Browsing by Author "González, B"

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    Deletions of mob and tra pJP4 transfer functions after mating of Ralstonia eutropha JMP134 (pJP4) with Escherichia coli harboring F′
    (2000) Clément, P; Springael, D; González, B
    One-tenth of Escherichia coli transconjugants resulting from the transfer of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 to E. coli XL1Blue, contained pJP4 derivatives with deletions (similar to 15-30 kb). The occurrence of these deletions is probably associated with the presence of Tn10 in the recipient. DNA endonuclease restriction analysis of the pJP4 deletion derivatives showed the absence of SphI and EcoRI fragments previously reported to hybridize with IncP Tra DNA probes. Moreover, these pJP4 deletion derivatives are not able to self-transfer, nor are they able to be mobilized. Accordingly, these pJP4 deletion derivatives lack transfer functions.
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    Efficient degradation of 2,4,6-trichlorophenol requires a set of catabolic genes related to tcp genes from Ralstonia eutropha JMP134(pJP4)
    (2003) Matus, V; Sánchez, MA; Martínez, M; González, B
    2,4,6-Trichlorophenol (2,4,6-TCP) is a hazardous pollutant. Several aerobic bacteria are known to degrade this compound. One of these, Ralstonia eutropha JMP134(pJP4), a well-known, versatile chloroaromatic compound degrader, is able to grow in 2,4,6-TCP by converting it to 2,6-dichlorohydroquinone, 6-chlorohydroxyquinol, 2-chloromaleylacetate, maleylacetate, and beta-ketoadipate. Three enzyme activities encoded by tcp genes, 2,4,6-TCP monooxygenase (tcpA), 6-chlorohydroxyquinol 1,2-dioxygenase (tcpC), and maleylacetate reductase (tcpD), are involved in this catabolic pathway. Here we provide evidence that all these tep genes are clustered in the R. eutropha JMP134 (pJP4) chromosome, forming the putative catabolic operon tcpRXABCYD. We studied the presence of tcp-like gene sequences in several other 2,4,6-TCP-degrading bacterial strains and found two types of strains. One type includes strains belonging to the Ralstonia genus and possessing a set of tep-like genes, which efficiently degrade 2,4,6-TCP and therefore grow in liquid cultures containing this chlorophenol as a sole carbon source. The other type includes strains belonging to the genera Pseudomonas, Sphingomonas, or Sphingopixis, which do not have tep-like gene sequences and degrade this pollutant less efficiently and which therefore grow only as small colonies on plates with 2,4,6-TCP. Other than strain JMP134, none of the bacterial strains whose genomes have been sequenced possesses a full set of tcp-like gene sequences.
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    Efficient turnover of chlorocatechols is essential for growth of Ralstonia eutropha JMP134(pJP4) in 3-chlorobenzoic acid
    (2003) Pérez-Pantoja, D; Ledger, T; Pieper, DH; González, B
    Ralstonia eutropha JMP134(pJP4) degrades 3-chlorobenzoate (3-CB) by using two not completely isofunctional, pJP4-encoded chlorocatechol degradation gene clusters, tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II). Introduction of several copies of each gene cluster into R. eutropha JMP222, which lacks pJP4 and thus accumulates chlorocatechols from 3-CB, allows the derivatives to grow in this substrate. However, JMP222 derivatives containing one chromosomal copy of each cluster did not grow in 3-CB. The failure to grow in 3-CB was the result of accumulation of chlorocatechols due to the limiting activity of chlorocatechol 1,2-dioxygenase (TfdC), the first enzyme in the chlorocatechol degradation pathway. Micromolar concentrations of 3- and 4-chlorocatechol inhibited the growth of strains JMP134 and JMP222 in benzoate, and cells of strain JMP222 exposed to 3 mM 3-CB exhibited a 2-order-of-magnitude decrease in viability. This toxicity effect was not observed with strain JMP222 harboring multiple copies of the tfdC(I) gene, and the derivative of strain JMP222 containing tfdC(I)D(I)E(I)F(I) plus multiple copies of the tfdC(I) gene could efficiently grow in 3-CB. In addition, tfdC(I) and tfdC(II) gene mutants of strain JMP134 exhibited no growth and impaired growth in 3-CB, respectively. The introduction into strain JMP134 of the xylS-xylXYZL genes, encoding a broad-substrate-range benzoate 1,2-dioxygenase system and thus increasing the transformation of 3-CB into chlorocatechols, resulted in derivatives that exhibited a sharp decrease in the ability to grow in 3-CB. These observations indicate that the dosage of chlorocatechol-transforming genes is critical for growth in 3-CB. This effect depends on a delicate balance between chlorocatechol-producing and chlorocatechol-consuming reactions.
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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
    (2002) Bobadilla, R; Varela, C; Céspedes, R; González, B
    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 chlorocatechol-accumulating 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 tfd(I)CDEF gene cluster was inserted in the chromosome of these strains using mini Tn5-derived vectors that allow expression of the Tfd enzymes driven by the lacI(q)/P-trc or tfdR/Ptfd-I regulatory systems, and therefore, responding to the inducers isopropyl-beta-D-thiogalactopyranoside (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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    Importance of different tfd genes for degradation of chloroaromatics by Ralstonia eutropha JMP134
    (2002) Plumeier, I; Pérez-Pantoja, D; Heim, S; González, B; Pieper, DH
    The tfdC(I)D(I)E(I)F(I), and tfdD(II)C(II)E(II)F(II) gene modules of plasmid pJP4 of Ralstonia eutropha JMP134 encode complete sets of functional enzymes for the transformation of chlorocatechols into 3-oxoadipate, which are all expressed during growth on 2,4-dichlorophenoxyacetate (2,4-D). However, activity of tfd(1)-encoded enzymes was usually higher than that of tfd(II)-encoded enzymes, both in the wild-type strain grown on 2,4-D and in 3-chlorobenzoate-grown derivatives harboring only one tfd gene module. The tfdD(II)-encoded chloromuconate cycloisomerase exhibited special kinetic properties, with high activity against 3-chloromuconate and poor activity against 2-chloromuconate and unsubstituted muconate, thus explaining the different phenotypic behaviors of R. eutropha strains containing different tfd gene modules. The enzyme catalyzes the formation of an equilibrium between 2-chloromuconate and 5-chloro- and 2-chloromuconolactone and very inefficiently catalyzes dehalogenation to form trans-dienelactone as the major product, thus differing from all (chloro)muconate cycloisomerases described thus far.
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    Molecular characterization of a deletion/duplication rearrangement in tfd genes from Ralstonia eutropha JMP134(pJP4) that improves growth on 3-chlorobenzoic acid but abolishes growth on 2,4-dichlorophenoxyacetic acid
    (2001) Clément, P; Pieper, DH; González, B
    Ralstonia eutropha JMP134(pJP4) is able to grow on minimal media containing the pollutants 3-chlorobenzoate (3-CB) or 2,4-dichlorophenoxyacetate (2,4-D). tfd genes from the 88 kb plasmid pJP4 encode enzymes involved in the degradation of these compounds. During growth of strain JMP134 in liquid medium containing 3-CB, a derivative strain harbouring a similar to 95 kb plasmid was isolated. This derivative, designated JMP134(pJP4-F3), had an improved ability to grow on 3-CB, but had lost the ability to grow on 2,4-D. Sequence analysis of pJP4-F3 indicated that the plasmid had undergone a deletion of similar to 16 kb, which included the tfdA-tfdS intergenic region, spanning the tfdA gene to a previously unreported IS1071 element. The loss of the tfdA gene explains the failure of the derivative to grow on 2,4-D. A similar to 23 kb duplication of the region spanning tfdR-tfdD(II)C(II)E(II)F(II)-tfdB(II)-tfdK-ISJP4-tfdT-tfdC(I)D(I)E(I)F(I)-tfdB(I), giving rise to a 51-kb-long inverted repeat, was also observed. The increase in gene copy number for the tfdCD(DC)EF gene cluster may provide an explanation for the derivative strain's improved growth on 3-CB. These observations are additional examples of the metabolic plasticity of R. eutropha JMP134, one of the more versatile pollutant-degrading bacteria.
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    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
    (2002) Ledger, T; Pieper, DH; Pérez-Pantoja, D; González, B
    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 (xyIXYZ) and the 1,2-dihydro-1,2-dihydroxytoluate dehydrogenase (xyIL) 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.
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    Poly-β-hydroxyalkanoates consumption during degradation of 2,4,6-trichlorophenol by Sphingopyxis chilensis S37
    (2003) Godoy, FA; Bunster, M; Matus, V; Aranda, C; González, B; Martínez, MA
    Aims: To analyse the possible effect of poly-beta -hydroxyalkanoate (PHA) consumption on 2,4,6-trichlorophenol (2,4,6-TCP) degradation during starvation by Sphingopyxis chilensis S37 strain, which stores PHAs and degrades 2,4,6-TCP.
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    Role of tfdCIDIEIFI and tfdDIICIIEIIFII gene modules in catabolism of 3-chlorobenzoate by Ralstonia eutropha JMP134(pJP4)
    (2000) Pérez-Pantoja, D; Guzmán, L; Manzano, M; Pieper, DH; González, B
    The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, a nd maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(II)F(II) (module II), putatively encoding these enzymes, To assess the role of both ya modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene, These constructs were introduced into R. eurtropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,3-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I, The R, eutropha JMP222 derivative containing tfdR-tfdC(I)D(I)E(I)F(I) grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD(II)C(II)E(II)F(II) In the case of P, putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB, These results indicate that efficient degradation of 3-CB by R, eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.
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    The copy number of the catabolic plasmid pJP4 affects growth of Ralstonia eutropha JMP134 (pJP4) on 3-chlorobenzoate
    (2002) Trefault, N; Clément, P; Manzano, M; Pieper, DH; González, B
    Ralstonia eutropha JMP134 (pJP4) grows on 3-chlorobenzoate (3-CB) or 2,4-dichlorophenoxyacetate (2,4-D). The copy number of chlorocatechol genes has been observed to be important for allowing growth of bacterial strains on chloroaromatic compounds. Despite the fact that two functional chlorocatechol degradation tfd gene clusters are harbored on plasmid pJP4, a single copy of the region comprising all tfd genes in strain JMP134-F was insufficient to allow growth on 3-CB, whereas growth on 2,4-D was only slightly retarded compared to the wild-type strain. Using competitive PCR, approximately five copies of pJP4 per genome were observed to be present in the wild-type strain, whereas only one copy of pJP4 was present per chromosome in strain JMP134-F. Therefore, several copies of pJP4 per chromosome are required for full expression of the tfd-encoded growth abilities in the wild-type R. eutropha strain. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
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    Tolerance to trichlorophenols in microorganisms from a polluted and a pristine site of a river.
    (1999) Godoy, F; Zenteno, P; Cerda, F; González, B; Martínez, M
    The effect of 2,4,5- and 2,4,6-trichlorophenol on the microbiota from a polluted and a pristine site of a river was studied. Bacterial metabolic activity measurements by epifluorescence microscopy showed that the polluted site contained more metabolically active cells than the pristine site. Total culturable bacterial counts and tolerant bacterial counts from both sites were not affected by incubation (for up to 5 days) with 200 ppm of chlorophenols. However, the incubation with 500 ppm of 2,4,5-trichlorophenol prevented detection of total and tolerant bacterial counts in the pristine site, and inhibited tolerants in the polluted site. None of 250 bacterial colonies directly isolated from these samples was able to grow on chlorophenols. However, bacteria able to grow on 2,4,6-trichlorophenol, were obtained by enrichment of water and sediments samples. (C) 1998 Elsevier Science Ltd. All rights reserved.