Recombination and Natural Competence in Lactobacillus casei

FINA MARTIN, JOAQUINA; PALOMINO, MARÍA MERCEDES; SANCHEZ RIVAS, CARMEN; RUZAL, SANDRA M; ALLIEVI, MARIANA CLAUDIA

Rosario

Congreso; IX Congreso Argentino de Microbiología General-SAMIGE; 2013

Sociedad Argentina de Microbiologia General

Improve the conditions of transformation in Lactobacillus understood as the ability to efficiently internalized nucleic acids has been a goal we reach first by debilitating the cell envelope as a result of the growth in high salt (Palomino et at, 2011). To obtain recombinants between the incoming DNA and genome remains for this species difficult. However this aspect has relevance both scientific (for the construction and analysis of mutants and allocation of functions) as technology (development of new improved strains) reasons which led to investigate this limitation. In a first step we proceeded in the search of natural competence in L. casei, understood as the capacity to take naked DNA from the environment and incorporate it into their genome by homologous recombination. Employing on line tools: SEED viewer(theseed.uchicago.edu/FIG/seedviewer.cgi?page=Home), STRING (string-db.org) y BLAST (ncbi.nlm.nih.gov/BLAST) functions were search for natural competence and recombination in the genome of L. casei BL23 (NC_010999.1). Codification was found for ComX (alternative sigma factor of the RNA polimerase, YP_001986877.1, Gene ID: 6404650), for genes with homology to comE(YP_001987489, Gene ID: 6406153), comG (YP_001987132.1, Gene ID: 6404762),involved in the DNA transport and processing and recombination RecA (recombinase, YP_001988726 GeneID: 6405340). It was found the presence of comX y recA by PCR of cromosomic DNA with specific primers. The presence of competence related functions suggests that L. casei BL23 could be naturally competent and transformable with naked DNA whenever the growth conditions that promote competition can be identify. We checked various conditions that could promote gene expression of messenger RNA of these genes, analyzed by qPCR and compared to early stationary phase growth condition: UV y Mitomicin C, Protoplasts and Starvation. It was observed an increase in the expression in the condition of starvation. The number of natural transformants obtained in that condition was verified. In order to investigate conditions that can induce homologous recombination in this bacterium, we analysed if the entry of DNA damaged by UV could be an alternative for induction, that it would lead to the induction of the SOS response involving the activation of RecA. For this purpose, L. casei BL23 was co-transformed with two plasmids: a replicative plasmid [pNZ273 (CmR)] subject or not to damage by UV that can measure the efficiency of transformation with or without DNA damaged and an integrative plasmid containing part of dltA gene for single cross-over recombination [pRV300+dltA (EmR)]. It was observed a decrease in the number of transformants CmR, in the case of the UV treatment of pNZ273 indicating effective damage to DNA, while the number of recombinants selected was significantly higher implying an increase in recombination events.