Characterization of Streptococcus thermophilus host range phage mutants

Abstract

To investigate phage-host interactions in Streptococcus thermophilus, a phage-resistant derivative (SMQ-301R) was obtained by challenging a Tn917 library of phage-sensitive strain S. thermophilus SMQ-301 with virulent phage DT1. Mutants of phages DT1 and MD2 capable of infecting SMQ-301 and SMQ-301R were isolated at a frequency of 10(-6). Four host range phage mutants were analyzed further and compared to the two wild-type phages. Altogether, three genes (orf15, orf17, and orf18) contained point mutations leading to amino acid substitutions and were responsible for the expanded host range. These three proteins were also identified in both phages by N-terminal sequencing and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The results suggest that at least three phage structural proteins may be involved in phage-host interactions in S. thermophilus.

FIG. 1.

(A) Diagrams of ORF15, ORF17, and ORF18 of phages DT1 and MD2. Putative conserved domains are displayed in white. The FLY motif of ORF17 is also identified. Collagen-like repeats are numbered and are represented by small dark-gray regions within ORF18. VR1, VR2, and VR3 indicate variable regions 1, 2, and 3. (B) Alignment of ORF15 (aa 1236 to 1265) where a mutation occurred in DT1.R2. (C) Alignment of ORF15 (aa 1405 to 1434) where a mutation occurred in DT1.R1. (D) Alignment of ORF17 (aa 313 to 352), including the FLY motif, where mutations were present in all the host range phage mutants. Amino acid substitutions are in bold. Numbers represent the positions on the amino acid sequence of DT1.

FIG. 2.

Protein profiles of phages DT1 and MD2 on Coomassie blue-stained SDS-PAGE gels. MW, molecular weight markers. The asterisk indicates that the protein functions are putative except for ORF18 of DT1 (protein 2).

FIG. 3.

Amino acid sequences of the ORF15 proteins of phages DT1 and MD2. The light gray region represents the protein identified in the phage structure by MALDI-TOF or LC-MS-MS. Underlined amino acid sequences, peptides identified by mass spectrometry. Variable region, the region that differs from the homologue proteins of other cos-type S. thermophilus phages. Dark-gray regions, CHAP and SLT domains. Boldfaced amino acids in DT1 indicate the amino acids that were mutated in phages DT1.R1 and DT1.R2.