Characterization of Streptococcus thermophilus Host Range Phage Mutants

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

Effects of mild-overexpression of Fst-Sm/srSm type I TA system on <i>S. mutans</i> persister formation.

<p>Oxacillin-treated (<b>A</b>), cefotaxime-treated (<b>B</b>), and vancomycin-treated (<b>C</b>) cells were removed at the indicated time points, serially diluted, spot plated onto THYE agar plates, and the number of CFU per ml was determined from plate counts. The curves presented are the averages and standard deviations of results from three independent cultures.</p

Bacterial strains and plasmids used in this study.

a<p>Em^r, erythromycin resistance; Cm^r, chloramphenicol resistance; Km^r, kanamycin resistance.</p

Characterization of the Fst-Sm/srSm TA system in <i>E. coli</i>.

<p>Cells of LMG194 containing pSK1 (Fst-Sm), pSK2 (Fst-Sm/srSm), and pSK8 (NT-Fst) were grown to mid-log phase, at which time arabinose (induced) and glucose (uninduced control) were added. After induction, appropriate dilutions were plated on LB agar for determination of the number of CFU per ml. The curves presented are the averages and standard deviations of results from three independent cultures.</p

<i>S. mutans</i> toxicity assay based on natural competence.

a<p>The transformation efficiency was expressed as the percentage of chloramphenicol-resistant transformants divided by the total number of recipient cells. All experiments were performed in triplicate from three independent experiments. Statistical significance: * WT(pSK9) vs. WT(pIB166); ^¶ΔIGR176(pSK9) vs. ΔIGR176(pIB166).</p

Primers used in this study.

a<p>Restriction sites are underlined.</p>b<p>Modified residues are shown in bold and underlined.</p

RNA half-life determination.

<p>Stability of <i>fst-Sm</i> mRNA (<b>A</b>) and <i>srSm</i> RNA (<b>B</b>) by Northern blot analysis. Total RNA was extracted from WT mid-log cells at the indicated times after addition of 300 µg/ml rifampicin. Time points of sampling are indicated above each lane. Biotin-labeled DNA probes were used for RNA detection. The probing for 5S RNA confirmed equal loading. Control RNA extraction represents total RNA extracted from cells cultivated without rifampicin at time-point 150-min (<i>fst</i>-<i>Sm</i> mRNA detection) and 60-min (<i>srSm</i> RNA detection). Blots shown represent results from three experiments.</p

Analysis of <i>S. mutans</i> IGR176 region.

<p>(<b>A</b>) Schematic representation of the location of the <i>fst-Sm</i>/<i>srSm</i> locus on the <i>S. mutans</i> chromosome. Arrows indicate the direction of transcription. The <i>fst-Sm</i> and <i>srSm</i> promoter sequences are indicated by P<i>_fst-Sm</i> and P<i>_srSm</i>, respectively. A predicted stem-loop bidirectional terminator is indicated between <i>srSm</i> and <i>fst-Sm</i>. Shown at the bottom are the boundaries of the intergenic region IGR176. (<b>B</b>) Nucleotide and amino acid sequences of the <i>S. mutans fst-Sm</i>/<i>srSm</i> type I TA locus located in the intergenic region IGR176 (from 211452 to 211769) of UA159 genome. The conserved APUU(A/V)GUU motif present in Fst-Sm peptide is boxed. Putative promoter sites of <i>fst-Sm</i> toxin (–35, –10) and <i>srSm</i> antitoxin (–10), ribosome binding site (RBS) of Fst-Sm toxin, and a factor-independent bidirectional terminator (double underlined) are indicated. The transcriptional start site (+1) of <i>fst-Sm</i> and <i>srSm</i> identified by 5′ RACE-PCR are indicated below the sequence. The regions encoding the DRI and DRII repeats are boxed. The primers CMT-497 and CMT-498 used in the RT-PCR experiments are underlined. (<b>C</b>) Proposed RNA:RNA interactions (in shaded regions) between <i>fst-Sm</i> mRNA and <i>srSm</i> RNA.</p