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Bernardo González - One of the best experts on this subject based on the ideXlab platform.
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Role of eukaryotic microbiota in soil survival and Catabolic performance of the 2,4-D herbicide degrading bacteria Cupriavidus necator JMP134
Antonie van Leeuwenhoek, 2007Co-Authors: Marlene Manzano, Ana C. Morán, Bruno Tesser, Bernardo GonzálezAbstract:Cupriavidus necator (formerly Ralstonia eutropha ) JMP134, harbouring the Catabolic Plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and Catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C.␣necator JMP134 could be isolated after soil enrichment. In␣native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5α (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with Catabolic bacteria.
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Molecular and population analyses of a recombination event in the Catabolic Plasmid pjp4
Journal of bacteriology, 2006Co-Authors: Juanita Larraín-linton, Rodrigo De La Iglesia, Francisco Melo, Bernardo GonzálezAbstract:Cupriavidus necator JMP134(pJP4) harbors a Catabolic Plasmid, pJP4, which confers the ability to grow on chloroaromatic compounds. Repeated growth on 3-chlorobenzoate (3-CB) results in selection of a recombinant strain, which degrades 3-CB better but no longer grows on 2,4-dichlorophenoxyacetate (2,4-D). We have previously proposed that this phenotype is due to a double homologous recombination event between inverted repeats of the multicopies of this Plasmid within the cell. One recombinant form of this Plasmid (pJP4-F3) explains this phenotype, since it harbors two copies of the chlorocatechol degradation tfd gene clusters, which are essential to grow on 3-CB, but has lost the tfdA gene, encoding the first step in degradation of 2,4-D. The other recombinant Plasmid (pJP4-FM) should harbor two copies of the tfdA gene but no copies of the tfd gene clusters. A molecular analysis using a multiplex PCR approach to distinguish the wild-type Plasmid pJP4 from its two recombinant forms, was carried out. Expected PCR products confirming this recombination model were found and sequenced. Few recombinant Plasmid forms in cultures grown in several carbon sources were detected. Kinetic studies indicated that cells containing the recombinant Plasmid pJP4-FM were not selectable by sole carbon source growth pressure, whereas those cells harboring recombinant Plasmid pJP4-F3 were selected upon growth on 3-CB. After 12 days of repeated growth on 3-CB, the complete Plasmid population in C. necator JMP134 apparently corresponds to this form. However, wild-type Plasmid forms could be recovered after growing this culture on 2,4-D, indicating that different Plasmid forms can be found in C. necator JMP134 at the population level.
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genetic organization of the Catabolic Plasmid pjp4 from ralstonia eutropha jmp134 pjp4 reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways
Environmental Microbiology, 2004Co-Authors: Nicole Trefault, R De La Iglesia, A M Molina, M A Manzano, Thomas Ledger, Danilo Perezpantoja, Miguel Angel Sanchez, M Stuardo, Bernardo GonzálezAbstract:: Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta Plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 Plasmid was able to capture chromosomal genes and form hybrid Plasmids with the IncP-1 alpha Plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants.
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The copy number of the Catabolic Plasmid pJP4 affects growth of Ralstonia eutropha JMP134 (pJP4) on 3‐chlorobenzoate
FEMS microbiology letters, 2002Co-Authors: Nicole Trefault, Pascale Clément, Marlene Manzano, Dietmar H. Pieper, Bernardo GonzálezAbstract: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.
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Deletions of mob and tra pJP4 transfer functions after mating of Ralstonia eutropha JMP134 (pJP4) with Escherichia coli harboring F'::Tn10.
Canadian journal of microbiology, 2000Co-Authors: Pascale Clément, Dirk Springael, Bernardo GonzálezAbstract: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 (~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.Key words: pJP4, mobilization functions, deletion.
Stephen K Farrand - One of the best experts on this subject based on the ideXlab platform.
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quorum dependent transfer of the opine Catabolic Plasmid paof64 95 is regulated by a novel mechanism involving inhibition of the trar antiactivator tram
MicrobiologyOpen, 2019Co-Authors: Margaret E Wetzel, Robert E Asenstorfer, Max E Tate, Stephen K FarrandAbstract:We previously described a Plasmid of Agrobacterium spp., pAoF64/95, in which the quorum-sensing system that controls conjugative transfer is induced by the opine mannopine. We also showed that the quorum-sensing regulators TraR, TraM, and TraI function similarly to their counterparts in other repABC Plasmids. However, traR, unlike its counterpart on Ti Plasmids, is monocistronic and not located in an operon that is inducible by the conjugative opine. Here, we report that both traR and traM are expressed constitutively and not regulated by growth with mannopine. We report two additional regulatory genes, mrtR and tmsP, that are involved in a novel mechanism of control of TraR activity. Both genes are located in the distantly linked region of pAoF64/95 encoding mannopine utilization. MrtR, in the absence of mannopine, represses the four-gene mocC operon as well as tmsP, which is the distal gene of the eight-gene motA operon. As judged by a bacterial two-hybrid analysis, TmsP, which shows amino acid sequence relatedness with the TraM-binding domain of TraR, interacts with the antiactivator. We propose a model in which mannopine, acting through the repressor MrtR, induces expression of TmsP which then titrates the levels of TraM thereby freeing TraR to activate the tra regulon.
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quorum dependent mannopine inducible conjugative transfer of an agrobacterium opine Catabolic Plasmid
Journal of Bacteriology, 2014Co-Authors: Margaret E Wetzel, Marilyn Miller, Gary J Olsen, Stephen K FarrandAbstract:The Ti Plasmid in Agrobacterium tumefaciens strain 15955 carries two alleles of traR that regulate conjugative transfer. The first is a functional allele, called traR, that is transcriptionally induced by the opine octopine. The second, trlR, is a nonfunctional, dominant-negative mutant located in an operon that is inducible by the opine mannopine (MOP). Based on these findings, we predicted that there exist wild-type agrobacterial strains harboring Plasmids in which MOP induces a functional traR and, hence, conjugation. We analyzed 11 MOP-utilizing field isolates and found five where MOP induced transfer of the MOP-Catabolic element and increased production of the acyl-homoserine lactone (acyl-HSL) quormone. The transmissible elements in these five strains represent a set of highly related Plasmids. Sequence analysis of one such Plasmid, pAoF64/95, revealed that the 176-kb element is not a Ti Plasmid but carries genes for catabolism of MOP, mannopinic acid (MOA), agropinic acid (AGA), and the agrocinopines. The Plasmid additionally carries all of the genes required for conjugative transfer, including the regulatory genes traR, traI, and traM. The traR gene, however, is not located in the MOP catabolism region. The gene, instead, is monocistronic and located within the tra-trb-rep gene cluster. A traR mutant failed to transfer the Plasmid and produced little to no quormone even when grown with MOP, indicating that TraRpAoF64/95 is the activator of the tra regulon. A traM mutant was constitutive for transfer and acyl-HSL production, indicating that the anti-activator function of TraM is conserved.
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Two Opines Control Conjugal Transfer of an Agrobacterium Plasmid by Regulating Expression of Separate Copies of the Quorum-Sensing Activator Gene traR
Journal of Bacteriology, 2002Co-Authors: Philippe Oger, Stephen K FarrandAbstract:Conjugal transfer of Ti Plasmids from Agrobacterium spp. is controlled by a hierarchical regulatory system designed to sense two environmental cues. One signal, a subset of the opines produced by crown gall tumors initiated on plants by the pathogen, serves to induce production of the second, an acyl-homoserine lactone quorum-sensing signal, the quormone, produced by the bacterium itself. This second signal activates TraR, and this transcriptional activator induces expression of the tra regulon. Opines control transfer because the traR gene is a member of an operon the expression of which is regulated by the conjugal opine. Among the Ti Plasmid systems studied to date, only one of the two or more opine families produced by the associated tumor induces transfer. However, two chemically dissimilar opines, nopaline and agrocinopines A and B, induce transfer of the opine Catabolic Plasmid pAtK84b found in the nonpathogenic Agrobacterium radiobacter isolate K84. In this study we showed that this Plasmid contains two copies of traR, and each is associated with a different opine-regulated operon. One copy, traRnoc, is the last gene of the nox operon and was induced by nopaline but not by agrocinopines A and B. Mutating traRnoc abolished induction of transfer by nopaline but not by the agrocinopines. A mutation in ocd, an upstream gene of the nox operon, abolished utilization of nopaline and also induction of transfer by this opine. The second copy, traRacc, is located in an operon of four genes and was induced by agrocinopines A and B but not by nopaline. Genetic analysis indicated that this gene is required for induction of transfer by agrocinopines A and B but not by nopaline. pAtK84b with mutations in both traR genes was not induced for transfer by either opine. However, expression of a traR gene in trans to this Plasmid resulted in opine-independent transfer. The association of traRnoc with nox is unique, but the operon containing traRacc is related to the arc operons of pTiC58 and pTiChry5, two Ti Plasmids inducible for transfer by agrocinopines A-B and C-D, respectively. We conclude that pAtK84b codes for two independently functioning copies of traR, each regulated by a different opine, thus accounting for the activation of the transfer system of this Plasmid by the two opine types.
Víctor De Lorenzo - One of the best experts on this subject based on the ideXlab platform.
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Subcellular Architecture of the xyl Gene Expression Flow of the TOL Catabolic Plasmid of Pseudomonas putida mt-2
mBio, 2021Co-Authors: Juhyun Kim, Angel Goñi-moreno, Víctor De LorenzoAbstract:ABSTRACT Despite intensive research on the biochemical and regulatory features of the archetypal Catabolic TOL system borne by pWW0 of Pseudomonas putida strain mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL Plasmid, and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL Plasmid and the cell nucleoid. Also, they colocalize with ribosome-rich domains of the intracellular milieu. This arrangement was maintained even when the xyl genes were artificially relocated to different chromosomal locations. The same held true when genes were expressed through a heterologous T7 polymerase-based system, which likewise led to mRNA foci outside the DNA. In contrast, rifampin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs exit from their initiation sites to move to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest the distinct subcellular motion of xyl mRNAs results from both innate properties of the sequences and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed within the background of current knowledge on the three-dimensional organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism. IMPORTANCE The transfer of information between DNA, RNA, and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware, i.e., the nucleoid, the RNA polymerase, and the ribosomes, are habitually taken for granted. In this work, we inspected the localization and fate of the transcripts that stem from the archetypal biodegradative Plasmid pWW0 of soil bacterium Pseudomonas putida strain KT2440 through the nonhomogeneous milieu of the bacterial cytoplasm. The results expose that—similarly to computers—the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code and accompanies the environmental lifestyle of this bacterium.
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The subcellular architecture of the xyl gene expression flow of the TOL Catabolic Plasmid of Pseudomonas putida mt-2
2020Co-Authors: Juhyun Kim, Angel Goñi-moreno, Víctor De LorenzoAbstract:ABSTRACT Despite intensive research on the biochemical and regulatory features of the archetypal Catabolic TOL system borne by pWW0 of Pseudomonas putida mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL Plasmid and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL Plasmid and the cell nucleoid. Also, they co-localize with ribosome-rich domains of the intracellular milieu. This arrangement was kept even when the xyl genes were artificially relocated at different chromosomal locations. The same happened when genes were expressed through a heterologous T7 polymerase-based system, which originated mRNA foci outside the DNA. In contrast, rifampicin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs intrinsically run away from their initiation sites to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest that the distinct subcellular motion of xyl mRNAs results both from innate properties of the sequence at stake and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed on the background of current knowledge on the 3D organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism. IMPORTANCE The transfer of information between DNA, RNA and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware i.e. the nucleoid, the RNA polymerase and the ribosomes, are habitually taken for granted. In this work we inspected the localization and fate of the transcripts that stem from the archetypal biodegradative Plasmid pWW0 of soil bacterium Pseudomonas putida KT2440 through the non-homogenous milieu of the bacterial cytoplasm. The results expose that—similarly to computers also—the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code that accompanies the environmental lifestyle of this bacterium.
Nicole Trefault - One of the best experts on this subject based on the ideXlab platform.
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genetic organization of the Catabolic Plasmid pjp4 from ralstonia eutropha jmp134 pjp4 reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways
Environmental Microbiology, 2004Co-Authors: Nicole Trefault, R De La Iglesia, A M Molina, M A Manzano, Thomas Ledger, Danilo Perezpantoja, Miguel Angel Sanchez, M Stuardo, Bernardo GonzálezAbstract:: Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta Plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 Plasmid was able to capture chromosomal genes and form hybrid Plasmids with the IncP-1 alpha Plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants.
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The copy number of the Catabolic Plasmid pJP4 affects growth of Ralstonia eutropha JMP134 (pJP4) on 3‐chlorobenzoate
FEMS microbiology letters, 2002Co-Authors: Nicole Trefault, Pascale Clément, Marlene Manzano, Dietmar H. Pieper, Bernardo GonzálezAbstract: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.
Jeffrey D. Saffer - One of the best experts on this subject based on the ideXlab platform.
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Complete Sequence of a 184-Kilobase Catabolic Plasmid from Sphingomonas aromaticivorans F199
Journal of bacteriology, 1999Co-Authors: Margaret F. Romine, Lisa C. Stillwell, Kwong Kwok Wong, Sarah J. Thurston, Ellen C. Sisk, Christoph Wilhelm Sensen, Terry Gaasterland, James K. Fredrickson, Jeffrey D. SafferAbstract:Sphingomonas aromaticivorans F199 was isolated from sediments collected 410 m below the land surface near Allendale, S.C., in 1988 (4, 21). It was established that this bacterium possessed the novel ability to degrade a variety of aromatic compounds including toluene, all isomers of xylene, p-cresol, naphthalene, biphenyl, dibenzothiophene, fluorene, salicylate, and benzoate (20, 22). In recent years, there have been many reports of other Sphingomonas strains that are capable of degrading aromatic compounds (12, 17, 30, 36, 40, 44, 45, 62–65, 68–70, 88, 91). Studies of Sphingomonas strains suggest that members of this genus are well adapted for the degradation of high-molecular-weight polycyclic aromatic hydrocarbons and other aromatic contaminants. The inability to detect Sphingomonas biodegradative genes via hybridization with Catabolic genes from phylogenetically distinct bacteria suggested that biodegradative genes from Sphingomonas sp. evolved independently from phylogenetically distinct bacteria such as those within the genus Pseudomonas (46, 47). Some Sphingomonas strains are further distinguished in that the genes necessary for degradation of one type of aromatic compound are distributed into multiple operons that also possess genes for the degradation of other aromatic compounds (107). This unusual gene arrangement suggests that a highly complex regulatory network is responsible for the expression of aromatic degradative pathways in some Sphingomonas spp. S. aromaticivorans F199 was shown to possess two Plasmids (20, 22), which are designated pNL1 (∼180 kbp) and pNL2 (∼480 kbp). We reported earlier that catechol meta ring cleavage activity, a central step in the catabolism of aromatic rings, was associated with the smaller Plasmid, pNL1 (86), and we described a physical map for this Plasmid. To further probe the Catabolic functions and accessory genes encoded on pNL1, we undertook the complete sequencing and annotation of this Plasmid. This approach has allowed a thorough genetic analysis of pNL1-associated Catabolic genes, a comparison of these genes with analogous chromosomally located ones in S. yanoikuyae B1, and the development of hypotheses regarding functions of pNL1-encoded genes.
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Physical mapping and characterization of a Catabolic Plasmid from the deep-subsurface bacterium Sphingomonas sp. strain F199.
Journal of bacteriology, 1995Co-Authors: Lisa C. Stillwell, Margaret F. Romine, Sarah J. Thurston, James K. Fredrickson, R P Schneider, Jeffrey D. SafferAbstract:A supercoiled 180-kb Plasmid, pNL1, has been isolated from the deep-subsurface, chemoheterotrophic Sphingomonas sp. strain F199, and a physical map was generated. Analysis of a pNL1-derived cosmid library indicated that catechol 2,3-dioxygenase activity was linked to two distinct regions of the Plasmid. Thus, the genes for aromatic catabolism in this Sphingomonas strain are, at least in part, Plasmid encoded.