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M Couturier

Publications and source records attributed to M Couturier.

At least 55 records · Page 3Linked to original sources
PubMed1994

Replicon typing of 71 multiresistant Serratia marcescens strains.

Replicon typing is the identification of plasmids by hybridization with specific DNA probes which contain the genes involved in plasmid maintenance. This new method has been used to classify plasmids into replicon (rep) groups which can often be correlated with incompatibility (Inc) groups. We studied 71 multiresistant Serratia marcescens strains with 19 rep probes constructed from reference plasmid replicons belonging to known Inc groups. These probes are known to react with enteric bacterial plasmids. However, they did not represent the totality of the thirty known Inc groups. For 52% of the studied strains, plasmids were identified and classified into groups FIB, FIC, FIIA, HI2, L/M, N, B/O, P, W, Y and Com9. Most (79%) of the plasmid preparations hybridized with a single rep probe, and 21% hybridized with two different probes. Electrophoretic analysis of DNA suggested that double hybridization could result from the presence of either two different Inc plasmids in the same strain (e.g. S37) or one single plasmid with a multireplicon (e.g. S113).

Bacterial Typing Techniques↗
PubMed1994

Lon-dependent proteolysis of CcdA is the key control for activation of CcdB in plasmid-free segregant bacteria.

The ccd locus contributes to the stability of plasmid F by post-segregational killing of plasmid-free bacteria. The ccdB gene product is a potent cell-killing protein and its activity is negatively regulated by the CcdA protein. In this paper, we show that the CcdA protein is unstable and that the degradation of CcdA is dependent on the Lon protease. Differences in the stability of the killer CcdB protein and its antidote CcdA are the key to post-segregational killing. Because the half-life of active CcdA protein is shorter than that of active CcdB protein, persistence of the CcdB protein leads to the death of plasmid-free bacterial segregants.

ATP-Dependent Proteases↗
PubMed1994

Detection of 4-quinolone resistance mutation in gyrA gene of Shigella dysenteriae type 1 by PCR.

To study 4-quinolone resistance, the N-terminal coding region of gyrA from nalidixic acid-susceptible and -resistant isolates of Shigella dysenteriae type 1 was amplified by PCR, cloned, and sequenced. DNA sequence analysis of gyrA from nalidixic acid-resistant isolates revealed a C-to-T transition at nucleotide position 248 leading to a Ser-83-to-Leu substitution which was absent in susceptible clinical isolates. Direct HinfI digestion of PCR-amplified DNA detected similar mutations. Thus, DNA gyrase A subunit mutation Ser-83 to Leu is implicated in 4-quinolone resistance in S. dysenteriae type 1.

4-Quinolones↗
PubMed1994

Cloning and characterization of the Inc A/C plasmid RA1 replicon.

The Inc A/C plasmids, like Inc P and Inc Q plasmids, have a broad host range. However, their maintenance functions remain to be studied. An autoreplicative region of 2.79 kb named RepA/C, able to replicate both in the family Enterobacteriaceae and in Pseudomonas spp., was isolated and sequenced. The stability, copy number, and incompatibility expression of this replicon were determined. RepA/C and a nonautoreplicative fragment of 16 kb of this replicon were used as probes and showed specific hybridizations with the Inc P3-A/C plasmids from Pseudomonas spp. and members of the Enterobacteriaceae. These probes could be used as tools for identification of the plasmids of this epidemiologically important Inc group.

Amino Acid Sequence↗
PubMed1993

The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase.

DNA topoisomerases perform essential roles in DNA replication, gene transcription, and chromosome segregation. Recently, we identified a new type of topoisomerase II poison: the CcdB protein of plasmid F. When its action is not prevented by CcdA protein, the CcdB protein is a potent cytotoxin. In this paper, using purified CcdB, CcdA and gyrase, we show that CcdB protein efficiently traps gyrase in a cleavable complex. The CcdA protein not only prevents the gyrase poisoning activity of CcdB but also reverses its effect on gyrase. The mechanism by which the CcdB protein induces DNA strand breakage is closely related to the action of quinolone antibiotics. However, the ATP dependence of the CcdB cleavage process differentiates the CcdB mechanism from quinolone-dependent reactions because the quinolone antibiotics stimulate efficient DNA breakage, whether or not ATP is present. We previously showed that bacteria resistant to quinolone antibiotics are sensitive to CcdB and vice versa. Elucidation of the mechanism of action of CcdB protein may permit the design of drugs targeting gyrase so as to take advantage of this new poisoning mechanism.

Adenosine Triphosphate↗
PubMed1993

Purification, circular dichroism analysis, crystallization and preliminary X-ray diffraction analysis of the F plasmid CcdB killer protein.

Large crystals of the Escherichia coli F plasmid CcdB killer protein were grown from solutions containing 32% ammonium sulphate. The crystals belong to space group P4(2)2(1)2 with a = b = 104.52 A and c = 88.45 A or P2(1)2(1)2(1) with a = 77.62 A, b = 93.28 A and c = 141.44 A. Both crystal forms diffract to 2.6 A resolution. Structure determination by multiple isomorphous replacement is under way.

Bacterial Proteins↗
PubMed1993

Simultaneous determination of the main molecular species of soybean phosphatidylcholine or phosphatidylethanolamine and their corresponding hydroperoxides obtained by lipoxygenase treatment.

A method for the simultaneous determination of the main molecular species of soybean phosphatidylcholine or phosphatidylethanolamine and their corresponding hydroperoxides is described. Hydroperoxides were formed by incubation of phospholipids with lipoxygenase at pH 9.2. Silicic acid column chromatography (silica Sep-Pak column) was used to separate the phospholipids into phosphatidylcholine and phosphatidylethanolamine. A single C-18 reverse-phase column was employed to separate the main molecular species of soybean phosphatidylcholine or phosphatidylethanolamine and their hydroperoxides by high-performance liquid chromatography. The mobile phase consisted of 5% 10 mM ammonium acetate at pH 5 and 95% methanol. The molecular species of phosphatidylcholine and phosphatidylethanolamine were detected at 205 nm; the eluate was mixed with a chemiluminescence reagent (isoluminol and microperoxidase) and monitored by fluorometry. Under the experimental conditions used, three individual molecular species of both soybean phosphatidylethanolamine and phosphatidylcholine (18:3/18:2, 18:2/18:2 and 16:0/18:2), together with their corresponding hydroperoxides, were identified and quantitated.

Chromatography, High Pressure Liquid↗
PubMed1993

Replicon typing characterization of plasmids encoding resistance to gentamicin and apramycin in Escherichia coli and Salmonella typhimurium isolated from human and animal sources in Belgium.

Escherichia coli and salmonella strains with plasmids conferring resistance to gentamicin and apramycin have been isolated with increasing frequency both from cattle and hospital patients in Belgium. The apramycin-gentamicin resistance plasmids were characterized in recipient strains by their profiles and molecular weights using agarose gel electrophoresis, by their antimicrobial resistance patterns and by replicon typing using a series of DNA probes specific for the genes controlling their systems of replication. Overall, most of the plasmids differed in their DNA electrophoretic patterns. Seventeen different antimicrobial resistance profiles were observed, and there were six different types of replicons. However, two replication genes predominated and had a preferential distribution in different bacterial species. The rep FIC.a plus rep Q multireplicon was found mainly in plasmids recovered from gentamicin- and apramycin-resistant E. coli while replicon of the type rep FIC.b largely prevailed in S. typhimurium. Identical replication genes were found in most animal and human strains, hence suggesting a high homology between apramycin-gentamicin plasmids in these communities. Finally, our results indicate that the rapid spread of apramycin-gentamicin-resistance in several species of Enterobacteriaceae isolated from animals and from humans in Belgium is not due to a single plasmid, but rather that the gene encoding AAC(3)-IV is carried by various replicons.

Animals↗
PubMed1993

Molecular characterization of resistance plasmids in epidemiologically unrelated strains of multiresistant Haemophilus influenzae.

Thirty-three epidemiologically unrelated strains of ampicillin-chloramphenicol-resistant isolates of Haemophilus influenzae (22 type b, 11 unencapsulated), isolated over 10 years in Belgium, were compared with 53 ampicillin-resistant chloramphenicol-susceptible isolates (22 type b, 31 unencapsulated). All ampicillin-chloramphenicol-resistant and 76% of ampicillin-resistant chloramphenicol-susceptible strains were resistant to tetracycline, kanamycin, or both. Resistance to these antibiotics was specified by a 37- to 44-MDa conjugative plasmid. The genetic relatedness of these plasmids and of those in multiresistance strains from Spain was investigated. Plasmids specifying ampicillin-chloramphenicol-tetracycline-kanamycin resistance in Belgium or in Spain had highly related restriction fragment patterns. By homoduplex analysis, they had similar molecular organization and contained a structure identical to Tn10-TnCm, a transposon previously identified in chloramphenicol-tetracycline-resistant H. influenzae. Plasmids coding for different resistance phenotypes had less resemblance by restriction endonuclease analysis; however, study of heteroduplex molecules indicated they shared a high proportion of core sequences. These findings support the hypothesis of independent transposition events resulting in resistance plasmids of close molecular organization.

Belgium↗
PubMed1993

Isolation and location on the R27 map of two replicons and an incompatibility determinant specific for IncHI1 plasmids.

Two replicons were isolated independently from different IncHI1 plasmids. One was isolated from R27, and a second was isolated from pIP522. We demonstrate, by DNA-DNA hybridization experiments, that these maintenance regions are different and that they are specific to, and carried by, all IncHI1 plasmids tested. In view of this specificity we decided to designate the replicon isolated from R27 as RepHI1A and the replicon isolated from pIP522 as RepHI1B. These two autoreplicative regions are not related to a third replicon present in all IncHI1 plasmids that bears homology with RepFIA and that expresses the characteristic incompatibility of IncHI1 subgroup plasmids toward F factor (D. Saul, D. Lane, and P. L. Bergquist, Mol. Microbiol. 2:219-225, 1988; D. E. Taylor, R. W. Hedges, and P. L. Bergquist, J. Gen. Microbiol. 131:1523-1530, 1985). These results demonstrate that all IncHI1 plasmids tested contain at least three replicons. An incompatibility (Inc) region that hybridizes specifically to all the IncHI1 plasmids was previously isolated (M. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Although this Inc locus is not located in an autoreplicative region of IncHI1 plasmids, we observed that this locus stabilizes a low-copy-number replicon. This Inc locus is probably a component of an active partition locus involved in the maintenance of IncHI1 plasmids. The nucleotide sequence of the Inc region contains direct repeats of 31 bp. In addition, this incompatibility determinant hybridizes specifically with IncHI1 plasmids but expresses incompatibility toward plasmids of both IncHI subgroups (IncHI1 and IncHI2). In this communication, we present the mapping of these maintenance elements on the R27 genome.

Base Sequence↗
PubMed1993

Comparison of obese and non-obese patients with gestational diabetes.

The aim of this study was to assess the effect of obesity on the outcome of gestational diabetes (GD). The age of the subject (31 +/- 5 years) and duration of pregnancy (29 +/- 5 weeks) were similar at the time of diagnosis for the 19 obese (34 +/- 6 kg/m2) and 19 non-obese (23.0 +/- 2.1 kg/m2) subjects. Before treatment, the area under the curve of the glucose tolerance test and the mean capillary blood glucose levels were similar in both groups; however, the baseline blood glucose levels of the obese patients (6.0 +/- 0.8 mmol/l) were higher than those of the non-obese patients (5.3 +/- 0.8 mmol/l; P < 0.01). During pregnancy, the total weight gain of the obese patients was less (10.6 +/- 5.0 vs. 15.2 +/- 4.7 kg, P < 0.006). Fifteen of the obese patients required treatment with insulin, while only six of the non-obese women required insulin (P < 0.005). During insulin therapy, the mean capillary blood glucose, glycosylated haemoglobin and fructosamine levels were similar in both groups. In conclusion, our data suggest that GD in obese patients is characterized by lower weight gain and higher baseline glucose with the result that insulin therapy is more frequently instituted.

Adult↗
PubMed1993

Effects of alpha-tocopherol on antioxidant enzyme activity in human fibroblast cultures.

The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (Cat) were determined in human fibroblast cultures at four concentrations of exogenous alpha-tocopherol: 0.2, 2.5, 10 and 50 micrograms/ml of culture medium, or without alpha-tocopherol. Relationships between alpha-tocopherol levels and the activities of SOD and GPx were identified. The cellular alpha-tocopherol level correlated with GPx activity (p < or = 0.01) and inversely correlated with SOD activity (p < or = 0.003), but only when alpha-tocopherol was added to the culture medium. The variations in the cellular GPx/SOD ratio depended on the level of cellular alpha-tocopherol (p < or = 0.001). Furthermore, there was a strong inverse correlation between SOD and GPx activity (p < or = 0.0001). Cat activity did not correlate either with cellular alpha-tocopherol concentration, or with SOD or GPx activity. These results underline the complex interplay between alpha-tocopherol and other antioxidant systems in human fibroblast cultures.

Catalase↗
PubMed1992

Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes.

In Escherichia coli, the miniF plasmid CcdB protein is responsible for cell death when its action is not prevented by polypeptide CcdA. We report the isolation, localization, sequencing and properties of a bacterial mutant resistant to the cytotoxic activity of the CcdB protein. This mutation is located in the gene encoding the A subunit of topoisomerase II and produces an Arg462----Cys substitution in the amino acid sequence of the GyrA polypeptide. Hence, the mutation was called gyrA462. We show that in the wild-type strain, the CcdB protein promotes plasmid linearization; in the gyrA462 strain, this double-stranded DNA cleavage is suppressed. This indicates that the CcdB protein is responsible for gyrase-mediated double-stranded DNA breakage. CcdB, in the absence of CcdA, induces the SOS pathway. SOS induction is a biological response to DNA-damaging agents. We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes. In addition, we observe that the CcdBS sensitive phenotype dominates over the resistant phenotype. This is better explained by the conversion, in gyrA+/gyrA462 merodiploid strains, of the wild-type gyrase into a DNA-damaging agent. These results strongly suggest that the CcdB protein, like quinolone antibiotics and a variety of antitumoral drugs, is a DNA topoisomerase II poison. This is the first proteinic poison-antipoison mechanism that has been found to act via the DNA topoisomerase II.

Amino Acid Sequence↗
PubMed1992

Replicon typing of virulence plasmids of enterotoxigenic Escherichia coli isolates from cattle.

Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle. The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins. The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repI1, repHI1, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY). The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate. The sizes of most of these virulence plasmids were from 65 to 95 MDa. The F41 probe failed to hybridize with any plasmid band. The virulence plasmids had multireplicon types typical of plasmids of the IncF groups. The most common basic replicon association was the triple RepFIA-RepFIB-RepFIC family association.

Animals↗
PubMed1991

The 41 carboxy-terminal residues of the miniF plasmid CcdA protein are sufficient to antagonize the killer activity of the CcdB protein.

The ccd operon of plasmid F encodes two genes, ccdA and ccdB, which contribute to the high stability of the plasmid by post-segregational killing of plasmid-free bacteria. The CcdB protein is lethal to bacteria and the CcdA protein is an antagonist of this lethal action. A 520 bp fragment containing the terminal part of the ccdA gene and the entire ccdB gene of plasmid F was cloned downstream of the tac promoter. Although the CcdB protein was expressed from this fragment, no killing of host bacteria was observed. We found that the absence of killing was due to the presence of a small polypeptide, CcdA41, composed of the 41 C-terminal residues of the CcdA protein. This polypeptide has retained the ability to regulate negatively the lethal activity of the CcdB protein.

Bacterial Proteins↗
PubMed1991

2-Deoxy-D-glucose uptake and fatty acid content in fibroblast cultures from children with syndromic paucity of interlobular bile ducts (Alagille syndrome).

2-Deoxy-D-glucose (2-DOG) uptake was studied in skin fibroblast cultures from control children and children with Alagille syndrome or syndromic paucity of interlobular bile ducts (PILBD). No significant differences in uptake were observed between patients and controls. However, as the scatter of the results was larger in the fibroblasts from patients, we attempted to establish for these patients a relationship between 2-DOG uptake and some biochemical parameters. We observed an inverse relationship between this uptake and the levels of plasma cholesterol and phospholipids (r = -0.85). Compared to controls, 2-DOG uptake was significantly lower in cultures from patients who had very high levels of cholesterol (P2 group), but not in cultures from patients with moderately increased levels of cholesterol (P1 group). The level of total cellular cholesterol in cultured cells from the P1 and P2 groups was not significantly different from the control level, but we found marked differences between the concentrations of fatty acids. In the cultures from patients (especially the P2 group), we observed a significant increase in total fatty acids; among the saturated fatty acids, this increase chiefly concerned the 18:0 (14%) and among the polyunsaturated the n - 3 fatty acids (55%). The high concentrations of 20:5, 22:5 and 22:6, which enhance membrane fluidity, might explain the decrease in 2-DOG uptake found in the cultures from patients (P2 group) with PILBD. The nature of these abnormalities might be connected with the genetic origin of Alagille syndrome.

Bile Ducts↗
PubMed1991

Epidemiological study of Salmonella enteritidis strains of animal origin in Belgium.

Since 1987, the number of cases of salmonellosis caused by Salmonella enteritidis has considerably increased in Western Europe. Comparison of endemic animal strains isolated in Belgium from 1976-84 with strains isolated from 1987 on shows that the strains which cause the current epidemic have no features distinguishing them from the previously-isolated strains and that furthermore, they do not constitute a bacterial clone. They belong to 13 different lysotypes and in most cases remain sensitive to antibiotics. Nevertheless, the lysotype 33 (which belongs to the phage type 4 has increased significantly. It encompasses 37% of the animal strains isolated in Belgium from 1987-9, but only 7% of the strains isolated from 1976-84. It is worth noting that the endemic as well as the epidemic strains contain a virulence plasmid sharing sequence similarities with the FIB and FIIA plasmid replicons and with the VirA and VirB virulence regions of the S. typhimurium virulent plasmid: pIP1350.

Animals↗