The biofilm phenotype of Pseudomonas aeruginosa enables this opportunistic pathogen to develop re... more The biofilm phenotype of Pseudomonas aeruginosa enables this opportunistic pathogen to develop resistance to the immune system and antimicrobial agents. Pseudomonas aeruginosa biofilms are generated under varying levels of shear stress, depending on the infection site. In the lung mucus of cystic fibrosis (CF) patients, P. aeruginosa forms matrix-enclosed microcolonies which cause chronic infections representing the major cause of mortality in CF patients. The lung mucus of CF patients is probably characterized by low fluid shear as the main shear-causing factor, i.e. mucociliary clearance, is absent. In this study, the influence of fluid shear on the growth behaviour of P. aeruginosa PA01 was investigated using a low-shear suspension culture device, the rotating wall vessel (RWV). Cultivation in low shear induced a self-aggregating phenotype of P. aeruginosa PA01, resulting in the formation of biofilms in suspension similar to what has been described in CF mucus. The addition of a ceramic bead to the culture medium in the RWV created a highershear condition which led to the formation of surfaceattached rather than suspension biofilms. In low-shear culture conditions, a significant increase of the rhl N-butanoyl-L-homoserine lactone (C4-HSL) directed quorum sensing (QS) system, and the psl polysaccha-ride synthetic locus was demonstrated using gene expression analysis. Accordingly, the low-shear condition induced a higher production of rhamnolipids, which is controlled by the C 4-HSL QS-system and is known to play a role in CF lung pathology. These results indicate that fluid shear has an impact on the growth phenotype of P. aeruginosa which might play a role in CF lung infections caused by this bacterium. Fig. 2. Biofilm formation of P. aeruginosa PA01 in low-shear (A) and high-shear (B) conditions using the RWV technology. The gas-permeable membrane on the backside of the vessel was stained with crystal violet. The decanted culture was collected in 50 ml falcon tubes (centre). The ceramic bead of the high-shear growth condition is visible on the bottom of the falcon tube.
International Journal of Environmental and Analytical Chemistry, Jan 25, 2007
Cupriavidus metallidurans strain CH34 is a multiple metal resistant -proteobacterium isolated fro... more Cupriavidus metallidurans strain CH34 is a multiple metal resistant -proteobacterium isolated from the sediments of a zinc metallurgical plant. This strain possesses a large diversity of heavymetal-resistance genes that are located mostly on two large plasmids, pMOL28 and pMOL30. The metal efflux ATPases constitute the major mechanism by which metal ions are removed and pumped out from the cytoplasmic pool of thiol groups. The genome of C. metallidurans CH34 contains eight such P1-ATPases. This high number of ATPases, compared with the P1-ATPase content in other micro-organisms, is a typical feature of the adaptation of C. metallidurans CH34 to metal-rich biotopes. We performed a phylogenetic analysis, and used quantitative PCR to study the metal-specific induction of the ATPases genes and, for some of them, their neighbouring genes. One of the ATPases, a homologue of the E. coli zntA gene, was characterized in detail by analysing the phenotypes of zntA mutants in different genetic backgrounds. In addition to the already-reported sensitivity to Zn(II), Cd(II), and Pb(II), zntA mutants also displayed a higher sensitivity to Tl(I) and Bi(III), indicating that the ZntA protein might play a role in the tolerance of very large cations. ]
Cupriavidus metallidurans CH34 est une bactérie résistante aux métaux lourds, isolée des sédiment... more Cupriavidus metallidurans CH34 est une bactérie résistante aux métaux lourds, isolée des sédiments d'une usine de métallurgie non-ferreuse en Belgique. Le génome de cette bactérie contient un chromosome (3.6 Mb), un mégaplasmide (2.6 Mb) et deux plasmides pMOL28 (171 kb) et pMOL30 (234 kb) Nous avons fait le catalogue des gènes impliqués dans la résistance aux métaux lourds et cherché à mesurer leur expression par deux approches transcriptomiques, dont les puces à ADN. L'analyse du génome montre au moins 170 gènes relatifs à la résistance aux ions métalliques localisés sur les 4 réplicons, principalement sur les deux plasmides. L'annotation des plasmides a mis en évidence la présence de trois îlots génomiques concentrant la plupart des résistances aux métaux . Les puces montrent la surexpression de 83 sur 164 gènes dans pMOL28, et de 143 sur 250 gènes dans pMOL30. Elles montrent aussi que les gènes présents sur les deux plasmides sont davantage surexprimés que ceux local...
International Journal of Environmental Analytical Chemistry, 2006
Cupriavidus metallidurans strain CH34 is a multiple metal resistant -proteobacterium isolated fro... more Cupriavidus metallidurans strain CH34 is a multiple metal resistant -proteobacterium isolated from the sediments of a zinc metallurgical plant. This strain possesses a large diversity of heavymetal-resistance genes that are located mostly on two large plasmids, pMOL28 and pMOL30. The metal efflux ATPases constitute the major mechanism by which metal ions are removed and pumped out from the cytoplasmic pool of thiol groups. The genome of C. metallidurans CH34 contains eight such P1-ATPases. This high number of ATPases, compared with the P1-ATPase content in other micro-organisms, is a typical feature of the adaptation of C. metallidurans CH34 to metal-rich biotopes. We performed a phylogenetic analysis, and used quantitative PCR to study the metal-specific induction of the ATPases genes and, for some of them, their neighbouring genes. One of the ATPases, a homologue of the E. coli zntA gene, was characterized in detail by analysing the phenotypes of zntA mutants in different genetic backgrounds. In addition to the already-reported sensitivity to Zn(II), Cd(II), and Pb(II), zntA mutants also displayed a higher sensitivity to Tl(I) and Bi(III), indicating that the ZntA protein might play a role in the tolerance of very large cations. ]
The stress response of bacterium Cupriavidus metallidurans CH34 into simulated microgravity R. Va... more The stress response of bacterium Cupriavidus metallidurans CH34 into simulated microgravity R. Van Houdt, P. De Boever, I. Coninx, A. Janssen, M.A. Benotmane, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. We have studied the response of Cupriavidus (formerly Ralstonia) metallidurans CH34 to simulated microgravity by culturing in a Rotating Wall Vessel (RWV) bioreactor. This bioreactor technology generates a unique Low-Shear Modeled Microgravity (LSMMG) environment and is exploited as analogue for in vivo medical and space environments. Cupriavidus and Ralstonia species are relevant model bacteria since they are often isolated from the floor, air and surfaces of spacecraft assembly rooms and not only contaminate the clean rooms but have also been found prior-to-flight on surfaces of space robots such as the Mars Odyssey Orbiter and even in-flight in ISS cooling water and Shuttle drinking water. In addition, C. metallidurans CH34 is also being used in fundamental space flight experiments aimed to gain a better insight in the bacterial adaptation to space. The first objective was to elucidate the stress response of C. metallidurans CH34 grown in LSMMG compared to a normal gravity control. Transcriptomic analysis revealed that a significant part of the heat shock response was induced in LSMMG. Transcription of d naK, encoding the major heat-shock protein and a prokaryotic homologue of the eukaryotic Hsp70 protein, was induced 6.4 fold in LSMMG. DnaK is assisted by partner chaperones DnaJ and GrpE for which transcription respectively were induced 2.0 and 2.6 fold. Transcription of other chaperones known to belong to the heat shock response was also induced in LSMMG: hslV and hsl U, encoding the HslVU protease, were induced respectively 5.5 and 3.4 fold; htpG, encoding a Hsp90 family chaperone, was induced 4.6 fold and clpB was induced 4.7 fold. Transcription of the Lon protease was induced 2.5 fold. It appears that C. metallidurans CH34 experiences growth in Low-Shear Modelled Microgravity as a stressful condition eliciting the need to express the heat-shock proteins which assist protein folding, assembly, transport, repair and degradation. Challenging cells grown in simulated gravity (LSMMG) to a heat-shock for 30 min at 50° C resulted indeed in a smaller reduction (1.7 log) in cultivable cells compared to the reduction observed for cells grown in normal earth gravity (Low-Shear Gravity LSG) (4.0 log). Next to genes involved in the heat shock response, 5 of the 11 copies of uspA, encoding a widely conserved protein belonging to a superfamily whose physiological function is unknown but which is induced in response to a variety of stresses, were induced from 2.7 to 8.7 fold. In addition, LSMMG resulted in the upregulation of various genes encoding site-specific tyrosine recombinases, site-specific serine recombinase and transposases possibly indicating that Low-Shear Modeled Microgravity could elicit an adaptive response by genetic rearrangements. Finally, the parA and parB genes from pMOL30, one of the two plasmids carried by CH34 and specialized in heavy metals resistance, were strongly induced in LSMMG respectively 19.6 and 7.0 fold. The overproduction of similar proteins was also detected in C. metallidurans cells, cultured in during space flight.
Leu+ mutants from Salmonella typhimurium leu-500 strain MA412 arise at high frequencies and mutan... more 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.
... Parameter Estimation Techniques for Modal Analysis. Date Published: 1979-02-01. Paper Number:... more ... Parameter Estimation Techniques for Modal Analysis. Date Published: 1979-02-01. Paper Number: 790221. DOI: 10.4271/790221. Author(s): DL Brown - University of Cincinnati RJ Allemang - University of Cincinnati Ray Zimmerman ...
Alcaligenes eutrophus CH34 DNA fragments encoding resistance to Cd2+, Co2+, Zn2+ (czc), or Hg2+ (... more Alcaligenes eutrophus CH34 DNA fragments encoding resistance to Cd2+, Co2+, Zn2+ (czc), or Hg2+ (merA) were cloned and used as probes in colony hybridization procedures with bacteria isolated from polluted environments such as a zinc factory area (desertified because of the toxic effects of zinc contamination) and from sediments from factories of nonferrous metallurgy in Belgium and mine areas in Zaire. From the different soil samples, strains could be isolated and hybridized with the czc probe (resistance to Cd2+, Co2+, and Zn2+ from plasmid pMOL30). Percentages of CFU isolated on nonselective plates which hybridized with the czc and the mercury resistance probes were, respectively, 25 and 0% for the zinc desert, 15 to 20 and 10 to 20% for the two Belgian factories, and 40 and 40% for the Likasi mine area. Most of these strains also carried two large plasmids of about the same size as those of A. eutrophus CH34 and shared many phenotypic traits with this strain. These findings indicated a certain correlation between the heavy-metal content in contaminated soils and the presence of heavy-metal-resistant megaplasmid-bearing A. eutrophus strains.
Escherichia coli argE and argH gene products were detected in Pseudomonas fluorescens argH122 car... more Escherichia coli argE and argH gene products were detected in Pseudomonas fluorescens argH122 carrying the E. coli FllO plasmid.
Two mathematical models to elucidate the mechanism of retromobilization (or retrotransfer), that ... more Two mathematical models to elucidate the mechanism of retromobilization (or retrotransfer), that is, the ability of conjugative plasmids to mobilize genes into the cell containing the conjugative plasmid, were developed. This study deals with retromobilization of nonconjugative plasmids (Tra-Mob'). Plasmid transfer was modeled by two mass action models. The first is based on the hypothesis that retromobilization of the Tra-Mob' vector occurs in one step, by means of the pilus formed by the Tra+ plasmid in the original host. In the second model, retromobilization is considered to be a two-step process involving two transfer events. The first step involves the transfer of the Tra+ plasmid from the recipient cell to the donor of the nonconjugative vector, and during the second encounter the nonconjugative vector is mobilized toward the recipient. Since the relationships between the number of transconjugants and the number of recipients for the two models are different, filter matings were performed for short time periods with different initial densities of the recipient population. Comparison of the numbers of transconjugants with the results of the mathematical equations confirmed the hypothesis that retromobilization is a one-step conjugation process.
Electroporation was used as a tool to explore the genetics of the heavy-metal-resistant strain Al... more 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 we,rs observed. Transfer of the nonselected markers pheB332 and aro-333, linked to lysA194, confirmed the intervention of homologous recombination. However, during transformation of ale::TnS-Tc, illegitimate recombination and transposition were also observed as an alternative for the inheritance of the Tn5-Tc markers.
The biofilm phenotype of Pseudomonas aeruginosa enables this opportunistic pathogen to develop re... more The biofilm phenotype of Pseudomonas aeruginosa enables this opportunistic pathogen to develop resistance to the immune system and antimicrobial agents. Pseudomonas aeruginosa biofilms are generated under varying levels of shear stress, depending on the infection site. In the lung mucus of cystic fibrosis (CF) patients, P. aeruginosa forms matrix-enclosed microcolonies which cause chronic infections representing the major cause of mortality in CF patients. The lung mucus of CF patients is probably characterized by low fluid shear as the main shear-causing factor, i.e. mucociliary clearance, is absent. In this study, the influence of fluid shear on the growth behaviour of P. aeruginosa PA01 was investigated using a low-shear suspension culture device, the rotating wall vessel (RWV). Cultivation in low shear induced a self-aggregating phenotype of P. aeruginosa PA01, resulting in the formation of biofilms in suspension similar to what has been described in CF mucus. The addition of a ceramic bead to the culture medium in the RWV created a highershear condition which led to the formation of surfaceattached rather than suspension biofilms. In low-shear culture conditions, a significant increase of the rhl N-butanoyl-L-homoserine lactone (C4-HSL) directed quorum sensing (QS) system, and the psl polysaccha-ride synthetic locus was demonstrated using gene expression analysis. Accordingly, the low-shear condition induced a higher production of rhamnolipids, which is controlled by the C 4-HSL QS-system and is known to play a role in CF lung pathology. These results indicate that fluid shear has an impact on the growth phenotype of P. aeruginosa which might play a role in CF lung infections caused by this bacterium. Fig. 2. Biofilm formation of P. aeruginosa PA01 in low-shear (A) and high-shear (B) conditions using the RWV technology. The gas-permeable membrane on the backside of the vessel was stained with crystal violet. The decanted culture was collected in 50 ml falcon tubes (centre). The ceramic bead of the high-shear growth condition is visible on the bottom of the falcon tube.
International Journal of Environmental and Analytical Chemistry, Jan 25, 2007
Cupriavidus metallidurans strain CH34 is a multiple metal resistant -proteobacterium isolated fro... more Cupriavidus metallidurans strain CH34 is a multiple metal resistant -proteobacterium isolated from the sediments of a zinc metallurgical plant. This strain possesses a large diversity of heavymetal-resistance genes that are located mostly on two large plasmids, pMOL28 and pMOL30. The metal efflux ATPases constitute the major mechanism by which metal ions are removed and pumped out from the cytoplasmic pool of thiol groups. The genome of C. metallidurans CH34 contains eight such P1-ATPases. This high number of ATPases, compared with the P1-ATPase content in other micro-organisms, is a typical feature of the adaptation of C. metallidurans CH34 to metal-rich biotopes. We performed a phylogenetic analysis, and used quantitative PCR to study the metal-specific induction of the ATPases genes and, for some of them, their neighbouring genes. One of the ATPases, a homologue of the E. coli zntA gene, was characterized in detail by analysing the phenotypes of zntA mutants in different genetic backgrounds. In addition to the already-reported sensitivity to Zn(II), Cd(II), and Pb(II), zntA mutants also displayed a higher sensitivity to Tl(I) and Bi(III), indicating that the ZntA protein might play a role in the tolerance of very large cations. ]
Cupriavidus metallidurans CH34 est une bactérie résistante aux métaux lourds, isolée des sédiment... more Cupriavidus metallidurans CH34 est une bactérie résistante aux métaux lourds, isolée des sédiments d'une usine de métallurgie non-ferreuse en Belgique. Le génome de cette bactérie contient un chromosome (3.6 Mb), un mégaplasmide (2.6 Mb) et deux plasmides pMOL28 (171 kb) et pMOL30 (234 kb) Nous avons fait le catalogue des gènes impliqués dans la résistance aux métaux lourds et cherché à mesurer leur expression par deux approches transcriptomiques, dont les puces à ADN. L'analyse du génome montre au moins 170 gènes relatifs à la résistance aux ions métalliques localisés sur les 4 réplicons, principalement sur les deux plasmides. L'annotation des plasmides a mis en évidence la présence de trois îlots génomiques concentrant la plupart des résistances aux métaux . Les puces montrent la surexpression de 83 sur 164 gènes dans pMOL28, et de 143 sur 250 gènes dans pMOL30. Elles montrent aussi que les gènes présents sur les deux plasmides sont davantage surexprimés que ceux local...
International Journal of Environmental Analytical Chemistry, 2006
Cupriavidus metallidurans strain CH34 is a multiple metal resistant -proteobacterium isolated fro... more Cupriavidus metallidurans strain CH34 is a multiple metal resistant -proteobacterium isolated from the sediments of a zinc metallurgical plant. This strain possesses a large diversity of heavymetal-resistance genes that are located mostly on two large plasmids, pMOL28 and pMOL30. The metal efflux ATPases constitute the major mechanism by which metal ions are removed and pumped out from the cytoplasmic pool of thiol groups. The genome of C. metallidurans CH34 contains eight such P1-ATPases. This high number of ATPases, compared with the P1-ATPase content in other micro-organisms, is a typical feature of the adaptation of C. metallidurans CH34 to metal-rich biotopes. We performed a phylogenetic analysis, and used quantitative PCR to study the metal-specific induction of the ATPases genes and, for some of them, their neighbouring genes. One of the ATPases, a homologue of the E. coli zntA gene, was characterized in detail by analysing the phenotypes of zntA mutants in different genetic backgrounds. In addition to the already-reported sensitivity to Zn(II), Cd(II), and Pb(II), zntA mutants also displayed a higher sensitivity to Tl(I) and Bi(III), indicating that the ZntA protein might play a role in the tolerance of very large cations. ]
The stress response of bacterium Cupriavidus metallidurans CH34 into simulated microgravity R. Va... more The stress response of bacterium Cupriavidus metallidurans CH34 into simulated microgravity R. Van Houdt, P. De Boever, I. Coninx, A. Janssen, M.A. Benotmane, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. We have studied the response of Cupriavidus (formerly Ralstonia) metallidurans CH34 to simulated microgravity by culturing in a Rotating Wall Vessel (RWV) bioreactor. This bioreactor technology generates a unique Low-Shear Modeled Microgravity (LSMMG) environment and is exploited as analogue for in vivo medical and space environments. Cupriavidus and Ralstonia species are relevant model bacteria since they are often isolated from the floor, air and surfaces of spacecraft assembly rooms and not only contaminate the clean rooms but have also been found prior-to-flight on surfaces of space robots such as the Mars Odyssey Orbiter and even in-flight in ISS cooling water and Shuttle drinking water. In addition, C. metallidurans CH34 is also being used in fundamental space flight experiments aimed to gain a better insight in the bacterial adaptation to space. The first objective was to elucidate the stress response of C. metallidurans CH34 grown in LSMMG compared to a normal gravity control. Transcriptomic analysis revealed that a significant part of the heat shock response was induced in LSMMG. Transcription of d naK, encoding the major heat-shock protein and a prokaryotic homologue of the eukaryotic Hsp70 protein, was induced 6.4 fold in LSMMG. DnaK is assisted by partner chaperones DnaJ and GrpE for which transcription respectively were induced 2.0 and 2.6 fold. Transcription of other chaperones known to belong to the heat shock response was also induced in LSMMG: hslV and hsl U, encoding the HslVU protease, were induced respectively 5.5 and 3.4 fold; htpG, encoding a Hsp90 family chaperone, was induced 4.6 fold and clpB was induced 4.7 fold. Transcription of the Lon protease was induced 2.5 fold. It appears that C. metallidurans CH34 experiences growth in Low-Shear Modelled Microgravity as a stressful condition eliciting the need to express the heat-shock proteins which assist protein folding, assembly, transport, repair and degradation. Challenging cells grown in simulated gravity (LSMMG) to a heat-shock for 30 min at 50° C resulted indeed in a smaller reduction (1.7 log) in cultivable cells compared to the reduction observed for cells grown in normal earth gravity (Low-Shear Gravity LSG) (4.0 log). Next to genes involved in the heat shock response, 5 of the 11 copies of uspA, encoding a widely conserved protein belonging to a superfamily whose physiological function is unknown but which is induced in response to a variety of stresses, were induced from 2.7 to 8.7 fold. In addition, LSMMG resulted in the upregulation of various genes encoding site-specific tyrosine recombinases, site-specific serine recombinase and transposases possibly indicating that Low-Shear Modeled Microgravity could elicit an adaptive response by genetic rearrangements. Finally, the parA and parB genes from pMOL30, one of the two plasmids carried by CH34 and specialized in heavy metals resistance, were strongly induced in LSMMG respectively 19.6 and 7.0 fold. The overproduction of similar proteins was also detected in C. metallidurans cells, cultured in during space flight.
Leu+ mutants from Salmonella typhimurium leu-500 strain MA412 arise at high frequencies and mutan... more 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.
... Parameter Estimation Techniques for Modal Analysis. Date Published: 1979-02-01. Paper Number:... more ... Parameter Estimation Techniques for Modal Analysis. Date Published: 1979-02-01. Paper Number: 790221. DOI: 10.4271/790221. Author(s): DL Brown - University of Cincinnati RJ Allemang - University of Cincinnati Ray Zimmerman ...
Alcaligenes eutrophus CH34 DNA fragments encoding resistance to Cd2+, Co2+, Zn2+ (czc), or Hg2+ (... more Alcaligenes eutrophus CH34 DNA fragments encoding resistance to Cd2+, Co2+, Zn2+ (czc), or Hg2+ (merA) were cloned and used as probes in colony hybridization procedures with bacteria isolated from polluted environments such as a zinc factory area (desertified because of the toxic effects of zinc contamination) and from sediments from factories of nonferrous metallurgy in Belgium and mine areas in Zaire. From the different soil samples, strains could be isolated and hybridized with the czc probe (resistance to Cd2+, Co2+, and Zn2+ from plasmid pMOL30). Percentages of CFU isolated on nonselective plates which hybridized with the czc and the mercury resistance probes were, respectively, 25 and 0% for the zinc desert, 15 to 20 and 10 to 20% for the two Belgian factories, and 40 and 40% for the Likasi mine area. Most of these strains also carried two large plasmids of about the same size as those of A. eutrophus CH34 and shared many phenotypic traits with this strain. These findings indicated a certain correlation between the heavy-metal content in contaminated soils and the presence of heavy-metal-resistant megaplasmid-bearing A. eutrophus strains.
Escherichia coli argE and argH gene products were detected in Pseudomonas fluorescens argH122 car... more Escherichia coli argE and argH gene products were detected in Pseudomonas fluorescens argH122 carrying the E. coli FllO plasmid.
Two mathematical models to elucidate the mechanism of retromobilization (or retrotransfer), that ... more Two mathematical models to elucidate the mechanism of retromobilization (or retrotransfer), that is, the ability of conjugative plasmids to mobilize genes into the cell containing the conjugative plasmid, were developed. This study deals with retromobilization of nonconjugative plasmids (Tra-Mob'). Plasmid transfer was modeled by two mass action models. The first is based on the hypothesis that retromobilization of the Tra-Mob' vector occurs in one step, by means of the pilus formed by the Tra+ plasmid in the original host. In the second model, retromobilization is considered to be a two-step process involving two transfer events. The first step involves the transfer of the Tra+ plasmid from the recipient cell to the donor of the nonconjugative vector, and during the second encounter the nonconjugative vector is mobilized toward the recipient. Since the relationships between the number of transconjugants and the number of recipients for the two models are different, filter matings were performed for short time periods with different initial densities of the recipient population. Comparison of the numbers of transconjugants with the results of the mathematical equations confirmed the hypothesis that retromobilization is a one-step conjugation process.
Electroporation was used as a tool to explore the genetics of the heavy-metal-resistant strain Al... more 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 we,rs observed. Transfer of the nonselected markers pheB332 and aro-333, linked to lysA194, confirmed the intervention of homologous recombination. However, during transformation of ale::TnS-Tc, illegitimate recombination and transposition were also observed as an alternative for the inheritance of the Tn5-Tc markers.
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Papers by Max Mergeay