Multiple Gene Clusters and Their Role in the Degradation of Chlorophenoxyacetic Acids in Bradyrhizobium sp. RD5-C2 Isolated from Non-Contaminated Soil.

Bradyrhizobium sp. RD5-C2, isolated from soil that is not contaminated with 2,4-dichlorophenoxyacetic acid (2,4-D), degrades the herbicides 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). It possesses tfdAα and cadA (designated as cadA1), which encode 2,4-D dioxygenase and the oxygenase large subunit, respectively. In the present study, the genome of Bradyrhizobium sp. RD5-C2 was sequenced and a second cadA gene (designated as cadA2) was identified. The two cadA genes belonged to distinct clusters comprising the cadR1A1B1K1C1 and cadR2A2B2C2K2S genes. The proteins encoded by the cad1 cluster exhibited high amino acid sequence similarities to those of other 2,4-D degraders, while Cad2 proteins were more similar to those of non-2,4-D degraders. Both cad clusters were capable of degrading 2,4-D and 2,4,5-T when expressed in non-2,4-D-degrading Bradyrhizobium elkanii USDA94. To examine the contribution of each degradation gene cluster to the degradation activity of Bradyrhizobium sp. RD5-C2, cadA1, cadA2, and tfdAα deletion mutants were constructed. The cadA1 deletion resulted in a more significant decrease in the ability to degrade chlorophenoxy compounds than the cadA2 and tfdAα deletions, indicating that degradation activity was primarily governed by the cad1 cluster. The results of a quantitative reverse transcription-PCR analysis suggested that exposure to 2,4-D and 2,4,5-T markedly up-regulated cadA1 expression. Collectively, these results indicate that the cad1 cluster plays an important role in the degradation of Bradyrhizobium sp. RD5-C2 due to its high expression.

2,4-Dichlorophenoxyacetic acid (2,4-D)- and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading gene cluster in the soybean root-nodulating bacterium Bradyrhizobium elkanii USDA94.

Herbicides 2,4-dichlorophenoxyacetic acid (2,4-D)- and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading Bradyrhizobium strains possess tfdAα and/or cadABC as degrading genes. It has been reported that root-nodulating bacteria belonging to Bradyrhizobium elkanii also have tfdAα and cadA like genes but lack the ability to degrade these herbicides and that the cadA genes in 2,4-D-degrading and non-degrading Bradyrhizobium are phylogenetically different. In this study, we identified cadRABCK in the genome of a type strain of soybean root-nodulating B. elkanii USDA94 and demonstrated that the strain could degrade the herbicides when cadABCK was forcibly expressed. cadABCK-cloned Escherichia coli also showed the degrading ability. Because co-spiked phenoxyacetic acid (PAA) could induce the degradation of 2,4-D in B. elkanii USDA94, the lack of degrading ability in this strain was supposed to be due to the low inducing potential of the herbicides for the degrading gene cluster. On the other hand, tfdAα from B. elkanii USDA94 showed little potential to degrade the herbicides, but it did for 4-chlorophenoxyacetic acid and PAA. The 2,4-D-degrading ability of the cad cluster and the inducing ability of PAA were confirmed by preparing cadA deletion mutant. This is the first study to demonstrate that the cad cluster in the typical root-nodulating bacterium indeed have the potential to degrade the herbicides, suggesting that degrading genes for anthropogenic compounds could be found in ordinary non-degrading bacteria.

Genes of Bacillus subtilis 168 that Support Growth of the Cyanobacterium, Synechococcus leopoliensis CCAP1405/1 on Agar Media.

Synechococcus leopoliensis CCAP1405/1 cannot grow on common solid media; however, the strain can grow when co-cultured with Bacillus subtilis 168. Gene-disruptant strains of B. subtilis 168 by pMUTINs at the following sites lost the ability to support the growth of S. leopoliensis CCAP1405/1 on agar media: yxeO and yufO (transporter), yxdK (histidine kinase), sdhC (succinate dehydrogenase), yvgQR (sulfite reductase), acoB (acetoin dehydrogenase), yusE (thioredoxin), yrdA (function unknown). Involvement of the assimilatory sulfate reduction pathway was the suggested reason for loss of the function.

Characterization of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid-degrading fungi in Vietnamese soils.

Sixty-nine fungal strains were isolated countrywide from 10 Vietnamese soils, in areas both with and without a history of exposure to Agent Orange, and their degrading activities on the phenoxy acid herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), as well as related compounds, were examined. Among taxonomically various fungi, 45, 12 and 4% of the isolates degraded phenoxyacetic acid (PA), 2,4-D and 2,4,5-T, respectively. While the PA-degrading fungi were distributed to all sites and among many genera, the 2,4-D-degraders were found only in order Eurotiales in class Eurotiomycetes. All of the 2,4,5-T-degrading fungal strains were phylogenetically close to Eupenicillium spp. and were isolated from southern Vietnam. As a degradation intermediate, the corresponding phenol compounds were detected in some strains. The degradation substrate spectrum for 26 compounds of Eupenicillium spp. strains including 2,4,5-T-degraders and -non-degraders seemed to be related to phylogenetic similarity and soil sampling location of the isolates. These results suggest that the heavily contaminated environments enhanced the adaptation of the phylogenetic group of Eupenicillium spp. toward to obtain the ability to degrade 2,4,5-T.

Growth of the cyanobacterium Synechococcus leopoliensis CCAP1405/1 on agar media in the presence of heterotrophic bacteria.

The cyanobacterium Synechococcus leopoliensis CCAP1405/1 does not grow on common solid media made of agar, agarose HT, noble agar, gelrite and gelatin, although it grows in liquid media with the same components. The inoculation of S. leopoliensis CCAP1405/1 at a high initial cell density allowed it to grow on the agar media, and co-inoculation with one of the heterotrophic bacterial strains belonging to a wide range of phylogeny, showed the same effects even at a low initial cell density of S. leopoliensis CCAP1405/1. The addition of thiosulfate and high concentrations of vitamin B(12), biotin and thiamine also supported growth on solid media, but catalase had no effect. On inorganic solid media, the autotrophic cyanobacterial strain supported the growth of heterotrophic bacteria, suggesting mutual interaction.

Construction of river model biofilm for assessing pesticide effects.

Due to the high importance of biofilms on river ecosystems, assessment of pesticides' adverse effects is necessary but is impaired by high variability and poor reproducibility of both natural biofilms and those developed in the laboratory. We constructed a model biofilm to evaluate the effects of pesticides, consisting in cultured microbial strains, Pedobacter sp. 7-11, Aquaspirillum sp. T-5, Stenotrophomonas sp. 3-7, Achnanthes minutissima N71, Nitzschia palea N489, and/or Cyclotella meneghiniana N803. Microbial cell numbers, esterase activity, chlorophyll-a content, and the community structure of the model biofilm were examined and found to be useful as biological factors for evaluating the pesticide effects. The model biofilm was formed through the cooperative interaction of bacteria and diatoms, and a preliminary experiment using the herbicide atrazine, which inhibits diatom growth, indicated that the adverse effect on diatoms inhibited indirectly the bacterial growth and activity and, thus, the formation of the model biofilm. Toxicological tests using model biofilms could be useful for evaluating the pesticide effects and complementary to studies on actual river biofilms.

Reductive dechlorination of chlorophenols in estuarine sediments of Lake Shinji and Lake Nakaumi.

Dechlorination of all mono- and dichlorophenol isomers in anaerobic sediment samples of estuarine Lake Shinji and Lake Nakaumi was examined to characterize the chlorophenol-dechlorinating microbial communities in the environments with different salinity levels. Dechlorination was observed only in 2-chlorophenol (2-CP), 3-chlorophenol (3-CP) and 2,6-dichlorophenol (2,6-DCP), and in 2-CP and 2,6-DCP in the Lake Shinji and Nakaumi sediment, respectively. In the sediment of Lake Shinji, the highest activity was observed without adding sodium chloride and sulfate, whereas in the Lake Nakaumi sediment, the highest activity was at 0.7 % of sodium chloride and 6.0 mM of sodium sulfate. The chlorophenols were degraded to benzoate via phenol in both sediments under methanogenic conditions. Benzoate then disappeared from the cultures. All microbial consortia enriched with each monochlorophenol dechlorinated 2-CP, but showed different substrate specificities for dichlorophenols as follows: 2-CP-enriched consortium dechlorinated 2,3-dichlorophenol and 2,6-DCP, 3-CP-enriched consortium dechlorinated all dichlorophenol isomers, and 4-chlorophenol-enriched consortium dechlorinated 2,4-dichlorophenol and 2,6-DCP. Maintenance of the population by halorespiration was suggested in the dechlorination of 2-CP.

2,4-Dichlorophenoxyacetic Acid (2,4-D)- and 2,4,5-Trichlorophenoxyacetic Acid (2,4,5-T)-Degrading Bacterial Community in Soil-Water Suspension during the Enrichment Process.

Changes in the bacterial community in soil-water suspensions during the enrichment period of 2,4-D and 2,4,5-T degraders were examined using denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The nucleotide sequences of almost all major bands at the degradation of 2,4-D and 2,4,5-T corresponded to those of 2,4-D- and 2,4,5-T-degrading isolates, and successions of diverse 2,4-D- and 2,4,5-T-degrading bacterial communities were demonstrated in the DGGE profile. These results suggested that 2,4-D- and 2,4,5-T-degrading isolates were responsible for the degradation of 2,4-D and 2,4,5-T in soil-water suspensions; however, one major band of a non-degrader was found in some cases. Therefore, to elucidate the relationship between the structures and functions of bacterial communities, the importance of a combined approach using both culture-dependent and -independent methods was demonstrated.

Chlorophenol hydroxylase activity encoded by TfdB from 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading Bradyrhizobium sp. strain RD5-C2.

The tfdB gene encoding chlorophenol hydroxylase and its homolog were found in 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading strain RD5-C2, which belongs to Bradyrhizobium sp. of alpha-Proteobacteria. The nucleotide and deduced amino acid sequence identities of the two genes, designated tfdBa and tfdBb, were 60% and 57% respectively. Their nucleotide sequences most closely matched those of previously reported tfdB, which consisted of those from 2,4-D-degrading beta- and gamma-Proteobacteria and Sphingomonas sp. in alpha-Proteobacteria, with 61-67% identity. The TfdBa expressed in Escherichia coli showed the highest activity for 2,4-dichlorophenol but a narrower range of activity for the other chlorophenols than previously reported TfdBs. In the case of TfdBb, however, no observable activity for any chlorophenols or phenol was detected, although production of a protein with an appropriate molecular size was observed. Based on codon usage patterns and the GC content of the genes, it probable that the tfdBa genes in the 2,4-D-degrading Bradyrhizobium sp. were obtained through horizontal gene transfer.

Root nodule Bradyrhizobium spp. harbor tfdAalpha and cadA, homologous with genes encoding 2,4-dichlorophenoxyacetic acid-degrading proteins.

The distribution of tfdAalpha and cadA, genes encoding 2,4-dichlorophenoxyacetate (2,4-D)-degrading proteins which are characteristic of the 2,4-D-degrading Bradyrhizobium sp. isolated from pristine environments, was examined by PCR and Southern hybridization in several Bradyrhizobium strains including type strains of Bradyrhizobium japonicum USDA110 and Bradyrhizobium elkanii USDA94, in phylogenetically closely related Agromonas oligotrophica and Rhodopseudomonas palustris, and in 2,4-D-degrading Sphingomonas strains. All strains showed positive signals for tfdAalpha, and its phylogenetic tree was congruent with that of 16S rRNA genes in alpha-Proteobacteria, indicating evolution of tfdAalpha without horizontal gene transfer. The nucleotide sequence identities between tfdAalpha and canonical tfdA in beta- and gamma-Proteobacteria were 46 to 57%, and the deduced amino acid sequence of TfdAalpha revealed conserved residues characteristic of the active site of alpha-ketoglutarate-dependent dioxygenases. On the other hand, cadA showed limited distribution in 2,4-D-degrading Bradyrhizobium sp. and Sphingomonas sp. and some strains of non-2,4-D-degrading B. elkanii. The cadA genes were phylogenetically separated between 2,4-D-degrading and nondegrading strains, and the cadA genes of 2,4-D degrading strains were further separated between Bradyrhizobium sp. and Sphingomonas sp., indicating the incongruency of cadA with 16S rRNA genes. The nucleotide sequence identities between cadA and tftA of 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were 46 to 53%. Although all root nodule Bradyrhizobium strains were unable to degrade 2,4-D, three strains carrying cadA homologs degraded 4-chlorophenoxyacetate with the accumulation of 4-chlorophenol as an intermediate, suggesting the involvement of cadA homologs in the cleavage of the aryl ether linkage. Based on codon usage patterns and GC content, it was suggested that the cadA genes of 2,4-D-degrading and nondegrading Bradyrhizobium spp. have different origins and that the genes would be obtained in the former through horizontal gene transfer.

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.