Modulation of competence for genetic transformation in Streptococcus pneumoniae

The spontaneous development of competence by cultures of Streptococcus pneumoniae in casein hydrolysate medium was strongly dependent on the initial pH of the culture medium. Cells growing in cultures beginning with a wide range of initial pH values (6.8 to 8.0) all developed competence, as measured by [3H]DNA uptake, [3H]DNA degradation and genetic transformation; but the initial pH of the medium affected both the timing of the occurrence of competence and the number of times the culture became competent. In cultures grown in media of lower initial pH, competence occurred only once, at high population densities, while in more alkaline media a succession of competence cycles occurred, beginning at lower cell densities. The critical population density required for the initiation of competence varied tenfold over the pH range studied. Successive competence cycles in an alkaline medium were not equivalent: while the percentage of competent cells in the first competence cycle was high (approximately 80%), that in the second competence cycle was lower (approximately 12%). Correspondingly, competence-specific proteins were less prominent in the labelled-protein pattern of the second competence cycle than in that of the first. These features of the physiology of competence control make it possible to adjust the expression of competence to suit various experimental requirements.     

LiCl treatment releases a nickase implicated in genetic transformation of Streptococcus pneumoniae.

When cells competent for genetic transformation of Streptococcus pneumoniae which could bind and enable entry of extracellular DNA molecules were treated with LiCl, they released a nickase that introduced nicks into a double-stranded DNA in the presence of EDTA. The nickase was specific for competent cells and coupled with DNA-binding activity. Furthermore, when noncompetent cells were treated with LiCl, they released the putative receptors for the competence activator.     

Identification of a deoxyribonuclease implicated in genetic transformation of Diplococcus pneumoniae.

A mutation of Diplococcus pneumoniae, end-1, reduces the major deoxyribonuclease activity of the cell, an endonuclease, to 10% of its normal value without impairing transformation. Further mutations, called noz, abolish the residual endonuclease activity and block transformation. The residual endonuclease is similar to the wild-type enzyme in size, charge, divalent cation dependence, inhibition by ribonucleic acid, and formation of oligonucleotide products. However, the mutant endonuclease is more temperature sensitive, which suggests that the end-1 mutation occurred in a structural gene for the enzyme. Genetic analysis showed that the noz mutations occur at the same genetic locus. A number of new end mutants were analyzed. Those that retained more than 1.4% of the normal endonuclease activity were essentially normal in transformation; those with less than 1% were defective. The transformation-defective end mutants appear to be blocked in the entry of deoxyribonucleic acid (DNA) since they carry out the prior step of binding DNA to the outside of the cell. The major endonuclease of the cell may act as a DNA translocase by attacking and degrading one strand of DNA, thereby facilitating entry of the complementary strand into the cell.     

Fate of DNA in eclipse complex during genetic transformation in Streptococcus pneumoniae

Uptake of DNA and genetic recombination proceeded normally in competent Streptococcus pneumoniae despite inhibition of DNA replication by 6-(p-hydroxyphenylazo)-uracil. Immediately after a brief uptake period, 68% of donor DNA label was in eclipse complex form, and 22% was in low-molecular-weight products; by the completion of integration at 10 min, 23% was integrated into the chromosome, and the rest was lost from the cell. Throughout the process, less than 1% was found as free single strands. The DNA in eclipse complex is therefore an intermediate in the integration process.     

Barriers to genetic exchange between bacterial species: Streptococcus pneumoniae transformation

Interspecies genetic exchange is an important evolutionary mechanism in bacteria. It allows rapid acquisition of novel functions by transmission of adaptive genes between related species. However, the frequency of homologous recombination between bacterial species decreases sharply with the extent of DNA sequence divergence between the donor and the recipient. In Bacillus and Escherichia, this sexual isolation has been shown to be an exponential function of sequence divergence. Here we demonstrate that sexual isolation in transformation between Streptococcus pneumoniae recipient strains and donor DNA from related strains and species follows the described exponential relationship. We show that the Hex mismatch repair system poses a significant barrier to recombination over the entire range of sequence divergence (0.6 to 27%) investigated. Although mismatch repair becomes partially saturated, it is responsible for 34% of the observed sexual isolation. This is greater than the role of mismatch repair in Bacillus but less than that in Escherichia. The remaining non-Hex-mediated barrier to recombination can be provided by a variety of mechanisms. We discuss the possible additional mechanisms of sexual isolation, in view of earlier findings from Bacillus, Escherichia, and Streptococcus.     

Regulation of natural genetic transformation and acquisition of transforming DNA in Streptococcus pneumoniae

The ability of pneumococci to take up naked DNA from the environment and permanently incorporate the DNA into their genome by recombination has been exploited as a valuable research tool for 80 years. From being viewed as a marginal phenomenon, it has become increasingly clear that horizontal gene transfer by natural transformation is a powerful mechanism for generating genetic diversity, and that it has the potential to cause severe problems for future treatment of pneumococcal disease. This process constitutes a highly efficient mechanism for spreading β-lactam resistance determinants between streptococcal strains and species, and also threatens to undermine the effect of pneumococcal vaccines. Fortunately, great progress has been made during recent decades to elucidate the mechanism behind natural transformation at a molecular level. Increased insight into these matters will be important for future development of therapeutic strategies and countermeasures aimed at reducing the spread of hazardous traits. In this review, we focus on recent developments in our understanding of competence regulation, DNA acquisition and the role of natural transformation in the dissemination of virulence and β-lactam resistance determinants.     

Two distinct functions of ComW in stabilization and activation of the alternative sigma factor ComX in Streptococcus pneumoniae

Natural genetic transformation in Streptococcus pneumoniae is controlled by a quorum-sensing system, which acts through the competence-stimulating peptide (CSP) for transient activation of genes required for competence. More than 100 genes have been identified as CSP regulated by use of DNA microarray analysis. One of the CSP-induced genes required for genetic competence is comW. As the expression of this gene depended on the regulator ComE, but not on the competence sigma factor ComX (sigma(X)), and as expression of several genes required for DNA processing was affected in a comW mutant, comW appears to be a new regulatory gene. Immunoblotting analysis showed that the amount of the sigma(X) protein is dependent on ComW, suggesting that ComW may be directly or indirectly involved in the accumulation of sigma(X). As sigma(X) is stabilized in clpP mutants, a comW mutation was introduced into the clpP background to ask whether the synthesis of sigma(X) depends on ComW. The clpP comW double mutant accumulated an amount of sigma(X) higher (threefold) than that seen in the wild type but was not transformable, suggesting that while comW is not needed for sigma(X) synthesis, it acts both in stabilization of sigma(X) and in its activation. Modification of ComW with a histidine tag at its C or N terminus revealed that both amino and carboxyl termini are important for increasing the stability of sigma(X), but only the N terminus is important for stimulating its activity.     

Constitutive competence for genetic transformation in Streptococcus pneumoniae caused by mutation of a transmembrane histidine kinase

Competence for DNA uptake and genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system. A competence-stimulating polypeptide (CSP) is secreted by the bacteria and acts back on the cells via a transmembrane histidine kinase. This enzyme phosphorylates a response regulator that activates synthesis of a SigH-like protein. The new sigma factor enables expression of a set of proteins transcribed from a novel promoter. A mutation called trt had been found that circumvented this regulation. The mutant cells are constitutively competent; that is, they can be transformed at low cell densities, in the presence of proteases that attack CSP, or during growth at low pH. In this work, cells containing trt were shown to be competent even in the presence of a comAB mutation that blocks secretion of CSP. The trt mutation was localized to comD, the gene encoding the transmembrane histidine kinase. A DNA segment of the trt mutant corresponding to comCDE was cloned, and it was shown to contain the trt mutation by its ability to confer constitutive competence. A two-step assay, which was based on transfer of trt to a wild strain and screening for transformability in the presence of trypsin, served to locate the trt mutation precisely. It corresponds to a GC-->AT transition, which changes Asp299 in the histidine kinase to Asn. This alteration in the carboxyl terminal half of the protein, which is cytoplasmically located and contains the phosphorylase activity, presumably alters the enzyme conformation so that it is permanently activated, independent of signals from the transmembrane domain. These results may help illuminate the mechanism by which external signals affect kinase action in two-component regulatory systems, and they may be of practical value in facilitating genetic studies by rendering pneumococcal strains permanently competent.     

Identification of Streptococcus pneumoniae mismatch repair genes by an additive transformation approach

Summary During transformation of Streptococcus pneumoniae, mismatch repair occurs on donor-recipient heteroduplexes harboring some mismatched base pairs. A few mutants defective in mismatch repair have been isolated and termed hex ^-. However, neither the number of genes involved nor their products have yet been identified. In an attempt to characterize such genes we have used an additive transformation approach — that is the inactivation of genes by insertion of chimeric plasmids. Pneumococcal DNA fragments were joined in vitro to a plasmid derivative of pBR325 that carries an erythromycin resistance determinant and does not replicate autonomously in S. pneumoniae. Ery-r transformants obtained with such a ligation mixture arise via homology-dependent integration of the chimeric plasmids into the chromosome. Hex ^- mutants have been selected among the ery-r population. Comparison of these mutants by Southern blot hybridization with a vector probe reveals that at least two genes are involved in mismatch repair.     

Peptide permeases modulate transformation in Streptococcus pneumoniae

To Identify elements participating In the process of transformation, a bank of genetically altered mutants of Streptococcus pneumoniae with defects in exported proteins was assessed for a decrease in transformation efficiency. One mutant consistentiy transformed 10-foid less than the parent strain. Sequence analysis and reconstitution of the altered locus revealed a gene, plpA (permease-like protein), which encodes a putative substrate-binding protein belonging to the family of bacterial permeases responsible for peptide transport. The derived amino acid sequence for this gene was 80% similar to AmiA, a peptide-binding protein homologue from pneumococcus, and 50% similar over 230 amino acids to SpooKA which is a regulatory element in the process of transformation and sporulation in Bacillus subtilis. PIpA fusions to alkaline phosphatase (PhoA) were shown to be membrane associated and labelled with [³H]-palmitic acid, which probably serves as a membrane anchor. Experiments designed to define the roles of the pIpA and ami determinants in the process of transformation showed that: (i) mutants with defects in plpA were >90% transformation deficient while ami mutants exhibited up to a fourfold increase in transformation efficiency; (ii) compared to the parental strain, the onset of competence in an ami mutant occurred earlier in logarithmic growth, whereas the onset was delayed in a plpA mutant; and (ill) the plpA mutation decreases the expression of a competence-regulated locus. Since the permease mutants would fail to bind specific ligands, it seems likely that the substrate-permease interaction modulates the process of transformation.     

Polarity of localised conversion in Streptococcus pneumoniae transformation

Summary 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 longpatch conversion by the length of DNA repair, gene requirements and sequence specificity, we devised expreriments to detect the reciprocal conversion, AmiA⁺ to AmiA^r. 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.     

Identification of the optochin-resistant Streptococcus pneumoniae strains

So far the prevalent method of the identification of pneumococci in the routine diagnostic practice has included three tests: sensitivity to optoxin, solubility in biliary salts and reaction with omniserum. At present optoxin-resistant pneumococcal strains, as well as those giving dual reaction to solubility in biliary salts, occur sufficiently often thatconsiderably complicates the identification of this infective agent. We have summarized experience in the isolation of S. pneumoniae strains, resistant to optoxin and not soluble in biliary salts; in addition, the prospects of introducing the complex approach to the identification of a given culture with the use of molecular diagnostic methods into daily practice are evaluated.     

Significant variation in transformation frequency in Streptococcus pneumoniae

The naturally transformable bacterium Streptococcus pneumoniae is able to take up extracellular DNA and incorporate it into its genome. Maintaining natural transformation within a species requires that the benefits of transformation outweigh its costs. Although much is known about the distribution of natural transformation among bacterial species, little is known about the degree to which transformation frequencies vary within species. Here we find that there is significant variation in transformation frequency between strains of Streptococcus pneumoniae isolated from asymptomatic carriage, and that this variation is not concordant with isolate genetic relatedness. Polymorphism in the signalling system regulating competence is also not causally related to differences in transformation frequency, although this polymorphism does influence the degree of genetic admixture experienced by bacterial strains. These data suggest that bacteria can evolve new transformation frequencies over short evolutionary timescales. This facility may permit cells to balance the potential costs and benefits of transformation by regulating transformation frequency in response to environmental conditions.     

Identification and characterization of IS1381, a new insertion sequence in Streptococcus pneumoniae.

A new insertion sequence (IS1381) was identified in the genome of Streptococcus pneumoniae R6 as an 846-bp segment containing 20-bp terminal inverted repeats and flanked by 7-bp direct repeats. The three sequenced copies of this element have two overlapping open reading frame (ORF) genes named orfA and orfB. However, significant variations between individual copies were found, suggesting that inactivating mutations have occurred in an original single ORF. Accordingly, the consensus IS1381 element derived from the comparison of the three available copies should contain a single ORF sufficient to encode a basic protein of 267 amino acids which exhibited high similarity to the putative transposases of ISL2 from Lactobacillus helveticus and of IS702 from the cyanobacterium Calothrix sp. strain PCC 7601. A minimum of five to seven copies were detected by hybridization experiments in the R6 genome. In remarkable contrast with the two previously reported pneumococcal insertion sequences, several copies of IS1381 have been detected in all of the clinical isolates tested so far. Interestingly, Streptococcus oralis NCTC 11427 (type strain), a close relative of pneumococcus, does not contain this element, but its occurrence in the type strain of Streptococcus mitis (NCTC 12261) suggests that this species has exchanged DNA with S. pneumoniae directly or through an intermediate species.     

Identification of a purC gene from Streptococcus pneumoniae.

A gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase was identified in Streptococcus pneumoniae as a 708-bp segment of the genome encoding a 27,001-Da protein with strong similarity to known PurC proteins. The S. pneumoniae purC gene, found immediately adjacent to the competence induction genes, comAB, was cloned and sequenced. The predicted protein product of purC displayed substantial (> 40%) identity to the entire sequence of the PurC proteins of Bacillus subtilis and Escherichia coli. Function of the S. pneumoniae gene product was demonstrated by complementation of E. coli purC mutations.     

Inhibition of transformation in Streptococcus pneumoniae by lysogeny.

Streptococcus pneumoniae R6X was lysogenized with bacteriophage 304 isolated after mitomycin induction of an ungrouped alpha-hemolytic streptococcus. Lysogenized pneumococci lost their capacity to undergo genetic transformation: transformability was restored after cells were spontaneously cured of their prophage. Both lysogens and nonlysogens produced activator substance (competence factor), and both bound deoxyribonucleic acid in a deoxyribonuclease-resistant form. However, nonlysogens retained deoxyribonucleic acid after washing, whereas lysogens did not. The latter did not liberate phage nor (unlike nonlysogens) degrade transforming deoxyribonucleic acid and contained normal levels of endonuclease.