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M Mergeay

Publications and source records attributed to M Mergeay.

At least 37 records · Page 2Linked to original sources
PubMed1997

Two-component regulatory system involved in transcriptional control of heavy-metal homoeostasis in Alcaligenes eutrophus.

The czc determinant, which mediates resistance to Co2+, Zn2+ and Cd2+ in Alcaligenes eutrophus CH34 by cation efflux, is regulated by a two-component regulatory system composed of the sensor histidine kinase CzcS and the response activator CzcR (in addition to other components previously described). Regulatory genes are arranged in an upstream regulatory region (URR) and a downstream regulatory region (DRR). Transcription of czcCBA and of the URR was regulated by heavy-metal cations. DNA sequencing of the region downstream of czcD revealed the presence of the czcR and czcS genes which together with czcD form the DRR. Regulation of the DRR was studied with a czcD::lacZ translational fusion and a czcS::lux transcriptional fusion. Expression of both genes is also regulated by heavy metals. The genes of the URR yielded three mRNAs of approx. 1200, 500 and 200 nucleotides, respectively. The genes czcCBA for the cation/proton antiporter CzcCBA were transcribed by one operon as a transcript of 6200 nucleotides.

Alcaligenes↗
PubMed1996

Identification of a partition and replication region in the Alcaligenes eutrophus megaplasmid pMOL28.

A 4.64 kb region of the 180 kb heavy metal resistance plasmid pMOL28 of Alcaligenes eutrophus CH34, previously shown to be able to replicate autonomously, was sequenced and analyzed. Three genes involved in plasmid maintenance were identified: parA28 and parB28 are involved in plasmid partitioning and stability, while repA28 encodes a protein required for replication. In addition to the par AB28 genes, a third locus, parS28, required in cis active partitioning was identified. The parABS28 locus of pMOL28 shows strong similarity in organization to the sop, par and rep regions, respectively, of the Escherichia coli F-factor, the E.coli P1 and P7 prophages and the Agrobacterium pTiB6S3 and pRiA4b plasmids. The ParAB28 proteins of pMOL28 also show similarity to the proteins encoded by two conserved open reading frames present in the replication regions of the Pseudomonas putida and Bacillus subtilis chromosomes. The functionality of the pMOL28 par region was examined by performing stability and incompatibility tests between pMOL28 and pMOL846 or pMOL850 which contain the 4.64 EcoRI replicon fragment of pMOL28, cloned in opposite orientations into pSUP202, which is itself unable to replicate in A. eutrophus. The RepA2 8 replication protein showed similarity to the RepL protein of P1, which is required for lytic replication of this E. coli phage. The replication origin of pMOL28, oriV28, seems to be located within the repA28 coding region, and pMOL28 replication may depend on transcriptional activation of oriV28.

Alcaligenes↗
PubMed1996

RP4::Mu3A-mediated in vivo cloning and transfer of a chlorobiphenyl catabolic pathway.

Chromosomal DNA fragments encoding the ability to utilize biphenyl as sole carbon source (Bph+) were mobilized by means of plasmid RP4::Mu3A from strain JB1 (tentatively identified as Burkholderia sp.) to Alcaligenes eutrophus CH34 at a frequency of 10(-3) per transferred plasmid. The mobilized DNA integrated into the recipient chromosome or was recovered as catabolic prime plasmids. Three Bph+ prime plasmids were transferred from A. eutrophus to Escherichia coli and back to A. eutrophus without modification of the phenotype. The transferred Bph+ DNA segments allowed metabolism of biphenyl, 2-, 3- and 4-chlorobiphenyl, and diphenylmethane. Genes involved in biphenyl degradation were identified on the prime plasmids by DNA-DNA hybridization and by gene cloning. Bph+ prime plasmids were transferred to Burkholderia cepacia, Pseudomonas aeruginosa, Comamonas testosteroni and A. eutrophus and the catabolic genes were expressed in those hosts. Transfer of the plasmid to the 3-chlorobenzoate-degrading bacterium Pseudomonas sp. B13 allowed the recipient to mineralize 3-chlorobiphenyl. Other catabolic prime plasmids were obtained from JB1 by selection on m-hydroxybenzoate and tyrosine as carbon sources. 16S rRNA sequence data demonstrated that the in vivo transfer of bph was achieved between bacteria belonging to two different branches of the beta-Proteobacteria.

Alcaligenes↗
PubMed1996

Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric iron-alcaligin E receptor.

Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells. However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus.

Alcaligenes↗
PubMed1995

The czc operon of Alcaligenes eutrophus CH34: from resistance mechanism to the removal of heavy metals.

The plasmid-borne czc operon ensures for resistance to Cd2+, Zn2+ and Co2+ ions through a tricomponent export pathway and is associated to various conjugative plasmids of A. eutrophus strains isolated from metal-contaminated industrial areas. The czc region of pMOL30 was reassessed especially for the segments located upstream and downstream the structural genes czc CBA. In cultures grown with high concentrations of heavy metals, czc-mediated efflux of cations is followed by a process of metal bioprecipitation. These observations led to the development of bioreactors designed for the removal of heavy metals from polluted effluents.

ATP-Binding Cassette Transporters↗
PubMed1994

Transfer and expression of PCB-degradative genes into heavy metal resistant Alcaligenes eutrophus strains.

Sites polluted with organic compounds frequently contain inorganic pollutants such as heavy metals. The latter might inhibit the biodegradation of the organics and impair bioremediation. Chromosomally located polychlorinated biphenyl (PCB) catabolic genes of Alcaligenes eutrophus A5, Achromobacter sp. LBS1C1 and Alcaligenes denitrificans JB1 were introduced into the heavy metal resistant Alcaligenes eutrophus strain CH34 and related strains by means of natural conjugation. Mobile elements containing the PCB catabolic genes were transferred from A. eutrophus A5 and Achromobacter sp. LB51C1 into A. eutrophus CH34 after transposition onto their endogenous IncP plasmids pSS50 and pSS60, respectively. The PCB catabolic genes of A. denitrificans JB1 were transferred into A. eutrophus CH34 by means of RP4::Mu3A mediated prime plasmid formation. The A. eutrophus CH34 transconjugant strains expressed both catabolic and metal resistance markers. Such constructs may be useful for the decontamination of sites polluted by both organics and heavy metals.

Alcaligenes↗
PubMed1994

Poisson-like fluctuation patterns of revertants of leucine auxotrophy (leu-500) in Salmonella typhimurium caused by delay in mutant cell division.

Leu+ mutants from Salmonella typhimurium leu-500 strain MA412 arise at high frequencies and mutant colonies appear over a broad range of time on selective plates. This observation suggested that these Leu+ mutants might be induced or "directed."= If such a mechanism was responsible, mutants should originate on selective plates rather than in the preceding culture in nonselective conditions and should give rise to Poisson-like fluctuation curves upon plating of sister cultures on selective medium. Poisson-like distribution profiles were indeed observed for Leu+ mutants of S. typhimurium MA412. However, an explanation for the observed Poisson-like fluctuation patterns without a need for selection-induced mutations was found. Microscopical analysis and cell mass/viable count measurements showed that the size of Leu+ mutant cells was often much larger than those of nonmutants. This size difference was a stable characteristic of a large proportion of Leu+ mutants, was observed both in stationary and growing culture and did not measurably affect the division rates of the cells in nutrient broth. As the transition from normal-sized nonmutant to oversized mutant cells during the nonselective culture phase of the fluctuation experiment may have been accompanied by a period with no or few completed cell division cycles, the number of mutant offspring may have been smaller than that of sibling nonmutants. Such underrepresentation of mutants in the final culture is expected to give rise to Poisson-like fluctuation patterns without invoking "directed" mutations.

Cell Division↗
PubMed1994

The sss gene product, which affects pyoverdin production in Pseudomonas aeruginosa 7NSK2, is a site-specific recombinase.

Pyoverdin production by Pseudomonas aeruginosa strain 7NSK2 was induced by Zn(II) in the presence of iron. A mutant was isolated in which Zn(II) no longer induced pyoverdin production. The sss gene which was inactivated in this mutant was cloned and sequenced. Its protein sequence showed 50% identity to the XerC protein of Escherichia coli, which is a member of the lambda integrase family of site-specific recombinases. An open reading frame was found upstream of sss whose protein sequence showed strong identity to DapF, the diaminopimelate epimerase. In E. coli, xerC is part of a multicistronic unit that also contains dapF. The sss gene of P. aeruginosa could restore site-specific recombination at cer in an E. coli xerC mutant and the E. coli xerC gene could complement a genomic sss mutation in P. aeruginosa.

Amino Acid Sequence↗
PubMed1994

Plasmids for heavy metal resistance in Alcaligenes eutrophus CH34: mechanisms and applications.

Alcaligenes eutrophus CH34 is the main representative of a group of strongly related strains (mostly facultative chemolithotrophs) that are well adapted to environments containing high levels of heavy metals. It harbors the megaplasmids pMOL28 and pMOL30 which carry resistance determinants to Co2+, Ni2+, CrO(4)2-, Hg2+, Tl+, Cd2+, Cu2+ and Zn2+. Among the best characterized determinants are the cnr operon (resistance to Co, Ni) on pMOL28 and the czc operon on pMOL30 (resistance to Co, Cd and Zn). Although the two systems reveal a significant degree of amino acid similarity in the structural genes, the regulation of the operons is different. The resistance mechanism in both cases is based on efflux. The efflux mechanism leads to a pH increase outside of the cytoplasmic membrane. Metals are sequestered from the external medium through the bioprecipitation of metal carbonates formed in the saturated zone around the cell. This latter phenomenon can be exploited in bioreactors designed to remove metals from effluents. The bacteria are immobilized on composite membranes in a continuous tubular membrane reactor (CTMR). The effluent continuously circulates through the intertubular space, while the external surface of the tubes is in contact with the growth medium. Metal crystals are eventually removed by the effluent stream and collected on a glass bead column. The system has been applied to effluents containing Cd, Zn, Co, Ni and Cu. By introducing catabolic plasmids involved in the aerobic degradation of PCBs and 2,4-D into metal-resistant A. eutrophus strains, the application range was widened to include effluents polluted with both organic and inorganic substances. Biosensors have been developed which are based on the fusion of genes induced by metals to a reporter system, the lux operon of Vibrio fischeri. Bacterial luciferases produce light through the oxidation of fatty aldehydes. The gene fusions are useful both for the study of regulatory genes and for the determination of heavy metal concentrations in the environment.

Alcaligenes↗
PubMed1994

Electroporation of Alcaligenes eutrophus with (mega) plasmids and genomic DNA fragments.

Electroporation was used as a tool to explore the genetics of the heavy-metal-resistant strain Alcaligenes eutrophus CH34. A 12.9-kb A. eutrophus-Escherichia coli shuttle vector, pMOL850, was constructed to optimize electroporation conditions. This vector is derived from the E. coli plasmid pSUP202 and contains the replication region of the A. eutrophus megaplasmid pMOL28. Electroporation was used to transform A. eutrophus CH34 derivatives with megaplasmids (sizes up to 240 kb), and transformants were selected for resistance to heavy metals. Electroporation was also performed with endonuclease-digested genomic DNA. Transformation of markers affecting lysine biosynthesis (lysA194) and biosynthesis of the siderophore alcaligin E were observed. Transfer of the nonselected markers pheB332 and aro-333, linked to lysA194, confirmed the intervention of homologous recombination. However, during transformation of ale::Tn5-Tc, illegitimate recombination and transposition were also observed as an alternative for the inheritance of the Tn5-Tc markers.

Alcaligenes↗
PubMed1994

Exogenous isolation of mobilizing plasmids from polluted soils and sludges.

Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with Escherichia coli (a member of the gamma subgroup of the Proteobacteria) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes czc: Co, Zn, and Cd), Alcaligenes eutrophus (a member of the beta subgroup of the Proteobacteria) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (E. coli CM120 harboring IncP [IncP1] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHI1, repH12, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present.

Journal Article↗
PubMed1993

Use of DNA probes and plasmid capture in a search for new interesting environmental genes.

Adaptation to a stressed environment leads to organisms bearing DNA, encoding defense mechanisms. These mechanisms can be heavy metal resistance, catabolism of organic xenobiotics or stress reactions. Genes responsible for these mechanisms can be used for monitoring changing environments and therefore it can be important to store such bacteria in a bank. DNA-probing will be presented by the use of DNA fragments (of Alcaligenes eutrophus) coding for heavy metal resistance or xenobiotic degradation. Some strains do not grow on petri dishes and accordingly cannot be isolated from soils. In order to isolate plasmids from such strains, coding for heavy metal resistances or xenobiotic degradations, an exogenous plasmid isolation method was developed. In this method, the endogenous population is conjugated with Pseudomonas or Alcaligenes strains bearing a retrotransfer plasmid like RP4. In that way new plasmids from various sources including non-culturable strains could be obtained. With these methods, a large number of specimens adapted to stressed situations can be isolated or constructed (in the case of the exogenous plasmid isolation method). They form a source of interesting genetic material that can be used to restore polluted areas in natural areas, if necessary with the aid of genetic engineering (in vitro or in vivo techniques). Full knowledge of such bacteria and their resistance mechanisms or degradation pathways, can lead to new constructions able to attack recalcitrant mixtures of different organics and to resist heavy metals.

Alcaligenes↗
PubMed1993

luxAB gene fusions with the arsenic and cadmium resistance operons of Staphylococcus aureus plasmid pI258.

pC101, a novel shuttle vector between Escherichia coli and Staphylococcus aureus carrying the lux genes encoding luciferase from Vibrio harveyi, selectable ampicillin and chloramphenicol markers and origins of replication for Gram-negative and Gram-positive bacteria has been constructed. The inducibility of the arsenic and cadmium operon from S. aureus plasmid pI258 to different ions has been tested in E. coli and in S. aureus with two fusions in pC101: an arsB-luxAB and a cadA-luxAB transcriptional gene fusion. Patterns of induction are influenced by the host strain and are slightly different from previous reports using the blaZ gene as reporter gene.

Adenosine Triphosphatases↗
PubMed1993

Chromosome mapping in Alcaligenes eutrophus CH34.

Mutants and mobilizing plasmids were developed as genetic tools in Alcaligenes eutrophus CH34. In order to map the chromosome, spontaneous and ethyl methane sulphonate (EMS)-induced mutants (mostly auxotrophs) were isolated. Another source of mutants was provided by the phenomenon of temperature-induced mortality and mutagenesis that is observed at 37 degrees C and is characteristic of many metallotolerant strains of A. eutrophus. Plasmid pULB113 (RP4::miniMu) was used to map the available mutations. Twenty-five loci were ordered in a circular map. pMOL50, a rearranged derivative of plasmid pMOL28, which was obtained in a survivor at 37 degrees C and displayed chromosome mobilizing activity (Cma+), was also used to mobilize chromosomal markers: resulting linkages were stronger than with pULB113, allowing confirmation of the circularity of the A. eutrophus CH34 chromosome with a small number of crosses.

Alcaligenes↗
PubMed1993

Characterization of a temperate phage hosted by Alcaligenes eutrophus strain A5.

Nineteen strains of Alcaligenes eutrophus were tested for the presence of prophages. One strain that lysed upon mitomycin C treatment produced a phage which could not form plaques on any of the strains available. DNA extracted from partially purified phage lysates was digested with various restriction enzymes which showed that the 42 kb long viral double-stranded DNA circularizes by means of cohesive ends. To our knowledge, this is the first description of a phage for the genus Alcaligenes.

Alcaligenes↗
PubMed1993

Construction and characterization of heavy metal-resistant haloaromatic-degrading Alcaligenes eutrophus strains.

Alcaligenes eutrophus strains exhibiting both plasmid-borne heavy metal resistance and haloaromatic-degrading functions were obtained by intraspecific conjugation. The strains which we constructed expressed catabolic and resistance markers together. Degradation of various polychlorinated biphenyl isomers and 2,4-D (2,4-dichlorophenoxyacetic acid) was observed in the presence of 1 mM nickel or 2 mM zinc, provided that the metal resistance determinant was present in the catabolizing strain. Such strains may be useful for decontamination of sites that are polluted with both organic compounds and heavy metals.

Alcaligenes↗