Evolution of Streptococcus pneumoniae and Its Close Commensal Relatives

Streptococcus pneumoniae is a member of the Mitis group of streptococci which, according to 16S rRNA-sequence based phylogenetic reconstruction, includes 12 species. While other species of this group are considered prototypes of commensal bacteria, S. pneumoniae is among the most frequent microbial killers worldwide. Population genetic analysis of 118 strains, supported by demonstration of a distinct cell wall carbohydrate structure and competence pheromone sequence signature, shows that S. pneumoniae is one of several hundred evolutionary lineages forming a cluster separate from Streptococcus oralis and Streptococcus infantis. The remaining lineages of this distinct cluster are commensals previously collectively referred to as Streptococcus mitis and each represent separate species by traditional taxonomic standard. Virulence genes including the operon for capsule polysaccharide synthesis and genes encoding IgA1 protease, pneumolysin, and autolysin were randomly distributed among S. mitis lineages. Estimates of the evolutionary age of the lineages, the identical location of remnants of virulence genes in the genomes of commensal strains, the pattern of genome reductions, and the proportion of unique genes and their origin support the model that the entire cluster of S. pneumoniae, S. pseudopneumoniae, and S. mitis lineages evolved from pneumococcus-like bacteria presumably pathogenic to the common immediate ancestor of hominoids. During their adaptation to a commensal life style, most of the lineages gradually lost the majority of genes determining virulence and became genetically distinct due to sexual isolation in their respective hosts

Naturlig genetisk transformasjon: utvikling av ett nytt genetisk verktøy for Streptococcus thermophilus

Lactic acid bacteria (LAB) have been used in fermentation of foods for hundreds or even thousands of years. During the last decade the genome sequences of a number of commercially important LAB species and strains have been published. These sequences provide new insights into the genetic and metabolic capacities of the species/strains concerned. To be able to fully exploit the wealth of information produced by the genomic revolution, efficient tools for genetic manipulation of bacterial genomes are required. Such tools have been lacking for Streptococcus thermophilus, an important bacterium for the dairy industry, which is used in the manufacture of yoghurt and Italian- and Swiss-type cheeses. As several species in the genus Streptococcus are competent for natural genetic transformation, we hypothesized that S. thermophilus possesses this property as well. Naturally transformable bacteria take up naked extracellular DNA and incorporate it into their genomes by homologous recombination. This mechanism provides an ideal tool for genetic engineering in bacteria. The major goal of the present work was therefore to determine whether S. thermophilus is naturally transformable. If so, a second goal was to investigate the usefulness of this mechanism as a tool for genetic engineering in S. thermophilus. Early on in the study, we identified and sequenced the gene encoding the S. thermophiles homologue of ComX, an alternative sigma factor controlling competence development in Streptococcus pneumonia. As we did not succeed in finding growth conditions that provoked spontaneous competence development in S. thermophilus, overexpression of comX was chosen as an alternative strategy. For this purpose, we used a pheromone-inducible twocomponent signal transduction system that regulates bacteriocin production in S. thermophilus. We found that overexpression of comX induced the competent state in the LMG 18311 strain, demonstrating for the first time that at least one strain of the species S. thermophilus is naturally transformable. Further investigations showed that the transformation efficiency of our system was high enough to allow genetic manipulation of the S. thermophilus genome without the use of a selectable marker. Instead, transformants could be identified by colony-lift hybridization with a specific oligonucleotide probe. The advantage of this procedure is that the bacterial genome can be altered at preselected sites without introduction of foreign DNA. In sum, the genetic tools developed in this thesis has opened up new research opportunities that will lead to a better understanding of the metabolism and physiology of S. thermophilus, and perhaps also to the development of novel starter strains with improved properties.Melkesyrebakterier har blitt benyttet til fermentering av mat i hundrevis og sannsynligvis tusenvis av år. I løpet av de siste ti årene har genomsekvensen til et betydelig antall kommersielt viktige arter og stammer av melkesyrebakterier blitt publisert. Dette har gitt ny innsikt i disse bakterienes genetiske og metabolske egenskaper og potensialer. For at forskerne skal kunne utnytte den stadig økende mengden av sekvensinformasjon på en best mulig måte, trengs det effektivt genetisk verktøy. Slikt verktøy har manglet for Streptococcus thermophilus, en viktig bakterie for meieriindustrien der den benyttes i produksjonen av yoghurt samt noen harde oster av italiensk og sveitsisk type. Siden flere arter i slekten Streptococcus er kompetente for naturlig genetisk transformasjon antok vi at det er en mulighet for at også S. thermophilus har denne egenskapen. Naturlig transformerbare bakterier tar opp ”nakent” ekstracellulært DNA og inkorporerer det i sitt eget genom ved hjelp av homolog rekombinasjon. Denne mekanismen representerer et ideelt verktøy for genetisk manipulasjon av bakterier. Hovedmålet med arbeidet som er presentert i denne avhandlingen var derfor å undersøke om S. thermophilus er naturlig transformerbar. Dersom dette skulle vise seg å være tilfelle, var neste mål å undersøke anvendeligheten av denne mekanismen som et verktøy for genetisk manipulasjon av S. thermophilus. Tidlig i studiet identifiserte og sekvenserte vi et gen fra S. thermophilus som koder for en homolog til ComX, den alternative sigma faktoren som kontrollerer kompetanseutvikling hos Streptococcus pneumoniae. Siden vi ikke klarte å finne vekstforhold som utløste spontan kompetanseutvikling hos S. thermophilus, valgte vi som en alternativ strategi å overuttrykke comX. Et induserbart tokomponent signaloverføringssystem som regulerer bakteriosinproduksjon hos S. thermophilus ble benyttet til dette formalet. Overuttrykk av comX induserte kompetanse i LMG 18311 stammen, og for første gang ble det vist at minst en stamme av arten S. thermophilus er naturlig transformerbar. Videre undersøkelser viste at transformasjonseffektiviteten vi oppnådde med vårt system var høy nok til at det var mulig å innføre mutasjoner direkte på genomet til S. thermophilus, uten å være avhengig av antibiotikaresistensgener eller andre markører til seleksjon av ønskede mutanter. Transformanter kunne i stedet identifiseres ved hjelp av kolonihybridisering med en spesifikk oligonukleotidprobe. Den store fordelen med denne framgangsmåten er at det bakterielle genomet kan endres på et hvilket som helst sted uten at det samtidig introduseres fremmed DNA. Det genetiske verktøyet som er utviklet i denne avhandlingen har gitt forskere som studerer S. thermophilus nye muligheter som på sikt vil resultere i bedre forståelse av denne bakteriens metabolisme og fysiologi, og kanskje også til utviklingen av nye starterstammer med forbedrede egenskaper.Norges Forskningsrå

Natural Genetic Transformation: a Novel Tool for Efficient Genetic Engineering of the Dairy Bacterium Streptococcus thermophilus

Streptococcus thermophilus is widely used for the manufacture of yoghurt and Swiss or Italian-type cheeses. These products have a market value of approximately $40 billion per year, making S. thermophilus a species that has major economic importance. Even though the fermentation properties of this bacterium have been gradually improved by classical methods, there is great potential for further improvement through genetic engineering. Due to the recent publication of three complete genome sequences, it is now possible to use a rational approach for designing S. thermophilus starter strains with improved properties. Progress in this field, however, is hampered by a lack of genetic tools. Therefore, we developed a system, based on natural transformation, which makes genetic manipulations in S. thermophilus easy, rapid, and highly efficient. The efficiency of this novel tool should make it possible to construct food-grade mutants of S. thermophilus, opening up exciting new possibilities that should benefit consumers as well as the dairy industry

A Hydrophobic Patch in the Competence-Stimulating Peptide, a Pneumococcal Competence Pheromone, Is Essential for Specificity and Biological Activity

Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic α-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the α-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified α-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions