The Small RNA MicF Represses ObgE and SeqA in Escherichia coli

Abstract

Small regulatory RNAs (sRNA) have been shown to play a large role in the management of stress responses in Escherichia coli and other bacteria. Upon fluctuations in nutrient availability and exposure to antimicrobials and superoxide-generating agents, the MicF sRNA in E. coli has been shown to regulate a small set of genes involved in the management of membrane permeability. Currently, it is unknown whether MicF acts on other processes to mediate the response to these agents. Using an sRNA interaction prediction tool, we identified genes in E. coli that are potentially regulated by MicF. Through subsequent analysis using a sfGFP-based reporter-gene fusion, we have validated two novel targets of MicF regulation: ObgE, a GTPase crucial for chromosome partitioning, and SeqA, a negative modulator of DNA replication. Importantly, the interaction between MicF and these target mRNAs is contingent upon the presence of the RNA chaperone protein, Hfq. Furthermore, our findings affirm the role of MicF's conserved 5' seed pairing region in initiating these regulatory interactions. Our study suggests that, beyond its established role in membrane permeability management, MicF exerts control over chromosome dynamics in response to distinct environmental cues, implicating a more multifaceted regulatory function in bacterial stress adaptation.

Keywords: Escherichia coli; RNA; gene regulation.

Figure 1

Regulation of mRNA::sfGFP reporter gene fusions by MicF. Target mRNA sequences were cloned as sfGFP fusions on a low-copy vector (pSC101) and transcribed from the constitutive promoter J23118. Four fusions were built for each target, with either 5, 10, 20, or 40 codons beyond the predicted MicF binding site fused to sfGFP. mRNA::sfGFP fusions were transformed into E. coli BW25113 with either a MicF overexpression plasmid (pMicF) or a control (pControl) on a medium-copy vector (p15A) also transcribed from the J23118 promoter. sfGFP fluorescence and OD₆₀₀ was measured for each condition. Each bar represents the ratio of average fluorescence/optical density (FL/OD₆₀₀) between cells harboring pMicF and pControl. mRNA::sfGFP fusions for ompF and lrp were included as references. Error bars were calculated from six biological replicates (see Figure S1).

Figure 2

MicF represses ObgE and SeqA expression. Western blot analysis of SeqA-3xFLAG (A,B) or ObgE-3xFLAG (C) protein expressed in E. coli BW25113 ΔmicF transformed with either a MicF overexpression plasmid (pMicF) or control (pControl) on a medium-copy vector (p15A) or high-copy vector (ColE1), as indicated. (D) Protein bands from (A–C) were analyzed via Image J and normalized to total protein in each lane determined from the Ponceau S stain. Bars show mean values and error bars represent the standard deviations of the three replicates. A two-tailed t-test was used to compare pControl and pMicF conditions. The significance is marked by asterisks above the pMicF bars indicating p < 0.01 (**) or p < 0.001 (***).

Figure 3

MicF’s regulation of obgE and seqA is dependent on the chaperone protein Hfq. Cell-free protein expression reactions were run with obgE::sfgfp or seqA::sfgfp plasmids with or without plasmids expressing MicF and Hfq. Shaded regions represent the standard deviations from three independent reactions calculated at each time point.

Figure 4

Compensatory mutations confirm MicF binding to obgE and seqA. (A) The sequence for the 5′ end of MicF, indicating the C to G mutations made for MicF-M1 (at C6) and MicF-M2 (at C15). (B) Bars show mean values of fluorescence/optical density (FL/OD₆₀₀) from cells with obgE::sfGFP, seqA::sfGFP, or their mutants in the presence of a control plasmid (pControl) or one that overexpresses a MicF variant. Plasmids were transformed into E. coli BW25113. Error bars represent the standard deviations of six biological replicates, shown as open circles.

Figure 5

Identifying the base pair regions required for MicF’s regulation of obgE and seqA. (A) The sequence for the 5′ end of MicF and its seed pairing region (in magenta). Lines indicate the nucleotides predicted to interact with the mRNAs of obgE and seqA. A more detailed representation of the predicted interactions between obgE and seqA is displayed in Figure S5. (B) Schematics of the MicF variants. The magenta box represents MicF’s 13-nucleotide seed region. SS is the SgrS scaffold used for the testing of truncated MicF segments. (C) Bars show mean values of fluorescence/optical density (FL/OD₆₀₀) from cells with obgE::sfGFP or seqA::sfGFP plasmids in the presence of a control plasmid (pControl) or one that overexpresses a MicF variant. Plasmids were transformed into E. coli BW25113. Error bars represent the standard deviations of six biological replicates, shown as open circles.

Figure 6

Contribution of the RNA degradosome in regulation by MicF. Bars show mean values of fluorescence/optical density (FL/OD₆₀₀) from cells with obgE::sfGFP, seqA::sfGFP, or ompF::sfGFP plasmids in the presence of a control plasmid (pControl) or one that overexpresses MicF (pMicF). Plasmids were transformed into strains of E. coli BW25113 mutated to remove either the C-terminal half of RNase E (rne-131), PNPase (Δpnp), or RhlB (Δrhlb). Error bars represent the standard deviations of six biological replicates, shown as open circles. A two-tailed t-test was performed between the pControl and pMicF conditions. Significance is marked by asterisks above the pMicF bar, indicating p < 0.001 (***).

Figure 7

Effect of chromosomally expressed MicF on mRNA::sfGFP fusions. (A). E. coli BW25113 wild type (WT) or ΔmicF was transformed with obgE::sfGFP or seqA::sfGFP plasmids and grown in LB or M9. (B) E. coli BW25113 wild type (WT) or ΔmicF was transformed with obgE::sfGFP or seqA::sfGFP plasmids and grown in M9 with or without H₂O₂. Bars show the mean values of fluorescence/optical density (FL/OD₆₀₀) and error bars represent the standard deviations of six biological replicates shown as open circles. A two-tailed t-test was performed between adjacent conditions in each graph. Significance is marked by asterisks above the light gray bar in the comparison indicating p < 0.01 (**) or p < 0.001 (***).

Figure 8

Proposed model for regulation of ObgE and SeqA by MicF. (A). Under oxidative stress MicF prevents translation of ObgE. (B). Under nutrient-rich conditions MicF prevents translation of SeqA.