Analysis of the luxS transcript in S. pyogenes . (A) Temporal analysis of luxS expression. Northern blot analysis of total RNA isolated from RDN29 (wild-type M1 strain) and EC478 (isogenic luxS-deficient mutant) cultures at (1) lag, (2) early-logarithmic, (3) mid-logarithmic, (4) mid-late-logarithmic, (5) late-logarithmic, (6) early-stationary and (7) stationary (O/N) phase of growth. Blots were hybridized with probes specific to luxS and rRNA16S (acting as loading control). The estimated sizes of transcripts are indicated. The blots shown are representative of four independent experiments. Identical luxS expression profiles were observed in RDN02 (wild-type M19 strain) and RDN306 (isogenic luxS-deficient mutant). (B) Determination of the transcriptional start site of luxS. Left: Primer extension analysis using total RNA isolated from RDN29 wild-type cultures at lag (lag) and early-logarithmic (el) phase and the γ-32P-radiolabelled reverse primer oliRN250. As a reference ladder, a DNA sequencing reaction using plasmid pEC83 as template and reverse primer oliRN250 is shown (lanes TGCA). The complementary base (C*) of the transcriptional start site (G), located 20 nucleotides upstream of the translational start codon (ATG), is indicated. The picture shown is representative of three independent experiments. Right: Sequence of the 5' part of the luxS locus. The putative -35 and -10 promoter regions and +1 transcriptional start site are represented in bold. The putative ribosomal binding site (RBS) and translational start codon (ATG) are underlined. The arrow indicates the direction of primer oliRN250, which was used for primer extension. An inverted repeat sequence located 97 bp downstream of the translation stop codon could serve as a rho-independent transcriptional terminator (not shown).