The <i>ospC</i> operator and mutagenesis strategy.

<p>(A) <i>ospC</i> operator mutations are linked to the kanamycin resistance cassette (<i>flgBp-aphI</i>). The sequence upstream of the <i>ospC</i> gene in <i>B. burgdorferi</i> strain 297 is shown (WT) with the dIR (solid arrows) and the overlapping pIR (dashed arrows). The nucleotides that have been changed are marked in bold. The strain nomenclature is as follows: dIR⁺ has the nucleotide sequence changed but the complementarity of the inverted repeats maintained, and dIR⁻ has the distal inverted repeat disrupted but the complementarity of the proximal IR intact. (B) PCR of genomic DNA from <i>ospC</i> operator mutants (lane 1, WT with <i>flgBp</i>-<i>aphI</i> cassette; lane 2, dIR⁺; lane 3, dIR⁻; and lane 4, no template control) using primers primers kanR 488R (a) and ospC D1572R+AgeI (b) to determine the orientation of the <i>flgBp</i>-<i>aphI</i> antibiotic resistance cassette.</p

Oligonucleotides used in this study^a.

a<p>Synthetic restriction sites are underlined.</p

<i>ospC</i> expression induced by relaxation of supercoiling is dependent on the dIR of the operator.

<p>qRT-PCR analyses of <i>flaB</i> (gray bars) and <i>ospC</i> (black bars) mRNA levels from strains grown to late log phase at 23°C in 10 ng ml⁻¹ coumermycin A₁ (Cou) or in DMSO as a solvent control. Values represent the mean and error bars the SE of the mean from three independent experiments. * = <i>P</i><0.05 by an unpaired <i>t</i>-test.</p

The role of the dIR in OspC synthesis mediated by relaxation of DNA supercoiling.

<p>Immunoblot analysis of whole-cell lysates from strains grown to late log phase at 23°C in 10 ng ml⁻¹ coumermycin A₁ (Cou) (+) or in DMSO as a solvent control (−). Membranes were probed with antibodies against OspC (upper panel) or FlaB (lower panel).</p

The dIR is required for OspC synthesis regulated by temperature.

<p>(A) Immunoblot analysis of whole-cell lysates from strains grown at 23°C and then temperature shifted to 34°C and grown to late log phase. The wild-type parental strain (297 WT) and the strain with a wild-type <i>ospC</i> operator linked to the antibiotic resistance cassette (WT) are controls. (B) Immunoblot analysis of whole-cell lysates from strains grown at 34°C and then temperature shifted to 23°C and grown to late log phase. Membranes were probed with antibodies against OspC (upper panels) or FlaB (lower panels).</p

Table_3_The Stringent Response-Regulated sRNA Transcriptome of Borrelia burgdorferi.XLSX

<p>The Lyme disease spirochete Borrelia (Borreliella) burgdorferi must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by Rel_Bbu globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that Rel_Bbu regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in B. burgdorferi. This is the first sRNA regulatory mechanism identified in any spirochete. Threefold more sRNAs were Rel_Bbu-upregulated than downregulated during nutrient stress and included antisense, intergenic and 5′ untranslated region sRNAs. Rel_Bbu-regulated sRNAs associated with genes known to be important for host infection (bosR and dhhp) as well as persistence in the tick (glpF and hk1) were identified, suggesting potential mechanisms for post-transcriptional regulation of gene expression.</p

Table_1_The Stringent Response-Regulated sRNA Transcriptome of Borrelia burgdorferi.xlsx

<p>The Lyme disease spirochete Borrelia (Borreliella) burgdorferi must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by Rel_Bbu globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that Rel_Bbu regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in B. burgdorferi. This is the first sRNA regulatory mechanism identified in any spirochete. Threefold more sRNAs were Rel_Bbu-upregulated than downregulated during nutrient stress and included antisense, intergenic and 5′ untranslated region sRNAs. Rel_Bbu-regulated sRNAs associated with genes known to be important for host infection (bosR and dhhp) as well as persistence in the tick (glpF and hk1) were identified, suggesting potential mechanisms for post-transcriptional regulation of gene expression.</p

Image_2_The Stringent Response-Regulated sRNA Transcriptome of Borrelia burgdorferi.TIFF

<p>The Lyme disease spirochete Borrelia (Borreliella) burgdorferi must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by Rel_Bbu globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that Rel_Bbu regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in B. burgdorferi. This is the first sRNA regulatory mechanism identified in any spirochete. Threefold more sRNAs were Rel_Bbu-upregulated than downregulated during nutrient stress and included antisense, intergenic and 5′ untranslated region sRNAs. Rel_Bbu-regulated sRNAs associated with genes known to be important for host infection (bosR and dhhp) as well as persistence in the tick (glpF and hk1) were identified, suggesting potential mechanisms for post-transcriptional regulation of gene expression.</p

Table_2_The Stringent Response-Regulated sRNA Transcriptome of Borrelia burgdorferi.XLSX

<p>The Lyme disease spirochete Borrelia (Borreliella) burgdorferi must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by Rel_Bbu globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that Rel_Bbu regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in B. burgdorferi. This is the first sRNA regulatory mechanism identified in any spirochete. Threefold more sRNAs were Rel_Bbu-upregulated than downregulated during nutrient stress and included antisense, intergenic and 5′ untranslated region sRNAs. Rel_Bbu-regulated sRNAs associated with genes known to be important for host infection (bosR and dhhp) as well as persistence in the tick (glpF and hk1) were identified, suggesting potential mechanisms for post-transcriptional regulation of gene expression.</p

Image_3_The Stringent Response-Regulated sRNA Transcriptome of Borrelia burgdorferi.TIFF

<p>The Lyme disease spirochete Borrelia (Borreliella) burgdorferi must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by Rel_Bbu globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that Rel_Bbu regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in B. burgdorferi. This is the first sRNA regulatory mechanism identified in any spirochete. Threefold more sRNAs were Rel_Bbu-upregulated than downregulated during nutrient stress and included antisense, intergenic and 5′ untranslated region sRNAs. Rel_Bbu-regulated sRNAs associated with genes known to be important for host infection (bosR and dhhp) as well as persistence in the tick (glpF and hk1) were identified, suggesting potential mechanisms for post-transcriptional regulation of gene expression.</p