Conservation of σ²⁸-Dependent Non-Coding RNA Paralogs and Predicted σ⁵⁴-Dependent Targets in Thermophilic <i>Campylobacter</i> Species

<div><p>Assembly of flagella requires strict hierarchical and temporal control via flagellar sigma and anti-sigma factors, regulatory proteins and the assembly complex itself, but to date non-coding RNAs (ncRNAs) have not been described to regulate genes directly involved in flagellar assembly. In this study we have investigated the possible role of two ncRNA paralogs (CjNC1, CjNC4) in flagellar assembly and gene regulation of the diarrhoeal pathogen <i>Campylobacter jejuni</i>. CjNC1 and CjNC4 are 37/44 nt identical and predicted to target the 5' untranslated region (5' UTR) of genes transcribed from the flagellar sigma factor σ⁵⁴. Orthologs of the σ⁵⁴-dependent 5' UTRs and ncRNAs are present in the genomes of other thermophilic <i>Campylobacter</i> species, and transcription of CjNC1 and CNC4 is dependent on the flagellar sigma factor σ²⁸. Surprisingly, inactivation and overexpression of CjNC1 and CjNC4 did not affect growth, motility or flagella-associated phenotypes such as autoagglutination. However, CjNC1 and CjNC4 were able to mediate sequence-dependent, but Hfq-independent, partial repression of fluorescence of predicted target 5' UTRs in an <i>Escherichia coli</i>-based GFP reporter gene system. This hints towards a subtle role for the CjNC1 and CjNC4 ncRNAs in post-transcriptional gene regulation in thermophilic <i>Campylobacter</i> species, and suggests that the currently used phenotypic methodologies are insufficiently sensitive to detect such subtle phenotypes. The lack of a role of Hfq in the <i>E</i>. <i>coli</i> GFP-based system indicates that the CjNC1 and CjNC4 ncRNAs may mediate post-transcriptional gene regulation in ways that do not conform to the paradigms obtained from the Enterobacteriaceae.</p></div

Bacterial strains used in this study.

<p>a. Kan^R, kanamycin antibiotic resistance cassette; Cm^R, chloramphenicol antibiotic resistance cassette; ^fdxApr, gene under control of the <i>fdxA</i> promoter.</p><p>Bacterial strains used in this study.</p

Inactivation and overexpression of CjNC1 and CjNC4 does not affect flagella-related phenotypes in <i>C</i>. <i>jejuni</i>.

<p>(a) The successful inactivation (indicated by Δ) and overexpression (indicated by ^ov) of CjNC4 is demonstrated using Northern hybridisation. The hybridisation of CjNC3 is shown as loading control. The first two lanes of the Northern hybridisation are also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141627#pone.0141627.g001" target="_blank">Fig 1C</a>, left panel. (b) Inactivation or overexpression of CjNC1 and CjNC4 does not change cell morphology or production of flagella, as shown by light microscopy (×10,000), with cells and flagella stained by modified Ryu staining [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141627#pone.0141627.ref044" target="_blank">44</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141627#pone.0141627.ref045" target="_blank">45</a>]. Cutouts show representative cells, the Δ<i>flaAB</i> non-motile mutant is included for comparison. (c) Inactivation or overexpression of CjNC1 or CjNC4 does not significantly affect motility on swarm plates, as measured by the diameter of the swarming zone on motility agar plates. Results shown are the average of at least three independent experiments, error bars indicate standard error of the mean. The asterisk indicates <i>P</i><0.05 compared to the wildtype strain (One-way ANOVA). Other phenotypes are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141627#pone.0141627.s002" target="_blank">S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141627#pone.0141627.s004" target="_blank">S4</a> Figs.</p

The CjNC1 and CjNC4 ncRNAs are predicted to target the 5' UTRs of σ⁵⁴-dependent genes, and are present in other thermophilic <i>Campylobacter</i> species.

<p>(a) Alignment of the CjNC4 ncRNA sequence with predicted target 5' UTRs of <i>C</i>. <i>jejuni</i> σ⁵⁴-dependent genes (<i>flaB</i>, <i>cj0243c</i>, <i>c0428</i>, <i>flaD</i>, <i>flgP</i>, <i>cj1650</i> and <i>flgE2</i>) and one σ⁷⁰-dependent gene (<i>cj0878</i>), all shown as RNA. The underlined residues indicated the transcription start site (TSS) and AUG startcodon, the predicted ribosome binding site is indicated in red, blue residues the conserved region of the CjNC1 and CjNC4 ncRNAs. Lines indicate complementarity, semicolons highlight U:G pairings, hyphens indicate a gap introduced for optimal alignment. (b) Schematic representation of the <i>C</i>. <i>jejuni</i> flagellum, modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141627#pone.0141627.ref016" target="_blank">16</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141627#pone.0141627.ref042" target="_blank">42</a>], with the predicted targets of CjNC1/CjNC4 indicated by a red oval. (c) Comparison of the genomic position and surrounding genes of CjNC1 (yellow) and CjNC4 (blue) orthologs in thermophilic <i>Campylobacter</i> species. Dashed lines indicate pseudogenes. Only two examples of the <i>C</i>. <i>lari</i> group are shown, but all have similar arrangements. (d) Alignment of CjNC1/CjNC4 orthologs of thermophilic <i>Campylobacter</i> species. Red residues show the predicted σ²⁸ promoter, underlined is the TSS in <i>C</i>. <i>jejuni</i> NCTC 11168, blue residues show the conserved region predicted to basepair with the 5' UTR targets, and the green residues highlight the complementary nucleotides predicted to form the stem-loop structure which could function as transcriptional terminator. Asterisks indicate conserved nucleotides. Thermophilic <i>Campylobacter</i> species included are: jejuni, <i>C</i>. <i>jejuni</i> NCTC 11168; coli, <i>C</i>. <i>coli</i> 15–537360, upsaliens, <i>C</i>. <i>upsaliensis</i> RM3195; insulaeni, <i>C</i>. <i>insulaenigrae</i> NCTC 12927; lari, <i>C</i>. <i>lari</i> NCTC 11845; peloridis, <i>C</i>. <i>peloridis</i> LMG 23910; volucris, <i>C</i>. <i>volucris</i> LMG 24379; subantarc, <i>C</i>. <i>subantarcticus</i> LMG 24374.</p

The CjNC1 and CjNC4 non-coding RNAs are paralogs, expressed from σ²⁸-dependent promoters.

<p>(a) Schematic representation of the genomic position, transcriptional orientation and surrounding genes of the CjNC1 and CjNC4 ncRNAs in <i>C</i>. <i>jejuni</i> NCTC 11168. Arrows indicate the position of transcription start sites of loci as mapped by dRNA-seq [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141627#pone.0141627.ref024" target="_blank">24</a>]. (b) Alignment and structure prediction of the <i>C</i>. <i>jejuni</i> NCTC 11168 CjNC1 and CjNC4 ncRNAs. The canonical -10 sequence of the σ²⁸-dependent promoter is indicated in red, the transcription start site (TSS) is underlined. The blue residues are the conserved region predicted to interact with target 5' UTRs, and the green residues highlight the complementary nucleotides predicted to form the stem-loop structure which could function as transcriptional terminator. Asterisks indicate conserved nucleotides. (c) Transcription of CjNC1 and CjNC4 is dependent on σ²⁸, as shown by comparing transcript levels in wildtype and <i>fliA</i> mutant of <i>C</i>. <i>jejuni</i> NCTC 11168, by Northern hybridisation (left) for CjNC4, and by RT-PCR for CjNC1 and CjNC4 (right), while transcription of the σ²⁸-dependent CjNC3 ncRNA is not affected by the inactivation of <i>fliA</i>. The RT-PCR utilised a tag-based primer attached by the reverse transcription process (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141627#sec002" target="_blank">Methods</a>) and hence cannot amplify genomic DNA, which is includes as control (gDNA). "Neg" represents the negative PCR-control. The addition of the tag adds 29 nt to the size of RT-PCR products.</p

Plasmids used in this study.

<p>a. <i>gfp</i>: gene encoding green fluorescent protein.</p><p>Plasmids used in this study.</p

CjNC1 and CjNC4 use sequence-dependent interactions to post-transcriptionally regulate predicted target 5' UTRs in an <i>E</i>. <i>coli</i> GPF reporter system.

<p>(a) Expression of the <i>cj0428</i>::<i>gfp</i> fusion in pXG10 requires more than just the methionine. The constructs with six or more amino acids of the N-terminus show repression by CjNC1 and CjNC4, while the nonsense ncRNA in pJV300 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141627#pone.0141627.ref037" target="_blank">37</a>] does not affect fluorescence. Results are shown as flurorescence in arbitrary units (top) and fold regulation (bottom). (b) Expression of <i>gfp</i> fusions of <i>cj0428</i>, <i>flgE2</i>, <i>cj1650</i> and <i>flgP</i> is repressed by CjNC1 and/or CjNC4, whereas the <i>cj0581</i>::<i>gfp</i> fusion is not affected by either CjNC1 or CjNC4. None of the fusions is affected by the nonsense ncRNA in pJV300. (c) Mutation strategy of the CjNC1 and CjNC4 ncRNAs, and corresponding regions in the <i>cj0428</i>, <i>flgE2</i> and <i>cj1650</i> 5' UTRs to test whether the observed effects are sequence-specific. The conserved sequence in the ncRNA is shown in blue, the mutations made in the ncRNA in green, the complementary mutations in the 5' UTR in pink. The RBS is indicated in underlined red, the AUG start codon is underlined. The mutated region was chosen to not alter the predicted RBS. (d) Alteration of an 8 nt sequence in CjNC1 and CjNC4 disrupts regulation of the <i>cj0428</i>, <i>flgE2</i> and <i>cj1650</i> 5' UTRs (left), while compensatory mutations in the 5' UTR restore regulation of the <i>cj0428</i>::<i>gfp</i> fusion (right). The compensatory mutations did not restore regulation of the <i>flgE2</i>::<i>gfp</i> and <i>cj1650</i>::<i>gfp</i> fusions, but were associated with a significant reduction in fluorescence (data not shown), and hence we cannot draw any conclusions about the effect of the compensatory mutations on regulation. Data shown are the average of two or more independent experiments, error bars indicate standard error of the mean. Asterisks indicate <i>P</i><0.05 compared to the control with no ncRNA (One-way ANOVA).</p