SEQUENCING AND COMPARATIVE ANALYSIS OF J-1 AND PL-1 PHAGE GENOMES: LESSONS FROM TAIL COMPONENTS

MARIA EUGENIA DIETERLE; CARLOS BATTHYANY; ESTEBAN LANZAROTTI; GRAHAM HATFULL; MARIANA PIURI

San Miguel de Tucumán

Simposio; IV Simposio Internacional de Bacterias Lácticas (SIBAL); 2013

Bacteriophages infecting lactic acid bacteria negatively impact on dairy fermentation processes with severe economic implications. Phage J-1 was originally isolated in 1965 from an abnormal fermentation of Yakult using the strain Lactobacillus casei ?Shirota? as starter. Two years later, phage PL-1 was isolated from a strain resistant to J-1. We have sequenced and annotated the complete genomes of both phages. The J-1 genome (KC171646) is 40.9 kb in length and contains 63 protein-coding genes. The PL-1 genome (KC171647) is 38.8 kb and contains 59 orfs. Interestingly, the DNA sequences of these genomes are almost identical. Compared to J-1, PL-1 has a deletion of 1,9 kb comprising 4 gene products that belong to the genetic switch region. The other most remarkable difference between J-1 and PL-1 is found in orf 16 and 17 that have been annotated as tail and host specificity proteins, respectively. Both proteins were identified by SDS-PAGE and detected by mass spectrometry (MS) analysis and unique peptides for each protein were distinguished. The N-term of gp16 is identical between both phages, after that, a region with no homology between each other is found, finally at the C-term both gene products are very similar. These conserved and variable regions were identified among other tail components of Lactobacillus phages. Structure comparison showed that the N-term domains of these proteins are related to baseplate distal tail proteins (Dit) of Lactoccocus phages and Bacillus phage SPP1. A model of the N-term domain of gp16 was built based on the crystals structures of ORF 46 of Tp901-1 and gp 19.1 of SPP1. Two differential regions corresponding to the N and C-term domains of SPP1 Dit could be superimposed with exception of the variable region between J-1 and PL-1, suggesting that gp16 could act as a hub where the tail-tube and the baseplate anchor. No structural homology with crystalized proteins could be detected for the C-term.Orf 17 of J-1 and PL-1 have an identity of 98%. By HHPred, the first 400 aminoacids showed homology to gp44 of Myoviriodae phage Mu. Downstream, four repetitions of collagen like repeats were present. More than 85% homology was found among orf 17 and other tail proteins of Lactobacillus phages Lc-Nu, A2 and Lrm1, however the number of collagen like repeats differs and variable regions were found. Since PL-1 was isolated from an abnormal fermentation process using a J-1 resistant strain, both proteins gp16 and gp17 are probably involved in determination of the host range. Previous experimental studies showed that these phages bind to a saccharide component. In order to show their involvement in host recognition, adsorption assays were performed in three different Lactobacillus casei/paracasei strains that are infected by both phages. No differences were found in L.casei BL23 strain however adsorption efficiencies varied in the analyzed L. paracasei subsp. paracasei 27092 and L. casei 27139 strains.