Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome

Abstract

Chlamydia trachomatis is an obligate intracellular pathogenic bacterium that has been refractory to genetic manipulations. Although the genomes of several strains have been sequenced, very little information is available on the gene structure of these bacteria. We used deep sequencing to define the transcriptome of purified elementary bodies (EB) and reticulate bodies (RB) of C. trachomatis L2b, respectively. Using an RNA-seq approach, we have mapped 363 transcriptional start sites (TSS) of annotated genes. Semi-quantitative analysis of mapped cDNA reads revealed differences in the RNA levels of 84 genes isolated from EB and RB, respectively. We have identified and in part confirmed 42 genome- and 1 plasmid-derived novel non-coding RNAs. The genome encoded non-coding RNA, ctrR0332 was one of the most abundantly and differentially expressed RNA in EB and RB, implying an important role in the developmental cycle of C. trachomatis. The detailed map of TSS in a thus far unprecedented resolution as a complement to the genome sequence will help to understand the organization, control and function of genes of this important pathogen.

Figure 1.

Characterization of the cDNA libraries. (A) Length distribution of reads after 5′ end-linker and polyA-tail clipping of four sequenced C. trachomatis cDNA libraries generated from either total RNA or total RNA enriched for primary transcripts of reticulate bodies (RB) or elementary bodies (EB), respectively. Shown are relative numbers of groups of sequence length in relation to the total number of reads per library. (B) Sequence read distribution of the four cDNA libraries grouped into different classes of RNAs. Transcripts antisense to ribosomal RNAs, transfer RNAs and messenger RNAs are not shown since they represent <1% of total sequences. Transcripts located in intergenic regions (IGR) can either be sRNA candidates, part of 5′- or 3′-UTRs of mRNAs or unannotated coding genes. The majority of the fraction of reads that could not be mapped to the chlamydial genome corresponds to contaminating host cell RNA, mainly mitochondrial RNAs.

Figure 2.

Validation of putative small RNAs by northern analysis. (A) Expression of eight out of twelve new candidate sRNAs could be confirmed by northern blotting whereas the length of the probed RNA corresponded to the calculated length from the sequencing data. The genomic location of the sRNAs is shown in (B). ctrR1 is not a primary transcript but presumably processed from a larger transcript containing tRNA:Thr. ctrR2, 3, 5–7 are located intergenic and primary transcripts. Two sRNAs are transcribed from ctrR4 and the longer transcript overlaps the gene 403 on the opposite strand. ctrR8 is located antisense to gene 807. ctrR0332 has previously been identified as ORF CTLon_0332, but represents two RNA species processed from a larger transcript lacking an ORF. Details on the genomic location and the sequence of ctrR0332 are given in Supplementary Figure S2. Two housekeeping RNAs (tmRNA and SRP RNA) and ihtA is the only so far reported chlamydial sRNA are shown in the right lanes. Genomic locations, sequence read numbers and hybridization probes are given in Supplementary Table S2.

Figure 3.

Transcriptome of the cryptic plasmid. (A) Calculated sequence graphs for C. trachomatis cryptic plasmid. Graphs show the number of sequence reads for every nucleotide of the plasmid up to the cut-off value of 10. Total RNA library reads are shown in grey and are overlaid by red graphs for the TSS enriched libraries. Annotated ORFs are shown as black bars for the plus strand (upper) and minus strand (lower), respectively. Note that total numbers for single transcripts are much higher than 10 but omitted for better visualization. TSS are indicated by horizontal arrows marked with the start positions. Hybridization probe binding sites used for northern detection are marked by vertical arrowheads and the probe name. (B) Northern analysis of C. trachomatis plasmid transcripts reveals a highly abundant small RNA pL2-sRNA1 of ∼100 nt in length antisense to pL2-07a. The region located antisense to the ORF pL2-02 encodes several transcripts in the range of ∼80–450 nt. Probe binding sites are marked by an arrow in (A). The names of the transcripts correspond to the first nucleotide of the probe binding site.