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.     

Localization of competence-induced proteins in Streptococcus pneumoniae.

Intracellular locations of 11 proteins associated with the development of competence in Streptococcus pneumoniae were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of subcellular fractions prepared from protoplasts. Controls showed that the competence-induced proteins were stable during the formation of protoplasts at 25 degrees C even though some had a half-life of only 8 min at 37 degrees C. Five competence-induced proteins p38, p27, p19.5, p16, and p14.5, were found in the cytoplasm. Two, p52 and p41, were associated with the membrane, and one, p10, was extracellular. Three others, p50, p36, and p29, were recovered in both cytoplasmic and membrane fractions. No competence-induced protein was detected in the periplasmic fraction except under conditions where leakage of all components was occurring, a phenomenon that was seen in many preparations. Similar fractionation of competent cells soon after uptake of [3H]DNA showed the "eclipse complex" of single-stranded DNA and p19.5 was associated approximately one-third with membranes and two-thirds with cytoplasmic fractions, with almost none in the periplasm. This result suggests strongly that at the time the donor DNA entered the cytosol it was in single-stranded form and it had not yet paired with the recipient DNA.     

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.     

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.     

Transformation in Streptococcus pneumoniae: formation of eclipse complex in a coiA mutant implicates CoiA in genetic recombination

CoiA is a transient protein expressed specifically during competence and required for genetic transformation in Streptococcus pneumoniae, but not for DNA uptake. It is widely conserved among Gram-positive bacteria but its function is unknown. Here we report that although the rate of DNA uptake was not affected in a coiA mutant, the internalized donor DNA did not recombine into the host chromosome to form a physical and genetic heteroduplex. Instead, DNA taken up by a coiA mutant accumulated in the form of a single-stranded (ss) DNA-protein complex indistinguishable from the eclipse complex formed as a recombination intermediate in wild-type competent cells. Internalized donor DNA in a dprA mutant did not accumulate either as ss DNA or as an eclipse complex. Together, these results establish that a coiA mutant exhibits a phenotype different from that of dprA or recA mutants, and that CoiA functions at a later step in promoting recombination during genetic transformation in Streptococcus pneumoniae.     

Choline-binding protein D (CbpD) in Streptococcus pneumoniae is essential for competence-induced cell lysis

Streptococcus pneumoniae is an important human pathogen that is able to take up naked DNA from the environment by a quorum-sensing-regulated process called natural genetic transformation. This property enables members of this bacterial species to efficiently acquire new properties that may increase their ability to survive and multiply in the human host. We have previously reported that induction of the competent state in a liquid culture of Streptococcus pneumoniae triggers lysis of a subfraction of the bacterial population resulting in release of DNA. We have also proposed that such competence-induced DNA release is an integral part of natural genetic transformation that has evolved to increase the efficiency of gene transfer between pneumococci. In the present work, we have further elucidated the mechanism behind competence-induced cell lysis by identifying a putative murein hydrolase, choline-binding protein D (CbpD), as a key component of this process. By using real-time PCR to estimate the amount of extracellular DNA in competent relative to noncompetent cultures, we were able to show that competence-induced cell lysis and DNA release are strongly attenuated in a cbpD mutant. Ectopic expression of CbpD in the presence or absence of other competence proteins revealed that CbpD is essentially unable to cause cell lysis on its own but depends on at least one additional protein expressed during competence.     

Use of a cloned DNA fragment to analyze the fate of donor DNA in transformation of Streptococcus pneumoniae

The integration of donor label into the recipient fragment is followed during transformation of Streptococcus pneumoniae. The method used involves gel analysis of restriction endonuclease-treated recipient DNA after recombination with a radioactively labeled homologous cloned fragment.     

Effect of mismatched base pairs on the fate of donor DNA in transformation of Streptococcus pneumoniae

Summary Investigation of the mechanism that discriminates against mismatched base pairs in transformation of Streptococcus pneumoniae of genotype hex ⁺ was based on the use of a radioactively labeled cloned fragment of pneumococcal DNA as donor in transformation. The fate of the donor label was followed by lysis of the transformed cells and separation by agarose gel electrophoresis of DNA fragments generated by restriction endonucleases. As a result of Hex action, most of the donor DNA fragment, which was a few kilobases in length, was lost when a mismatched base pair occurred between donor and recipient DNA. This was not observed in hex ^- recipient cells. Kinetic studies of mismatch-induced donor DNA loss showed that the process is faster in strain 800, an R6 derivative, than in DP 1601, a strain of different origin. In the latter strain, the amount of donor label that becomes double stranded rises substantially, indicating extensive formation of donorrecipient heteroduplex structures, before falling to the expected level. At 30°C the process is essentially completed 15 min after entry.     

Specificity of DNA uptake in genetic transformation of gonococci

Genetic transformation of gonococci to streptomycin resistance was inhibited by homologous DNA or by DNA from related $\text{Neisseriae}$, but not by high concentrations of heterologous DNAs. Gonococci were capable of adsorbing large quantities (up to about 50 μg per 10⁸ cells) of both homologous and heterologous DNA, which could not be eluted by strong shearing forces. Treatment with externally added DNase removed virtually all the heterologous DNA while a small fraction of the homologous DNA, not influenced by the presence of excess heterologous DNA, remained cell-bound in a form resistant to nuclease treatment. Competing homologous DNA suppressed nuclease-resistant binding. These findings suggest that gonococci have two types of DNA binding components at their surface. Competence of gonococci for genetic transformation undergoes a rapid decay if the cells are incubated with homologous (but $\text{not}$ with heterologous) DNA.     

DNA sequences required to induce localized conversion in Streptococcus pneumoniae transformation

Summary 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-ATTCAT5-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.     

Prophage spontaneous activation promotes DNA release enhancing biofilm formation in Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is able to form biofilms in vivo and previous studies propose that pneumococcal biofilms play a relevant role both in colonization and infection. Additionally, pneumococci recovered from human infections are characterized by a high prevalence of lysogenic bacteriophages (phages) residing quiescently in their host chromosome. We investigated a possible link between lysogeny and biofilm formation. Considering that extracellular DNA (eDNA) is a key factor in the biofilm matrix, we reasoned that prophage spontaneous activation with the consequent bacterial host lysis could provide a source of eDNA, enhancing pneumococcal biofilm development. Monitoring biofilm growth of lysogenic and non-lysogenic pneumococcal strains indicated that phage-infected bacteria are more proficient at forming biofilms, that is their biofilms are characterized by a higher biomass and cell viability. The presence of phage particles throughout the lysogenic strains biofilm development implicated prophage spontaneous induction in this effect. Analysis of lysogens deficient for phage lysin and the bacterial major autolysin revealed that the absence of either lytic activity impaired biofilm development and the addition of DNA restored the ability of mutant strains to form robust biofilms. These findings establish that limited phage-mediated host lysis of a fraction of the bacterial population, due to spontaneous phage induction, constitutes an important source of eDNA for the S. pneumoniae biofilm matrix and that this localized release of eDNA favors biofilm formation by the remaining bacterial population.     

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.     

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.