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.

CRISPR interference confers adaptive, sequence-based immunity against viruses and plasmids and is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from spacer-repeat units. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here, our studies of crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp. reveal a unique crRNA maturation pathway in which crRNAs are transcribed from promoters that are embedded within each repeat, yielding crRNA 5’ ends formed by transcription and not by processing. Although crRNA 3’ end formation involves RNase III and trans-encoded tracrRNA, as in other Type II CRISPR systems, this processing is dispensable for interference. The meningococcal pathway is the most streamlined CRISPR/cas system characterized to date. Endogenous CRISPR spacers limit natural transformation, which is the primary source of genetic variation that contributes to immune evasion, antibiotic resistance, and virulence in the human pathogen N. meningitidis.

SummaryThe recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoidstructuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an antiLambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.

The translation of many heat shock and virulence genes is controlled by RNA thermometers. Usually, they are located in the 5'-untranslated region (5'-UTR) and block the Shine-Dalgarno (SD) sequence by base pairing. Destabilization of the structure at elevated temperature permits ribosome binding and translation initiation. We have identified a new type of RNA thermometer in the 5'-UTR of the Salmonella agsA gene, which codes for a small heat shock protein. Transcription of the agsA gene is controlled by the alternative sigma factor sigma(32). Additional translational control depends on a stretch of four uridines that pair with the SD sequence. Mutations in this region affect translation in vivo. Structure probing experiments demonstrate a temperature-controlled opening of the SD region in vitro. Toeprinting (primer extension inhibition) shows that ribosome binding is dependent on high temperatures. Together with a postulated RNA thermometer upstream of the Yersinia pestis virulence gene lcrF (virF), the 5'-UTR of Salmonella agsA might be the founding member of a new class of RNA thermometers that we propose to name 'fourU' thermometers.

SummaryWhereas about 70 small non-coding RNAs have been found in the Escherichia coli genome, relatively little is known about regulatory RNAs from Gram-positive bacteria. Here, we demonstrate that the recently identified small untranslated RNA SR1 from the Bacillus subtilis genome is a regulatory RNA involved in finetuning of arginine catabolism. 2D protein gel electrophoresis indicated three possible SR1 targets that are regulated by the transcriptional activator AhrC, which was shown to be the primary target of SR1. In vitro pairing studies and an in vivo reporter gene test demonstrated a specific interaction between SR1 and ahrC mRNA. This interaction did not lead to degradation of ahrC mRNA, but inhibited translation at a postinitiation stage. Our data show that the Hfq chaperone was not required for the stabilization of SR1 in vivo.