MicL, a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein

Abstract

In enteric bacteria, the transcription factor σ(E) maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σ(E)-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σ(E) activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σ(E) regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σ(E) to repress the synthesis of all abundant outer membrane proteins in response to stress.

Keywords: Hfq; copper; cutC; outer membrane homeostasis; sRNA; σE.

Figure 1.

MicL expression is regulated by σ^E. (A) Schematic of the genomic context of MicL; its processed transcript, MicL-S; and cutC (see Supplemental Fig. S1B). (B) MicL levels increase following σ^E overexpression. Cells harboring either vector or a σ^E expression plasmid growing exponentially in EZ rich defined medium were induced with 1 mM IPTG for 1 h. RNA was extracted and probed for the 3′ end of MicL and 5S RNA. (C) MicL levels increase in stationary phase. Total RNA was extracted at the indicated times during growth in EZ rich defined medium and probed for MicL and 5S RNA. (D) The micL promoter is similar to a logo for σ^E promoter sequences (Rhodius et al. 2012). (E) P_micL is σ^E dependent. Cells carrying either the vector control or the pTrc-RpoE plasmid, expressing GFP from the indicated minimal promoters (−65 to +20 relative to transcription start site), and growing exponentially in LB were induced with 1 mM IPTG, and GFP fluorescence was monitored. Promoter activity was measured by normalizing GFP fluorescence by OD (see the Materials and Methods). (F) MicL-S is a processed transcript. RNA isolated following induction of MicL for 3 h from an IPTG-inducible promoter was left untreated, incubated in buffer, or incubated in buffer with 5′ monophosphate-dependent terminator exonuclease (TEX). MicL-S levels were subsequently probed.

Figure 2.

lpp is the sole target of MicL. (A) MicL interacts with Hfq. Extracts were prepared from wild-type cells after 16 h of growth in LB medium and subjected to immunoprecipitation with α-Hfq or preimmune serum. MicL was probed in the immunoprecipitated samples (0.5 μg of RNA loaded) as well as on total RNA isolated from wild-type and the isogenic hfq-1 mutant cells (5 μg of RNA loaded). (B) MicL expression reduces lpp mRNA levels ∼20-fold. mRNA-seq was performed in exponential phase after 20 min of MicL induction from pBR′-MicL at 30°C in EZ rich defined medium and compared with a similarly treated vector control strain. Expression level is in reads per kilobase per million (RPKM). (C) MicL expression reduces translation on lpp mRNA ∼10-fold. Ribosome profiling was performed in exponential phase after 20 min of MicL induction from pBR′-MicL at 30°C in EZ rich defined medium and compared with profiles taken before MicL induction. Relative translation is in RPKM. Other genes (fepA and fiu) close to the fivefold cutoff are repressed by growth (Supplemental Fig. S4F).

Figure 3.

MicL repression of lpp is physiologically important. (A) Expression of MicL phenocopies the dibucaine sensitivity of Δlpp. Wild-type or Δlpp cells carrying pBR*-MicL, pBR*-MicL-S, or empty vector were spotted at the indicated dilutions on LB plates containing 1.4 mM dibucaine with or without 1 mM IPTG. (B) MicL represses lpp RNA levels following σ^E overexpression. Wild type and a ΔcutC strain with either control vector or pRpoE growing exponentially in LB (OD₆₀₀ ∼0.1) were induced with 1 mM IPTG for 2 h. Total RNA was isolated and probed for lpp, MicL, and 5S RNA. (C) lpp mRNA and Lpp protein levels in wild-type and ΔcutC mutant backgrounds. At the indicated times, total RNA was extracted from wild type and the ΔcutC mutant strain grown in LB. Total RNA was probed to examine lpp, MicL, and 5S RNA levels, and Lpp and GroEL protein levels were examined by immunoblotting protein samples taken at the same time points. For B and C, the intensity of the lpp RNA or protein band for each strain was quantified using ImageJ software, and the ratios between the corresponding samples for the ΔcutC mutant and wild-type strains are given.

Figure 4.

MicL base-pairs with lpp. (A) MicL and MicL-S, but not MicA and RybB, repress an lpp-lacZ translational fusion. β-Galactosidase activity of the lpp-lacZ fusion preceded by a P_BAD promoter was assayed in strains with control vector, pBR-MicL, pBR-MicL-S, pBR-MicA, and pBR-RybB plasmids after 3 h of induction with 0.2% arabinose (for fusion) and 1 mM IPTG (for sRNA) (final OD₆₀₀ ∼1.0) in LB. Average values and standard deviations from four independent experiments are shown. (B) Predicated structure of MicL-S. Nucleotides predicted to comprise the core of base-pairing with lpp are shaded. (C) Predicted MicL and lpp base-pairing core with mutations designed to disrupt interaction. (D) Effect of disruption and restoration of base-pairing on MicL repression of lpp-lacZ. Plasmids carrying wild-type MicL-S or the MicL-S-1 derivative were transformed into strains containing lpp-lacZ or lpp-1-lacZ, which carries compensatory mutations to restore base-pairing with MicL-S-1. β-Galactosidase activity was assayed as in A. (E) MicL-S but not MicL-S-1 lowers lpp RNA and Lpp protein levels. The lpp-lacZ fusion strain was transformed with pBR-MicL-S or pBR-MicL-S-1 and induced as in A. Samples were collected after 3 h, and levels of lpp, the MicL-S and 5S RNA, or the Lpp and GroEL proteins were probed.

Figure 5.

MicL repression of lpp is dependent on translation. (A) Diagrammatic representation of the derivatives carrying early stop codon mutations lpp-1 (ATG to TAG at the first codon), lpp-2 (AAA to TAA at the second codon), and lpp-4 (ACT to TAA at the fourth codon). (B) The pBR*-MicL-S plasmid was transformed into wild-type and lpp translation-defective cells, MicL-S was induced with 1 mM IPTG in LB for 3 h, and RNA was extracted (final OD₆₀₀ ∼1.0) and probed for lpp, MicL-S, and 5S RNA. The intensity of the lpp band from each strain was quantified using ImageJ software, and the fold changes listed below are calculated for the corresponding samples with and without IPTG. Immunoblot analysis for Lpp confirmed that translation was eliminated in the stop codon mutants (data not shown). (C) Translation efficiency of Lpp is unchanged after MicL expression. Translation efficiency per gene after 20 min of MicL induction is plotted versus translation efficiency before MicL induction. Translation efficiency was calculated as the number of ribosome footprints per gene/mRNA reads per gene from the ribosome profiling and mRNA-seq data.

Figure 6.

Copper sensitivity of ΔcutC is due to loss of MicL. (A) Sensitivity of wild-type strains and variants with P_micL mutant (-10C-T/-35A-G), cutCΔ5′ (which preserves MicL), ΔcutC, and Δlpp to 4 mM Cu(II)Cl₂. Three microliters of each strain in exponential phase was spotted on LB supplemented with 4 mM Cu(II)Cl₂ at the indicated dilutions (Tetaz and Luke 1983; Gupta et al. 1995). (B) Sensitivity of wild-type cells, ΔcutC, and Δlpp transformed with pBR* control vector, pBR*-MicL-S, and pBR*-MicL to 4 mM Cu(II)Cl₂ using conditions in A with the exception that the medium was additionally supplemented with kanamycin. Some differences in sensitivity between A and B may be due to a synthetic effect between copper and the kanamycin used for plasmid selection in B.

Figure 7.

MicL and Lpp are part of an envelope protective regulatory loop. (A) Overexpression of Lpp increases σ^E activity. Cells with either control vector or pTrc-Lpp were induced with either 50 μM or 1 mM IPTG (at the time indicated). σ^E activity was measured from a σ^E-dependent rpoHp3-lacZ reporter. The σ^E activity for the vector control strain treated with 50 μM or 1 mM IPTG was similar at all points (data not shown). (B) Overexpression of MicL lowers σ^E activity. Cells with empty vector or pBR*-MicL were induced with 1 mM IPTG when overnight cultures were diluted to OD₆₀₀ ∼0.01. σ^E activity was measured as in A. Notably, MicL overexpression lowers Lpp protein levels to an extent similar to that observed in ribosome profiling (∼10-fold) (cf. Fig. 2C; Supplemental Fig. S10B). The inset provides the average and standard deviation for increased σ^E activity for all pBR* and pBR*-MicL points, normalized to pBR* at each time point. (C) Shutoff of σ^E activity leads to cell death and can be rescued by concomitant expression of MicA or MicL from derivatives of the pEG plasmid. σ^E activity is shut off by overexpressing the σ^E-negative regulators RseA/B from pTrc-RseAB. Aliquots (2 μL) of cells growing exponentially in LB with ampicillin (amp) and cm were plated at the indicated dilutions on LB plates ± 1 mM IPTG, which induces both RseA/B and the sRNA (MicL or MicA).

Figure 8.

Model of the envelope protective σ^E–MicL–Lpp loop. σ^E transcribes genes encoding proteins that relieve folding stress and sRNAs that inhibit new synthesis of the abundant proteins of the OM (OMPs and Lpp). Defects in lipoprotein transport inhibit proper OM assembly of both LPS and OMPs, which then bind to RseB and DegS, respectively, inducing RseA cleavage and σ^E activation. In response, σ^E activates the sRNA MicL to specifically down-regulate synthesis of Lpp, the major lipoprotein. (Inset) σ^E is held inactive by RseA in the inner membrane. RseB binds to RseA and prevents DegS from cleaving RseA.