High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni isolates

Abstract

Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA-seq (dRNA-seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA-seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA-seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA-seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter, which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level.

Figure 1. Differential RNA–seq and SuperGenome-based annotation of transcriptional start sites.

(A) cDNA reads of −/+TEX libraries for the four C. jejuni strains were mapped to the SuperGenome, generated by whole genome alignment. All y-axes were set to same scale. Horizontal bars at the top and bottom indicate the highly variable LOS (lipooligosaccharide modification), FLA (flagellin modification), and CPS (capsule) gene loci as well as the integrated genome elements (CMLP1, CJIE2, CJIE3, and CJIE4) for strain RM1221. (B) Example region in the SuperGenome with mapped dRNA–seq reads of −/+TEX libraries of the four strains. TSS are indicated by black arrows. Red and blue arrows indicate an internal TSS within kpsM, which is missing in two of the strains, and a primary TSS which was only detected in strain 81116, respectively. All y-axes were set to same scale, which reflects a relative expression score. Note that gene CJE1630 (dotted arrow) is only annotated in RM1221 and that homologs of Cj1456c (white arrows) have different lengths in the four strains.

Figure 2. Transcriptome features of multiple C. jejuni strains.

(A) Venn diagram showing the overlap among detected TSS between the four C. jejuni strains. The total numbers of TSS that were detected in each strain are indicated in brackets. TSS detected in single strains are marked by circles. See also Table 2. (B) Motif searches in the −50 to +1 sequences upstream of the 8,019 detected TSS in all four C. jejuni strains reveal promoter consensus sequence motifs for the three sigma factors, σ⁷⁰ (RpoD), σ²⁸ (FliA), and σ⁵⁴ (RpoN). The numbers of sequences which contain the motif are indicated as well as the distances to the associated TSS. (C) dRNA–seq reads mapped to the SuperGenome between the murC and dapE genes in the four strains. In three strains, the cjeI gene is inserted together with a TSS (red arrow) for transcription of the downstream genes.

Figure 3. SNP–dependent promoter usage in C. jejuni.

(A) dRNA–seq reads mapped to the pnk-recN operon in the SuperGenome. Red and black arrows indicate a primary (pTSS) and an internal TSS (iTSS), respectively. Corresponding alignments of the promoter region of the two TSS for pnk are shown. The iTSS within pnk is not expressed in two of the strains (81–176 and 81116) which carry a T to C exchange at the conserved “T” residue of the −10 box. (B) dRNA–seq reads mapped to the Cj0005c-Cj0004c operon, which encodes for a molydopterin containing oxidoreductase and monoheme cytochrome c. Promoter alignment of the primary TSS of Cj0005c shows disruptions in the A/T rich cyclic pattern upstream of the conserved σ⁷⁰ −10 box shows in 81116. (C) Intact cells from the four C. jejuni strains were assayed for cytochrome c oxidoreductase activity in the absence (dark grey bars) and presence (light grey bars) of sulphite. The reduction of cytochrome c was measured as the increase in the absorbance at 550 nm. Strains NCTC11168, 81–176, and RM1221 showed a significant increase (p<0.0001) over basal levels (control without cells) of cytochrome c reduction when the samples were supplemented with sulphite.

Figure 4. Small RNAs in Campylobacter jejuni.

(A) (Left) Left and right flanking genes and orientation of sRNA candidates in C. jejuni NCTC11168. Arrows indicating the genes are not drawn to scale. Small RNA candidates were termed “CJncXXX” and numbered in steps of ten according to their genome position. (Right) Expression analysis of the housekeeping and candidate sRNAs during different growth phases in the four C. jejuni strains. Specifically, total RNA (15 µg per lane) was extracted at mid-exponential (E), stationary (S), and overnight (O) growth phase and analyzed by Northern blot using labeled DNA probes complementary to the sRNAs (see Table S14). The blots were probed for the housekeeping 5S rRNA as loading control. Note that the oligonucleotide probe for CJnc170 does not detect the homolog in strain RM1221 due to point mutations. (B) Genomic locations and Northern blots for sRNA candidates in the pVir and pTet plasmids of C. jejuni strain 81–176.

Figure 5. Conservation of Campylobacter jejuni sRNA candidates in Epsilonproteobacteria.

Conservation of sRNA candidates in different C. jejuni strains and other representative Epsilonproteobacteria was analyzed by BLAST searches. The color intensity of the boxes represents the sequence identity to sRNAs of C. jejuni NCTC11168 or in case of the plasmid encoded sRNAs to strain 81–176. Identity values below 40% as well as the lack of an ortholog are symbolized by empty boxes. Housekeeping sRNAs are marked in bold.

Figure 6. A minimal CRISPR in Campylobacter jejuni.

(A) cDNAs reads of TEX−/+ libraries mapped to the CRISPR loci in C. jejuni NCTC11168. TSS are marked by black arrows. A −10 box for each crRNA is present in the 3′ part of the corresponding upstream located repeat (blue box). The lower panel shows the potential base-pairing of C. jejuni TracrRNA with the repeat sequence. (B) Northern blot analysis of TracrRNA and crRNAs. Total RNA (15 µg per lane) extracted from C. jejuni strains at mid-exponential (E), stationary (S), and overnight (O) growth phase was analyzed on a Northern blot using labeled DNA probes complementary to TracrRNA (two upper panels) and crRNA2 in NCTC11168 (see Table S14). Both the mature (∼62 nt) and processed (∼74 nt) TracrRNA forms as well as intermediate (∼103 nt) and mature (∼38 nt) crRNA forms for crRNA2 in NCTC11168 were detected. 5S rRNA served as loading control. (C) Conservation of CRISPR loci in different Campylobacter jejuni strains. In strain RM1221, cas9 contains a premature-stop mutation. In strain 81–176 the whole CRISPR locus is replaced by two genes (green arrows) with low G/C-content. (D) Subclassification of type-II CRISPR-loci. cas9, cas1, and cas2 are indicated with blue arrows and tracrRNA by a red box, respectively. TSS upstream of the repeat (black squares)-spacer (green diamonds) units in type-II A and B CRISPR loci , or within each spacer in the case of the minimal type-II C CRISPR systems of Campylobacter jejuni (CJ) and Neisseria meningitidis (NM) are marked by black arrows. Representative species for each type-II class are listed (SP: Streptococcus pyogenes, ST: S. thermophilus, TD: Treponema denticola, LI: Listeria innocua, and LP: Legionella pneumophila).