2,4-Dichlorophenoxyacetic acid-degrading bacteria contain mosaics of catabolic genes

Abstract

DNA from 32 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria from diverse locations was probed with the first three genes of the well-known 2,4-D degradation pathway found in Alcaligenes eutrophus JMP134(pJP4). The majority of strains did not show high levels of homology to the first three genes of the 2,4-D degradation pathway, tfdA,-B, and-C. Most strains showed combinations of tfdA-, Band nd C-like elements that exhibited various degrees of homology to the gene probes. Strains having the same genomic fingerprints (as determined by repetitive extragenic palindromic PCR) exhibited the same hybridization pattern regardless of the geographic origin of the strain, with the exception of a strain isolated from Puerto Rico. This strain had the same genomic fingerprint as that of numerous other strains in the collection but differed in its hybridization against the tfdA gene probe. Members of the ␤ subdivision of the Proteobacteria class, specifically Alcaligenes, Burkholderia, and Rhodoferax species, carried DNA fragments with 60% or more sequence similarity to tfdA of pJP4, and most carried fragments showing at least 60% homology to tfdB. However, many strains did not hybridize with tfdC, although they exhibited chlorocatechol dioxygenase activity. Members of the ␣ subdivision of the Proteobacteria class, mostly of the genus Sphingomonas, did not hybridize to either tfdA or tfdC, but some hybridized at low stringency to tfdB. The data suggest that extensive interspecies transfer of a variety of homologous degradative genes has been involved in the evolution of 2,4-D-degrading bacteria.

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

“…Type 4 @U's are found in two species, Rhodoferax fermentens (B6-9, TFD31) and Variovorux parudoxus TVl. What is common to these strains is the observation of one or two large plasmids where the hybridization signal is detected [24,25]. Organisms carrying type 4 sequences have been found in various strains.…”

“…Hybridizations were performed as described by Fulthorpe et al [24] using <fdA and [fdB probes from A. eut~ophus JMPI 34 under medium stringency conditions (20% formamide in hybridization solution). Internal fragments from the #dA and (fdB genes used as molecular probes have been described by Holben et al [22].…”

“…Amongst them. two strains (TFD31 and B6.9) did not show any similarity to the ffdB gene probe in previous hybridization studies [24]. The Hue111 enzyme was chosen for the PCR-RFLP analysis of the $dB-like fragment because of the high frequency of its restriction sites on the A. eutrophus JMP134 (fdB sequence.…”

“…In recent work. Fulthorpe et al [24] showed that similarities amongst different tfdA and tfdB gene sequences could be as low as 60%. The detection of sequences presenting low similarity to a probe requires reduction in stringency during hybridization and leads to non-specific binding.…”

Diversity of tfdC genes: distribution and polymorphism among 2,4-dichlorophenoxyacetic acid degrading soil bacteria

“…The tfdCII gene of R. eutropha was found to be more closely related to the tfdC gene of P. putida PaW85 than to the canonical tfdC gene of R. eutropha JMP134. This data indicated that the P. putida PaW85 tfdC gene type could be more widely distributed than the canonical tfdC from R. eutropha JMP134 and it could explain why recent studies, using pJP4-based probes [16,18,27], failed to detect tfdC genes in 2,4-D degrading strains isolated from soil. The tfdC gene of strain RASC, isolated in the USA, is closely related to those of P.…”