Table 1.
Strains used in this study.
Fig 1.
Identification of the promoter for the mef(E)/mel(msr(D)) operon.
a) Identification of the operon transcriptional start site in the chromatogram of the nucleotide sequence of 5’ RACE PCR products. Poly-thymine runs indicate the 5’ end of a transcript molecule. Numbered arrows indicate the predicted 5’ termini. b) Comparison of the predicted mef(E)/mel promoter sequence with putative promoters of other mef-containing elements mef(A) and mef(I) found in S. pneumoniae. The extended-10 and the-35 promoter sequences are underlined and labeled. The deleted sequence (Δ-41-(-5) of mutations in the native mef(E) locus (XB30) or the mef(E)-lacZ reporter locus (XB05) is indicated; ‘+1’ indicates the transcriptional start site. Shading indicates dissimilar nucleotides compared to the mef(E) promoter region. c) The top panel illustrates the genetic organization of mef-containing elements in S. pneumoniae. The homology of the 3.2 “mef cassette” was extensive from RBS1 through mel(msr(D)) as indicated by shading. Blue arrows, macrolide resistance genes; red arrow, chloramphenicol resistance gene catQ; green open arrows, transposon-related genes; open arrows, unrelated genes. The bottom panel illustrates the series of mutations generated in the 5’ regulatory region of the native mef(E)/mel locus of GA17457 and the reporter locus, a mef(E)-lacZ fusion inserted into bgaA in XZ7042. The annotated red line represents the 327 base 5’ mef(E)/mel regulatory mRNA region with the mef(E)/mel promoter shown as a bent arrow. Each pair of converging, colored solid arrows represents one of four pairs of inverted repeats. Black arrow, mef(E)L. closed arrow heads, distal 17 base inverted repeats (dIR1 and dIR2);open arrow heads indicate 12 base direct repeats (DRs). The descriptive name of each mutation and the resulting native-locus and reporter mutant designations are indicated in the right-hand columns; nc, not created.
Fig 2.
β-galactosidase activity of promoter mutations in the mef(E)-lacZ reporter locus.
XZ7042 (squares) is the reporter strain generated by insertion of a mef(E)-lacZ fusion into the pneumococcal β-galactosidase gene bgaA; XZ7049 (diamonds), promoterless lacZ strain; XB05 (circles), strain containing the mef(E) promoter region deletion (Δ-41-(-5)) in the mef(E)-lacZ fusion in the bgaA locus. Dashed lines and open symbols, non-inducing conditions; solid lines and closed symbols, inducing conditions (treated with erythromycin at a concentration equal to 10-1 erythromycin MIC). Bars represent the standard error of the mean (SEM) of experiments performed in duplicate.
Table 2.
Influence of regulatory mutations on resistance to erythromycin and expression of mef(E).
Fig 3.
Structure of the mef(E)/mel 5′ attenuator structure(transcriptionally inactive).
The structure of the 327 nucleotide 5′ mRNA region predicted by RNAFold and visualized with Visualization Applet for RNA (VARNA) [Version 3.8; [43]]. Inverted repeats are shaded with colors consistent with Fig. 1C. The amino acid sequence of the leader peptide Mef(E)L is indicated. The minimum free energy values from each stem-loop predicted by RNAFold analyses of the nucleotides involved in each duplex. Red arrows direct repeats. Dashed arrows, distal inverted repeats; RBS1, mef(E)L ribosomal binding site; RBS2 mef(E) ribosomal binding site.
Table 3.
Putative leader peptide sequences of inducible macrolide resistance genes.
Fig 4.
Structure of the mef(E)/mel 5′ anti-attenuator structure (transcriptionally active).
Structure generated by RNAFold of the 249 bases remaining after removing of the 64 nucleotides from the 5′ terminus predicted to be unavailable for base paring due to macrolide-induced ribosomal stalling. Annotations are the same as stated for Fig. 1C.
Fig 5.
The sequence and predicted structure of the 5’ end of mef(E)/mel transcripts with the Δ+54–92 and Δ+63–80 mutations. Nucleotides are numbered relative to the mef(E)/mel transcriptional start (+1). The inverted repeats are annotated as described for Fig. 3. A single asterisk signifies a stop codon of the full-length mef(E)L. Double asterisks indicate the stop codon generates by the Δ+54–92 mutation. The dashed arrow indicates the location of the first unit of the distal inverted repeats (dIR1).
Fig 6.
Stem-loop R3/R4 is required for mef(E)/mel expression.
The predicted secondary structures of the Δ+162–189 mef(E)/mel regulatory region in (a) non-inducing and (b) inducing conditions. Disruption of R3/R4 by mutation was predicted to not disrupt the R5/R6 transcriptional terminator in inducing or non-inducing conditions, indicating that mutants with the Δ+162–189 deletion were uninducible. Annotations are consistent with those described in the Fig. 1C legend. Inducing conditions include growth with exposure to erythromycin at a concentration equal to 10-1 the erythromycin MIC.
Fig 7.
Visualization of mef(E) attenuator by RNA-Seq.
Whole transcriptome analysis by RNA-Seq of mRNA from the wild type strain GA17457, non- induced or exposed to spiramycin, the antimicrobial peptide, LL-37, or erythromycin. The number of sequence reads correlated to the mef(E)/mel transcript is shown on the graph. Dashed lines below each sample indicate transcripts predicted by Integrated Genome Viewer (IGV). The mef(E) and mel open reading frames are shown below.
Fig 8.
Comparison of the Rho-independent terminator in the Mega element in S. pneumoniae with predicted terminators from mobile elements found in other Gram-positive bacteria species.
The nucleotide sequence of the stem-loop portion (not including the poly-uracil tract) of the predicted Rho-independent transcriptional terminators are aligned and single nucleotide polymorphisms are shaded gray. The predicted free energy (ΔG) for each terminator is indicated The Rho-independent terminator structure from the Mega element is shown below in dot-bracket notation. Abbr., Mega (macrolide efflux genetic assembly), S. pneumoniae GA17457; Tn2009, S. pneumoniae GA08825 Tn916-like element (Genbank accession no. AILK01000006); Tn2010, S. pneumoniae GA47628 Tn916-like element (accession no. AILC01000010); 5612IQ, S. pneumoniae 5612IQ complex (accession no. AJ971089); Sdys_eq, S. dysgalactiae subspecies equisimilis G51 mef(A) element (accession no. AM168138); Tn1207.1, S. pneumoniae England14–1 phage-related element (accession no. AILI01000002); Tn1207.3, S. pyogenes 2812A phage-related element (accession no. AY657002); CkluΦ, Clostridium kluyveri DSM 555 phage-related element (accession no. CP000673); ΦMGAS10394, S. pyogenes MGAS10394 phage-related element (access no. CP000003); Cper_IFI, C. perfringens IFI mef(A) element (access no. EU553549); S. pyogenes MB56Spyo045 Φm46.1-like (access no. JF501521); ΦM46.1, S. pyogenes Φm46.1 (access no. FM864213).