Sequence analysis and molecular characterization of the Lactococcus lactis temperate bacteriophage BK5-T

Abstract

The Lactococcus lactis temperate bacteriophage BK5-T is one of twelve type phages that define L. lactis phage species. This paper describes the nucleotide sequence and analysis of a 21-kbp region of the BK5-T genome and completes the nucleotide sequence of the genome of this phage. The 40,003-nucleotide linear genome encodes 63 open reading frames. Sequence runoff experiments showed that the cohesive ends of the BK5-T genome contained a 12-bp 3' single-stranded overhang with the sequence 5'-CACACACATAGG-3'. Two major BK5-T structural proteins, of approximately 30 and 20 kDa, were identified, and N-terminal sequence analysis determined that they were encoded by orf7 and orf12, respectively. A 169-bp fragment containing a 37-bp direct repeat and several smaller repeat sequences conferred resistance to BK5-T infection when introduced in trans to the host cell and is likely a part of the BK5-T origin of replication (ori).

FIG. 1

Genetic map of the BK5-T genome. The map is linearized at the cohesive ends and arbitrarily split at the phage attP site located between orf31 and orf32. The grey-boxed areas indicate ORFs previously determined by Boyce et al. (8). The horizontal arrows indicate ORFs and their orientation while the numbers above the arrows indicate ORF designation. Open-headed right-angled arrows indicate positions of putative promoter sequences and their direction of transcription initiation and open-headed vertical arrows indicate the positions of palindromic sequences that may be involved in termination of transcription. Bracketed regions below the map indicate the ORFs associated with the functional modules as described in the text. Putative functions assigned to the BK5-T ORFs are described above the relevant ORFs.

FIG. 2

Comparison of the gene location near the immunity region of L. lactis phages. The relative locations of the ORFs in BK5-T (7), rlt (43), and TP901-1 (25) are indicated. Horizontal arrows indicate individual ORFs, with the ORF name shown within the arrows and the direction of the arrowhead indicating its orientation. The global amino acid percentage of identity between ORFs is shown between the broken lines. The horizontal hatched area illustrates the discordant genetic arrangement of BK5-T orf36 with the rlt and TP901-1 homologues orf8 and orf9, respectively.

FIG. 3

(A) Comparison of the ORF49, ORF269, and ORF235 amino acid sequences from L. lactis phages BK5-T, φ31.1, and ul36.1, respectively. The asterisks indicate identical amino acid residues and the periods indicate residues conserved across all three sequences. Amino acid residues within the putative helix-turn-helix DNA binding motif and the longest direct repeat sequence are indicated by the shaded regions and in bold, respectively. The positions of the four repeat sequences (and their designations) found in all three ORFs are indicated by the horizontal lines with matching arrowheads. (B) The nucleotide sequences of the repeat sequences (DR1 to DR3 and IR1) in BK5-T orf49 are shown in the top strand. The sequence below represents the homologous region from orf235 and orf269 from L. lactis phages ul36.1 and φ31.1, respectively, which are identical. The colons indicate identical nucleotides between BK5-T orf49 and the other L. lactis phage sequences. The nucleotide positions of the four repeat sequences (DR1 to DR3 and IR1) in BK5-T are indicated by the corresponding arrows underneath the repeat label.

FIG. 4

SDS-PAGE analysis of the BK5-T structural proteins. The N-terminal amino acid sequence of the marked proteins is indicated, together with the position of the N-terminal sequence within the corresponding BK5-T ORFs and their proposed functions. The size and location of the molecular mass markers used (Benchmark ladder; Life Technologies Inc., Rockville, Md.) are given in the left margin.

FIG. 5

Delineation of the putative BK5-T origin of replication. The thick solid bar represents the internal orf49 sequence generated by PCR that contained the putative ori. Horizontal arrows shown underneath the bar indicate the positions of the four repeat sequences (DR1 to DR3 and IR1). The plasmid designations of the clones containing deletion fragments are listed on the left and the EOP values (averaged from at least four different experiments) are given on the right. The horizontal thin lines indicate the DNA fragments cloned in the plasmid constructs. The plasmids were introduced into lactococcal host strain H2 (Table 1).

FIG. 6

The 130-bp intergenic cosB sequence. The cosN sequence is shown in bold. Underlined residues indicate runs of at least four identical bases. The C₃TC₅ and G₇ sequences are indicated in bold italics.