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. Many species have evolved into diverse strains with phenotypic and genotypic variations that facilitate adaptation to different ecological niches and, in the case of pathogens, to different hosts. Whereas comparison of genome sequences reveals differences and similarities among strains, the consequences of genomic variations can be tracked by studying the functional output from the genome. RNA sequencing has been revolutionizing transcriptome analyses of both pro- and eukaryotes. However, the bioinformatics-based analysis is still lagging behind, and transcriptome features are often manually annotated, which is laborious and time-consuming. This is even more compounded for the analyses of multiple strains. Here we compared the primary transcriptomes of four isolates of Campylobacter jejuni, the leading cause of bacterial gastroenteritis in humans, and provide genome-wide transcriptional start site (TSS) maps using a novel automated annotation method. Our comparative RNA–seq showed that most TSS are conserved in multiple strains, but we also observed SNP–dependent promoter usage. Furthermore, we identified a novel minimal RNA–based CRISPR immune system as well as strain-specific small RNA repertoires. Our automated, comparative TSS annotation will facilitate and improve transcriptome annotation for a wider range of organisms and provides insights into the contribution of transcriptome differences to phenotypic variation among closely related species.

Bacteria can become dormant or form spores when they are starved for nutrients. Here, we find that non-sporulating Bacillus subtilis cells can survive deep starvation conditions for many months. During this period, cells adopt an almost coccoid shape and become tolerant to antibiotics. Unexpectedly, these cells appear to be metabolically active and show a transcriptome profile very different from that of stationary phase cells. We show that these starved cells are not dormant but are growing and dividing, albeit with a doubling time close to 4 days. Very low nutrient levels, comparable to 10,000-fold diluted lysogeny broth (LB), are sufficient to sustain this growth. This extreme slow growth, which we propose to call ‘oligotrophic growth state’, provides an alternative strategy for B. subtilis to endure nutrient depletion and environmental stresses. Further work is warranted to test whether this state can be found in other bacterial species to survive deep starvation conditions.

Cellular RNAs guide CRISPR-Cas9: The Cas9 nuclease widely used for genome editing is derived from natural bacterial defense systems that protect against invading viruses. Cas9 is directed by RNA guides to cut matching viral DNA. Jiao et al. discovered that RNA guides can also originate from cellular RNAs unassociated with viral defense (see the Perspective by Abudayyeh and Gootenberg). They rendered this process programmable, linking the presence of virtually any RNA to cutting of matching DNA by Cas9. This capability is the basis of a new CRISPR diagnostic method developed by the authors that can detect many biomarkers at once. Named LEOPARD, this method can detect, for example, RNAs from severe acute respiratory syndrome coronavirus 2 and other viruses, thereby translating a new CRISPR discovery into a powerful diagnostic tool. Science , abe7106, this issue p. 941 ; see also abi9335, p. 914

The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.