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

Abstract

SummaryBacteriophage sk1 is a small isometric-headed lytic phage belonging to the 936 species. It infects Lactococcus lactis, a commonly used dairy starter organism. Nucleotide sequence data analysis indicated that the sk1 genome is 28 451 nucleotides long and contains 54 open reading frames (ORFs) of 30 or more codons, interspersed with three large intergenic regions. The nucleotide sequence of several of the sk1 ORFs demonstrated significant levels of identity to genes (many encoding proteins of unknown function) in other lactococcal phages of both small isometric-headed and prolate-headed morphotype. Based on this identity and predicted peptide structures, sk1 genes for the terminase, major structural protein and DNA polymerase have been putatively identified. Genes encoding holin and lysin were also identified, subcloned into an Escherichia coli expression vector, and their function demonstrated in vivo. The sk1 origin of replication was located by identifying sk1 DNA fragments able to support the maintenance in L. lactis of a plasmid lacking a functional Gram-positive ori. The minimal fragment conferring replication origin function contained a number of direct repeats and 179 codons of ORF47. Although no similarity between phage sk1 and coliphage at the nucleotide or amino acid sequence level was observed, an alignment of the sk1 late region ORFs with the structural and packaging genes revealed a striking correspondence in both ORF length and isoelectric point of the ORF product. It is proposed that this correspondence is indicative of a strong conservation in gene order within these otherwise unrelated isometric-headed phages that can be used to predict the functions of the sk1 gene products.

Sequence analysis of the lactococcal bacteriophage bIL170: insights into structural proteins and HNH endonucleases in dairy phages The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are bIL170, AF009630; bIL120, AY054975; bIL15, AY054976; bIL191, AY054977; bIL77, AY054978.

“…As expected, since they belong to the 936 group that is known to be homogeneous at the DNA level, the two entirely sequenced phages, bIL170 and sk1 (Chandry et al, 1997), share the same overall genetic organization and are highly similar at the nucleotide level (more than 84 % nt identity over 80 % of the shortest genome length). However, regions of high levels of DNA homology are interspersed with regions of lower or no similarity (Fig.…”

“…The c2 group has the same features, whereas the P335 group A.-M. Crutz-Le Coq and others contains mainly temperate phages that are not necessarily closely related at the DNA sequence level (Chopin et al, 2001). In the 936 group, only a few genomic segments (essentially encoding the holin, lysin, major structural protein, structure-specific endonuclease and ori fragment) have been studied experimentally (Bidnenko et al, 1998 ;Chandry et al, 1997 ;Chung et al, 1991 ;Kim & Batt, 1991a). In the c2 and P335 groups, a number of structural proteins have been experimentally characterized as well as endolysins, origins of replication and transcriptional regulators, or the lysogeny module in the case of temperate phages (see Table 2 for selected references).…”

“…Among lactococcal phages of the 936 group, the complete genome sequence of phage sk1 has been determined (Chandry et al, 1997) and DNA portions larger than 5 kb have been sequenced for two other phages, F4-1 and bIL41 (Chung et al, 1991 ;Kim & Batt, 1991b ;Parreira et al, 1996b). To further our knowledge of the genetics and evolution of this group of virulent lactococcal phages, we undertook the sequence analysis of phage bIL170.…”

Temporal transcription of the lactococcal temperate phage TP901-1 and DNA sequence of the early promoter region

“…A different approach is to increase the knowledge of the molecular biology of the attacking phages and through this to design new phage resistance modules, Molecular knowledge of selected phage functions, such as lysis functions, origin of replication, site-specific integration functions and transcriptional regulators has been obtained from several lactococcal bacteriophages since 1989 (Shearman et al, 1989;Hill et al, 1990;Lillehaug & Birkeland, 1993 ;O'Sullivan et al, 1993;Christiansen et al, 1994;van de Guchte et al, 1994a, b ;Arendt et al, 1994;Birkeland, 1994;Boyce et al, 1995a, b;Nauta et al, 1996; O'Sullivan et al, 1996), and these gene cassettes are expected to serve as important genetic tools for the future general engineering of industrial lactococcal starter strains, in addition to the phage resistance aspect. More recently, DNA sequence analysis of entire lactococcal phage genomes has been performed and so far the published reports encompass three virulent phages, the prolateheaded related phages c2 and bIL67, and the small isometric-headed phage skl, and one temperate phage, rlt, also having a small isometric head (Lubbers et al, 1995;Schouler et al, 1994;Chandry et al, 1997;van Sinderen et al, 1996). TP901-1 is a temperate lactococcal phage and like r l t it belongs to the P335 class of small isometric phages (Jarvis et al, 1991;Braun et al, 1989).…”

Viral genomes are part of the phylogenetic tree of life

“…Furthermore, approximately two-thirds of predicted protein-coding genes (~600 open reading frames (ORFs)) in the Mimivirus genome have no functional similarity to any known proteins, suggesting that Mimivirus diverged early in evolution 9 . This finding is by no means exclusive to Mimivirus; approximately 60% of the ORFs of the bacteriophage sk1 genome have no known functional homologues 10 , as is the case with 94% of the white spot syndrome virus genome ORFs 11 .…”