Theoretical outcomes and applications of taxonomic studies on the genera Enterococcus and Lactobacillus

The present thesis, titled “Theoretical outcomes and applications of taxonomic studies on the genera Enterococcus and Lactobacillus”, concerns taxonomy of bacteria. This scientific discipline aims at studying all living organisms with the final goal of characterizing and arranging them in an orderly manner on the basis of their similarity and relationships. The classification scheme based on the real genealogical relationships of living organisms is considered the most stable. However, as a consequence of their simple structure, bacteria investigation has been strongly influenced by scientific and technical advances. Microbes certainly play a crucial role in several scientific area, such as medicine, genetics, ecology and they are largely employed in food production industry, pharmacology and environmental science. Thus, it is necessary to study existing biodiversity in order to use microorganisms for human activities, to discover new biotechnological applications and to deep the risks linked to the presence of unwanted or even dangerous bacteria. Moreover, taxonomy assigns a unique name to each described organism, in order to provide information exchange by using a common terminology and to provide tools or methods to improve the correct identification of new isolates. During this project different taxonomic aspects have been faced concerning some species belonging to the genera Lactobacillus ed Enterococcus, which include relevant bacteria for ecological and applicative purpose, in particular, for fermented food production where they are largely employed as natural component or as starter. Some species are marked as common inhabitant of the gastro-intestinal tract of human and animals and they can modulate the microflora composition, nutrient assimilation as well as the general health status of the host. Due to their presence in so different environments and their application in several human area, their study appear particularly interesting and necessary. The present project has been carried out by using different approaches based on the aspect to be evaluated, the organism and the taxonomic rank under investigation (i.e. the comparison of distinct or strictly correlated species and the analysis of strains belonging to the same taxonomic unit). In the first part of this dissertation (chapter 2) is presented an evaluation of the phylogenetic relationships among enterococci by using a comparative sequence analysis of six molecular markers (16S rDNA, recA, hsp60, pheS, atpA, rpoA) and synteny of the region flanking recA gene. These approaches allowed to underline the presence of distinct groups of closely related species and to hypothesize their speciation order. The obtained gene sequence data of three target genes, 16S rRNA, recA e hsp60, were analysed in order to design diagnostic probes in correspondence of species-specific polymorphisms. The designed probes, employed to develop a prototype of spotted DNA-microarray, permitted to distinguish, on the basis of the obtained hybridization profiles, the Enterococcus species which are more frequently detected in clinical samples and in several food production chains, e.g. E. durans and E. hirae, usually hardly differentiated by the use of other molecular and phenotypic methods. In the second part (chapters 3 and 4) other two aspects, involving closely related species (L. ingluviei e L. thermotolerans, L. vaccinostercus e L. durianis) have been faced. These species showed highly conserved molecular markers and similar phenotypic properties. Thus, their degree of similarity has been determined by DNA-DNA hybridization, that is the unique valid method to circumscribe a bacterial species. Obtained results permitted to evidence that these closely related taxa belong to a single species, and they were wrongly classified with different names. As a consequence of these evidences, the classification scheme of Lactobacillus genus and the nomenclature of these organisms were corrected, in order to allow in the future a more clear and unambiguous identification of natural isolates. In the third part of the thesis (chapters 5 and 6) it is reported an exploration of microbial biodiversity at inter-specific level, in particular of two relevant species largely employed in several food production processes, i.e. L. plantarum and L. delbrueckii. Concerning the L. plantarum species, the gene-content of strains of different isolation origin was evaluated by using commercial DNA-microarray representing the complete genome of WCFS1 strain, sequenced in the 2003. Hybridization experiments highlighted an consistent genetic heterogeneity which seems correlatine with the phenotypic profiles of the considered strains. A further evaluation of these observations, by the application of powerful bioinformatic tools, will permit to match specific genetic determinants and physiological properties in a functional genomic perspective. In chapter 6 the presence of two distinct metabolic pathways, responsible for lactose transport and degradation in L. delbrueckii species, is reported for the first time: one involves a permease and the other a phosphoenol-pyruvate dependent system. The presence/absence and transcription regulation of the first or of the second metabolic pathway seemed linked to ecology, technological properties and evolution of the four subspecies, L. delbrueckii subsp. lactis, bulgaricus, delbrueckii and indicus. The presented observations represent an original contribution to appraise the flexibility of this bacterial species and functionality of single isolates made them able to survive in different habits (milk, vegetable) and to be employed in several food industry processes (e.g. yogurt or cheese production). In conclusion, the reported taxonomic studies have contributed to improve the classification scheme of the investigated bacteria and removing some ambiguous aspects of the nomenclature system. Moreover, it is highlight the applications of basic research studies such as a deep evaluation of functional traits and criteria, helpful to the selection of organism of technological relevance. Finally, the observed heterogeneity furnish a more clear view of genetic flexibility which force these organisms to rapidly evolve and to easily adapt to the several environments.