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Sigma E Controls Biogenesis of the Antisense RNA Mica Klas I Published online 31 January 2007 Nucleic Acids Research, 2007, Vol. 35, No. 4 1279–1288 doi:10.1093/nar/gkl1154 Sigma E controls biogenesis of the antisense RNA MicA Klas I. Udekwu* and E. Gerhart H. Wagner Department of Cell & Molecular Biology, Uppsala university, Biomedical Center, Box 596, S-75124 Uppsala, Sweden Received October 30, 2006; Revised November 29, 2006; Accepted December 18, 2006 Downloaded from https://academic.oup.com/nar/article/35/4/1279/1748494 by guest on 28 September 2021 ABSTRACT In addition, a second level of posttranscriptional control is frequently involved. In Escherichia coli, a growing Adaptation stress responses in the Gram-negative number of small non-coding RNAs (sRNAs) have been bacterium Escherichia coli and its relatives involve a implicated in the regulation of stress responses and growing list of small regulatory RNAs (sRNAs). virulence traits (1–3). Many of these are conserved in Previous work by us and others showed that the enteric relatives, and quite a few sRNAs have since been antisense RNA MicA downregulates the synthesis of identified in other bacteria as well. the outer membrane protein OmpA upon entry into Most of the sRNAs are antisense RNAs that inhibit stationary phase. This regulation is Hfq-dependent (or, less frequently, activate) the translation of target and occurs by MicA-dependent translational inhibi- mRNAs or promote their degradation. The ubiquitous tion which facilitates mRNA decay. In this article, we RNA-binding protein Hfq (4) is often required for investigate the transcriptional regulation of the regulatory activity, though its mechanism of action is micA gene. Induction of MicA is dependent on the not yet fully understood. alarmone ppGpp, suggestive of alternative p factor In contrast to the cis-encoded antisense RNAs in plasmids, most chromosomally encoded sRNAs are involvement, yet MicA accumulates in the absence S trans-encoded (5). Their genes do not overlap target of the general stress/stationary phase p .We genes, and thus complementarity to the target mRNA is identified stress conditions that induce high MicA limited and often non-contiguous. Therefore, the location levels even during exponential growth—a phase in of an sRNA gene does not automatically identify the which MicA levels are low (ethanol, hyperosmolarity target gene. Assigning functions for new sRNAs has been and heat shock). Such treatments are sensed as based on screening for downstream effects in strains envelope stress, upon which the extracytoplasmic lacking the sRNA, or overexpressing it. Microarray sigma factor pE is activated. The strict dependence analyses, two-dimensional protein gels, phenotypic tests of micA transcription on pE is supported by three and bioinformatics-aided complementarity searches have observations. Induced overexpression of pE resulted in the identification of targets (6). increases micA transcription, an "rpoE mutant Conceptually, sRNAs are expected to be under appro- displays undetectable MicA levels and the micA priate transcriptional control, so that their induction promoter has the consensus pE signature. Thus, matches requirements for their regulatory activity. This appears to be borne out by observations: for instance, MicA is part of the pE regulon and downregulates its RyhB is under control of the Fur repressor. When iron target gene, ompA, probably to alleviate membrane concentration is low, Fur repression of the ryhB promoter stress. is abolished, and synthesized RyhB inhibits the synthesis of several iron-binding proteins (7). A similar logic guides the expression characteristics of several other sRNAs. INTRODUCTION Many sRNAs are upregulated immediately before or Bacteria meet adverse environmental conditions by rapid upon entry into stationary phase (8,9). This stress adaptive changes in gene expression patterns, thereby response in E. coli has been studied extensively and mounting appropriate responses. This most often involves is characterized by major physiological changes arising transcriptional regulation, by repressors or activators, from orchestrated alterations in gene expression (10), the often under the control of two-component regulatory majority of which are dependent on the stress/stationary systems, and/or by alternative sigma (s) factors. phase sS. This transcription factor, in turn, is under *To whom correspondence should be addressed. Tel: þ46 18 471 4579; Fax: þ46 18 530396; Email: [email protected] Correspondence may also be addressed to E. Gerhart H. Wagner. Tel: þ46 18 471 4579; Fax: þ46 18 530396; E-mail: [email protected] ß 2007 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1280 Nucleic Acids Research, 2007, Vol. 35, No. 4 intricate control involving several environmental signals accumulation of MicA were unknown. In this work, that converge on the expression of its gene, the translation we investigated the transcriptional regulation of MicA or stability of its mRNA and the activity/stability under a variety of stress conditions. We find that of the protein itself (11). Among these regulators are transcription of MicA is strictly dependent on sE, two antisense sRNAs, DsrA and RprA, that are induced making it the first sRNA in E. coli shown to be an by different signals and enhance translation of the rpoS exclusive member of the sE regulon. mRNA (12). Transcriptome and proteome analyses have been used to chart the sS regulon, which includes transcriptional regulators important for the scavenging MATERIALS AND METHODS of nutrients, such as Crp and the two-component Ntr and Promoter sequence alignment Pho systems, and many genes whose products help in adaptation to stationary phase. Recently, microarray data Selected micA upstream sequences from several have contributed to the understanding of the transcrip- enterobacterial genomes (gamma proteobacteriaceae sub- Downloaded from https://academic.oup.com/nar/article/35/4/1279/1748494 by guest on 28 September 2021 tomes of other s factors, such as the heatshock sH (13) division) were aligned. We aligned the sequence and the extracytoplasmic stress sE (14). Interestingly, ranging from the known E. coli micA gene À55 to þ4 sH responds not only to heat stress, but is itself trans- sequence (þ1 being the transcription start site (8; K.U., criptionally upregulated in a sE-dependent manner (15), unpublished)) with corresponding regions in related perhaps indicative of some regulatory ‘hierarchy’. genomes. Alignment to the consensus rpoE recognition The extracytoplasmic function (ECF) sE is specifically module was based on previous research (14,19). Sequences induced in response to conditions that culminate in were obtained from the NCBI database, and BLAST changes within the periplasm (16). Its intracellular levels (http://www.ncbi.nlm.nih.gov/BLAST) and accession are high, almost as high as those of the housekeeping numbers indicated are listed in the Figure 3 legend. s70 (17). sE is sequestered at the cytoplasmic membrane by an anti-s factor, RseA. Upon accumulation of Bacterial strains and plasmids misfolded proteins in the periplasm, ethanol exposure, Bacterial strains and plasmids are listed in Table 1. The heatshock and hyperosmotic stress, the distress signals are E. coli strain MC4100relAþ was used as wildtype unless perceived by the protease DegS. Successive cleavages of otherwise stated. To construct plasmid p30-luc, we cleaved RseA by DegS and RseP, a second protease, release the out the promoter region of pZE12-luc (23) with the active sE into the cytoplasm to compete for RNA restriction enzymes EcoRI and XhoI. The micA promoter polymerase. More than 80 genes are induced in response region was amplified using primers KU30-LmicA to sE upregulation (14,18,19). sE is an essential protein in (50-GAA CCT CGA GCT ATC TAA CAA CGG CCA E. coli (20), which likely testifies to the importance of the TTT A; XhoI site underlined) and KU32-pMic (50-GAT periplasmic and outer membrane compartments for many AAG AAT TCA TAT ACT CAG ACT CGC CT; EcoRI life processes. site underlined). The resulting 106 bp DNA fragment We and others have studied the sRNA MicA (earlier: containing micA (À85) to (À1) flanked by XhoI and SraD (8)). MicA accumulates upon entry into stationary EcoRI sites was digested, purified and ligated into the phase and, aided by Hfq, downregulates the translation linearized plasmid. Plasmid p30-luc thus carried the luc of the outer membrane protein OmpA, concomitantly gene under transcriptional control of micA’s À85 to À1 inducing the degradation of the ompA mRNA (21,22). The region. A promoterless control plasmid (pZE12b-luc) was signals and effectors that control transcription and constructed by treating the EcoRI- and XhoI-cleaved Table 1. Strains and plasmids used in this study Strain or plasmid Description Source or reference Strain MC4100relA1 araD139, Á(argF-lac)205, flb-5301, pstF25, rpsL150, deoC1, relA1 (21) MC4100relAþ relAþ derivative of MC4100 (21) MC4100relAþ rpoS::Tn10 Sigma S deficient derivative of MC4100relAþ Shoshy Altuvia MC4100 relA1spoT ppGpp-null derivative of MC4100 Thomas Nystro¨m MC4100 relA1 cyaÀ cAMP – deficient MC4100 B.E. Uhlin CAG16037 araD Á(ara-leu)7697, Á(codB-lacI), galK16, galE15, mcrA0 (40) relA1, rpsL150, spoT1, mcrB9999, hsdR2[rpoH P3::lacZ] CAG22216 CAG16037 ÁrpoE (41) LMG194 F– ÁlacX74, galE, thi, rpsL, ÁphoA, Áara714, leu::Tn10 Invitrogen GW 986 LMG194 (pACYC184) This work GW 969 LMG194 (pAC-rpoE4) This work GW 962 MC4100relAþ (p30-luc) This work GW 982 MC4100 relAþ (pZE12b-luc) This work Plasmid pAC-rpoE4 rpoE gene with arabinose-responsive promoter (19) pZE12-luc p15A plasmid carrying pLac-luc gene (23) pZE12b-luc pZE12-luc derivative lacking the pLac promoter This work p30-luc micA promoter (À85 to þ1) transcriptionally fused to luc gene This work Nucleic Acids Research, 2007, Vol. 35, No. 4 1281 pZE12-luc with Mung bean nuclease prior to re-ligation.
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