Res. Microbiol. 151 (2000) 407–411 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0923250800001765/REV Molecular biology of Streptococcus pneumoniae: an everlasting challenge Michel Sicard*, Anne Marie Gasc, Philippe Giammarinaro, Jacques Lefrançois, Frank Pasta, Mustapha Samrakandi Laboratoire de microbiologie et de génétique moléculaire du C.N.R.S. et Université Paul Sabatier, 118, route de Narbonne 31062 Toulouse cedex, France Abstract – Streptococcus pneumoniae is a model for elucidating: 1) recombination steps of DNA, from its discovery to polarity of integration; 2) long-patch mismatch repair, short-patch repair triggered by A/G and exclusion of deletions; 3) resistance to β-lactam antibiotics; and 4) factors of virulence. Several of these topics remain a challenge for future investigations. © 2000 Éditions scientifiques et médicales Elsevier SAS Streptococcus pneumoniae / recombination / antibiotic resistance / virulence 1. Introduction At the beginning of the century, pneumococcus was the major cause of death by bacterial infection. In the USA, more than 50 000 people died every year. Even today throughout the world, millions of people are killed every year by this virulent bacteria . The mortality of septic shock has been reduced during the last 30 years, but only from 70 to 45% for patients admitted to intensive care for septicemia, with an increasing proportion of Gram-positives (45%) . Septic shock from invasive pneumococci can kill, at a high frequency (10–15%) and within a few days or hours, patients who had been in good health. These pneumococci are frequently not even resistant to antibiotics. This is why these bacteria have been studied for more than 80 years, resulting in several major contributions to biology and requiring a worldwide effort to resolve many scientific mysteries concerning public health. 2. DNA recombination It is common practice to quote the famous publication of Avery and his collegues in 1944 * Correspondence and reprints [email protected] for the identification of nucleic acids as genetic determinants. But it should be remembered that it took 9 years to be accepted by the scientific community, thanks to the efforts of several scientists such as Rollin Hotchkiss who intensively purified DNA in order to refute the criticisms of the biochemist A. Mirsky, who argued that traces of proteins could account for the transforming activity. In fact, it is likely that the criticisms of Mirsky are responsible for the failure to confer the Nobel prize upon Avery. Even in 1951, some geneticists did not accept that bacteria contain genes, since “Mendel laws cannot be demonstrated” in these organisms . When Watson and Crick defined the structure of DNA accounting for its fantastic information content required for genes, the replication and the mutation of this molecule , these debates became only of historical interest. The field of genetics had developed over a 40-year period by hybridization of eucaryotes, but was unable to answer fundamental questions such as: what is the structure, the nature, the mode of replication, recombination, mutation and expression of the gene? Unexpectedly, bacteria solved these genetic mysteries. The discovery that genes are DNA was the first molecular biology experiment. Before 1950 it was generally admitted that crossing over, a key process in genetics, occurred 408 M. Sicard et al. / Res. Microbiol. 151 (2000) 407–411 between genes that could not be split. As it was possible to handle millions of microorganisms and select rare events, intragenic recombination was easily demonstrated even between two base pairs. As early as 1951, Harriett EphrussiTaylor isolated four partially capsule-deficient mutants. Rare normal encapsulated pneumococci were obtained by reciprocal transformation as one of the first cases of intragenic recombination. In the middle 1950s, to explain recombination, two hypotheses were put forward: breakage and reuniting of DNA molecules without DNA replication or copy-choice in which the replication machinery would shift from one parental chromatid to the other. In 1960, Maury Fox demonstrated that pneumococcal transformation occurred without significant DNA synthesis showing that the model of breakage and reunion between DNA molecules was correct . Almost at the same time , Sandy Lacks observed that donor DNA is singlestranded inside the recipient bacteria before its integration in the chromosome. This was the first evidence for hybrid DNA during recombination, a step proposed much later to account for gene conversion in fungi. Obviously, most of our understanding of the steps and enzymes of recombination results from experiments on Escherichia coli. However, concerning some specific items, Streptococcus pneumoniae contributed significantly to this knowledge. For example, what is the process of recombination of long heterologies? Are they excluded, or integrated as point mutations, or are they submitted to some replication or repair process? In pneumococci the efficiency of integration of deletions or insertions is fairly high as long as their size is much smaller than the donor DNA molecule. This is accounted for by a normal pairing between the surrounding homologous sequences. However, we have observed that, when these deletions are transformed in a two-point cross, the frequency of wild-type recombinants is tremendously increased (more the ten-fold). Franck Pasta was able to show that this observation is accounted for by the exclusion of the donor heterologous DNA at a frequency of 20% . Taking advan- Figure 1. Model accounting for the preponderance of 5’ site recombinants. tage of the interruption in recombination by deletion, he compared the transformation efficiencies of the segments 5’- and 3’-ward from this deletion. Using the refined tools of molecular genetics of the ami locus, artificial heteroduplexes containing the deletion, PCR amplification of the DNA from individual transformants, restriction enzyme analysis to detect which strand and which side of the deletion had recombined, Franck Pasta found that in vivo the 5’ side of donor single-strand DNA is strongly (80%) favored by recombination  (figure 1). Therefore, the strand exchange is polarized from 5’ to 3’. It will be interesting to see if this polarity can be found in other organisms. 3. DNA mismatch repair A major contribution of pneumococcal studies was the discovery and understanding of mismatch repair. One of the original observations was the isolation by Harriett EphrussiTaylor in 1959 of an optochin-resistant mutant transforming eight-fold less than the standard reference marker. She proposed that this mutation was a long deletion or insertion, or as an alternative, that there were some weak points on the DNA molecule near the position of this mutation. How to test these possibilities? A genetic map of such a gene could give an answer but it was not possible to make it. We decided to change a locus and to find a gene where wild-type recombinants could be recovered, as well as mutant strains. We were able to find such mutations that confer resistance to amethopterin but could not grow in a defined M. Sicard et al. / Res. Microbiol. 151 (2000) 407–411 medium containing an excess of isoleucine versus leucine and valine . Having constructed such a system, hundreds of mutants were isolated either spontaneously or by mutagenic treatments. We could perform a one-point cross in both directions and two-point crosses to build a genetic map. Unexpectedly, the poorly transformable mutants were not long heterologies, as they were spread all along the gene. Jean-Pierre Claverys, Pedro García and AnneMarie Gasc showed that they were single-site transitions or ± 1- to 3-base deletions . Sandy Lacks found the same results in the amylomaltase locus . In 1966 Harriett Ephrussi-Taylor  proposed that low efficiency markers were excised and eventually one tenth of them would integrate by a repair process. This explanation resulted from the observation that optochinresistant mutations disappeared after their entry into the cell and by analogy to the excision repair of E. coli DNA after UV irradiation. Much work has been devoted to confirming this daring hypothesis by J.M. Louarn, J.P. Claverys, G. Tiraby and Anne-Marie Gasc in my laboratory and in Sandy Lacks laboratory, especially by the isolation of strains unable to discriminate between high and low efficiency mutants, opening the way for cloning and sequencing the hexA and hexB genes. Gérard Tiraby demonstrated in 1973 that this repair system was also antimutagenic . Later it was found in other bacteria, in yeast and in man where these genes protect against specific cancers. It should be emphasized how long it took from the original unusual observations of low efficiency markers by several scientists in 1959, and the studies of the first discriminating strain by Green and Ravin in 1959 , to the characterization of the specificity of the repair system in 1981, and eventually to the description of the anticancerous properties of these repair genes in 1993. At the same time as Peggy Lieb described the short-patch repair system of the A/G mismatch, Lefèvre observed a 12-base-long repair for this mismatch in pneumococcus  that is coded by a mutY homologue, since Franck Pasta, who 409 cloned and mutated it, found that it complements the E. coli mutY gene. 4. β-Lactamine resistance Penicillin-resistant mutants were obtained by Rollin Hotchkiss in 1951 , i.e. 16 years before the first hospital-resistant isolates. Most resistant mutants could be accounted for by a modification of penicillin-binding proteins (PBPs) as demonstrated by Regine Hakenbeck, Alex Tomasz and their collegues . By serial transformation of the wild type by DNA from highly resistant strains, we showed that genes coding for PBP 2x and PBP 3 yielded increased resistance. Moreover, transformants to higher resistance were not modified in their PBP pattern. Eric Guenzi in Regine Hakenbeck’s laboratory showed that this is due to mutations in two genes that encode a sensor protein belonging to the family of signal-transducing histidine kinases and a regulator responding to an environmental signal . Philippe Giammarinaro in my laboratory has shown that Ca++ is the environmental signal of this system (ciaR/ciaH) which is likely to affect cell-wall structure and competence genes . An unexpected observation was that a mutation of this system not only increases cefotaxime resistance but blocks competence. Indeed, competence is not fully understood, even though the startling discovery by Pakula in Poland in 1962  of a competence hormone and the characterization of its structure by Don Morrison, Sive Harvestein and their collegues in 1995  tremendously contributed to the knowledge of DNA uptake in pneumococcus. In my laboratory, Jacques Lefrançois , using electropermeation, found that it is not sufficient to introduce chromosomal DNA inside the cell to obtain recombinants even if we induce natural competence. The integration of markers in the chromosome in naturally competent cells must require DNA processing during entry, which is still to be discovered. While it is well demonstrated that the dissemination of penicillin-resistant isolates is directly related to the use of this antibiotic, the 410 M. Sicard et al. / Res. Microbiol. 151 (2000) 407–411 process of bacterial migration is not well understood. Using a very discriminating method, FIGE analysis, Anne-Marie Gasc in my laboratory has shown that most of the 9V French isolates, which were either resistant or susceptible to penicillin, are clonal . What could be the selective advantage of this clone in being able to invade Spain, France and other countries in a few years? 5. Factors of virulence In the early 1920s, compelling evidence had accumulated that virulent pneumococci are encapsulated, appearing as smooth colonies on plates, whereas nonencapsulated strains are not virulent and are rough. At the same time, unexpectedly, Oswald Avery and Michael Heidelberger were able to show that this capsule was not a protein but polysaccharides that protect the bacteria from phagocytosis . In 1949, using intermediate mucoid colonies of type III, Harriett Ephrussi-Taylor in Avery’s laboratory reported that there is a direct relationship between virulence and the size of the capsule . An average three-fold reduction in the amount of polysaccharide is enough to shift by a factor of six to seven logs the quantity of bacteria required to kill approximately 50% of mice . Since then, scores of genes involved in the biosynthesis of several capsular types have been characterized. Why is virulence so highly sensitive to relatively small differences in the amount of capsular polysaccharides? How does the capsule protect against phagocytosis? Why is the capsule a barrier to DNA uptake? Deficiency in several proteins was reported to attenuate virulence without suppressing it, in contrast to capsule deficiency that blocks virulence. However, significant conclusions require isolation of well-characterized mutants, comparison between isogenic strains and reproducibility in other laboratories. The refined molecular genetics tools now available should be intensively used. Autolysin coded by lytA is one of these factors that partially affects virulence in mouse models [27, 28]. Virulence of lytA– strains can be further reduced by the addition of a mutation of a gene involved in calcium transport . The effects of autolysin, however, seemtobemediatedbythereleaseofpneumolysin, another important virulence factor. Virulence is reduced in pneumolysin-negative strains as well as in autolysin-negative strains . Recent studies suggested that pneumolysin stimulates nitric oxide production from macrophages more efficiently than crude cell-wall preparation . It was possible to isolate pneumolysin mutants affected in either one of their dual functions: lysis or complement activation. Strains producing competent activation-deficient pneumolysin are fully virulent, whereas lysis-deficient pneumolysin significantly reduces virulence . Although pneumolysin-deficient mutants are still partially virulent, it seems that inflammation, a major step during invasion, is triggered by this molecule. Obviously, other factors of virulence remain to be discovered. A survey of virulence-negative mutants was performed by Daniel Simon and coworkers  by random inactivation of genes. Mutation of several new Figure 2. Hybridization using cap3A probes on type 3 DNA restricted by EcoRI. Lane 1: original encapsulated strain; lane 2: the same strain after five passages on mice; lane 3: the same encapsulated strain transferred 35 times on Petri dishes; lanes 4 and 5: two capsule-deficient strains isolated on Petri dishes after 35 transfers. M. Sicard et al. / Res. Microbiol. 151 (2000) 407–411 genes reduced septicemia, opening the way to future investigation. With Mustapha Samrakandi we initiated a program to detect genomic modifications resulting from loss of virulence when virulent strains were subcultured in vitro by serial transfers. This procedure as well as prolonged storage in the refrigerator had already been described in the 1920s as selecting virulentdeficient mutants. By FIGE or restriction enzyme analysis using several probes (PspA, lytA, recA, ply, nanaA, hyal, cap3A), deletions or amplifications could not be detected. A modification was, however, observed in a capsule-deficient strain obtained by this method (figure 2). 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