Sequencing and analysis of the prolate-headed lactococcal bacteriophage c2 genome and identification of the structural genes

Abstract

The 22,163-bp genome of the lactococcal prolate-headed phage c2 was sequenced. Thirty-nine open reading frames (ORFs), early and late promoters, and a putative transcription terminator were identified. Twenty-two ORFs were in the early gene region, and 17 were in the late gene region. Putative genes for a DNA polymerase, a recombination protein, a sigma factor protein, a transcription regulatory protein, holin proteins, and a terminase were identified. Transcription of the early and late genes proceeded divergently from a noncoding 611-bp region. A 521-bp fragment contained within the 611-bp intergenic region could act as an origin of replication in Lactococcus lactis. Three major structural proteins, with sizes of 175, 90, and 29 kDa, and eight minor proteins, with sizes of 143, 82, 66, 60, 44, 42, 32, and 28 kDa, were identified. Several of these proteins appeared to be posttranslationally modified by proteolytic cleavage. The 175-and 90-kDa proteins were identified as the major phage head proteins, and the 29-and 60-kDa proteins were identified as the major tail protein and (possibly) the tail adsorption protein, respectively. The head proteins appeared to be covalently linked multimers of the same 30-kDa gene product. Phage c2 and prolate-headed lactococcal phage bIL67 (C. Schouler, S. D. Ehrlich, and M.-C. Chopin, Microbiology 140:3061-3069, 1994) shared 80% nucleotide sequence identity. However, several DNA deletions or insertions which corresponded to the loss or acquisition of specific ORFs, respectively, were noted. The identification of direct nucleotide repeats flanking these sequences indicated that recombination may be important in the evolution of these phages. Several poorly conserved ORFs and a poorly conserved module containing several structural genes that might be involved in phagespecific properties, such as host range determination, were identified.

Typing of bacteriophages by randomly amplified polymorphic DNA (RAPD)-PCR to assess genetic diversity

“…The two phages infecting S. aureus FH5 and vB_SauS-phiIPLA35 (F35) belonged to morphotype B1 and morphotype B2, respectively, and their complete genome sequence was available (García et al, 2007(García et al, , 2009). Finally, the lytic L. lactis phage FC2 belonging to the morphotype B2 (Lubbers et al, 1995) was chosen as representative of phages infecting a different genus within Gram-positive bacteria.…”

Analysis of the DNA sequence, gene expression, origin of replication and modular structure of the Lactococcus lactis lytic bacteriophage sk1

“…The sequence of sk1 ORF50 is 93% identical to the homologous ORF from bIL66, but there are no homologues of sk1 ORF48 and ORF49 in bIL66. Comparison of the genomes of the prolate-headed L. lactis phage c2 and bIL67 has shown that deletion/insertion of discrete ORFs is associated with the presence of direct repeats flanking the deleted DNA (Lubbers et al, 1995). No direct repeats were observed in the sk1 regions flanking ORF48 and ORF49, suggesting that a similar mechanism of deletion/insertion had not occurred in sk1.…”

“…sk1 ORF53 shares homology with an ORF in the prolate-headed phages c2 and bIL67 (Table 1). Lubbers et al (1995) suggested that this ORF could be the phage holin, but the sk1 holin is encoded by ORF19 in the late region of the phage (see below).…”

“…In recent years, extensive nucleotide sequence data have been determined for a number of lactococcal phages. For prolate-headed lytic phages of the c2 species, the complete sequences of the c2 (22 163 bp) and bIL67 (22 195 bp) genomes have been reported (Schouler et al, 1994;Lubbers et al, 1995). The genomes contain 39 and 37 ORFs respectively, and share 80% nucleotide sequence identity, which is interrupted at several locations by deletions or insertions that cause the loss or acquisition of specific ORFs.…”

“…Functions were proposed for 16 ORFs in c2 and nine ORFs in bIL67. A comparison of the genome sequences allowed mechanisms of lactococcal phage evolution to be proposed (Lubbers et al, 1995). Transcription analyses of c2-infected L. lactis cells revealed two phases of phage DNA transcription, early and late, with 22 of the 39 ORFs being expressed in the early phase (Beresford et al, 1993).…”