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Article

The Small RNA MicF Represses ObgE and SeqA in Escherichia coli

by
Aaron Y. Stibelman
1,2,
Amy Y. Sariles
1 and
Melissa K. Takahashi
1,*
1
Department of Biology, California State University Northridge, Northridge, CA 91330, USA
2
Department of Biosciences, Rice University, Houston, TX 77005, USA
*
Author to whom correspondence should be addressed.
Microorganisms 2024, 12(12), 2397; https://doi.org/10.3390/microorganisms12122397
Submission received: 30 October 2024 / Revised: 17 November 2024 / Accepted: 19 November 2024 / Published: 22 November 2024
(This article belongs to the Section Molecular Microbiology and Immunology)
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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.

1. Introduction

Bacteria encounter a wide range of pressures in their natural environment such as nutrient availability, oxidative stress, and the presence of antimicrobials. Small regulatory RNAs (sRNAs) aid in the adaptation to these conditions by enabling bacteria to swiftly transition between different physiological states [1,2]. These regulatory RNAs also allow bacteria to efficiently allocate resources and maximize their survival in diverse environmental niches. sRNAs modulate the expression of trans-encoded mRNA by base pairing at locations near the 5′ untranslated region (UTR). The hybridization of sRNAs may alter mRNA translation directly by affecting ribosome accessibility or indirectly by modifying mRNA stability [3,4,5]. An important aspect of sRNA-based gene regulation is that it occurs through imperfect base-pairing interactions that allow a single sRNA to act with regulatory plasticity, targeting genes across diverse pathways, linking different biological processes, and facilitating connections within unique cellular networks [2,5,6].
MicF was amongst the earliest chromosomally encoded sRNAs discovered and initially was only associated with the repression of the nonspecific outer membrane protein, OmpF [7,8]. MicF is a 93-nucleotide sRNA with a well-studied secondary structure [9]. MicF’s conservation across γ-proteobacteria [10,11] underscores its pivotal role in responding to various extracellular stresses. MicF expression is activated by the transcription factors Rob, MarA, and SoxS, and repressed by H-NS and the leucine-responsive transcription factor, Lrp [12]. In response to antibiotics or oxidative stress, among other things, MicF reduces outer membrane permeability, enabling survival [13,14,15,16]. Conversely, under nutrient-limiting conditions, MicF expression is suppressed to maximize OmpF production [17]. More recently, additional targets of MicF regulation in Escherichia coli were identified, including the mRNAs lrp, cpxR, phoE, and oppA [18,19]. These genes expanded MicF’s role in membrane permeability management and suggested a larger role for MicF in metabolism via repression of Lrp, which is responsible for the regulation of approximately 10% of genes in E. coli [20].
To further explore the regulatory plasticity of MicF, we used sRNA target prediction tools to identify new candidates for MicF regulation. Using mRNA-sfGFP fusions, Western blot analysis, and cell growth experiments, we identify two additional targets of MicF regulation, obgE and seqA. The repression of both genes by MicF suggests two additional roles for MicF regarding chromosome replication during conditions of nutrient abundance and oxidative stress.

2. Materials and Methods

2.1. Growth Conditions

All strains were grown in Lysogeny broth (LB), LB agar (1.5%) plates, MOPS EZ Rich Defined Media (Teknova M2105, Hollister, CA, USA), or M9 at 37 °C. The M9 media contained: 1 × M9 salts, 1 mM thiamine hydrochloride, 0.4% glycerol, 0.2% casamino acids, 2 mM magnesium sulfate, and 0.1 mM calcium chloride. When necessary, media was supplemented with antibiotics (carbenicillin: 100 μg/mL, chloramphenicol: 34 μg/mL, kanamycin: 50 μg/mL).

2.2. Strains

The E. coli strains used in this study are listed in Table S1. Deletion strains were constructed using the lambda red recombination method [21]. DNA fragments containing a KanR cassette flanked by two FRT sites were amplified from pKD4 with the appropriate flanking sequences and electroporated into BW25113 for the construction of ΔmicF and rne-131. The construction of the C-terminal 3xFLAG-tagged strains were carried out similarly using the following modification. The DNA fragments contained a 3xFLAG tag followed by a FRT flanked CmR cassette. DNA fragments were PCR amplified and electroporated into the ΔmicF strain. Removal of KanR or CmR was achieved using the helper plasmid pCP20 following Datsenko and Wanner [21]. All deletions and insertions were confirmed using PCR and Sanger DNA sequencing. The primers used for creating the deletion strains or 3xFLAG tag strains are listed in Table S1.

2.3. Plasmids

All plasmids used in this study are listed in Table S3, with key sequences found in Table S2. Plasmids were constructed using inverse PCR or Gibson assembly. Gene sequences were PCR amplified from purified genomic DNA. New England Biolabs (Ipswich, MA, USA) Turbo E. coli cells was used for the transformation of constructed plasmids. All plasmids were verified using Sanger DNA sequencing.

2.4. CopraRNA and TargetRNA2

The web-accessible programs CopraRNA and TargetRNA2 were used to identify target genes regulated by MicF in E. coli. The organisms included in the CopraRNA query were E. coli NC 000913, Citrobacter koseri NC009792, Citrobacter rodentium NC_013716, Escherichia fergusonii, NC 011740, and Salmonella enterica subsp. enterica serovar Typhimurium NC 003197. The CopraRNA program was run under default conditions. The CopraRNA default consists of sequences extracted around the start codon (200 nucleotides upstream, 100 nucleotides downstream), a dynamic setting for p-value, and no p-value filtering or consensus prediction. For the TargetRNA2, the MicF sequence was obtained from Genbank NC_000913 and screened against the genome of E. coli str. K-12 substr. MG1655.

2.5. Fluorescence Measurement and Culturing Conditions

Plasmids were transformed into chemically competent E. coli strains, plated on LB-agar plates containing chloramphenicol and carbenicillin, and incubated overnight at 37 °C. Individual colonies from each condition were inoculated into 300 μL of LB with corresponding antibiotics in a 2 mL 96-well block (Corning Costar 3961, Corning, NY, USA) sealed with a breathe-easier membrane (USA Scientific 9126-2100, Madison, FL, USA) and grown for 17 h overnight at 37 °C while shaking at 100 rpm (Labnet Vortemp 56, Edison, NJ, USA). Four microliters of the overnight culture were added to 296 μL of MOPS media that was pre-warmed at 37 °C for 30 min. The cultures were grown under the same conditions as above for 2.5 or 3 h depending on the strain used. This resulted in cultures with an OD600 of 1–2.2. One hundred microliters of each culture were transferred to a 96-well plate (Corning Costar 3631, Corning, NY, USA), and OD600 and bulk sfGFP fluorescence (485 nm excitation, 520 nm emission) were measured using a Biotek Synergy H1 plate reader. Each experiment included two sets of controls: a media blank and E. coli transformed with control plasmids. The OD600 and FL for each culture were first corrected by subtracting the mean value of the media blank. The ratio of the corrected FL and OD600 (FL/OD600) was calculated for each culture. The E. coli cultures transformed with control plasmids were used to correct for autofluorescence. The average FL/OD600 from the control cultures was subtracted from FL/OD600 from each condition.
For experiments assessing endogenously expressed MicF, reporter plasmids were transformed into chemically competent E. coli strains, plated on LB-agar plates containing chloramphenicol, and incubated overnight at 37 °C. Individual colonies from each condition were inoculated into 300 μL of LB or M9 and grown overnight as above. Subcultures grown in LB were grown for 2.5 h, while subcultures grown in M9 were grown for 4 h.

2.6. Determining Cell Doubling Time

E. coli strains were isolated on LB-agar plates containing appropriate antibiotics if necessary. Six individual colonies from each condition were inoculated into 300 μL of LB with corresponding antibiotics, if necessary, in a 2 mL 96-well block (Costar 3961) sealed with a breath-easier membrane and grown for 17 h overnight at 37 °C while shaking at 100 rpm (Labnet Vortemp 56). Four microliters of the overnight culture were added to 296 μL of LB (no antibiotics for all strains) and grown under the same conditions for one hour. From that point, 2 μL from each culture was removed every 20 min and used to determine the CFU/mL via dilutions and plating. The doubling time for an individual colony was determined by taking the log base 2 of each CFU/mL value, then determining the slope log2(CFU/mL) vs. time and taking the inverse of that slope value. Doubling times were averaged across the six colonies. For the ΔmicF pMicF pSeqA condition, seqA was transcribed from the inducible promoter pLux [22] and induced with 10 μM N-acyl homoserine lactone (Cayman Chemical 10011207, Ann Arbor, MI, USA).

2.7. Protein Purification and Western Blot

E. coli cultures were grown overnight at 37 °C while shaking at 275 rpm in LB supplemented with the appropriate antibiotics. Cells were diluted 100-fold in LB and grown to an OD600 of approximately 0.5. Cell pellets corresponding to 1 mL of each culture were resuspended using 4× Laemmli buffer and heated for 4 min at 95 °C. For each sample, 10 μL was separated on a 10% SDS-PAGE gel and transferred to a 0.2 μM PVDF membrane (Sigma Aldrich 0301004001, St. Louis, MO, USA) at 40 mA overnight at 4 °C with stirring. Protein expression was visualized using monoclonal anti-FLAG antibodies and HRP (ThermoFisher Scientific MA1-91878-HRP, Waltham, MA, USA) and developed using a Pierce Fast Western Kit SuperSignal West Pico Mouse (ThermoFisher Scientific 35060, Waltham, MA, USA). Blots were developed in SuperSignal West Pico Working Solution for five minutes and imaged using an iBright 1500 (ThermoFisher Scientific, Waltham, MA, USA). The images were processed and quantified using Image J (https://imagej.net/ij/ accessed on 29 October 2024, version 1.54g) software. The intensities of the MicF target protein bands were normalized with the intensities of the corresponding total protein visualized via Ponceau S staining.

2.8. Cell-Free Protein Expression to Determine Hfq Dependency

PURExpress (New England Biolabs, E6800, Ipswich, MA, USA) reactions were prepared according to manufacturer’s protocol. Plasmids were provided at the following concentrations: mRNA::sfgfp (1 nM), MicF (4 nM), and Hfq (4 nM). SUPERase-In RNase inhibitor (Thermo Fisher Scientific AM2696, Waltham, MA, USA) was added to each reaction (0.25 μL/10 μL reaction). Eight microliters of each reaction was added to a 384-well plate (Thermo Scientific 142761, Waltham, MA, USA), covered with a seal (VWR 60941-078, Radnor, PA, USA), and placed on a Biotek SynergyH1 plate reader. The temperature was controlled at 37 °C, and sfGFP fluorescence was measured from the bottom of the plate every five minutes (485 nm excitation, 520 nm emission).

3. Results

3.1. Identification of MicF Regulated mRNA Targets

To identify novel E. coli mRNA as candidates for regulation by MicF, we utilized two sRNA target prediction tools, CopraRNA [23] and TargetRNA2 [24]. Both tools have previously been shown to accurately predict in vivo mRNA targets of bacterial sRNAs by generating RNA-RNA interactions after accounting for the intramolecular accessibility of each RNA and the phylogenetic conservation of each interaction. When evaluating the top results from both algorithms (Tables S4 and S5), only CopraRNA predicted known targets of MicF regulation in E. coli (ompF, lrp, oppA). Therefore, we restricted our selection of candidate mRNA targets to those predicted by the CopraRNA algorithm. We selected five of the top 10 hits (obgE, seqA, hofQ, mgrB, hypB) for experimental validation by excluding those that have previously been validated (lrp [18], oppA [19], ompF [25]), invalidated (murG [18]), or had uncharacterized function (ysaB). We cross-referenced the five candidate mRNAs with published experimental data sets and found that none of the targets were identified using RIL-seq (RNA interaction by ligation and sequencing) [26], while two of the targets (obgE and seqA) were identified using MAPS (MS2 affinity purification coupled with RNA sequencing) [19].
To evaluate MicF’s ability to regulate the mRNA targets predicted by CopraRNA, we built translational fusions of the mRNAs with superfolder green fluorescent protein (sfGFP) following the work of Urban and Vogel [27] and Corcoran et al. [11]. When testing an sRNA’s ability to regulate an mRNA, it is common to truncate the coding sequence (CDS) of the target when fusing it to the reporter protein [27,28]. As the length of the fused coding sequence could impact both sRNA binding and sfGFP protein stability, we built four variants for each target that included either 5, 10, 20, or 40 codons downstream of the predicted MicF interaction site. In the case of mgrB, a maximum of only 34 codons were included due to the small size of its CDS. For the predicted monocistronic targets (seqA and mgrB), the entire 5’ UTR was incorporated into the fusion. For targets predicted within an operon (obgE, hofQ, and murG), a truncated lacZ sequence was placed upstream of the target mRNA to mimic the transcription and translation of a polycistronic mRNA [27]. MicF was predicted to interact with obgE downstream of the stop codon of the previous gene in its operon, thus the stop codon was placed at the end of the lacZ sequence, followed by the obgE 5’ UTR. For hofQ and murG, MicF was predicted to interact with the sequence within the CDS of the previous gene, thus the sequence starting 30 nucleotides upstream of the predicted interaction site was fused to the lacZ sequence.
The expression of each candidate mRNA target was evaluated in the presence and absence of MicF by transforming each mRNA::sfGFP plasmid into E. coli BW25113 along with either a control plasmid (pControl) or one that constitutively transcribes MicF (pMicF). The known targets ompF::sfGFP and lrp::sfGFP were included for comparison. Of the five genes tested, obgE::sfGFP and seqA::sfGFP had variants that were repressed at least two-fold by MicF (Figure 1). The 20-codon fusion for seqA was the only one of its variants that was repressed by MicF. Upon folding the seqA fusion mRNA’s in NUPACK [29,30], we discovered that the other three fusion lengths were predicted to form secondary structures that would block the region MicF was predicted to bind (Figure S2). Although several mgrB::sfGFP variants showed increased expression in the presence of MicF, we chose to only pursue targets that exhibited at least a two-fold change in expression. The obgE 5-codon and the seqA 20-codon fusions were used for all subsequent experiments.
To confirm that MicF represses the expression of ObgE and SeqA in vivo, we performed a Western blot analysis by chromosomally inserting a 3xFLAG epitope at the C-terminal end of either the obgE or seqA CDS in their native locus and used anti-FLAG antibodies to observe the protein levels in a strain of E. coli with the MicF gene deleted. The production of ObgE::3xFLAG and SeqA::3xFLAG was compared between a strain complemented with MicF from a medium-copy plasmid (pMicF) and a strain transformed with a control plasmid (pControl). The Western blot analyses confirmed that MicF negatively regulates ObgE, as its expression was decreased in the presence of MicF (Figure 2C,D). For SeqA, no significant difference was observed when MicF was overexpressed from the medium-copy plasmid (Figure 2A,D). However, a decrease in expression was observed when MicF was overexpressed from a high-copy plasmid (Figure 2B,D).

3.2. The RNA Chaperone Protein Hfq Is Required for MicF’s Inhibition of obgE and seqA

Due to their intrinsically weak posttranscriptional interactions with mRNA, many sRNA require the RNA chaperone protein Hfq to more stably interact with their targets [2,4]. Hfq supports RNA-RNA interactions by binding to both molecules and causing an increase in their local concentrations [31,32]. Additionally, as Hfq binds to each RNA, it facilitates the melting of intramolecular secondary structures, further promoting and identifying the correct sRNA-mRNA hybridization [33,34,35]. Since Hfq is required for regulation of MicF’s known targets [11,27], we investigated its necessity for the repression of obgE and seqA. We transformed the mRNA::sfGFP fusions with pMicF or pControl into a Δhfq strain of E. coli BW25113. In the Hfq deleted strain, MicF’s repression of both obgE::sfGFP and seqA::sfGFP was completely lost (Figure S3). However, it has been shown that MicF is unstable in a Δhfq strain [27,36]. Therefore, we utilized a cell-free protein expression system (PURExpress) that is manufactured from purified components [37] to assess the importance of Hfq. RNA degradasome components are not included in the system and the degradation of RNA is further limited with the addition of an RNase inhibitor (see Section 2). Plasmids for each mRNA::sfGFP construct were expressed in reactions with or without plasmids expressing MicF and Hfq. Repression of obgE::sfGFP was only observed when both MicF and Hfq were present in the reaction (Figure 3). Repression of seqA::sfGFP was observed with MicF alone and was further enhanced by the presence of Hfq (Figure 3). Together these results suggest that, like its other targets, MicF’s inhibition of obgE and seqA expression is dependent on the presence of Hfq.

3.3. MicF’s 13-Nucleotide Seed Pairing Region Is Required for the Repression of obgE and seqA

To confirm that the regulation of obgE and seqA was occurring due to the binding of MicF, we introduced two different mutations into the MicF sequence along with compensatory mutations to the predicted binding regions within the obgE::sfGFP and seqA::sfGFP constructs. MicF-M1 changed the sixth nucleotide of MicF from a C to a G, while MicF-M2 changed the fifteenth nucleotide of MicF from a C to a G (Figure 4A). Repression of obgE::sfGFP and seqA::sfGFP was no longer observed in the presence of pMicF-M1 or pMicF-M2. For obgE, both mutant targets (obgE-M1’::sfGFP and obgE-M2’::sfGFP) were not repressed by wild-type MicF but were repressed by the respective MicF mutant. For seqA, the M2 mutant was not repressed by wild-type MicF and was repressed by MicF-M2. However, seqA-M1’::sfGFP was not repressed by either wild-type MicF or MicF-M1 (Figure 4B). We note that the fluorescence from cells expressing the seqA-M1’::sfGFP construct along with pControl was approximately 2.5 times lower than the seqA::sfGFP construct, and the predicted secondary structure of the seqA-M1’::sfgfp mRNA indicated that the C to G mutation may induce a structure that sequesters the availability of the predicted MicF binding region (Figure S4).
To further explore the binding between MicF and the two mRNAs, we examined the importance of MicF’s seed pairing region. A seed pairing region is an unstructured region that initiates base pairing with target mRNAs and is a feature of many sRNAs [38]. The seed pairing region of MicF consists of a conserved 13 nucleotides at the 5′ end that can fully regulate the mRNAs lrp and ompF when fused to an unrelated sRNA backbone [11,39]. To explore the role of MicF’s seed pairing region in the repression of obgE and seqA, we designed a synthetic sRNA composed of two parts: the first 13 nucleotides of MicF and the Hfq binding scaffold of an unrelated sRNA (SgrS) that has been used in the engineering of synthetic Hfq-dependent sRNAs [40,41,42] (Figure 5B). To test these constructs, we transformed E. coli BW25113 cells with plasmids expressing one of two MicF variants. The first is composed of the seed pairing region of MicF fused to the 5′ end of the SgrS scaffold (MicF(1–13) SS), and the second has the first 13 nucleotides of MicF deleted (MicF Δ(1–13)). The repression of both obgE::sfGFP and seqA::sfGFP was eliminated in the absence of MicF’s seed pairing region. However, the seed pairing region alone was not capable of fully repressing either gene (Figure 5C).
Next, we sought to identify the portion of MicF that would be sufficient for the repression of obgE and seqA. CopraRNA predicts that MicF binds to obgE in several different regions through nucleotide 51 of MicF, while only nucleotides 5–19 are predicted to interact with seqA (Figure S5). We built two new plasmid constructs where nucleotides 1–51 or 1–19 were fused to the 5′ end of the SgrS scaffold, pMicF(1–51) SS and pMicF(1–19) SS, respectively. While pMicF(1–19) SS sufficiently repressed seqA::sfGFP, pMicF(1–51) SS only moderately repressed obgE::sfGFP (Figure 5C). To further investigate obgE regulation, we considered the possibility that not all predicted interactions take place. CopraRNA does not account for Hfq binding, which is predicted to occur somewhere within the region that spans nucleotides 28 to 93 of MicF [43]. Thus, in MicF(1–51) SS, nucleotides 28–51 may interfere or compete with the SgrS scaffold and reduce its accessibility to Hfq. Since one way sRNAs regulate expression is through occlusion of the ribosome binding site (RBS), we used the RBS Calculator [44] to predict the RBS of obgE and fused the nucleotides of MicF that were predicted to bind through the RBS to the SgrS scaffold (pMicF(1–30) SS). Using this variant, we observed complete repression of obgE::sfGFP (Figure 5C), suggesting that regulation of obgE may occur through an interference with ribosome accessibility.

3.4. The Repression of obgE and seqA Is Not Influenced by RNase E Mediated Decay

While the first 19 nucleotides of MicF were sufficient to repress seqA::sfGFP, those nucleotides are predicted to bind within the CDS of seqA, downstream of both the RBS and start codon (Figure S5). This suggests an alternative mechanism for seqA repression that does not involve ribosome occlusion. Since sRNAs may control protein expression by modifying the stability of the mRNA transcript, we explored whether MicF exacerbates the decay of the seqA transcript by inducing its degradation by RNase E. In Salmonella, MicF represses lpxR through a dual mechanism involving the inhibition of ribosome accessibility and the stimulation of degradation by RNase E [11]. Therefore, we also probed MicF’s ability to induce the degradation of the obgE transcript by RNase E.
In E. coli, RNase E is one of the major enzymes involved in the formation of the multi-protein RNA degradosome. The N-terminal half of RNase E contains its endoribonuclease activity while the C-terminal half is natively unstructured and serves as a scaffold for the binding of the other degradosome components: PNPase, RhlB, and enolase [45]. The C-terminal domain of RNase E also interacts with Hfq, and this interaction is necessary for the sRNA-mediated degradation of mRNA [46]. To determine whether MicF’s repression of obgE and seqA involves Hfq-dependent RNase-E-mediated decay, we utilized a strain of E. coli where the entire C-terminal scaffold of RNase E is deleted (rne-131) [47,48]. Prior research has shown that MicF is stably expressed in the RNase-E-mutant strains rne-131 [19] and rne-701 [27]. The rne-701 strain has another C-terminal truncation that prevents interaction with Hfq and degradosome assembly. In the rne-131 strain, neither obgE::sfGFP nor seqA::sfGFP was fully repressed by MicF (Figure 6).
The rne-131 truncation also prevents PNPase and RhlB from associating with RNase E. PNPase is a 3′ exoribonuclease, while RhlB is a DEAD-box RNA helicase. Both aid in the decay of the RNA intermediates created by RNase E and have reduced activity when not associated to RNase E [45]. Additionally, a complex between PNPase, Hfq, and the sRNA protects some sRNAs from degradation by RNase E [49,50]. Prior research demonstrated that the regulation of ompF by MicF does not depend on RNase E degradation [27]. We do observe a reduction in the fold repression of ompF::sfGFP in the rne-131 strain (12-fold, Figure 6) as compared to the wild-type strain (30-fold, Figure S1). The effect seen on the regulation of all three mRNAs could be due to the reduced activity of PNPase and RhlB or the general reduction in RNA degradation associated with the rne-131 strain. To assess this directly, we tested MicF regulation in strains that had an intact RNase E but deleted PNPase (Δpnp) or RhlB (Δrhlb).
If PNPase protects MicF from RNase E degradation, we would expect a decrease in MicF concentration and a reduction in regulation in the Δpnp strain. Furthermore, we may observe an increase in FL/OD from the mRNA::sfGFP fusions due to a decrease in chromosomally expressed MicF. While we do observe a decrease in regulation for all three target mRNAs, we also observe a decrease in FL/OD from the mRNA::sfGFP fusions in the pControl condition (Figure 6). Since Hfq-RNA complexes compete for binding to RNase E with RhlB [46,51], in the Δrhlb strain we would expect an increase in regulation if the mechanism were RNase E dependent. However, we observe a small decrease in regulation for all three target mRNAs (Figure 6). Together these results suggest RNase E is not involved in MicF’s repression of obgE::sfGFP and seqA::sfGFP and the observed reduction in regulation in the rne-131 strain could be due to a general reduction in RNA degradation.

3.5. Chromosomally Expressed MicF Represses obgE and seqA sfGFP Fusions

To evaluate the regulation of obgE and seqA without overexpressing MicF from a plasmid, we transformed our mRNA::sfGFP constructs into E. coli BW25113 wild-type cells or ΔmicF cells and grew them under conditions known to vary in MicF concentrations. First, we took advantage of MicF being highly expressed in cells grown in nutrient-rich media such as LB and poorly expressed in cells grown in nutrient-poor media such as M9 [18,52]. For both obgE::sfGFP and seqA::sfGFP, we observe an increase in FL/OD in ΔmicF cells when grown in LB as compared to wild-type cells. Furthermore, no change is observed in ΔmicF cells when grown in M9 (Figure 7A).

3.6. The obgE sfGFP Fusion Is Repressed by MicF upon Exposure to Hydrogen Peroxide

The concentration of MicF is also known to increase in the presence of hydrogen peroxide due to activation by SoxRS [53]. To determine whether MicF influences obgE or seqA expression in response to hydrogen peroxide, we compared BW25113 wild-type and ΔmicF cells transformed with the mRNA::sfGFP fusions when grown in M9 with or without hydrogen peroxide. A decrease in FL/OD was observed for obgE::sfGFP in the presence of hydrogen peroxide; however, no difference was noted for seqA::sfGFP (Figure 7B).

3.7. Overexpression of MicF Results in an Increase in Cell Doubling Time

In E. coli, the primary role of SeqA is to regulate the start of chromosome replication through its sequestration of hemimethylated origins of replication from the replication initiator protein DnaA [54]. Following replication, SeqA also aids in the cohesion of sister chromosomes prior to their segregation [55]. Mutants deficient in SeqA produce filamentous cells with unsegregated DNA and their doubling times are increased by approximately 20–30% [56,57]. To observe the effect of MicF overexpression, we measured the doubling time of several different strains (Table 1). An ANOVA was performed on the results, and the Holm method was used to control for the family-wise error rate (Table 2). ΔmicF cells (no plasmid) and ΔmicF cells complemented with a control plasmid (pControl) or a different sRNA (pMicC) had comparable doubling times to that of wild-type cells. Furthermore, ΔmicF cells that overexpressed MicF (ΔmicF pMicF) had increased doubling times when compared to the wild type. Although the average doubling time of ΔmicF pMicF was not as high as that of the ΔseqA cells, the two data sets were not statistically different. To determine if the increase in doubling time was due to MicF’s repression of seqA, we simultaneously overexpressed MicF and SeqA from a plasmid in the ΔmicF cells (ΔmicF pMicF pSeqA). The overexpression of SeqA resulted in doubling times comparable to that of the wild type, thus linking the observed phenotype to MicF’s repression of seqA.

4. Discussion

Here, we experimentally validated two additional mRNA targets of MicF (obgE and seqA) that were predicted by CopraRNA. Notably, these two targets were also identified by MAPS [19]. ObgE is an essential GTPase in E. coli that has roles in chromosome partitioning [58,59] and in the cellular response to amino acid starvation [60]. SeqA serves as a negative modulator during the initiation of chromosome replication [54] that also facilitates the segregation of newly replicated sister chromosomes [55]. We found that MicF represses the expression of both genes.
Our experiments confirmed that MicF regulates both genes through an Hfq-dependent antisense mechanism that requires the 5′ seed pairing region of MicF. Unlike the regulation of OmpF and Lrp [11], the seed pairing region alone was not sufficient to repress obgE::sfGFP and seqA::sfGFP (Figure 5). The regulation of ObgE included the first 30 nucleotides of MicF that are predicted to bind through the RBS and start codon of obgE. The seed region itself is not involved in the pairing with the RBS or start codon (Figure S5), which suggests its role is to initiate binding between the two RNAs. Part of MicF’s seed pairing region is predicted to bind to seqA, although the first 19 nucleotides were required to completely repress seqA::sfGFP. In the case of SeqA, MicF is only predicted to bind within the CDS and not the RBS or start codon. It is still possible that MicF inhibits translation initiation even though it does not bind to the RBS or start codon. Prior research investigating RybB repression of ompN in Salmonella demonstrated that translational control could occur if base pairing took place within a five-codon window [61]. MicF is predicted to bind to the seqA CDS starting within the fourth codon.
Besides occluding translation initiation, sRNAs are known to repress protein expression by promoting mRNA decay by RNase E or RNase III [62,63]. RNase III is known to degrade dsRNA or intramolecular duplexes formed within a single RNA; however, in either case the duplex must be of sufficient length, approximately 22 base pairs [64]. In the case of seqA, the predicted binding interaction is only 15 base pairs long, which makes it a less likely target. We assessed the role of RNase E; however, neither the regulation of seqA::sfGFP nor obgE::sfGFP appeared to be dependent on RNase E (Figure 6). We acknowledge that the native mRNAs were not assessed and our results only provide insight to the mRNA-sfGFP fusions. Given these limitations, we propose that MicF represses the translation of both ObgE and SeqA by preventing the formation of the translation initiation complex (Figure 8).
The regulation of ObgE and SeqA introduces a new role for MicF beyond the regulation of the outer membrane. ObgE is known to play a critical role in chromosome partitioning and is potentially used in a replication checkpoint response [59,65]. Notably, chromosome partitioning defects have been observed with a modest reduction in ObgE concentrations [58]. Thus, repression of ObgE by MicF, although small, could contribute to cell cycle arrest. This helps to explain a role of MicF when activated by SoxS. Transcription of soxS is activated by SoxR, which is induced by superoxide-generating agents or nitric oxide [66]. Early work showed that MicF repressed OmpF due to the activation of the soxRS locus, although the importance of OmpF suppression under oxidative stress conditions was not clear [15]. Strains deficient in soxRS were shown to be hypersensitive to chloramphenicol and nalidixic acid, which does suggest a direct role for repression of OmpF, since one way these antibiotics enter the cell is through OmpF [67,68]. However, elimination of OmpF did not increase the resistance of E. coli to menadione, another agent known to induce soxRS [15]. Other work showed only a modest increase in Lrp expression in a ΔmicF strain that was treated with paraquat [69]. Here, we observed repression of obgE::sfGFP in the presence of hydrogen peroxide (Figure 8A). Perhaps the role of MicF during oxidative stress is to suppress chromosome partitioning and work together with the OxyS sRNA to induce cell cycle arrest and provide time for DNA damage repair [70].
SeqA has also been tied to replication arrest; however, this occurs under conditions with high SeqA concentrations [71,72]. Instead, we propose a role for suppression of SeqA in high-nutrient conditions where MicF is already known to repress OmpF and Lrp [12,18]. Initiation of chromosome replication by DnaA is blocked by the binding of SeqA to hemimethylated oriC. This prevents immediate re-initiation and ensures a single round of replication per division cycle. However, under nutrient-rich conditions and faster growth rates it is known that E. coli can initiate a new round of replication before the previous round is complete [54,73]. The DnaA concentration increases with increasing growth rate [74] and a parallel decrease in SeqA concentration would aid in the initiation of another round of replication. Repression of SeqA under these conditions (Figure 8B) would complement MicF’s repression of OmpF and Lrp.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms12122397/s1. This article contains supporting information: Table S1: Strains used in this study; Table S2: Important DNA sequences; Table S3: Plasmids used in this study; Table S4: Top 15 CopraRNA results; Table S5: Top 15 TargetRNA2 results, and Figure S1: FL/OD600 measurements for data in Figure 1; Figure S2: NUPACK RNA structure predictions; Figure S3: Regulation of obgE::sfGFP or seqA::sfGFP in a Δhfq strain; Figure S4: Predicted secondary structure for seqA-M1’::sfGFP; Figure S5: CopraRNA predicted MicF-mRNA interaction for obgE and seqA. Ref. [75] can be found in Supplementary Materials.

Author Contributions

Conceptualization, A.Y.S. (Aaron Y. Stibelman) and M.K.T.; Methodology, A.Y.S. (Aaron Y. Stibelman), A.Y.S. (Amy Y. Sariles) and M.K.T.; Formal analysis, A.Y.S. (Aaron Y. Stibelman), A.Y.S. (Amy Y. Sariles) and M.K.T.; Investigation, A.Y.S. (Aaron Y. Stibelman), A.Y.S. (Amy Y. Sariles) and M.K.T.; Writing—Original Draft Preparation, A.Y.S. (Aaron Y. Stibelman) and M.K.T.; Writing—Review and Editing, A.Y.S. (Amy Y. Sariles); Visualization—A.Y.S. (Aaron Y. Stibelman) and M.K.T. All authors have read and agreed to the published version of the manuscript.

Funding

Funding for this research was provided by California State University Northridge start-up funds to M.K.T. The research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number TL4GM118977 to A.Y.S. (Amy Y. Sariles). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Data Availability Statement

Data is contained within the article or Supplementary Material. Dataset available on request from the authors.

Acknowledgments

We would like to thank Cristian Ruiz Rueda, Gilberto Flores, and Mariano Loza Coll (California State University Northridge) for experimental protocols and constructive feedback on this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Microorganisms 12 02397 g001
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 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).
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Microorganisms 12 02397 g002
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 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 (***).
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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 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.
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Microorganisms 12 02397 g004
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 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.
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Microorganisms 12 02397 g005
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 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.
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Microorganisms 12 02397 g006
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 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 (***).
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Microorganisms 12 02397 g007
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 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 (***).
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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.
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.
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Table 1. Average doubling time of E. coli variants 1.
Table 1. Average doubling time of E. coli variants 1.
StrainDT (min) ± STD
Wild type (BW25113)22.3 ± 1.6
ΔseqA29.7 ± 2.5
ΔmicF23.9 ± 2.4
ΔmicF pMicF27.6 ± 1.8
ΔmicF pMicF pSeqA25.1 ± 0.6
ΔmicF pControl24.1 ± 1.9
ΔmicF pMicC24.1 ± 1.8
1 Data from six biological replicates.
Table 2. Pairwise t-test with Holm p-value correction for doubling time data in Table 1.
Table 2. Pairwise t-test with Holm p-value correction for doubling time data in Table 1.
Wild TypeΔseqAΔmicFΔmicF pMicFΔmicF pMicF pSeqAΔmicF pControl
ΔseqA1.70 × 10−6 *
ΔmicF10.00014 *
ΔmicF pMicF0.00036 *0.768820.02492 *
ΔmicF pMicF pSeqA0.972550.00024 *10.03959 *
ΔmicF pControl0.972550.00024 *10.03959 *1
ΔmicF pMicC0.972550.00024 *10.03959 *11
* p-value < 0.05.
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Stibelman, A.Y.; Sariles, A.Y.; Takahashi, M.K. The Small RNA MicF Represses ObgE and SeqA in Escherichia coli. Microorganisms 2024, 12, 2397. https://doi.org/10.3390/microorganisms12122397

AMA Style

Stibelman AY, Sariles AY, Takahashi MK. The Small RNA MicF Represses ObgE and SeqA in Escherichia coli. Microorganisms. 2024; 12(12):2397. https://doi.org/10.3390/microorganisms12122397

Chicago/Turabian Style

Stibelman, Aaron Y., Amy Y. Sariles, and Melissa K. Takahashi. 2024. "The Small RNA MicF Represses ObgE and SeqA in Escherichia coli" Microorganisms 12, no. 12: 2397. https://doi.org/10.3390/microorganisms12122397

APA Style

Stibelman, A. Y., Sariles, A. Y., & Takahashi, M. K. (2024). The Small RNA MicF Represses ObgE and SeqA in Escherichia coli. Microorganisms, 12(12), 2397. https://doi.org/10.3390/microorganisms12122397

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