Novel insights into the interplay between peripheral reactions encoded by xyl genes and the chlorocatechol pathway encoded by tfd genes for the degradation of chlorobenzoates by Ralstonia eutropha JMP134

Abstract

Many bacteria can grow on chloroaromatic pollutants because they can transform them into chlorocatechols, which are further degraded by enzymes of a specialized ortho-cleavage pathway. Ralstonia eutropha JMP134 is able to grow on 3-chlorobenzoate by using two pJP4-encoded, ortho-cleavage chlorocatechol degradation gene clusters (tfdC I D I E I F I and tfdD II C II E II F II ). Very little is known about the acquisition of new catabolic genes encoding enzymes that lead to the formation of chlorocatechols in R. eutropha JMP134. The effect on the catabolic properties of an R. eutropha JMP134 derivative that received the xylS-xylXYZL gene module, encoding the xylS-regulated expression of the broad-substrate-range toluate 1,2-dioxygenase (xylXYZ) and the 1,2-dihydro-1,2-dihydroxytoluate dehydrogenase (xylL) from pWW0, which allows the transformation of 4-chlorobenzoate into 4-chlorocatechol, was studied. Such a derivative could efficiently grow on 4-chlorobenzoate. Unexpectedly, this derivative also grew on 3,5-dichlorobenzoate, a substrate for XylXYZL but not an inducer of the XylS regulatory protein. The ability to grow on 4-chlorobenzoate or 3,5-dichlorobenzoate was also observed in derivatives of strain JMP134 containing the xyl gene module but lacking xylS, indicating the presence of an xylS-like element in R. eutropha with an inducer profile different from that of the pWW0-encoded regulator. Growth on 4-chlorobenzoate was also observed after introduction of the xyl gene module into strain JMP222, a JMP134 derivative lacking pJP4, but only if multiple copies of tfdC I D I E I F I or tfdD II C II E II F II were present. However, only the derivative containing multiple copies of tfdD II C II E II F II was able to grow on 3,5-dichlorobenzoate. These observations indicate that although the acquisition of new catabolic genes actually enhances the catabolic abilities of R. eutropha JMP134, these new properties are strongly influenced by the dosage of the tfd genes, the presence of a chromosomal xylS-like regulatory element and the different contributions of the tfd gene clusters. the chlorocatechol pathway allow bacteria to grow on chloroaromatic compounds. Therefore, it has been proposed that a wise combination of peripheral reactions with a chlorocatechol pathway should produce new or expanded catabolic properties in bacteria (Reineke, 1998). This hypothesis has been shown to be correct in several cases (e.g. Brinkmann & Reineke, 1992 ;Klemba et al., 2000 ; Ravatn et al., 1998 ;Reineke & Knackmuss, 1979, 1980Rojo et al., 1987) and has given some insight into how a catabolic pathway may evolve in chloroaromatic-degrading bacteria. KeywordsRalstonia eutropha JMP134 is well known for its catabolic properties toward chloroaromatic compounds. This bacterium grows on 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-CB), as well as other chloroaromatic pollutants (Cle! ment et al., 1995 ;Don & Pemberton, 1981 ; Pieper et al., 1988). The key catabolic abilities of R. eutropha JMP134 are encoded on the plasmid pJP...

Efficient Turnover of Chlorocatechols Is Essential for Growth of Ralstonia eutropha JMP134(pJP4) in 3-Chlorobenzoic Acid

“…Introduction of multiple copies of the tfdC I D I E I F I or tfdD II C II E II F II genes into strain JMP222, a derivative of strain JMP134 lacking pJP4 and therefore unable to grow in 3-CB, allows the derivatives to grow in this compound (27). However, we observed that derivatives of strain JMP222 harboring single chromosomal copies of each tfd gene module or one copy of the tfdC I D I E I F I module and one copy of the tfdD II C II E II F II module did not grow in 3-CB (23). A tfd gene dosage effect has been shown to be important for efficient growth in 3-CB of strain JMP134 derivatives harboring a rearranged form of the pJP4 plasmid (5).…”

“…This enzyme system efficiently catalyzes the conversion of 3-CB to 3-CC and 4-CC (33). By using pSPM100, a pUT derivative containing the xylSxylXYZL genes, several R. eutropha transconjugants containing chromosomal insertions of the xyl gene cluster were obtained (23). One of the derivatives, strain JMP134::X(pJP4), exhibited significantly less growth in 3-CB than the wild-type strain (Fig.…”

“…The effect of the presence of single copies of these tfd gene modules was also tested. The mini Tn5-derived suicide plasmid vector pBSL202 was used to insert tfdR-P tdf-I tfdC I D I E I F I (pR1TFD), tfdR-P tdf-II tfdD II C II E II F II (pR2TFD), or both gene clusters (pR12TFD) into the chromosome of strain JMP222, as recently reported (23). The presence of each gene module in the chromosome of JMP222 transconjugants was confirmed by PCR and Southern blot analysis (23).…”

“…The mini Tn5-derived suicide plasmid vector pBSL202 was used to insert tfdR-P tdf-I tfdC I D I E I F I (pR1TFD), tfdR-P tdf-II tfdD II C II E II F II (pR2TFD), or both gene clusters (pR12TFD) into the chromosome of strain JMP222, as recently reported (23). The presence of each gene module in the chromosome of JMP222 transconjugants was confirmed by PCR and Southern blot analysis (23). None of the transconjugants, including strains JMP222::tfdR-P tdf-I tfdC I D I E I F I (JMP222::R1TFD), JMP222::tfdR-P tdf-II tfdD II C II E II F II (JMP222::R2TFD), and JMP222::tfdR-P tdf-I tfdC I D I E I F I -tfdR-P tdf-II tfdD II C II E II F II (JMP222::R12TFD), was able to proliferate in liquid cultures containing 0.5 to 10 mM 3-CB ( Fig.…”

“…The two pJP4-encoded chlorocatecholdegrading tfd modules were cloned into the medium-copy-number plasmid vector pBBR1MCS-2 to obtain pBBR1M-I and pBBR1M-II, as described elsewhere (27). To allow insertion into the chromosome, the tfdR-P tfd-I tfdC I D I E I F I gene module, the tfdR-P tfd-II tfdD II C II E II F II gene module, and both modules were cloned into the mini Tn5-derived plasmid vector pBSL202 to obtain pR1TFD, pR2TFD, and pR12TFD, respectively, as reported elsewhere (23).…”