tfdA gene probe from A. eutrophus JMP134. Significant variations in the gene sequences were confirmed by PCR-RFLP analysis. DNA amplification using the tfdB primer set produced a 1.1 kb fragment from 19 strains. Amongst them, two did not show any similarity to the tfdB gene probe. The size and restriction pattern of the products obtained from A. eutrophus JMP134 were in accordance with the expected size calculated from the A. eutrophus tfdA and tfdB gene sequence and their theoretical PCR-RFLP patterns. Some strains which did not amplify using the tfdA primer set did however amplify with the tfdB primer set. These results suggest the independent evolution of these two genes in the construction of the 2,4-D metabolic pathway. Our tfdA and tfdB primer sets could be used for the detection of similar sequences in bacteria and soils. Moreover, PCR-RFLP patterns could also be used to select subsets of strains for sequencing to study the phylogeny of the tfdA and tfdB genes.     

The potential of soil microorganisms to mineralize atrazine as predicted by MCH-PCR followed by nested PCR

The potential of soil microorganisms to mineralize atrazine was studied in soil samples collected from fields with various histories of atrazine application. In contrast to many previous studies, which showed no atrazine mineralization activity, all the tested soils mineralized atrazine regardless of their atrazine application history. However, the delay before mineralization and the variation in the subsequent mineralization rate were in agreement with the initial copy number of the atrazine dechlorinaze gene, and the proliferation rate of the degraders. Soils from corn fields, which had up to 100 copies of the atzA gene per gram of soil, had a lag period of 4-5 days before atrazine mineralization started, and final mineralization percentages ranged from 40% to 54%. However, soils from fields that were never amended with atrazine had much longer lag periods (more than 17 days), which decreased after enrichment of the degrader population with high concentrations of atrazine for 15 days. Generally the mineralization rate and the atzA gene copy number increased after the enrichment period. The atrazine mineralization potential was measured by PCR of genes from the atrazine mineralization pathway. Magnetic capture hybridization was the most efficient of the two tested methods for purifying target DNA of PCR inhibitors, without reducing the copy number of the required fragment. Nested PCR proved to be the most effective method for predicting the exact potential of the soil to mineralize the pollutant even without enrichment of a small population with the target genes. This method can complement microcosm studies and eliminate futile efforts when the potential to mineralize the pollutant does not exist in the soil.