Genome analysis of two virulent Streptococcus thermophilus phages isolated in Argentina

GUGLIELMOTTI, D. M.; DEVEAU, H.; BINETTI, A.G.; REINHEIMER, J. A.; MOINEAU, S.; QUIBERONI, A.

San Miguel de Tucumán

Simposio; III International Symposium on Lactic Acid Bacteria. II Argentinean LAB Net Meeting International; 2009

Centro de Referencias para Lactobacilos (CERELA-CONICET)

Two Streptococcus thermophilus phages (ALQ13.2 and Abc2) were isolated from breakdowns of cheese manufactures in Argentinean dairy plants. Primers previously designed to sequence the genome of the pac-type phages 2972 and 858 were used for direct sequencing of the conserved regions of phage ALQ13.2. Then, new primers were designed to complete the sequencing of both strands. To sequence the genome of phage Abc2, a similar approach was taken except that primers previously designed to sequence the genome of the cos-type phage DT1 were used as starting point. DNA sequence analysis, contig assembly and editing were carried out using Staden Package and BioEdit Sequence Alingment Editor. Open Reading Frame (ORF) Finder graphical analysis tool and Heuristic GenMark were used to define potential coding regions. Protein sequence comparisons were performed using BLASTP 2.2.18 from NCBI. CsCl-purified phage particles were analyzed for structural protein composition by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The bands of interest were excised from the gel, and identified by liquid-chromatography tandem mass spectrometry (LC-MS/MS). According to the complete nucleotide sequence analysis, they have linear double-stranded DNA, of 35,525 bp for pac-type phage ALQ13.2, and 34,882 bp for the cos-grouped fAbc2. Forty-four and forty-eight open reading frames (ORF) were deduced for ALQ13.2 and Abc2, respectively. Comparative genomic analysis exposed they share many similarities with the eight correspondent cos- and pac- phages previously studied. Particularly, a region of Abc2 genome was highly similar to a region of phage 7201, which was unique until now. Proteomic studies for pac- phage ALQ13.2 showed ten structural proteins; three corresponded to major and seven to minor structural proteins. For Abc2, SDS-PAGE analysis revealed seven proteins bands, two related to major structural proteins, as expected for a cos-belonging phage. Similarities with the remaining eight complete sequenced S. thermophilus phages demonstrated that phage diversity is not extended for this species. These data reinforce also the current classification of all S. thermophilus phages on the basis of the DNA packaging mechanism (cos and pac) and structural protein composition, disregarding of whether they derive from different type of dairy samples or they were isolated in very distant locations.