Genome sequencing and functional characterization of Lactobacillus phages J1 and PL1 shed light on the understanding of these two well known viruses

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

Olympia

Congreso; 20th Biennial Evergreen International Phage Meeting; 2013

Evergreen State College

Bacteriophages of lactic acid bacteria are the main cause of fermentation failure in the dairy industry. Phage J1 was originally isolated in 1965 from an abnormal fermentation of Yakult using the strain Lactobacillus casei ?Shirota? as starter. Two years later, phage PL1 was isolated from a strain resistant to J1. Since its isolation, both phages have been well characterized but their genome sequences were not available. We have sequenced and annotated the complete genomes of phages J1 and PL1 using 454 Sequencing and DNA Master program. J1 genome is 40,931bp in length and contains 63 protein-coding genes. The PL1 genome is 38,880 bp in length and contains 59 orfs. The genome organization shared the structure observed in other Lactobacillus phages and can be divided in the following modules: packaging, structural proteins, lysis, integration, genetic switch region and replication. Interestingly, the DNA sequences of these genes are almost identical although PL1 has a 1.9 Kb deletion at the genetic switch module. We have constructed a vector based on the constitutive l-ldh promoter from L. casei BL23 for protein expression in this host. This useful tool allowed functional analysis of several differential gene products located in this module. Even though J1 was originally described as lytic, a gene that codes for a putative integrase (int) of the tyrosine-recombinase family and a phage attachment site (attP) were found indicating that this phage could have a lysogenic life cycle in the right host. To demonstrate functionality of this gene product we have designed an integrative vector carrying the attP-int of J1. We are currently studying if the phage integration occurs at the expected place in our lab model strain Lactobacillus casei BL23. Other remarkable features were revealed from the sequences. As a consequence of ribosomal frameshifting, two head proteins with the same NH2 termini are coded. At the end of orf 6 a slippery (CCCAAAA) sequence was found. Wobbling of the ribosome on that sequence could generate a -1 frameshift that would originate a second protein 86 aminoacids longer (orf 7) than the product of orf 6. We identified these two proteins by SDS-PAGE analysis and mass spectrometry (MS). Data suggests that the shortest form is at least 2.5 times more represented than the longest form. This work reveals new and interesting characteristics of these two widely studied Lactobacillus phages.