Characterization of prophage like elements in the genome of Lactobacillus casei BL23, induction and genome sequencing

MARIA EUGENIA DIETERLE; CHARLES BOWMAN; DEBORAH JACOBS-SERA; DANIEL RUSSELL; GRAHAM F. HATFULL; MARIANA PIURI

Mar del Plata

Congreso; VIII Congreso de Microbiología General; 2012

SAMIGE

Using a bioinformatic approach, we identified the presence of prophage like elements (PLE) in the genome of L.casei BL23. We found at least four PLEs where three of them seem to be complete with six identifiable modules: integration, genetic switch, replication, packaging, structural and cell lysis. L.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, we evaluate these PLEs more deeply. 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 found 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. 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 recently sequenced the DNA that comes from the induction using 454 Sequencing. A first analysis showed that with mitomycin C PLE2 was induced but also parts of the PLE3 genome were found. Our results indicate the possibility of induction of a prophage in a starter bacteria and do not discard recombination between prophage sequences present in the bacterial genome that could lead to the appearance of new phages with novel properties and host range.