amiA Locus Research Articles

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.

Cloning of the amiA locus of Streptococcus pneumoniae and identification of its functional limits

Mutations in the amiA locus of the Gram-positive bacterium Streptococcus pneumoniae confer a complex phenotype including resistance to various antineoplastic drugs. As a first step towards the understanding of the molecular organization and the function(s) of this locus, we have cloned DNA fragments carrying its 5' -and 3'-extremities. We have isolated and characterized a down-promoter mutation and have located the functional limits of the locus. The amiA locus is between 5.8 and 7.5 kb long strongly suggesting that it encodes several proteins.

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 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.

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.

Electric transmembrane potential mutation and resistance to the cationic and amphiphilic antitumoral drugs derived from pyridocarbazole, 2-N-methylellipticinium and 2-N-methyl-9-hydroxyellipticinium, in Streptococcus pneumoniae.

delta psi-reduced amiA mutants of Streptococcus pneumoniae were shown to be resistant to the positively charged antitumoral drugs 2-N-methylellipticinium (NME) and 2-N-methyl-9-hydroxyellipticinium (NMHE). Conversely, mutants selected for their resistance to NMHE were mapped within the amiA locus and exhibited the pleiotropic AmiA- phenotype. This shows that delta psi is a critical parameter in determining resistance to these drugs in S. pneumoniae and suggests that they are accumulated within this bacterium in response to delta psi. As a consequence NME and NMHE appear to be valuable tools for selecting delta psi-reduced mutants in S. pneumoniae.

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.

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.

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.

Amplification of a chimeric plasmid carrying an erythromycin-resistance determinant introduced into the genome of Streptococcus pneumoniae

Hybrid plasmid molecules carrying an insert of pneumococcal DNA can integrate into the pneumococcal genome by homologous recombination. The resulting structure is a duplication of the pneumococcal DNA insert bracketing a vector genome. To select for plasmid integration, the vector plasmid was marked with an erythromycin (ery) resistance determinant ( ery r) originating from pVA736, a streptococcal plasmid. Experiments with pR27 and pR28, two plasmids carrying the same insert of pneumococcal DNA but in opposite orientations, led to the following observations: (i) In one orientation of the ery region with respect to the amiA locus, cells exhibited a low-level resistance to ery; when these cells were grown in the presence of ery, amplification of the integrated plasmid occurred and cells became resistant to a high level of antibiotic, (ii) In the opposite orientation, a high level of resistance was observed, without need for amplification. These results indicate that, in the orientation conferring a high-level resistance without amplification, the ery region is transcribed both from its own promoter and from the promoter of the amiA locus. In the opposite orientation, a low level of transcription from the ery r promoter could account for a strong selective pressure for the amplified state, which then allows for rapid growth in the presence of ery.

Relation between the transforming activity of a marker and its proximity to the end of the DNA particle

Transforming pneumococcal DNA is inactivated by treatment with restriction enzymes. For mutations belonging to the same locus (amiA locus), the extent of inactivation depends strongly upon the mutations and the enzymes. Two EcoRI and one BamHI restriction sites have been located within the amiA locus. After treatment of donor DNA with either one of these enzymes, the lowest transforming activity is observed for mutations that map near restriction sites. This effect of proximity to the nearest end of the DNA fragment extends over a distance of 1,400 nucleotides. The curve of transforming activity versus DNA size obtained with endonuclease-generated DNA fragments is very similar to that obtained previously with mechanically sheared DNA. Both curves show a striking slope change for donor DNA size around 2,700 base pairs, i.e. twice the length found for the extent of the 'end effect'. We suggest that for donor DNA fragments larger than 2,700 base pairs the transforming activity depends mainly upon the size of donor whereas for donor DNA fragments shorter than 2,700 base pairs both a size-dependent phenomenon and the 'end effect' contribute to reduce drastically the transforming activity.

Base specificity of mismatch repair in Streptococcus pneumoniae.

DNA sequence analysis was undertaken to investigate the structural basis of mutations showing different integration efficiencies in Streptococcus pneumoniae. Wild type, mutant and revertant sequences at two sites in the amiA locus were determined. It appears that markers which transform efficiently or inefficiently can result from single base pair changes. A low efficiency (LE) marker corresponds to a C:G to T:A change and a high efficiency (HE) marker to a G:C to T:A change. In the latter case, two mismatches, G/A and T/C, can exist at the heteroduplex stage in transformation; only T/C appears to be recognized by the hex system which controls transforming efficiencies in pneumococcus. Each of the recognized mismatches, T/G and C/A, which result from transitional change, and T/C appears to involve at least one pyrimidine. It is proposed that the mismatch repair system of S. pneumoniae is directed against mismatched pyrimidines. DNA sequence analysis also reveals that short deletions (33 or 34 bases long) behave as very high efficiency markers, confirming that deletions are not recognized by the hex system.

Cloning of Streptococcus pneumoniae DNA: Its use in pneumococcal transformation and in studies of mismatch repair

EcoRI fragments of the amiA locus in Streptococcus pneumoniae were cloned either into a derivative of X or into pBR325 plasmid. Mutations in the amiA locus confer resistance to aminopterin. Pneumococcal DNA fractions were enriched for the desired EcoKI fragments by agarose gel electrophoresis. Recombinant clones were detected directly by transformation with DNA from X plaques or from single-colony lysates containing pBR325. The use of cloned DNA in pneumococcal transformation has revealed a number of features pertinent to transformation in general, and also to the mismatch repair process. High transformation levels can be achieved, from 40 to 80% of a competent culture. These high levels of transformation with cloned DNA made in a foreign host are taken to confirm the absence of restriction effects on transformation in S. pneumoniae. At saturation, similar transformation levels are obtained with hybrid phage or hybrid plasmid DNAs, but the DNA amount required is 20 to 25 times lower for hybrid plasmid than for hybrid phage, probably because plasmid DNA is 10 times shorter than phage DNA. There is no “end effect” with intact hybrid DNA i.e. similar transformation levels are achieved for markers whatever their map position on the cloned pneumococcal fragment. Cloned DNA has been used to study the action of the mismatch repair process ( hex system). The presence of two mismatches in the same cell is not enough to saturate the hex system, and is not enough to kill the colony-forming center (cfc).

Characterization of an amber suppressor in Pneumococcus.

Partial revertant has been isolated, with resistance to aminopterin intermediate between wild type and mutant. This phenotype is the result of a mutation at a gene unlinked to the amiA locus. This suppressor mutation (su+) has no phenotypic characteristics by itself except a slow growth. 9 amiA mutants (belonging to 6 sites) are affected by su+ out of the 30 investigated mutants (i.e. 22 sites). The efficiency of suppression is site dependent. Two sites out of 14 mutants belonging to the thymidylate synthetase gene are suppressible. Thymidylate synthetase activity is partially restored by su+. Optochin mutants can also be suppressed. Thus su+ is not gene specific but site specific. Moreover when the str-41 allele conferring resistance to streptomycin is introduced by transformation, the suppression effect is restricted. All these properties are characteristic of an informational suppressor. The t-RNA extracted from the suppressor strain su+ but not the wild type restored the synthesis of coat protein coded by RNA from an amber mutant of bacteriophage f2. Attempts to detect ochre suppression activity gave negative results. It is suggested that the su+ gene is amber specific. Thus su+ can provide insight into the nature of suppressible mutations which should be point mutations. Both low efficiency and high efficiency mutants are affected by su+; this is additional evidence that both categories contain point mutations.

GENETIC STUDIES OF ACRIDINE-INDUCED MUTANTS IN STREPTOCOCCUS PNEUMONIAE

The mutagenic properties of acridines on pneumococcus are described. All seven acridines tested were mutagenic at the amiA locus conferring a resistance to 10(-5) M aminopterin. The effects of quinacrine were more specifically investigated. It was observed that: mutants can be obtained only by treatment of exponentially growing cells; a sharp maximum mutagenic effect occurs at a concentration slightly lower than the bacteriostatic value; and the amount of quinacrine required to yield the maximum mutagenic effect decreases with the pH of the medium. Moreover, the number of mutants detected after quinacrine treatment varies from locus to locus. The majority of quinacrine-induced mutants are readily reverted by quinacrine, but not by nitrosoguanidine treatment. This suggests that in pneumococcus quinacrine induces mainly frameshift mutations. A further study of the revertants obtained by quinacrine treatment of quinacrine-induced mutants strengths this interpretation: most of the revertants result from a mutation at the same site; some partial revertants exhibiting an intermediate resistance to aminopterin were found to contain two very closely linked mutated sites, each mutation conferring the maximum level of resistance to aminopterin. Thus, the majority of quinacrine-induced mutants at the amiA locus of pneumococcus consists of frameshift mutations. Nearly all of the isolated mutants induced by quinacrine as well as other acridines belong to the low efficiency class of transformation. It was concluded that the mismatch resulting from the pairing between the wild type and the frameshift-containing sequence is recognized by the excision-repair system involved in the discrimination function in a way similar to that in which it recognizes mismatched base pairs between a transition mutation and the wild-type sequence.

Genetic recombination in DNA-induced transformation of Pneumococcus. IV. The pattern of transmission and phenotypic expression of high and low-efficiency donor sites in the amiA locus.

Genetic recombination in DNA-induced transformation of Pneumococcus. IV. The pattern of transmission and phenotypic expression of high and low-efficiency donor sites in the amiA locus.

Genetic studies of recombining DNA in pneumococcal transformation.

The results of genetic fine structure experiments, performed on the amiA locus of Pneumococcus are summarized. The peculiar feature of transformation genetics is that a given donor marker mutation transforms with an efficiency characteristic of the mutated site. In spite of this difficulty, mapping procedures have been devised and quantitative recombination studies performed. It is concluded from these studies that transformation, in this locus, is the consequence of frequent, and essentially random exchanges occurring between donor DNA and the chromosomal DNA of the recipient cell. The average length of uninterrupted donor DNA polynucleotide strand which could be inserted into the chromosome of a transformed cell is estimated, from genetic data, to be probably not greater than 3.10(5) daltons (for a double-stranded insertion). It is proposed, on the basis of genetic evidence, that following essentially random exchanges between donor DNA and recipient chromosome, a revision process, specific for certain types of mutated sites, occurs. The revision process appears to remove preferentially donor DNA sequences from the primary recombinant structure, and allow repair along the chromosomal template, leading to low efficiency in the genetic integration of these sites. A mechanism for this "destruction-choice" process is presented, and evidence in support of this mechanism discussed.