Genetic studies of cefotaxime resistance in Streptococcus pneumoniae.
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Bacterial Proteins↗
Biomedical subjects
A M Gasc
Publications and source records attributed to A M Gasc.
Electrotransformation of Streptococcus pneumoniae: evidence for restriction of the DNA on entry.
Electrotransformation is a method generally used in biotechnology to introduce recombinant DNA into a wide range of bacteria. However the mechanism of DNA entry is poorly understood. We report that in Streptococcus pneumoniae, a naturally transformable species, electrotransformation efficiently introduces a plasmid replicon. DNA is strongly restricted by the restriction-modification systems DpnI and DpnII which degrade methylated and nonmethylated DNA respectively at GATC sequences. This suggests that in electrotransformation double-strand DNA penetrates into these bacteria without a single-strand step in contrast to natural transformation. Single-strand DNA by itself is able to electrotransform very weakly and linearized double-stranded plasmid DNA yields barely detectable levels of transformants.
DNA Methylation↗
Structural organization of the Streptococcus pneumoniae chromosome and relatedness of penicillin-sensitive and -resistant strains in type 9V.
Fragmentation of Streptococcus pneumoniae genomic DNA with low-frequency-cleavage restriction endonucleases and separation of the fragments by field-inversion gel electrophoresis (FIGE) provides a DNA-fingerprint of a strain. This method enables us to construct a physical and genetic map of the R6 laboratory strain what will be presented. The origin of replication containing several Dna boxes was located in the dnaA region. It was of interest to compare the profiles of subclones. Two clones of strain R36A (R6 and C13) were cultivated separately for more than 15,000 generations in two laboratories. FIGE profiles differed by only one band. Another R36A descendant, isolated in 1958 by Ravin, strain Rx was of interest since it was deficient in Dpn restriction enzymes and methylases and in the hex B function. Its origin was questionable; its profile is identical to others R6 descendants, demonstrating that Rx is derived from R36A. FIGE analysis was carried out on several penicillin-resistant strains of type 9V because penicillin-resistance in this type increased recently. The profiles of a collection of a number of these resistant isolates were very similar, showing that they result from a clone. The profiles of penicillin sensitive isolates of the same type are very similar to the resistant isolates. This suggests that the 9V type has spread recently from a clone, and the resistance genes have mutated and were selected when penicillin was extensively used.
Blotting, Southern↗
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↗
A two-component signal-transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae.
Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin-binding protein (PBP) variants with decreased affinities for beta-lactam antibiotics. Cefotaxime-resistant laboratory mutants, selected after several steps on increasing concentrations of this beta-lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, ciaR and ciaH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal-transducing proteins). Two hydrophobic peptide regions divided the histidine kinase CiaH into two putative domains: an N-terminal extracellular sensor part, and an intracellular C-terminal domain with the conserved His-226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime-resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime-resistant mutants were each located in the C-terminal half of CiaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated ciaH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal-transducing system cia is involved in early steps of competence regulation.
Amino Acid Sequence↗
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↗
Genetic studies of cefotaxime resistance in Streptococcus pneumoniae: relationship to transformation deficiency.
A laboratory pneumococcal strain resistant to cefotaxime was studied by DNA-induced transformation in order to characterize its genetic structure. At least three independent genes were required to confer the highest level of resistance to this beta-lactam antibiotic. The accumulation of mutations in these three genes accounted for three levels of resistance. Mutation of the gene encoding penicillin-binding protein 2x was very likely responsible for the first step of resistance, which was a prerequisite for sequential increase in resistance. Additionally, strains highly resistant to cefotaxime were defective for natural transformation. Revertants of these strains were frequently observed. Such strains had recovered full transformability, suggesting a correlation between the inability to be transformed and a high level of resistance to cefotaxime. The possibility of electrotransforming these highly resistant strains suggests that natural transformation is probably blocked at the DNA-uptake level.
Cefotaxime↗
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↗
Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae.
A physical map of the Streptococcus (Diplococcus) pneumoniae chromosome, which is circular and 2,270 kbp in circumference, has been constructed. The restriction enzymes ApaI, SmaI, and SacII were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). The digests produced 22, 20, and 29 fragments, respectively. The order of the fragments was deduced from Southern blot hybridization of isolated labeled fragments to separated fragments of the various restriction digests. Genetic markers were correlated with the physical map by transformation of recipient cells with FIGE-isolated DNA fragments derived from genetically marked S. pneumoniae strains. In addition, markers were mapped by the hybridization of cloned genes to FIGE-separated restriction fragments. Six rRNA gene (rrn) clusters were mapped by hybridization to rrn-containing fragments of Haemophilus influenzae.
Base Sequence↗
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↗
Conversion of deletions during recombination in pneumococcal transformation.
Genetic analysis of 16 deletions obtained in the amiA locus of pneumococcus is described. When present on donor DNA, all deletions increased drastically the frequency of wild-type recombinants in two-point crosses. This effect was maximal for deletions longer than 200 bases. It was reduced for heterologies shorter than 76 bases and did not exist for very short deletions. In three-point crosses in which the deletion was localized between two point mutations, we demonstrated that this excess of wild-type recombinants was the result of a genetic conversion. This conversion extended over several scores of bases outside the deletion. Conversion takes place during the heteroduplex stage of recombination. Therefore, in pneumococcal transformation, long heterologies participated in this heteroduplex configuration. As this conversion did not require an active DNA polymerase A gene it is proposed that the mechanism of conversion is not a DNA repair synthesis but involves breakage and ligation between DNA molecules. Conversion of deletions did not require the Hex system of correction of mismatched bases. It differs also from localized conversion. It appears that it is a process that evolved to correct errors of replication which lead to long heterologies and which are not eliminated by other systems.
Chromosome Deletion↗
DNA sequences required to induce localized conversion in Streptococcus pneumoniae transformation.
In pneumococcal transformation a particular point mutation belonging to the amiA locus is able markedly to enhance recombination frequency when crossed with any other markers of this gene. This results from a polarized conversion of the mutation towards the wild-type sequence. In this report, by site-directed oligonucleotide mutagenesis, we have generated a series of mutants showing various degrees of conversion. We have found that the substitution 5'-ATTCAT----5'-ATTAAT is a sufficient signal for localized conversion. Changing individual bases within this sequence results in decreased conversion frequencies to levels that depend on the mutation, suggesting that there is a family to related sequences which may act as a substrate for a conversion system. Moreover, the length over which this conversion occurs has been estimated to be 12 base pairs on the average.
Base Sequence↗
Inhibition of DNA repair by neighbouring mismatched bases in Streptococcus pneumoniae.
A set of pneumococcal strains containing immediately adjacent or nearby double mutations at the amiA locus, conferring resistance to amethopterin, has been isolated by oligonucleotide site-specific mutagenesis. Repair of these double mutations has been measured by transformation of wild-type strains with DNA extracted from these strains. In several transformations we have observed an inhibition of repair by neighbouring mismatches. This inhibition ranges from mild to severe depending upon the interfering mismatch. Unrepaired mismatches can strongly inhibit repair of an adjacent repairable mutation. This suggests that the repair-complex proteins attach not only to repairable mismatches but also to some mismatches known to escape the repair system.
DNA Repair↗
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↗
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↗
Localized conversion in Streptococcus pneumoniae recombination: heteroduplex preference.
In pneumococcal transformation the frequency of recombinants between point mutations is generally proportional to distance. We have recently described an aberrant marker in the amiA locus that appeared to enhance recombination frequency when crossed with any other allele of this gene. The hyperrecombination that we have observed in two-point crosses could be explained by two hypotheses: the aberrant marker induces frequent crossovers in its vicinity or the mutant is converted to wild type. In this report we present evidence showing that, in suitable three-point crosses, this hyperrecombination does not modify the recombination frequency between outside markers, suggesting that a conversion occurs at the site of this mutation. To estimate the length over which this event occurs, we isolated very closely linked markers and used them in two-point crosses. It appears that the conversion system removes only a few base pairs (from three to 27) around the aberrant marker. This conversion process is quite different from the mismatch-repair system controlled by hex genes in pneumococcus, which involves several thousand base pairs. Moreover, we have constructed artificial heteroduplexes using separated DNA strands. It appears that only one of the two heteroduplexes is specifically converted. The conversion system acts upon 5'..ATTAAT..3'/3'.. TAAGTA..5'. A possible role of the palindrome resulting from the mutation is discussed.
Base Sequence↗
Hyperrecombination at a specific DNA sequence in pneumococcal transformation.
In pneumococcal transformation, recombination frequency between point mutations is usually proportional to physical distances. We have identified an aberrant marker belonging to the amiA locus that appeared to markedly enhance recombination frequency when crossed with any other markers of this gene. This mutation results from the C-to-A transversion in the sequence A-T-T-C-A-T----A-T-T-A-A-T. This effect is especially apparent for short distances as small as 27 base pairs. The hyperrecombination does not require the wild-type function of the pneumococcal gene for an ATP-dependent DNase (which is homologous to the product of the Escherichia coli recBC genes) or of the hex genes, which correct certain mismatched bases in transformation. The hyperrecombination is affected by the presence of nearby mismatched bases that trigger an excision-repair system. It is proposed that the mutation that shows hyperrecombination is sometimes converted to the wild-type allele at the heteroduplex stage of transformation.
Bacterial Proteins↗
Mismatch repair in Streptococcus pneumoniae: relationship between base mismatches and transformation efficiencies.
Genetic transformation in Streptococcus pneumoniae involves the insertion of single-stranded pieces of donor DNA into a recipient genome. Efficiencies of transformation strongly depend on the mutations (markers) carried by donor DNA. Markers are classified according to their transforming efficiencies into very high, high, intermediate, and low efficiency. The last is approximately 1/20th as efficient as the first. This marker effect is under the control of the Hex system, which is thought to correct mismatches at the donor-recipient heteroduplex stage in transformation. To investigate this effect, wild type, mutant, and revertant DNA sequences at five genetic sites within the amiA locus were determined. The results show that low-efficiency markers arise from transitional changes A . T to G . C. The transversion A . T to T . A corresponds to an intermediate-efficiency marker. Transversions G . C to T . A and G . C to C . G lead to high-efficiency markers. Among the eight possible mismatches that could exist transiently at the heteroduplex stage in transformation, only two--namely, A/G and C/C--are not corrected by the Hex system. It is noteworthy that the four possible base pairs (A . T, T . A, G . C, and C . G) have been encountered at the very same site (amiA6 site), which constitutes a good illustration of the marker effect. DNA sequence analysis also reveals that short deletions (33 or 34 bases long) are integrated with very high efficiencies. These results confirm that the Hex system corrects point mismatches harbored in donor-recipient heteroduplexes thousands of bases long. The correction pattern of the Hex system toward multiple-base mismatches has also been investigated. Its behavior toward double-base mismatches is complex, suggesting that neighboring sequences may affect the detection of mispaired bases.
Base Sequence↗