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. 2022 Mar 8;119(10):e2117930119.
doi: 10.1073/pnas.2117930119. Epub 2022 Mar 3.

Dual-function AzuCR RNA modulates carbon metabolism

Medha Raina  1 , Jordan J Aoyama  1   2 , Shantanu Bhatt  1 , Brian J Paul  1 , Aixia Zhang  1 , Taylor B Updegrove  3 , Juan Miranda-Ríos  1 , Gisela Storz  1
Affiliations

Dual-function AzuCR RNA modulates carbon metabolism

Medha Raina et al. Proc Natl Acad Sci U S A. .

Abstract

SignificanceWhile most small, regulatory RNAs are thought to be "noncoding," a few have been found to also encode a small protein. Here we describe a 164-nucleotide RNA that encodes a 28-amino acid, amphipathic protein, which interacts with aerobic glycerol-3-phosphate dehydrogenase and increases dehydrogenase activity but also base pairs with two mRNAs to reduce expression. The coding and base-pairing sequences overlap, and the two regulatory functions compete.

Keywords: Hfq; ProQ; catabolite repression; glycerol dehdrogenase; sRNA.

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Conflict of interest statement

Competing interest statement: M.R. and C.V. are coauthors on a review published in 2018.

Figures

Fig. 1.
Fig. 1.
Discordance between AzuC protein and AzuCR RNA levels. (A) Diagram of the AzuCR RNA, overexpression constructs generated, and sequence of the promoter and coding region. Light blue boxes and font denote AzuC coding sequence, yellow box and highlighted text denote region of AzuR base pairing with target mRNAs, and gray arrow and bold black font denote the rest of the AzuCR RNA sequence. The start of the AzuCR transcript is indicated in green font (position 1988001 of the E. coli K-12 genome) and the 3′ end of the transcript is in red font. The ribosome binding site and the start and stop codons of the AzuC ORF are indicated by black boxes. Potential σ70-10 and -35 sequences are underlined, the predicted CRP binding sites are highlighted in light gray (17), and the region targeted by the FnrS sRNA is highlighted in dark gray. (B) Immunoblot blot analysis of AzuC-SPA levels (Top) in a azuC-SPA::kan strain (GSO351) and Northern blot analysis of AzuCR RNA levels (Bottom) in MG1655. Cultures were grown in M63 medium supplemented with glucose, glycerol, or galactose at pH 7.0 or glucose or glycerol at pH 5.5. Samples were taken at OD600 ∼0.5 and 1.5. α-FLAG antibody was used to detect the SPA tag. The membrane was stained with Ponceau S stain to control for loading. The AzuCR RNA and 5S RNA were detected by oligonucleotide probes specific to each of these transcripts.
Fig. 2.
Fig. 2.
Membrane-associated AzuC protein copurifies with GlpD. (A) Helical wheel projection generated using NetWheels (37) showing amphipathic nature of AzuC. Mutations introduced in C are indicated. (B) Fractionation of AzuC-SPA strain. A culture expressing AzuC-SPA (GSO351) was grown in M63 glucose medium to OD600 ∼0.5, and cells were fractionated into a soluble, inner membrane, and pellet fractions, which were compared to the whole-cell lysate. The Upper panel shows AzuC-SPA as detected with α-FLAG antibody. The Lower panel shows the outer membrane OmpA control detected with α-OmpA antibody. (C) Microscopy of AzuC-GFP. AzuC-GFP (GSO1008) and AzuCI6L7 to E6E7-GFP (GSO1009) cells were grown in M63 glucose medium to OD600 ∼0.5 to observe membrane localization by fluorescent microscopy. Left panels are fluorescent images showing GFP labeled AzuC, and Right panels are the corresponding brightfield images. Insets provide a 2.3× enlargement of a few cells. (D) GlpD copurifies with AzuC-SPA. Cells expressing AzuC-SPA (GSO351) or AcrZ-SPA (GSO350) from the chromosome were grown in M63 glucose medium to OD600 ∼1.0 or in LB to OD600 ∼0.6, respectively. The cell lysates were split and passed over calmodulin beads. Eluants from each column were subjected to SDS/PAGE followed by Coomassie blue staining. The bands enriched in the eluant from the calmodulin beads and indicated by the arrows were excised from the gel, and proteins were identified by mass spectrometry. (E) AzuC-SPA copurifies with GlpD-HA-His6. Cells expressing either AzuC-SPA (GSO351) or GlpD-HA-His6 (GSO1011) from the chromosome were grown in M63 glucose or M63 glycerol media, respectively, to OD600 ∼1.0 and mixed in a 1:1 ratio. As a control, cells expressing MgtA-HA (GSO785) grown in N medium supplemented without added MgSO4 to OD600 ∼0.5, were mixed with the AzuC-SPA (GSO351) cells in the same ratio. The mixed cells were homogenized, cell lysates (L) were applied to α-HA beads and the flow-through (FT) samples were collected. The beads were washed (W), after which the bound proteins were eluted (E) and examined on immunoblots using either α-HA antibodies to detect MgtA-HA or GlpD-HA-His6 (Upper) or α-FLAG antibodies to detect AzuC-SPA (Lower).
Fig. 3.
Fig. 3.
AzuC increases GlpD activity and membrane association. (A) Effect of AzuC overexpression on GlpD activity. WT or ΔazuCR::kan (GSO193) (Top), or ΔazuCR::kan transformed with pKK, pKK-AzuC, or pKK-AzuCL3STOP (Middle), or ΔazuCR ΔglpD::kan (GSO1015) transformed with the same plasmids (Bottom) were grown in M63 glucose medium to OD600 ∼1.0. Cells were then washed and resuspended in M63 glycerol medium, pH 5.5 for 3 h. MTT was added, and A570, reflecting the reduction of MTT to formazan, which is coupled to the oxidation of glycerol-3-phosphate to DHAP, was measured. Lines correspond to the average of three biological replicates, and error bars represent 1 SD. (B) Effect of ΔazuCR on GlpD-YFP membrane localization. WT (GSO1116) and ΔazuCR (GSO1117) cells expressing GlpD-YFP (38) grown in M63 glycerol pH 5.5 medium to OD600 ∼1.5 were examined by fluorescence microscopy. MG1655 cells grown in the same medium and mixed 1:1 with cells from either of the two GlpD-YFP strains prior to microscopy served as a control. Example images and quantitation of the membrane signal for 40 cells are shown (circles represent average of 10 cells for 4 different samples). Insets provide a 2.1× enlargement of a few cells. (C) Effect of AzuC overexpression on E. coli cell morphology. ΔazuCR::kan (GSO193) transformed with pKK, pKK-AzuC, pKK-AzuCL3STOP, or pKK-GlpD were grown in M63 glucose medium to OD600 ∼1.0. Cells were washed and resuspended in M63 glycerol medium, pH 5.5 for 3 h prior to microscopy. Example images are shown.
Fig. 4.
Fig. 4.
AzuC and AzuCR overexpression lead to different growth phenotypes. OD600 of the ΔazuCR::kan strain (GSO193) transformed with pKK, pKK-AzuC, pKK-AzuCL3STOP, pRI, pRI-AzuCR, or pRI-AzuCRL3STOP in M63 medium with glucose pH 7.0, glycerol pH 7.0, glycerol pH 5.5, or galactose pH 7.0, was measured 16 h after dilution. Bars correspond to the average of three biological replicates with circles corresponding to individual data points and error bars representing 1 SD. Representative growth curves are shown in SI Appendix, Fig. S4A.
Fig. 5.
Fig. 5.
AzuCR represses cadA and galE expression. (A) AzuCR-cadA base pairing predicted by TargetRNA2 (27). AzuCR coordinates are relative to the +1 of the transcript while cadA coordinates are relative to the first nucleotide of the start codon. (B) Effect of AzuCR and AzuCRL3STOP overexpression on a cadA-gfp fusion in WT and ΔazuCR backgrounds. WT and ΔazuCR::kan (GSO193) cells were cotransformed with a reporter plasmid expressing a cadA-gfp translational fusion and either the empty pRI vector, AzuCR, or AzuCRL3STOP. (C) Test of AzuCR-cadA base pairing. ΔazuCR::kan (GSO193) cells were cotransformed with the WT cadA-gfp translational fusion reporter plasmid or a M1 derivative along with the empty pRI vector, AzuCRL3STOP, or mutant AzuCRL3STOP-M1. The mutations in the cadA-gfp translational fusion and AzuCRL3STOP are indicated (A). (D) AzuCR-galE base pairing predicted by IntaRNA (28). AzuCR coordinates are relative to the +1 of the transcript while galE coordinates are relative to the first nucleotide of the start codon. (E) Effect of AzuCR and AzuCRL3STOP overexpression on a galE-gfp fusion in WT and ΔazuCR backgrounds. WT and ΔazuCR::kan (GSO193) cells were cotransformed with a reporter plasmid expressing a galE-gfp translational fusion and either the empty pRI vector, AzuCR, or AzuCRL3STOP. (F) Test of AzuCR-galE base pairing. ΔazuCR::kan (GSO193) cells were cotransformed with the WT galE-gfp translational fusion reporter plasmid or a M2 derivative along with the empty pRI vector, AzuCRL3STOP, or mutant AzuCRL3STOP-M2. The mutations in the galE-gfp translational fusion and AzuCRL3STOP are indicated in D. For (B, C, E, and F), cells were grown in LB for 3 h before measuring the fluorescence corresponding to GFP expression. Bars correspond to the average of three biological replicates with circles corresponding to individual data points and error bars representing 1 SD. (G) AzuCR coimmunoprecipitation with Hfq and ProQ. Extracts from MG1655 (GSO982), Δhfq::cat-sacB (GSO954), and ΔproQ::kan (GSO956) cells grown in M63 glucose were incubated with α-Hfq or α-ProQ antiserum. Total and RNA chaperone-bound RNA was extracted and subjected to Northern analysis using an oligonucleotide probe specific for AzuCR. (H) Effect of Δhfq::kan (GSO955), ΔproQ::kan (GSO956) and Δhfq ΔproQ::kan (GSO959) double mutant on cadA-gfp and galE-gfp expression upon overexpression of AzuCR or AzuCRL3STOP. Bars correspond to the average of three biological replicates with circles corresponding to individual data points and error bars representing 1 SD. (I) Immunoblot blot analysis of AzuC-SPA levels (Top) and Northern blot analysis of AzuCR RNA levels (Bottom) in WT (GSO351) and Δhfq (GSO1007) cells grown with different carbon sources. The WT or Δhfq derivatives of the azuC-SPA::kan (GSO351) strain (Top) or unmarked (MG1655) strain (Bottom) were grown and analyzed as in Fig. 1B.
Fig. 6.
Fig. 6.
AzuC and AzuR activities compete. (A) Effect of cadA, cadAcontrol, galE, and galEcontrol on AzuC-SPA levels in cells (GSO351) transformed with the respective overexpression plasmid and grown in M63 medium supplemented with glucose or galactose. Samples were taken at OD600 ∼0.5 and α-FLAG antibody was used to detect the SPA tag. The membranes stained with Ponceau S stain serves as a loading control. (B) Model for the different functions of the AzuCR RNA. For growth in M63 glycerol, pH 5.5, the RNA can be translated to give the 28-amino acid amphipathic AzuC protein, which increases the activity of GlpD glycerol-3-phosphate dehydrogenase. Under anaerobic conditions, translation of AzuC, as well as GlpD, is blocked by the FnrS sRNA. The RNA also can act as the AzuR base-pairing sRNA to repress synthesis of CadA and GalE.
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