Analysis of duplicated gene sequences associated with tfdR and tfdS in Alcaligenes eutrophus JMP134

Matrubutham, Udayakumar; Harker, Alan R.

doi:10.1128/jb.176.8.2348-2353.1994

Cited by 64 publications

(55 citation statements)

References 33 publications

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“…ment et al, 1995 ;Don & Pemberton, 1981 ;Pieper et al, 1988). The key catabolic abilities of R. eutropha JMP134 are encoded on the plasmid pJP4 (Don & Pemberton, 1981), and the catabolic enzymes and genes of this plasmid have been studied extensively (Don et al, 1985 ;Kasberg et al, 1995 ;Kuhm et al, 1990 ;Laemmli et al, 2000 ;Leveau et al, 1999 ;Matrubutham & Harker, 1994 ;Pe! rez-Pantoja et al, 2000 ;Perkins et al, 1990 ;Pieper et al, 1988Pieper et al, , 1989Pieper et al, , 1993Seibert et al, 1993 ;Streber et al, 1987 ;Vollmer et al, 1999).…”

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confidence: 99%

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

et al. 2002

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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...

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Section: Abbreviationsmentioning

confidence: 99%

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

et al. 2002

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“…The tfdS gene is oriented divergently from the tfdA gene (40,48). The tfdR gene is organized in a fashion similar to that of tfdS, with a tfdD-like gene (tfdD II ) on plasmid pJP4 (27). The TcbR, ClcR, TfdR, and partial TfdT proteins have a very high degree of identity in their overlapping predicted amino acid sequences (45,48).…”

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confidence: 99%

“…The binding of the proteins to their target promoter DNA has been reported and described meticulously (4,25). Binding of the TfdR protein to the tfdD II promoter region has also been demonstrated (27), but regulation of this gene by TfdR has not yet been established.…”

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confidence: 99%

“…These genes encode enzymes for the conversion of 2,4-dichlorophenoxyacetic acid (2,4-D) (40) and 2,4-dichlorophenol (29) which, in concert with the CC-degradative pathway encoded by tfdCDEF, enable host strain R. eutropha JMP134(pJP4) to grow on 2,4-D. The identical tfdR and tfdS genes, which code for the TfdR protein, are located about 8 kb away from the tfdCDEF operon on plasmid pJP4 (27,48). The tfdS gene is oriented divergently from the tfdA gene (40,48).…”

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The tfdR gene product can successfully take over the role of the insertion element-inactivated TfdT protein as a transcriptional activator of the tfdCDEF gene cluster, which encodes chlorocatechol degradation in Ralstonia eutropha JMP134(pJP4)

Leveau

Meer

1996

J Bacteriol

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The tfdT gene is located upstream of and transcribed divergently from the tfdCDEF chlorocatechol-degradative operon on plasmid pJP4 of Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134. It is 684 bp long and encodes a 25-kDa protein. On the basis of its predicted amino acid sequence, the TfdT protein could be classified as a LysR-type transcriptional regulator. It has the highest degree of similarity with the proteins TcbR, ClcR, and TfdR, which are involved in the regulation of chloroaromatic breakdown. Despite this homology, the TfdT protein failed to activate the expression of its presumed target operon, tfdCDEF. This failure could be attributed to the inability of TfdT to bind the tfdC promoter region, an absolute requirement for transcriptional activation. Sequence analysis downstream of the tfdT gene revealed the presence of an insertion element-like element. We postulate that this element disrupted the tfdT open reading frame, leading to a premature termination and the production of a truncated, disfunctional TfdT protein. As an alternative to the inactivated TfdT protein, we propose that the product of the tfdR gene (or its identical twin, tfdS), located elsewhere on plasmid pJP4, can successfully take over the regulation of tfdCDEF expression. The TfdR protein was capable of binding to the tfdC promoter region and activated tfdCDEF gene expression by a factor of 80 to 100 when provided in cis as a tfdR-tfdCDEF hybrid regulon. Although to a lesser extent, induction of tfdCDEF expression was also observed when no functional TfdR protein was provided, implying cross-activation by chromosomally encoded regulatory elements in R. eutropha JMP134(pJP4).

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“…The regulation of the tfd genes is not well understood. Recent works indicate that tfdR, a LysR-type transcriptional activator, may play the main role in expression of both chlorocatechol-catabolism tfd clusters (Matrubutham and Harker, 1994;Leveau and Van der Meer, 1996;Leveau et al 1999).…”

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Engineering bacterial strains through the chromosomal insertion of the chlorocatechol catabolism tfdICDEF gene cluster, to improve degradation of typical bleached Kraft pulp mill effluent pollutants

Bobadilla¹,

Varela²,

Céspedes³

et al. 2002

Electron. J. Biotechnol.

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Chloroaromatic pollutants from bleached Kraft pulp mill effluents (BKME) are difficult to degrade, because bacterial strains present in BKME aerobic treatments, only partially degrade these compounds, accumulating the corresponding chlorocatechol intermediates. To improve the catabolic performance of chlorocatecholaccumulating strains, we introduced, by chromosomal insertion, the tfd I CDEF gene cluster from Ralstonia eutropha JMP134 (pJP4). This gene cluster allows dechlorination and channelling of chlorocatechols into the intermediate metabolism. Two bacterial strains, R. eutropha JMP222 and Pseudomonas putida KT2442, able to produce chlorocatechols from 3-chlorobenzoate (3-CB) were used. Acinetobacter lwoffii RB2 isolated from BKME by its ability to grow on guaiacol as sole carbon source and shown to be able to produce the corresponding chlorocatechols from the BKME pollutants 4-, and 5-chloroguaiacol, was also used. The Bobadilla, R. et al. 163tfd I CDEF gene cluster was inserted in the chromosome of these strains using miniTn5-derived vectors that allow expression of the Tfd enzymes driven by the lacI q /P trc or tfdR/P tfd-I regulatory systems, and therefore, responding to the inducers isopropyl-ß-Dthiogalactopyranoside (IPTG) or 3-CB, respectively. Crude extracts of cells from strains JMP222, KT2442 or RB2 engineered with the tfd genes, grown on benzoate and induced with IPTG or 3-CB showed Tfd specific activities of about 15% -80% of that of the strain JMP134. Dechlorination rates for 3-CB or chloroguaiacols correlated with levels of Tfd enzymes. However, none of the strains containing the chromosomal copy of the tfd I CDEF cluster grew on monochloroaromatics as sole carbon source. Experiments with BKME aerobic treatment microcosms showed that the catabolic performance of the engineered bacteria was also lower than the wildtype R. eutropha strain JMP134.

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Analysis of duplicated gene sequences associated with tfdR and tfdS in Alcaligenes eutrophus JMP134

Cited by 64 publications

References 33 publications

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

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

The tfdR gene product can successfully take over the role of the insertion element-inactivated TfdT protein as a transcriptional activator of the tfdCDEF gene cluster, which encodes chlorocatechol degradation in Ralstonia eutropha JMP134(pJP4)

Engineering bacterial strains through the chromosomal insertion of the chlorocatechol catabolism tfdICDEF gene cluster, to improve degradation of typical bleached Kraft pulp mill effluent pollutants

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