E. Gerhart H. Wagner

Novel small RNA-encoding genes in the intergenic regions of Escherichia coli

BACKGROUND Small, untranslated RNA molecules were identified initially in bacteria, but examples can be found in all kingdoms of life. These RNAs carry out diverse functions, and many of them are regulators of gene expression. Genes encoding small, untranslated RNAs are difficult to detect experimentally or to predict by traditional sequence analysis approaches. Thus, in spite of the rising recognition that such RNAs may play key roles in bacterial physiology, many of the small RNAs known to date were discovered fortuitously. RESULTS To search the Escherichia coli genome sequence for genes encoding small RNAs, we developed a computational strategy employing transcription signals and genomic features of the known small RNA-encoding genes. The search, for which we used rather restrictive criteria, has led to the prediction of 24 putative sRNA-encoding genes, of which 23 were tested experimentally. Here we report on the discovery of 14 genes encoding novel small RNAs in E. coli and their expression patterns under a variety of physiological conditions. Most of the newly discovered RNAs are abundant. Interestingly, the expression level of a significant number of these RNAs increases upon entry into stationary phase. CONCLUSIONS Based on our results, we conclude that small RNAs are much more widespread than previously imagined and that these versatile molecules may play important roles in the fine-tuning of cell responses to changing environments.

Antisense RNAs in bacteria and their genetic elements.

Antisense RNA-mediated regulation is widespread in bacteria. Most antisense RNA control systems have been found in plasmids, phages, and transposons. Fewer examples were identified in bacterial chromosomes. This chapter summarizes our current knowledge about antisense RNAs with respect to their occurrence, their biological roles, and their diverse mechanisms of action. Examples of cis- or trans-encoded antisense RNAs are discussed, and their properties compared. Most antisense RNAs are posttranscriptionally acting inhibitors of target genes, but a few examples of activator antisense RNAs are known. The implications of RNA structure on topologically and kinetically favored binding pathways are addressed, and solutions that have evolved to permit productive interactions between intricately folded RNAs are discussed. Finally, we describe how particular properties of individual antisense/target RNA systems match their respective biological roles.

The Small RNA IstR Inhibits Synthesis of an SOS-Induced Toxic Peptide

More than 60 small RNAs (sRNA) have been identified in E. coli. The functions of the majority of these sRNAs are still unclear. For the few sRNAs characterized, expression and functional studies indicate that they act under stress conditions. Here, we describe a novel E. coli chromosome locus that is part of the SOS response to DNA damage. This locus encodes two sRNAs, IstR-1 and IstR-2, and a toxic peptide, TisB, encoded by tisAB mRNA. Transcription of tisAB and istR-2 is SOS regulated, whereas IstR-1 is present throughout growth. IstR-1 inhibits toxicity by base-pairing to a short region in the tisAB mRNA. This antisense interaction entails RNase III-dependent cleavage, thereby inactivating the mRNA for translation. In the absence of the SOS response, IstR-1 is present in high excess over its target. However, SOS induction leads to depletion of the IstR-1 pool, concomitant with accumulation of tisAB mRNA. Under such conditions, TisB exerts its toxic effect, slowing down growth. We propose that the inhibitory sRNA prevents inadvertent TisB synthesis during normal growth and, possibly, also limits SOS-induced toxicity. Our study adds the SOS regulon to the growing list of global regulatory circuits controlled by sRNA genes.

H‐NS‐mediated repression of CRISPR‐based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO

The 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 nucleoid‐structuring (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 anti‐Lambda 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.

Small RNAs in Bacteria and Archaea: Who They Are, What They Do, and How They Do It

Small RNAs are ubiquitously present regulators in all kingdoms of life. Most bacterial and archaeal small RNAs (sRNAs) act by antisense mechanisms on multiple target mRNAs, thereby globally affecting essentially any conceivable trait-stress responses, adaptive metabolic changes, virulence etc. The sRNAs display many distinct mechanisms of action, most of them through effects on target mRNA translation and/or stability, and helper proteins like Hfq often play key roles. Recent data highlight the interplay between posttranscriptional control by sRNAs and transcription factor-mediated transcriptional control, and cross talk through mutual regulation of regulators. Based on the properties that distinguish sRNA-type from transcription factors-type control, we begin to glimpse why sRNAs have evolved as a second, essential layer of gene regulation. This review will discuss the prevalence of sRNAs, who they are, what biological roles they play, and how they carry out their functions.

A small SOS-induced toxin is targeted against the inner membrane in Escherichia coli

We previously reported on an SOS‐induced toxin, TisB, in Escherichia coli and its regulation by the RNA antitoxin IstR‐1. Here, we addressed the mode of action of TisB. By placing the tisB reading frame downstream of a controllable promoter on a plasmid, toxicity could be analysed in the absence of the global SOS response. Upon induction of TisB, cell growth was inhibited and plating efficiency decreased rapidly. The onset of toxicity correlated with a drastic decrease in transcription, translation and replication rates. Cellular RNA was degraded, but in vitro experiments showed that TisB did not affect translation or transcription directly. Thus, these effects are downstream consequences of membrane damage: TisB is predicted to be hydrophobic and membrane spanning, and Western analyses demonstrated that this peptide was strictly localized to the cytoplasmic membrane fraction. Membrane damage and cell killing under tisB multicopy expression are also seen by live/death staining and the formation of ghost cells. This is reminiscent of another toxin, Hok of plasmid R1, which also targets the membrane. The biological significance of the istR/tisB locus is still elusive; deletion of the entire locus gave no fitness phenotype in competition experiments.

Antisense RNAs everywhere

In recent years, systematic searches of both prokaryote and eukaryote genomes have identified a staggering number of small RNAs, the biological functions of which remain unknown. Small RNA-based regulators are well known from bacterial plasmids. They act on target RNAs by sequence complementarity; that is, they are antisense RNAs. Recent findings suggest that many of the novel orphan RNAs encoded by bacterial and eukaryotic chromosomes might also belong to a ubiquitous, heterogeneous class of antisense regulators of gene expression.

Sigma E controls biogenesis of the antisense RNA MicA

Adaptation stress responses in the Gram-negative bacterium Escherichia coli and its relatives involve a growing list of small regulatory RNAs (sRNAs). Previous work by us and others showed that the antisense RNA MicA downregulates the synthesis of the outer membrane protein OmpA upon entry into stationary phase. This regulation is Hfq-dependent and occurs by MicA-dependent translational inhibition which facilitates mRNA decay. In this article, we investigate the transcriptional regulation of the micA gene. Induction of MicA is dependent on the alarmone ppGpp, suggestive of alternative σ factor involvement, yet MicA accumulates in the absence of the general stress/stationary phase σS. We identified stress conditions that induce high MicA levels even during exponential growth—a phase in which MicA levels are low (ethanol, hyperosmolarity and heat shock). Such treatments are sensed as envelope stress, upon which the extracytoplasmic sigma factor σE is activated. The strict dependence of micA transcription on σE is supported by three observations. Induced overexpression of σE increases micA transcription, an ΔrpoE mutant displays undetectable MicA levels and the micA promoter has the consensus σE signature. Thus, MicA is part of the σE regulon and downregulates its target gene, ompA, probably to alleviate membrane stress.

PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids

The replication of CoIE1‐related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for pcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5′‐end processing by RNAse E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST‐ PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.

Kinetic aspects of control of plasmid replication by antisense RNA

Plasmids are replicating DNA molecules that are present in defined numbers of copies per cell. They encode systems that control their replication such that given steady-state values for their copy numbers are maintained. This is a special type of control, since it requires the genome to measure its concentration continuously and adjust its rate of replication to parallel the rate of growth of the cell mass. In this review we discuss the quantitative kinetic properties of copy-number control of the R1 plasmid, in which the control device is an antisense RNA that controls the synthesis of a protein that is rate-limiting for replication of the plasmid.