Characterization of the cro-ori region of the Streptococcus thermophilus virulent bacteriophage DT1

Abstract

The virulent cos-type Streptococcus thermophilus phage DT1 was previously isolated from a mozzarella whey sample, and its complete genomic sequence is available. The putative ori of phage DT1 is characterized by three inverted and two direct repeats located in a noncoding region between orf36 and orf37. As the replication ability of the putative ori and flanking genes could not be established, its ability to confer phage resistance was tested. When ori is cloned on a high-copy-number plasmid, it provides protection to S. thermophilus strains against phage infection during milk fermentation. This protection is phage specific and strain dependent. Then, a detailed transcriptional map was established for the region located between the cro-like gene (orf29) and the ori. The results of the Northern blots indicated that the transcription of this region started 5 min after the onset of phage infection. Comparative analysis of the expression of the cro-ori region in the three S. thermophilus cos-type phages DT1, Sfi19 (virulent), and Sfi21 (temperate) reveals significant differences in the number and size of transcripts. The promoter upstream of orf29 was further investigated by primer extension analysis, and its activity was confirmed by a chloramphenicol acetyltransferase assay, which showed that the phage promoter is more efficient than the constitutive bacterial promoter of the S. thermophilus operon encoding the general proteins of the phosphoenolpyruvate:sugar phosphotransferase system. However, the phage promoter is less efficient than the pts promoter in Lactococcus lactis and in Escherichia coli.

FIG. 1.

Schematic illustration of the phage DT1 genome analyzed in this study and its replication in S. thermophilus SMQ-301 infected cells. (A) XbaI restriction map of the phage DT1 genome. The bent arrow marks the position of promoter P1. (B) Agarose gel electrophoresis of DNA from SMQ-301(pNZ123). (C) Agarose gel electrophoresis of DNA from SMQ-301(pFB3). NB, uninfected cells before addition of DT1; NE, uninfected cells at the end of the experiment. Lane numbers correspond to the time (in minutes) after the beginning of the infection. Plasmid pFB3 is indicated by an arrow.

FIG. 2.

Northern blots of total RNA isolated from S. thermophilus SMQ-301 at the indicated time (in minutes) after infection with phage DT1. The letters A to G on the left side represent the probes used (see Table 2 and Fig. 3 for details). The estimated sizes of the observed transcripts are indicated on the right.

FIG. 3.

Comparison of the transcriptional maps of the cro-ori regions of S. thermophilus phages DT1, Sfi19, and Sfi21. The DNA probes used in the Northern blot experiments are indicated by the letters A to G and black lines above the transcripts. The arrows indicate the direction of transcription. The estimated sizes of the observed transcripts are also indicated. Bent arrows designate promoter sequences. Deduced proteins showing between 70% and 80% amino acid identity are in grey, while those sharing more than 90% amino acid identity are in black.

FIG. 4.

(A) Mapping of the transcription start site from the P1 promoter by primer extension (PE). Nucleotide position +1 is indicated. The nucleotide sequence of pFB1 was determined with the same primer (lanes T, C, G, and A). (B) Partial nucleotide sequence of the P1 and PTS promoter regions. Putative −35 and −10 boxes and the putative ribosome binding sites (RBS) are indicated.