Unexpected properties of sRNA promoters allow feedback control via regulation of a two-component system

Abstract

Two-component systems (TCS) and small regulatory RNAs (sRNAs) are both widespread regulators of gene expression in bacteria. TCS are in most cases transcriptional regulators. A large class of sRNAs act as post-transcriptional regulators of gene expression that modulate the translation and/or stability of target-mRNAs. Many connections have been recently unraveled between these two types of regulators, resulting in mixed regulatory circuits with poorly characterized properties. This study focuses on the negative feedback circuit that exists between the EnvZ-OmpR TCS and the OmrA/B sRNAs. We have shown that OmpR directly activates transcription from the omrA and omrB promoters, allowing production of OmrA/B sRNAs that target multiple mRNAs, including the ompR-envZ mRNA. This control of ompR-envZ by the Omr sRNAs does not affect the amount of phosphorylated OmpR, i.e. the presumably active form of the regulator. Accordingly, expression of robust OmpR targets, such as the ompC or ompF porin genes, is not affected by OmrA/B. However, we find that several OmpR targets, including OmrA/B themselves, are sensitive to changing total OmpR levels. As a result, OmrA/B limit their own synthesis. These findings unravel an additional layer of control in the expression of some OmpR targets and suggest the existence of differential regulation within the OmpR regulon.

Figure 1.

OmpR-P activates omrA and omrB transcription in vitro. (A) In vitro transcription reactions were performed using plasmid templates carrying regions −114 to +50 of either the OmrA or OmrB promoter, or −437 to +50 of ompC promoter (relative to P1 transcription start site), fused to 5S to give a 200 nt transcript. When indicated, OmpR was added at a molar ratio of 25, 50, 125 and 250 with the template DNA, and OmpR-P at a molar ratio of 25, 50 and 125. Products were separated on a sequencing gel and a portion of a representative gel is shown where transcripts driven from P_omrA, P_omrB or P_ompC promoter and the internal control RNA1 are visible. The ladder corresponds to radiolabeled pBR322/MspI digestion products and indicated sizes refer thus to double-stranded DNA fragments. Quantification of 5S rRNA fusion transcripts was performed after normalization to RNA1 and the level is arbitrarily set at 1 for control without OmpR for each promoter. The graph below represents the average activation of transcription from P_omrA, P_omrB or P_ompC in presence of increasing OmpR or OmpR-P concentrations, as calculated from two independent experiments. Error bars indicate standard deviations and, when not visible, are smaller than the symbols. (B) Similar in vitro transcription reactions were performed using templates carrying a short version of omrA promoter (P_omrA-60, carries nts −60 to +50 relative to TSS) or the sgrS promoter (from nts −70 to +50). OmpR-P was used at a molar ratio of 25-, 50- and 125-fold with template DNA, and OmpR at 250-fold. P_omrA was used here as a positive control for activation by 50- or 125-fold OmpR-P. Full gels are shown in Supplementary Figure S1.

Figure 2.

Transcription of OmrA and OmrB sRNAs is strongly dependent on EnvZ-OmpR TCS. (A) The effect of various mutants in envZ or ompR on OmpR phosphorylation in vivo was analyzed by western blot following protein separation in presence of Phos-Tag. Samples were taken from strains DJ480 (wt), MG1670 (envZ390), MG1671 (envZ473), MG1672 (ompR107), MG1673 (ompR472) and MG1035 (ΔompR::cm) grown in LB to exponential phase, before or after addition of 10 mM procaine for 10 min. A non-specific band obtained from cross-hybridization with an anti-EF-Tu antibody is shown as a loading control. (B) The activation of promoter fusions to omrA, omrB, ompC or ompF was calculated as the ratio between the activity of those fusions in ompR⁺ and ompR⁻ strains. The average and the standard deviations from at least two independent experiments are shown. Strains used here are, for ompR⁺ and ompR⁻ respectively, MG1004 and MG1811 (P_omrA fusion), MG1005 and MG1812 (P_omrB fusion), MG1863 and MG1891 (P_ompC fusion) and MG1690 and MG1810 (P_ompF fusion). In this experiment, the β-galactosidase activities in the ompR⁺ strain were, in Miller units, 19.1 (P_omrA fusion), 49.4 (P_omrB), 6010 (P_ompC) and 3270 (P_ompF). (C) The β-galactosidase activity of the same promoter fusions in wt strains or in the same set of envZ-ompR mutants used in (A) was measured in LB medium in exponential phase. wt, envZ390, envZ473, ompR107 and ompR472 strains are respectively MG1892, MG1893, MG1894, MG1895 and MG1896 (P_ompC fusion), MG1690, MG1692, MG1694, MG1696, MG1698 (P_ompF fusion), MG1004, MG1299, MG1300, MG1301, MG1302 (P_omrA fusion) and MG1005, MG1303, MG1304, MG1305 and MG1306 (P_omrB fusion). Numbers above the bars indicate repression- or activation-fold (in gray or black respectively) in expression relative to the wt strain; note that right-hand panel uses log scale.

Figure 3.

Control of ompR expression by OmrA/B changes OmpR, but not OmpR-P levels. (A) Seed-pairing interaction between OmrA/B 5′ end and ompR-envZ mRNA (26). Shine-Dalgarno sequence and start codon of ompR are in bold, and mut2 changes in OmrA/B or ompR mRNA are shown in gray. (B) The β-galactosidase activity of an ompR-lacZ translational fusion, wt or with the mut2 change, expressed from a P_BAD inducible promoter was measured in omrAB⁺ or omrAB⁻ strains in a medium supplemented or not with 20 mM procaine. Strains used in this experiment are MG2174, MG2175, MG2176 and MG2177. As a control, the activity of a P_BAD-phoP-lacZ translational fusion (strain MG1425) was measured in the same media. Activities are expressed as % of the wt fusions in omrAB⁺ cells in the absence of procaine; these reference activities are, in Miller units, 2238 (ompR fusion) and 542 (phoP fusion). (C) In vivo phosphorylation of OmpR was analyzed upon overproduction of OmrA/B or of their mut2 derivatives in strain DJ480. An extract from a ΔompR::cm strain (MG1035) was loaded on the same gel. A non-specific band revealed with the anti-OmpR antibody was used as a loading control.

Figure 4.

Different patterns of robustness in the OmpR regulon. (A) The activation of OmrA, OmrB, ompC or ompF promoter fusions (strains MG1004, MG1005, MG1892 and MG1690 respectively) was calculated as the ratio between the β-galactosidase activities of strains transformed by pOmpR and the pHDB3 empty vector respectively. Activities in presence of pHDB3 were, in Miller units, 33 (P_omrA), 87 (P_omrB), 7313 (P_ompC) and 3510 (P_ompF). (B) Western blot analysis of OmpR levels from cells carrying ompR-envZ operon expressed from its own promoter (wt ompR, strain MG1988) or from the Tet-P_lac-ompR construct (strain MG2000). Cells were grown to exponential phase in LB supplemented or not with IPTG at the indicated concentrations. As controls, the level of OmpR was also analyzed from strain MG1004 transformed with pHDB3 or pOmpR, and from the ΔompR strain MG1811. Immunoblot detection of EF-Tu is shown as a loading control. (C) The β-galactosidase activity of the same promoter fusions as in panel (A) was followed when ompR-envZ expression was modulated from the Tet-P_lac-ompR allele by increasing IPTG concentrations. Results of a representative experiment are shown here and two additional independent repeats are shown in Supplementary Figure S6. Strains used in this experiment are MG2000, MG2002, MG2004 and MG2006. (D) Levels of OmrA, OmrB sRNAs and of ompC mRNA were followed by northern blot when changing OmpR levels. SsrA is used as a loading control. Strains are MG1004 (wt) and MG2000 (Tet-P_lac-ompR).

Figure 5.

Feedback control in OmrA/B expression. (A) Scheme depicting the experiment performed in (B): the β-galactosidase activity of promoter fusions to OmrA, OmrB, ompC and ompF was measured upon overproduction of OmrA, OmrB or of their mut2 derivatives, in strains with a wt copy of ompR (gray bars) or with a mutant version carrying the ompRmut2 compensatory change to OmrA/Bmut2 (black bars). Strains used here are MG1014, MG1666, MG1017, MG1667, MG1901, MG1902, MG1686, MG1688 (panel B, see strain table for details). (C) Activity of the same set of promoter fusions was compared in omrAB⁺ and omrAB⁻ strains in the ompR107 background. Values are represented as % of wt strain for each fusion; the activities of the wt strains were, in Miller units, 135 (P_omrA fusion), 173 (P_omrB), 547 (P_ompF) and 5042 (P_ompC). Strains used are MG1301, MG1908, MG1305, MG1909, MG1696, MG1910, MG1895 and MG1911.

Figure 6.

Expression of omrA and omrB can be activated by non-phosphorylated OmpR. (A) The β-galactosidase activity of promoter fusions to omrA, omrB, ompC or ompF was measured in LB in exponentially growing cells with varying OmpR levels and/or phosphorylation. Activities are shown relative to that of wt cells. Strains used in this experiment are MG1690, MG1810, MG2043, MG2044 (P_ompF), MG1892, MG2018, MG2045, MG2046 (P_ompC), MG1004, MG1811, MG2039, MG2040 (P_omrA), MG1005, MG1812, MG2041 and MG2042 (P_omrB). The β-galactosidase activities of the different fusions in the wt strains were, in Miller units, 22 (P_omrA fusion), 63 (P_omrB), 11292 (P_ompC) and 3618 (P_ompF). (B) Activity of the same omrA, omrB and ompC transcriptional fusions was measured in wt cells or upon OmpR overproduction in ΔenvZ and ΔackA-pta background, both in omrAB⁺ or omrAB⁻ strains. Strains are MG1892, MG2203, MG2046 and MG2206 (P_ompC fusion), MG1004, MG2201, MG2040 and MG2204 (P_omrA), and MG1005, MG2202, MG2042 and MG2205 (P_omrB).

Figure 7.

Robustness and non-robustness of other OmpR targets. (A) The same RNA samples used in Figure 4D were analyzed by northern blot probed for ompF and for SsrA as a loading control. (B) MicF sRNA is involved in the post-transcriptional control of ompF upon OmpR overproduction. The β-galactosidase activity of P_tet-ompF-lacZ translational fusion was measured with and without IPTG induction of ompR overexpression in micF⁺ and micF⁻ cells (strains MG2127 and MG2167 respectively). (C) bolA mRNA levels were analyzed by northern blot in strains MG1004 (wt) transformed with pHDB3 or pOmpR, MG1811 (ΔompR) transformed with pHDB3 and in MG2000 (Tet-P_lac-ompR) with or without IPTG. SsrA levels were probed as well and used as a loading control. (D) Expression of a dtpA-lacZ promoter fusion was followed in a set of strains with various OmpR levels (MG2138 (wt), MG2169 (ΔompR) and MG2170 (Tet-P_lac-ompR)).

Figure 8.

Robustness and differential regulation within the OmpR regulon. (A) Regulatory circuit centered on the EnvZ-OmpR-OmrA/B feedback motif. Transcriptional and post-transcriptional regulations are in black and gray respectively, with arrows indicating activations and perpendicular bars indicating repressions. sRNAs are in gray. For simplicity, only gene products relevant to this study are indicated. Note that direct control has not been demonstrated in some aspects of this circuit (for instance, MicC control by OmpR, or OmpR autoregulation). (B) Differential regulation of robust or non-robust OmpR targets by the non phosphorylated form of OmpR. See text for more details.