Temperate Streptococcus thermophilus phages expressing superinfection exclusion proteins of the Ltp type

Abstract

Lipoprotein Ltp encoded by temperate Streptococcus thermophilus phage TP-J34 is the prototype of the wide-spread family of host cell surface-exposed lipoproteins involved in superinfection exclusion (sie). When screening for other S. thermophilus phages expressing this type of lipoprotein, three temperate phages-TP-EW, TP-DSM20617, and TP-778-were isolated. In this communication we present the total nucleotide sequences of TP-J34 and TP-778L. For TP-EW, a phage almost identical to TP-J34, besides the ltp gene only the two regions of deviation from TP-J34 DNA were analyzed: the gene encoding the tail protein causing an assembly defect in TP-J34 and the gene encoding the lysin, which in TP-EW contains an intron. For TP-DSM20617 only the sequence of the lysogeny module containing the ltp gene was determined. The region showed high homology to the same region of TP-778. For TP-778 we could show that absence of the attR region resulted in aberrant excision of phage DNA. The amino acid sequence of mature LtpTP-EW was shown to be identical to that of mature LtpTP-J34, whereas the amino acid sequence of mature LtpTP-778 was shown to differ from mature LtpTP-J34 in eight amino acid positions. LtpTP-DSM20617 was shown to differ from LtpTP-778 in just one amino acid position. In contrast to LtpTP-J34, LtpTP-778 did not affect infection of lactococcal phage P008 instead increased activity against phage P001 was noticed.

Keywords: Streptococcus thermophilus; TP-778L; TP-DSM20617; TP-EW; TP-J34; prophage; superinfection exclusion.

Figure 1

Comparison of TP-J34, TP-J34L, and TP-EW genomic DNAs. Agarose gel (A) and corresponding Southern blot (B) of HindIII-cleaved DNAs of TP-J34 (lane 2), TP-J34L (lane 3), and TP-EW (lane 4) hybridized with DIG-labeled 1 kb probe generated from 1.7 kb HindIII fragment of TP-J34L. Lanes 1 and 5: unlabeled and Dig-labeled λ-DNA, respectively. Sizes of restriction fragments of λ-DNA are shown in the right margin. Agarose gel (C) of PCR-products generated from TP-J34 (lane 2) and TP-J34L (lane 3) DNA with primer pair D8+ und D12+. Lane 1: DNA molecular weight marker IV (Roche Diagnostics GmbH, Mannheim, Germany), sizes are indicated in the left margin. Sizes of PCR products are shown in the right margin.

Figure 2

Transmission electron micrographs of S. thermophilus phages TP-778L (A) propagated lytically on the prophage-cured derivative strain J34-2, phage TP-EW (B) and TP-DSM60217 (C) induced by mitomycin C from lysogenic S. thermophilus host strains EW and DSM20167, respectively.

Figure 3

(A) Alignment of gene maps and functional gene regions of TP-J34 and TP-778L. On the genetic maps, genes, and direction of transcription are indicted by arrows (very small genes are shown as boxes, the directions of transcription correspond to adjacent genes). Numbers or gene abbreviations refer to orfs or genes as listed in Tables 3, 4. A scale indicating nucleotide positions is shown above the TP-J34 map. Approximate positions of functional regions (modules) are indicated by horizontal bars below the TP-778L map. Positions of CRISPR spacer sequences are indicated by dots above and below the maps of TP-J34 and TP-778L, respectively. (B) Dot plots of the TP-J34 nucleotide sequence compared to those of other S. thermophilus phages, including TP-778L. The horizontal line of each dot plot represents the 45,605 bp of TP-J34 DNA, whereas the vertical lines represent the numbers of bp for each phage, as indicated within each dot plot. Temperate (t) and virulent (v) phages are indicated.

Figure 4

Comparison of the genetic structure of the TP-J34 DNA region containing the triple repeat sequences R1–R3 with that of TP-J34L and TP-EW, respectively. The bp numbers indicate the first bp of a repeat. “a” and “b” denote the regions with similarities to sequences within the repeats (marked as “a” and “b”). Sequences exclusively found within the three repeats are indicated as “int.” HindIII restriction sites flanking the 4.4 and 1.7 kb fragment of TP-J34 and TP-J34L/TP-EW, respectively are shown. Gene 48 start and stop are marked by solid triangles.

Figure 5

Southern blot with DIG-labeled 1 kb probe of HindIII-cleaved phage and chromosomal DNA of eleven S. thermophilus strains relysogenized with TP-J34. Lane a: TP-J34; lane b: TP-J34L; lane c: J34; lane d: J34-6 (no prophage, negative control); lane M: DIG-labeled, HindIII-cleaved λ DNA. Other lanes (from left to right): J34-RL2; J34-6-RL2a; J34-6-RL2b; J34-6-RL2c; J34-6-RL2d; J34-6-RL2e; J34-6-RL2f; J34-6-RL4a; J34-6-RL4; J34-6-RL4b; J34-6-RL4c. The sizes of the λ DNA bands are indicated in the right margin.

Figure 6

Mechanism of excision of TP-778 prophage from its host's genome to yield phage TP-778L. Prophage and host DNA are shown by black and green line, respectively. Genes are indicated by arrows. Binding sites of primers 4 and 3 (Bruttin et al., are shown). The region of predicted cross-over is indicated by a cross.

Figure 7

Alignment of DNA sequences of S. thermophilus phages TP-EW, S3b (Acc. No. AF148561.1), ST3 (Acc. No. AF148565.1), J1 (Acc. No. AF148566.1), and DT1 (Acc. No. NC_002072) in the region surrounding the group-I-intron, present in all phage DNAs. The splice site is indicated by the vertical arrow. Sequence differences are indicated. Two 6-bp inverted repeats are indicated by horizontal arrows above the DNA sequence. The numbers flanking the TP-EW sequence correspond to the nt positions within the lys gene of this phage.

Figure 8

Alignment of amino acid sequences of different Ltp proteins. The cleavage site between signal sequence and mature protein is indicated. The first Cys of the mature TP-J34 lipoprotein is marked as +1. The two repeat regions are underlined. Amino acids identical to those of TP-J34 are indicated by “-.”