SummaryInheritable bacterial defence systems against phage infection and foreign DNA, termed CRISPR (clustered regularly interspaced short palindromic repeats), consist of cas protein genes and repeat arrays interspaced with sequences originating from invaders. The Cas proteins together with processed small spacer-repeat transcripts (crRNAs) cause degradation of penetrated foreign DNA by unknown mechanisms. Here, we have characterized previously unidentified promoters of the Escherichia coli CRISPR arrays and cas protein genes. Transcription of precursor crRNA is directed by a promoter located within the CRISPR leader. A second promoter, directing cas gene transcription, is located upstream of the genes encoding proteins of the Cascade complex. Furthermore, we demonstrate that the DNA-binding protein H-NS is involved in silencing the CRISPR-cas promoters, resulting in cryptic Cas protein expression. Our results demonstrate an active involvement of H-NS in the induction of the CRISPRcas system and suggest a potential link between two prokaryotic defence systems against foreign DNA.

Background6S RNA from E. coli is known to bind to RNA polymerase interfering with transcription initiation. Because 6S RNA concentrations are maximal at stationary phase and binding occurs preferentially to the holoenzyme associated with σ70 (Eσ70) it is believed that 6S RNA supports adjustment to stationary phase transcription. Previous studies have also suggested that inhibition is specific for σ70-dependent promoters characterized by a weak -35 recognition motif or extended -10 promoters. There are many exceptions to this precept, showing that other types of promoters, including stationary phase-specific (σ38-dependent) promoters are inhibited.ResultsTo solve this apparent ambiguity and to better understand the role of 6S RNA in stationary phase transition we have performed a genome-wide transcriptional analysis of wild-type and 6S RNA deficient cells growing to mid-log or early stationary phase. We found 245 genes at the exponential growth phase and 273 genes at the early stationary phase to be ≥ 1.5-fold differentially expressed. Up- and down-regulated genes include many transcriptional regulators, stress-related proteins, transporters and several enzymes involved in purine metabolism. As the most striking result during stationary phase, however, we obtained in the 6S RNA deficient strain a concerted expression reduction of genes constituting the translational apparatus. In accordance, primer extension analysis showed that transcription of ribosomal RNAs, representing the key molecules for ribosome biogenesis, is also significantly reduced under the same conditions. Consistent with this finding biochemical analysis of the 6S RNA deficient strain indicates that the lack of 6S RNA is apparently compensated by an increase of the basal ppGpp concentration, known to affect growth adaptation and ribosome biogenesis.ConclusionsThe analysis demonstrated that the effect of 6S RNA on transcription is not strictly confined to σ70-dependent promoters. Moreover, the results indicate that 6S RNA is embedded in stationary phase adaptation, which is governed by the capacity of the translational machinery.

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In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5′-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5′-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3′-UTR of transcripts.