New findings on the adaptation of Lactobacillus casei group to cheese environment

Abstract

Cheese represents a very complex environment, where the components initially present in milk are modified by technological processing, which lead to substantial changes in the biochemical properties of the initial matrix, and directs the development of the microbiota. Lactobacillus casei group is a species of technological interest, particularly in Italian long ripened cooked hard cheese, where it becomes a prevalent species of the microbiota involved in the maturation of the curd until the end of ripening. The aim of this thesis was to study the adaptation of Lb. casei group to cheese environment, using a comprehensive approach involving the use of in vitro and in situ model systems, as well as confirmation of the results in real cheese. Two systems potentially involved in adaptation of Lb. casei group to cheese environment, are spxB gene, which encodes for a pyruvate oxidase (POX), and a plasmid-encoded toxin-antitoxin system, involved in plasmid maintenance. POX catalyses the oxidation of pyruvate to acetyl-phosphate, which is then converted to acetate with the production of ATP from an acetate kinase (ACK), potentially representing an alternative metabolic pathway for bacterial growth during cheese manufacturing. spxB gene was found to be widespread in dairy isolates of Lb. casei group, and its sequence heterogeneity provides differentiation among isolates through high-resolution melting technique. Combining the taxonomic potential of spxB gene and high-throughput sequencing allowed to describe the population dynamics of Lb. casei group in ripening cheese up to the sequence-type level. Furthermore, activation of spxB was also measured in response to various oxygen concentrations in an expressly designed miniaturised cheese model, as well as bacterial growth and metabolite production. All the considered approaches have underlined the relevance of spxB gene in adaptation to cheese environment, as well as its suitability to be used to identify the species within Lb. casei group. The toxin antitoxin (TA) system detected in dairy Lb. rhamnosus consists of a plasmid-located two component system, coding for a stable toxin, and an unstable antitoxin, and failure in transmitting the plasmid to newborn cells causes its post-segregational killing. A novel type I toxin-antitoxin system from dairy Lb. rhamnosus is described for the first time, and an in-depth bioinformatics analysis reveals the wide distribution of this system on plasmids harboured by Lb. casei group. Transcriptional analysis showed that TA system is induced in cheese-based media, and the transcript can be detected in cheese as well, suggesting its importance in adaptation to this environment. The role of TA systems is relevant for plasmid stability and maintenance, but also in dormancy and apoptosis of the cell, making it one of the most versatile global regulatory system in bacteria. The projects presented highlight the importance of using a combined approach, integrating the results of experiments performed in model systems and real cheese, to describe the drivers of bacterial adaptation during cheese manufacturing.