induction and characterization of prophages in the genome of a probiotic L.casei strain

DIETERLE MARÍA EUGENIA; FINA MARTIN JOAQUINA; CHARLES BOWMAN; HATFULL GRAHAM; PIURI MARIANA

Zurich

Conferencia; EMBO meeting: Virus of microbes: Structures and function: from molecules to communities; 2014

EMBO

Lactic acid bacteria (LAB) have many applications in food and industrial fermentations, as well as significant probiotic properties. Lactobacillus casei BL23 is a well studied probiotic strain and shows a high similarity with L.casei BD-II that was isolated from a homemade koumiss in China. Due to the risk involved in dairy industry by prophage induction and the generation of new virulent phages, we screened for prophage like elements (PLE) in the recently sequenced model strain of L.casei BL23. Using a bioinformatic approach, we identified the presence of PLEs in the genome of L. casei BL23. We found at least four PLEs where three of them seem to be complete (named PLE1, PLE2 and PLE3) with six identifiable modules: integration, genetic switch, replication, packaging, structural and cell lysis. All of them showed a mosaic nature with homologies to Streptococcus, Lactococcus, Lactobacillus and Listeria phages or strains. Interestingly, we have also found that 30% of the phage J-1 genome (bacteriophage that infects several Lactobacillus casei/paracesei strains) is present in PLE2. By means of a PCR technique we were able to show the presence of the circularized form of the excised complete phages in a subpopulation of cells and obtained the sequence of the phage (attP) and the bacteria (attB) attachment sites. We could also established the hybrids sites (attL and attR) flanking each PLE at the bacterial genome. PLE1 and PLE2 integration complement the 3′ end of a tRNA carrying the anticodon specific for leucine and arginine respectively while PLE3 integrates in an intergenic region. Several protocols were tested for induction. A plateau of the cell growth was observed 7 hours post-induction with 0,1 ug/ml of mitomycin C without a complete clearance. Partially lysed cultures were concentrated and examined by electron microscopy. Complete phage particles were observed but many heads or disassociated tails were also visible. The prophage induced has a typical morphology of the Siphoviridae, with an isometric head approximately 62 nm in diameter and a tail approximately 178 nm in long. It seems to be defective since many particles were dissociated and a sensitive Lactobacillus host could still not be identified. Surprisingly, the restriction enzyme patterns obtained after DNA extraction did not match with any of the PLEs founded suggesting that more than one phage is being induced or recombination events occurred between different prophages and/or with the own bacterial genome. To reveal this, we have sequenced the DNA that comes from the induction using 454 Sequencing. Data analysis showed that with mitomycin C, PLE2 was induced but PLE3 genome was also found at lower levels. We are currently doing real time PCR using L.casei BL23 DNA as a template to find differences at the replication level between PLEs when mitomycin C is added. Mass spectrometry of viral particles is also being carried out to decipher the nature of the virion. Our results indicate the possibility of induction of a prophage in a starter bacteria and do not discard recombination between prophages present in the bacterial genome that could lead to the appearance of new phages with novel properties and host range.