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Biomedical subjects

A M Sicard

Publications and source records attributed to A M Sicard.

At least 19 recordsLinked to original sources
PubMed1995

Relatedness of penicillin-resistant Streptococcus pneumoniae serogroup 9 strains from France and Spain.

Pulsed-field gel electrophoresis of the genomic DNA of penicillin-resistant strains of Streptococcus pneumoniae was carried out. Eleven clinical strains of serogroup 9 from different French towns and Paris hospitals were tested. The restriction enzymes Apal and Smal were used to digest intact chromosomes, and the fragments were resolved by field-inversion gel electrophoresis (FIGE). Five strains were similar using Apal and Smal. Four others were closely related when using Apal, and five others were closely related when using Smal. These results suggest that 10 of these strains are genetically related and have a clonal origin. The profile of the eleventh strain was completely different. Thus, in a given serotype the spreading of penicillin resistance can result from both clonal and independent events. Five strains had similar FIGE profiles to strains first isolated in Spain, suggesting that a resistant strain had spread from Spain to France.

DNA, Bacterial↗
PubMed1994

Pulsed field gel electrophoresis for molecular epidemiology of penicillin resistant Streptococcus pneumoniae strains.

The emergence of strains of Streptococcus pneumoniae resistant to penicillin and other antibiotics has become a major concern for antimicrobial therapy of pneumococcal infections. The spread of that resistance over the world increases the need for their epidemiological surveillance: specific epidemiological markers are required. In this study, pulsed field gel electrophoresis of genomic DNA was carried out on sixteen resistant isolates of S. pneumoniae from different parts of the world and fifteen resistant isolates from Toulouse. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes and the fragments were resolved by field inversion gel electrophoresis (FIGE). Each digest produced 10 to 19 fragments for comparison between strains. The polymorphism obtained with FIGE was greater than that obtained with serotyping which appeared to be not a good criterion for genetic relatedness. Three common clones could be recognized among the penicillin-resistant isolates. Two clones were found in Spain and in Toulouse and were associated with serotypes 6B and 9V, respectively. The third clone was isolated in South Africa and in Spain and contained serotype 23F isolates and one serotype 19F strain. The FIGE profiles observed in this study also demonstrated that serogroup 23 multiresistant strains isolated in Toulouse are genetically closely related and might have originated from the same Spanish 23F clone. These results underline the importance of the geographic spread of resistant clones in the increase in the incidence of penicillin-resistant pneumococci. They indicate that pulsed field gel electrophoresis should be an effective tool for the typing of resistant S. pneumoniae strains capable of tracing their origin.

DNA, Bacterial↗
PubMed1993

The role of penicillin-binding protein 3 (PBP 3) in cefotaxime resistance in Streptococcus pneumoniae.

A pneumococcal strain, with a reduced amount of penicillin-binding protein 3 (PBP 3), permitted an analysis of the role of this protein in cefotaxime resistance. We observed that reduced amounts of PBP 3 sensitize the bacteria to high temperature, to excess glycine and to some D-amino acids. These phenotypes suggest that the amount of PBP 3 may influence the membrane properties of the bacteria. The strain with reduced PBP 3 was transformed to cefotaxime resistance. We show that the PBP 3 mutation, in certain genetic backgrounds, decreases the level of resistance to cefotaxime by a factor of 2. Models are presented to explain this result.

Bacterial Proteins↗
PubMed1993

DNA fingerprinting of Streptococcus pneumoniae strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis of genomic DNA was carried out on Streptococcus pneumoniae strains to determine its value in the epidemiological survey of pneumococcal infections. Twenty-one clinical strains were chosen to cover a broad range of diversity according to geographic location, penicillin susceptibility, serotype, and multilocus enzyme electrophoresis (MLEE) pattern. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). Each digest produced 10 to 19 fragments for comparison between strains. All the strains, including strains of the same serotype and strains with the same MLEE profile, had different FIGE patterns. In some cases, the restriction patterns differed by only a few fragment bands, and two isolates differed only in the location of a single DNA fragment. The polymorphism obtained with FIGE was greater than those obtained with serotyping and MLEE analysis. The stability of the FIGE profiles was established by testing of two independent clones derived from pneumococcus strain R36A. These results indicated that pulsed-field gel electrophoresis should be an effective tool for the typing of S. pneumoniae strains, capable of subdividing serotypes or MLEE types and of tracing the origin of pneumococcal strains.

DNA Fingerprinting↗
PubMed1991

Conversion of large heterologies in Streptococcus pneumoniae.

In genetic transformation, long deletions dramatically increase the frequency of wild-type recombinants in 2-point crosses. In 3-point crosses in which the deletion was localized between 2 point mutations we demonstrated that this hyper-recombination was the result of genetic conversion extending over several scores of bases outside the deletion. As this conversion did not require an active DNA polymerase A gene, it was proposed that the mechanism of conversion involves breakage and ligation between DNA molecules. A similar hyper-recombination was observed when donor DNA carried an insertion. These results suggest that long heterologies participated in recombination so that surrounding homologous regions are almost completely paired and that these long heterologies are converted. It appears that it is a process that evolved to correct errors of replication which lead to long deletions and which are not eliminated by other systems.

Chromosome Deletion↗
PubMed1990

Isolation and characterization of a restriction and modification deficient mutant of Brevibacterium lactofermentum.

In order to facilitate genetic engineering in amino-acid producing bacteria we have isolated two restriction-deficient Brevibacterium lactofermentum strains. They have been selected for their ability to obtain a high yield of plaques from CL31 phage which was grown on Corynebacterium lilium. These mutant strains do not restrict either phage DNA by transfection or DNA from the shuttle vector pBLA extracted from Escherichia coli by protoplast transformation. These mutants have also lost modification activity. We also report the presence of a restriction modification system in C. lilium ATCC 15990.

Bacteriophages↗
PubMed1990

Transfer of plasmid DNA to Brevibacterium lactofermentum by electrotransformation.

The Escherichia coli-Brevibacterium lactofermentum shuttle vector pBLA was introduced into intact cells of B. lactofermentum by electrotransformation. Several parameters of this procedure such as voltage and cell concentration were analysed. Optimal conditions gave an efficiency of 10(6) transformants per microgram of DNA. Two recalcitrant strains could be electrotransformed when an ampicillin pretreatment step was used. Electrotransformation experiments using DNAase or different structural forms of plasmid DNA showed that the electrotransformation process is quite different from natural transformation involving competence development. Restriction-modification-proficient B. lactofermentum could be efficiently electrotransformed with pBLA DNA isolated from E. coli. This restriction-modification system therefore seems to be overcome by electrotransformation. Thus electrotransformation may efficiently replace the protoplast bacterial transformation method.

Ampicillin↗
PubMed1989

Repair of single- and multiple-substitution mismatches during recombination in Streptococcus pneumoniae.

The use as genetic markers, during transformation of Streptococcus pneumoniae, of 19 sequences differing from wild type, located throughout the amiA locus, enabled us to examine the fate of 24 single- and 11 multiple-mismatches during recombination. Tentative mismatch ranking as a function of decreasing repair efficiency by the Hex mismatch repair system is G/T = A/C = G/G (maximum repair: 90-95%) greater than C/T (mostly 75 to 90% repair) greater than A/A (from 50 to 90% repair) greater than T/T (50-65% repair) greater than A/G (from 0 to 20% repair) greater than C/C. No indication of correction of the latter has been obtained. Over the limited number of samples examined, we observed no influence of the base composition of the surrounding sequence on correction efficiency for both transition mismatches and for G/G and C/C. Variations in the surrounding sequence affect repair of A/G and C/T, and, even more strongly, of A/A and T/T. No simple correlation to the G:C content of the surrounding sequence is apparent from our results, in contrast to the conclusion drawn for the Mut mismatch repair system of Escherichia coli. Examination of the fate of multiple mismatches suggests that C/C may sometimes impede recognition of otherwise corrected mismatches.

Base Composition↗
PubMed1988

General organization of the genes specifically involved in the diaminopimelate-lysine biosynthetic pathway of Corynebacterium glutamicum.

We utilized diaminopimelate-lysine mutants of Escherichia coli K12 to clone the genes specifically involved in the Corynebacterium glutamicum diaminopimelate-lysine anabolic pathway. From a cosmid genomic bank of C. glutamicum strain AS019, we isolated cosmids pSM71, pSM61 and pSM531, that are respectively able to complement dapA/dapB, dapD, and lysA mutants of E. coli. DNA hybridization analysis indicates that these complementing genes are located on the chromosome of C. glutamicum in at least three separate transcription units. Subcloning of parental cosmids in dapA, dapD, and lysA mutants of E. coli localized these genes, respectively, within 1.4, 3.4, and 1.8 kb fragments, cloned in an E. coli/C. glutamicum shuttle vector. Enzymatic analysis in C. glutamicum identified the dapA-complementing gene as L-2,3-dihydrodipicolinate synthetase (dapA), and the lysA-complementing gene as meso-diaminopimelate decarboxylase (lysA). In contrast, complementation of E. coli dapD8, presumably lacking L-delta 1-tetrahydrodipicolinate synthetase (dapD), led us to clone a diaminopimelate-lysine anabolic gene of C. glutamicum which does not exist in E. coli: meso-diaminopimelate dehydrogenase. Although meso-diaminopimelate is crucial in lysine formation and in cell wall biosynthesis, expression of the genomic copies of the cloned genes, which encode activities involved at key branching points of the diaminopimelate-lysine pathway of C. glutamicum, appears constitutive with regard to the addition of diaminopimelate and/or lysine during cell growth.

Amino Acids, Diamino↗
PubMed1988

Nucleotide sequence of the lysA gene of Corynebacterium glutamicum and possible mechanisms for modulation of its expression.

Sequence analysis localized the lysA gene of Corynebacterium glutamicum strain AS019 within a 1.35 kb open reading frame, potentially encoding a 445 amino acid product. Immediately downstream from this gene we found a potential rho-independent transcription terminator, while the 5' flanking region (300 bp) harbors unusual topological and structural features, located in the vicinity of a potential ribosome binding site. Within this upstream region, enzymatic and genetic analyses indicated the occurrence of a promoter responsible for significant, although weak, expression of the encoded enzymatic activity. The same significant expression level was observed with a plasmid harboring an additional 0.5 kb of genomic information upstream from lysA, while its full expression apparently requires 2 kb of additional genomic information located immediately upstream from the cloned gene. The upstream sequence requirement apparently associated with the full expression of the lysA gene of C. glutamicum shows some similarity with the Escherichia coli system.

Amino Acid Sequence↗
PubMed1987

Mismatch repair during pneumococcal transformation of small deletions produced by site-directed mutagenesis.

The genetic behaviour of short non-homologous regions has been studied during transformation of Streptococcus pneumoniae. Amethopterin-resistant mutants belonging to the amiA locus were used for these investigations. Five mutants deleted for 1-5 bp were obtained by oligonucleotide-directed mutagenesis. Their efficiency of transformation was measured using recipient strains either able to excise and repair mismatched bases (Hex+) or Hex- derivatives. Deletions or insertions of 1 and 2 bp are fully recognized by the Hex system, and are efficiently repaired whereas 3-bp deletions or insertions are only partially excised and repaired. The efficiency of repair is inversely related to the size of the non-homology. Markers with 5-bp deletions or insertions are poorly repaired and thus transform at very high frequency: similar results are obtained in reciprocal crosses. It is proposed that 1- or 2-bp deletions or insertions are included in the heteroduplex structure as transition mutations. The Hex system would detect only small deviations from the normal DNA structure.

Chromosome Deletion↗
PubMed1987

Polarity of localised conversion in Streptococcus pneumoniae transformation.

Localised conversion in pneumococcal transformation is a process that spans a few nucleotides when the 5'-ATTAAT/3'-TAAGTA configuration occurs at the pairing step. It was first observed in two-point crosses between an amiA mutation (amiA36) carrying this sequence and other closely linked mutants of the locus. The yield of the amiA resistance allele conversion to wild type is 20%. In order to characterize this process, which differs from long-patch conversion by the length of DNA repair, gene requirements and sequence specificity, we devised experiments to detect the reciprocal conversion, AmiA+ to AmiAr. For this purpose we examined the suppressibility by a pneumococcal informational suppressor of several nonsense mutations at the locus. Amber (UAG) and ochre (UAA) mutations are suppressed whereas UGA is not suppressed. In this genetic background, where amiA36 is partly suppressed, it was possible to select for double mutants in a cross between amiA36 and a closely linked non-suppressible marker. Direct isolation of such double mutants was also performed without any screening in crosses between amiA36 and the same linked marker in cloned DNA. The frequency of double mutants was very low (1/175) suggesting that there is no conversion of wild-type to mutant alleles. Thus conversion is a polarized process changing specifically A to C.

Base Sequence↗
PubMed1987

Structural characteristics of the Corynebacterium lilium bacteriophage CL31.

Bacteriophage CL31 was isolated on a Corynebacterium lilium strain. Out of 30 strains tested, only CL31 was able to form plaques on Corynebacterium glutamicum ATCC 13287, Brevibacterium lactofermentum ATCC 21086, and Arthrobacter sp. strain SI55, but at a very low frequency. This phage belongs to group B of Bradley's classification (D. E. Bradley, Bacteriol. Rev. 31:230-314; 1967). Its head is 53 nm in diameter, and its tail is 396 nm in length. The phage capsid contains three major proteins, of 12.5, 29.0, and 37.0 kilodaltons, and five minor ones (23.9, 26.0, 27.0, 40.0, and 55.4 kilodaltons). CL31 DNA is a linear molecule of 48 kilobases with cohesive ends. Restriction mapping was performed for endonucleases BglII, EcoRI, SalI, and KpnI. The expression of CL31 genes in Escherichia coli was studied by the maxicell technique; 12 different proteins were detected.

Bacteriophages↗
PubMed1987

Gene conversion in Streptococcus pneumoniae.

In pneumococcus, correction of mutations by the repair of mismatched bases results in gene conversion, i.e. transfer of genetic information from one strand of DNA to the other. Three different correction systems act upon a variety of mutations: 1) A long-patch process (a few Kb) is efficient in the elimination of transitions, mostly +/- 1 base-pair mutations and less efficient in eliminating some transversions without affecting fairly long deletions. Neighboring sequences may interfere with this process. It is directed by at least two genes. 2) A localized conversion system acts on a six base-pair heteroduplex structure such as 5'ATTAAT/3'TAAGTA by specifically converting the mutated A base to the wild type C base. Modifications of this configuration by site-directed mutagenesis lead to reduced conversion. 3) Fairly long deletions are eliminated during recombination by events that extend several scores of bases around the heterologus region. Although only the first conversion system has been shown to participate efficiently in protecting Streptococcus pneumoniae against spontaneous mutation, the two other processes may also eliminate mutations of different natures.

DNA Damage↗
PubMed1986

Frame-shift mutants induced by quinacrine are recognized by the mismatch repair system in Streptococcus pneumoniae.

We describe the isolation of amethopterin-resistant mutants induced by quinacrine treatment of exponentially growing cultures of Streptococcus pneumoniae. Only mutants located by recombination analysis in a few hundred base pairs were further studied. They were cloned and their DNA sequences show that most of them are +/-1-base frame-shift mutants. They are excised and repaired to a degree similar to transition mutants (low efficiency class), suggesting that the mismatches resulting from a transition or a +/-1-base mutation are similar substrates for the Hex mismatch repair system.

Base Sequence↗
PubMed1986

A shuttle vector system for Brevibacterium lactofermentum.

We have constructed a shuttle vector that replicates in Escherichia coli, Corynebacterium glutamicum and Brevibacterium lactofermentum, by fusion of a 4.4-kb cryptic plasmid isolated from B. lactofermentum and a derivative of pBR322. Resistance to erythromycin which is expressed in all three bacteria has been a useful selective marker. The frequency of homospecific transformation was 1.5 X 10(5) transformants/micrograms of hybrid plasmid DNA.

Brevibacterium↗
PubMed1985

Transfection of Corynebacterium lilium protoplasts.

A protoplast transfection system has been developed for a lysine-producing bacterium, Corynebacterium lilium, using the DNA of phage CL31. Phage CL31 is lytic and specific to C. lilium and has a genome of approximately 48 kb. The transfection procedure involves a polyethylene-glycol-mediated introduction of the DNA into lysozyme-treated cells and has a maximum efficiency of 3 X 10(4) transfectants per microgram DNA.

Bacteriophages↗