# A small regulatory RNA controls antibiotic adaptation in Staphylococcus aureus by modulating efflux pump expression

Kam Pou Ha, Etornam Kofi Kumeko, Philippe Bouloc

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Kam Pou Ha, Etornam Kofi Kumeko, Philippe Bouloc. A small regulatory RNA controls antibiotic adaptation in Staphylococcus aureus by modulating efflux pump expression. Antimicrobial Agents and Chemotherapy, 2025, 69 (5), pp.01176-24. 10.1128/aac.01176-24. hal-05023012

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Submitted on 14 Nov 2025

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Distributed under a Creative Commons Attribution 4.0 International License
# A small regulatory RNA controls antibiotic adaptation in *Staphylococcus aureus* by modulating efflux pump expression

Kam Pou Ha,¹ Etornam Kofi Kumeko,¹ Philippe Bouloc¹

**AUTHOR AFFILIATION** See affiliation list on p. 13.

**ABSTRACT** *Staphylococcus aureus* is an opportunistic pathogen that poses a considerable burden to healthcare settings worldwide, aided by its ability to thrive in different environmental growth conditions and survive exposure to antibiotics. Small regulatory RNAs (sRNAs) are decisive in enhancing bacterial fitness by modulating gene expression in response to changing environmental conditions. We investigated the role of sRNAs in the adaptation of *S. aureus* to antibiotics. By assessing the fitness of a library of sRNA mutants, we identified that RsA sRNA is required for optimal bacterial growth when exposed to low concentrations of fluoroquinolone, a class of antibiotics targeting DNA replication. We also found that in the absence of RsA, *S. aureus* is less susceptible to β-lactam antibiotics, which act on the cell wall. RsA has been reported to prevent the expression of MgrA, a master regulatory protein controlling the expression of efflux pumps. Here, we show that RsA affects the sensitivity of *S. aureus* to fluoroquinolone and β-lactam antibiotics through MgrA. RsA has two forms, a short one commonly referred to in RsA studies, and a long form about twice the length, of which less is known. Interestingly, our phenotype was only restored when complemented with the long form of the gene or when it was supplied in two parts, the short form and the missing part to obtain the long form. This work demonstrates the role of regulatory RNAs in the adaptation of *S. aureus* to antibiotic resistance and highlights their value as potential therapeutic targets for manipulating individual sRNA responses to promote the efficacy of existing antibiotics.

**KEYWORDS** *Staphylococcus aureus*, small regulatory RNA, RsA, fitness, antibiotic susceptibility, norfloxacin, ciprofloxacin, cefazolin, β-bactam antibiotics, MgrA

Antibiotic resistance refers to the ability of bacteria to grow in antibiotic concentrations that are considered inhibitory (1). Resistance involves the acquisition of resistance genes from other bacteria or by genetic mutation (1–3), leading to mechanisms such as enzymatic inactivation of antibiotics, alteration of antibiotic targets, or efflux pumps that expel antibiotics from bacterial cells (2, 3). By contrast, antibiotic tolerance is the prolonged survival of bacteria to bactericidal antibiotics for extended periods without necessarily acquiring genetic changes (1, 4). In an extreme subset of tolerance, also called persistence, bacterial cells may enter a dormant or near-dormant state to protect cellular processes against the action of antibiotics (5). Antibiotic persistence and tolerance, collectively termed recalcitrance, represent transient phenotypic changes in the bacterial population that increase survival in the presence of usually lethal antibiotic concentrations (6). Recalcitrance has been demonstrated as a prime starting point for the development of antibiotic resistance (7–9).

Although antibiotic resistance mechanisms have been well studied, global efforts have primarily focused on methicillin-resistant *Staphylococcus aureus*, which represent only a subset of the overall *S. aureus* burden (10). Indeed, treatment failure is common even when *S. aureus* isolates are sensitive to antibiotics (6), and both susceptible and

**Editor** Benjamin P. Howden, The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

Address correspondence to Philippe Bouloc, philippe.bouloc@i2bc.paris-saclay.fr.

The authors declare no conflict of interest.

See the funding table on p. 13.

Received 5 August 2024

Accepted 6 March 2025

Published 3 April 2025

Copyright © 2025 Ha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
resistant *S. aureus* infections lead to prolonged illness and increased mortality rates (10, 11). Therefore, understanding how *S. aureus* modulates its sensitivity to antibiotics in the absence of resistance could help in the development of novel treatments to reduce the incidence of both resistance and recalcitrance.

Small regulatory RNAs (sRNAs) are key players in the post-transcriptional regulation of target genes (12). They enable rapid environmental adaptation by base-pairing with their target mRNAs to modulate mRNA translation and stability (12). sRNAs act on almost all cellular functions and as a result, have emerged as important regulators of virulence (13), metabolic adaptation (14) and antibiotic resistance (15, 16). Although many sRNAs have been identified in *S. aureus*, their functions and targets are still mostly unknown (17, 18). The best-described sRNA in *S. aureus* is RNAIII, a large 514-nucleotide sRNA that is involved in virulence (19). Other described sRNAs include RsAE, which is associated with the TCA cycle (20, 21) and arginine catabolism (14), and IsrR, which plays a key role in the iron-sparing response (22, 23). Although sRNAs play important roles in regulating antibiotic-resistance processes such as cell envelope modifications and drug efflux pumps in gram-negative species (15, 24–26), their impact on antibiotic resistance and recalcitrance in gram-positive species has received less attention (15, 16). In addition, the role of sRNAs in antibiotic adaptation is poorly documented.

To find sRNAs required for *S. aureus* to adapt to sub-lethal antibiotic concentrations, we used a fitness assay with sRNA mutants grown in the presence of the fluoroquinolone antibiotic norfloxacin. We identified a single sRNA, RsAA, the loss of which increased sensitivity of the bacterium to fluoroquinolone antibiotics. Further tests revealed that the loss of RsAA also decreased the sensitivity of *S. aureus* to β-lactam antibiotics and that both effects were mediated by RsAA via the master regulatory protein MgrA. Taken together, our results demonstrate that sRNAs contribute toward antibiotic adaptation in *S. aureus*.

# RESULTS

## RsAA is required for growth in the antibiotic norfloxacin

To determine whether sRNAs are required for *S. aureus* to adapt to the presence of the fluoroquinolone antibiotic norfloxacin, we employed a well-established technique that enables the competitive fitness of a sRNA mutant library to be assessed (17, 22, 27, 28). This technique, referred to as the fitness competition assay, uses a library of DNA-tagged deletion mutants in the HG003 strain (Table S1) and enables subtle phenotypes to be screened more easily. Three mutants were independently constructed for each sRNA gene, which was then used to establish three independent libraries (Table S4). Each library was restricted to 48 mutants of *"bona fide"* sRNAs, defined as those that are genetically independent with their own promoter and terminator, to prevent any interference from nearby coding sequences (17). sRNA gene sequences were replaced with specific DNA tags to enable each mutant to be identified and the proportion of each mutant within the total population to be calculated. Using indexed PCR primer pairs, up to 40 samples could be tested in one DNA-seq run (17). Mutants that disappeared or accumulated under a given stress condition indicated a potential role for the corresponding sRNAs under this growth condition.

The sRNA libraries were exposed to a sub-lethal concentration of norfloxacin (0.25 µg/mL at 0.5× MIC, see Table S5; Fig. S1) and grown over 3 days with two serial dilutions. Samples were taken at different time points and growth stages, and the proportion of each mutant within the population was determined (Fig. 1A). Results were normalized to the same inoculum grown in the same medium without antibiotic and sampled at the same growth phases. Among the 48 tested mutants, the strain containing Tag075 displayed the greatest fitness disadvantage, decreasing by 28-fold [log2(FC)= −4.8] in the presence of norfloxacin after 72 h, when compared to the control condition (Fig. 1B). This strain, in which the *rsaA* gene has been replaced by Tag075 (Δ*rsaA*), progressively disappeared from the library under the selective pressure of norfloxacin under sub-lethal concentrations. Neither *rnall*, *rsaC*, *rsaD*, *rsaE*, *rsaG*, *isrR*
A

1. Growth of DNA-tagged mutant libraries +/- antibiotic

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2. Sampling at different growth phases

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3. DNA-tag amplification

4. Tag identification and counting by DNA-seq

B

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C

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<figure><img src="image_7.png" /><figcaption>FIG 1 S. aureus RsAA is required for optimal growth in the presence of norfloxacin. (A) Experimental protocol scheme to identify mutants with altered fitness in media containing the antibiotic norfloxacin. (B) Evolution of mutant proportions in libraries (for composition, see Table S4) grown in the presence of 0.25 µg/mL norfloxacin (0.5x MIC, see Table S5; Fig. S1 for MIC and growth data) normalized to the same libraries grown in the absence of antibiotic. Error bars indicate the standard deviation from three independent libraries. Left: data with the complete 48 mutant libraries. Tag075 corresponding to the ΔrsaA mutant is highlighted by a red box. For Tag-mutant association, see Table S1. Right: selected data for ΔrsaA are shown. Color code corresponds to the sampling time color from Fig. 1A. (C) Growth of WT and ΔrsaA strains serially diluted 10-fold and spotted onto BHI agar ±0.6 µg/mL norfloxacin (n = 3). BHI, Brain Heart Infusion; WT, wild-type.</figcaption></figure>

nor rsaOG mutants (17), present in the libraries tested, were impacted by the presence of norfloxacin, indicating that their corresponding sRNAs are unlikely to contribute to adaptation to norfloxacin under the tested conditions.

To assess whether the sensitivity of the *rsaA* mutant to norfloxacin also occurred in the absence of the 47 other sRNA strains, monocultures of *ΔrsaA* were grown on agar plates containing norfloxacin. Results from these spot tests revealed that *ΔrsaA* is 100-fold more sensitive to norfloxacin when compared to its parental strain HG003, referred to here as the wild-type (WT) strain (Fig. 1C). In contrast, no difference in growth was observed between either strain when grown in the absence of the antibiotic. Therefore, the loss of RsA leads to greater sensitivity of S. aureus to norfloxacin, and this is independent of any effects on growth in a mixed culture.
Taken together with the fitness competition results, these findings suggested that the
RsaA sRNA is required for optimal growth of *S. aureus* in the presence of norfloxacin.

S. aureus rsaA norfloxacin-dependent phenotype is complemented only by the long form of the rsaA gene

To confirm that the increased sensitivity of the *rsaA* mutant to norfloxacin was solely due to the loss of RsaA, the *ΔrsaA* mutant was transformed with a plasmid containing a WT copy of the gene. Since there are two versions of the RsaA transcript, a short form of 144 base pairs in length and a long version of 282 base pairs (29, 30) (Fig. 2A; Fig. S2), individual plasmids were constructed to reflect complementation with each form of the gene: pRsaA<sub>L</sub> and pRsaA<sub>S</sub> for the long and short forms, respectively.

When the strains were spotted onto agar containing norfloxacin, the loss of RsaA reduced the growth of *S. aureus* by 100-fold when compared to the WT strain, but no improvement in growth occurred when the *ΔrsaA* mutant was transformed with a plasmid containing the short form of the gene (pRsaA<sub>S</sub>), indicating that RsaA<sub>S</sub> expressed from pRsaA<sub>S</sub> is not enough to restore the WT phenotype (Fig. 2B). This phenotype was also observed when the same strains were grown in liquid media containing norfloxacin (Fig. 2C), with the difference that growth of *ΔrsaA* was 3.12-fold slower than the WT strain after 20 h rather than 100-fold, which reflects the higher sensitivity of spot tests when compared to growth curves. Unexpectedly, we found that all the cultures followed unusual growth kinetics, in that the cells died after 4 h and recovered after 7 h (Fig. 2C), which did not occur when the strains were grown without norfloxacin (Fig. S3). Given that norfloxacin has been reported to induce prophage activity in other bacterial species (32–34), it is plausible that similar activity could occur in *S. aureus*, particularly since the *S. aureus* HG003 strain contains three prophages: Φ11, Φ12, and Φ13 (35). Nevertheless, in both solid and liquid cultures, complementation of *ΔrsaA* with pRsaA<sub>L</sub> restored the WT phenotype, demonstrating that the long form of the gene (*rsaA*<sub>L</sub>) is required for optimal growth of *S. aureus* in this antibiotic.

Loss of RsaA sensitizes *S. aureus* to ciprofloxacin

Norfloxacin belongs to the fluoroquinolone class of antibiotics that cause DNA damage by blocking DNA replication (36), which leads to cell death. To determine whether the phenotype was specific to norfloxacin or could also be observed in another antibiotic of the same functional class, the spot test experiment was repeated with ciprofloxacin. As observed in Fig. 2D, the *ΔrsaA* mutant was more sensitive to ciprofloxacin than the WT strain, and this phenotype was restored when complemented with the long form of the gene. Therefore, RsaA<sub>L</sub>, provided here in multicopy form through pRsaA<sub>L</sub>, leads to a phenotype that affects fluoroquinolones in general.

Combined, these data indicate that RsaA is involved in protecting *S. aureus* against the action of fluoroquinolone antibiotics and that this function requires the long form of the gene to be present.

Loss of RsaA reduces sensitivity of S. aureus to β-lactam antibiotics

Having demonstrated that RsaA is required for the optimal growth of *S. aureus* in the presence of fluoroquinolone antibiotics, we tested whether a similar effect could occur for other antibiotic classes. For this, the β-lactam and glycopeptide antibiotic classes, represented by cefazolin and vancomycin respectively, were selected for testing due to their clinical relevance.

When the strains were grown in the presence of these two antibiotics, it was revealed that vancomycin had no effect on the growth of the *ΔrsaA* mutant when compared to the WT strain (Fig. S4). By contrast, in the presence of cefazolin, *S. aureus* growth was enhanced in the absence of RsaA by 10-fold in spot tests (Fig. 3A) and by 1.2-fold in liquid culture after 20 h (Fig. 3B), when compared to the WT strain. Furthermore, the WT phenotype was restored only when *ΔrsaA* was complemented by the long form of the gene.
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**FIG 2** The long form of RsaA restores the wild-type (WT) phenotype, but not the short form. (A) Schematic diagram of long and short forms of RsaA in *S. aureus*. Blue and white arrows represent genes, and dark blue sticks represent transcription terminators. Gene annotations refer to NCTC8325 nomenclature (CP00025.1) retrieved from Genbank (31). The predicted secondary structure of RsaAL, along with the experimentally confirmed structure of RsaAS (30), can be found in Fig. S2. (B) Growth of WT, *ΔrsaA*, and *ΔrsaA* containing pCN38 (empty vector), pRsaAS (*rsaA* short form), or pRsaAL (*rsaA* long form), serially diluted 10-fold and spotted onto BHI agar +0.6 µg/mL norfloxacin (n = 3). Spot tests in BHI broth alone are shown in Fig. S3. (C) Growth curves of WT, *ΔrsaA*, and *ΔrsaA* derivatives as in (B) grown in BHI broth containing 0.5 µg/mL norfloxacin (n = 3). OD600 measurements in BHI broth alone are shown in Fig. S3. Error bars represent the standard deviation of the mean. (D) Growth of WT, *ΔrsaA* mutant, empty vector (pCN38), and mutants complemented with RsaA short form (pRsaAS) or long form (pRsaAL), serially diluted 10-fold and spotted onto BHI agar +0.15 µg/mL ciprofloxacin (n = 3). BHI, Brain Heart Infusion.
<figure><img src="image_12.png" /><figcaption>**FIG 3** The ΔrsaA mutant is less sensitive to β-lactam antibiotics when compared to the WT (A) Growth of WT, ΔrsaA, and ΔrsaA containing pCN38 (empty vector), pRsaA~S~ (rsaA short form), or pRsaA~L~ (rsaA long form), serially diluted 10-fold and spotted onto BHI agar +0.3 µg/mL cefazolin (n = 3). Spot tests in BHI broth alone are shown in Fig. S3. (B) Growth curves of WT, ΔrsaA, and ΔrsaA derivatives as in (A) grown in BHI broth containing 0.125 µg/mL cefazolin (n = 3). OD~600~ measurements in BHI broth alone are shown in Fig. S3. Error bars represent the standard deviation of the mean. (C) Growth of WT, ΔrsaA mutant, and ΔrsaA derivatives as in (A), serially diluted 10-fold and spotted onto BHI agar +0.6 µg/mL cefalexin, 0.15 µg/mL cloxacillin, or 0.03125 µg/mL penicillin G (n = 3). Spot tests in BHI broth alone are shown in Fig. S3. BHI, Brain Heart Infusion; WT, wild-type.</figcaption></figure>

As a follow-up, we repeated the spot tests on a selection of other β-lactam antibiotics: cefalexin, cloxacillin, and penicillin G. Results demonstrated that the growth of the ΔrsaA mutant is increased in the presence of β-lactams when compared to the WT strain (Fig. 3C). Taken together, these data demonstrate that the loss of RsaA reduces the sensitivity of S. aureus to β-lactam antibiotics. This is opposite to the phenotype found for fluoroquinolone antibiotics, in which the loss of RsaA made S. aureus more sensitive to these antibiotics instead. Nevertheless, in both cases, the long form of RsaA is required for this process.

## RsaA acts on MgrA to fully mediate its effect on norfloxacin and partly for cefazolin

RsaA is known to repress the translation of the master regulatory protein, MgrA (30). The latter regulates S. aureus virulence by promoting genes involved in capsule synthesis and inhibiting genes related to biofilm formation (37, 38). Separately, MgrA has also been shown to act on several efflux pumps, including the fluoroquinolone efflux pumps NorA (39, 40), NorB (40–42), and NorC (43), and a β-lactam efflux pump, AbcA (42). Since MgrA is reported to inhibit these NorA efflux pumps and promote AbcA (39–43), this would align
with our observed phenotypes in the *ΔrsaA* mutant, in which MgrA is no longer being repressed. To test this hypothesis, we performed spot tests on single and double mutants of *ΔrsaA* and *ΔmgrA* in the presence of norfloxacin or cefazolin.

As shown in Fig. 4A, the loss of RsaA and MgrA led to opposite effects on *S. aureus* growth when compared to the WT strain, supporting previous reports of RsaA as a repressor of MgrA. While *ΔrsaA* was more sensitive to norfloxacin than WT *S. aureus*, the *ΔmgrA* mutant showed reduced sensitivity, and the reverse was true of these two strains concerning cefazolin. When the double mutant was tested, its growth in norfloxacin was restored to WT levels, indicating that the increased sensitivity of *ΔrsaA* was most likely caused by derepression of MgrA. In the presence of cefazolin, the growth of *ΔrsaA* *ΔmgrA* was very slightly increased compared to *ΔmgrA* but did not reach *ΔrsaA* levels, again indicating that the phenotype is likely due to the derepression of MgrA. However, in this case, growth also did not reach WT levels, suggesting a minor role for another RsaA-mediated mechanism.

To further investigate the link between RsaA, the derepression of MgrA, and its subsequent effect on *S. aureus* sensitivity to norfloxacin and cefazolin, we generated a

<figure><img src="image_13.png" /><figcaption>**FIG 4** The increased sensitivity of the ΔrsaA mutant to norfloxacin is mediated by MgrA. Growth of WT, ΔrsaA, ΔmgrA, and ΔrsaA ΔmgrA strains, serially diluted 10-fold and spotted onto BHI agar containing (A) 0.6 µg/mL norfloxacin or (B) 0.3 µg/mL cefazolin (n = 3). (B) Growth of WT, ΔmgrA, and ΔmgrA complemented with empty vector (pCN38) or long form (pRsaAL), serially diluted 10-fold and spotted onto BHI agar containing (C) 0.6 µg/mL norfloxacin or (D) 0.3 µg/mL cefazolin (n = 3). Spot tests in BHI alone are shown in Fig. S5. BHI, Brain Heart Infusion; WT, wild-type.</figcaption></figure>
*ΔmgrA* mutant that overproduced *RsaA*<sub>L</sub> (*ΔmgrA* p*RsaA*<sub>L</sub>) and repeated the spot tests (Fig. 4B). These results revealed that the overproduction of *RsaA*<sub>L</sub> has no additional effect on the growth of *ΔmgrA* in the presence of norfloxacin, demonstrating that *RsaA* acts solely through *MgrA* to mediate its effect on norfloxacin. By contrast, the overproduction of *RsaA*<sub>L</sub> led to a slightly increased sensitivity of *ΔmgrA* to cefazolin, indicating that while *RsaA* mostly acts through *MgrA* to mediate its effect on cefazolin, another mechanism may be involved.

## The full-length *rsaA* gene is required for expression of the short RsAA form

Having confirmed that the long form of the *rsaA* gene was required for mediating the sensitivity of *S. aureus* to growth in norfloxacin and cefazolin, we wanted to determine why it was required. To investigate this, we first searched for possible open-reading frames in the *RsaA*<sub>L</sub> transcript that could translate to a small protein with a regulatory function. This identified one candidate that spanned most of the long-form region; however, mutation of the predicted ORF to block expression had no effect on antibiotic phenotype in spot tests (Fig. S6). Next, we examined whether additional interactions are present between *mgrA* mRNA and *RsaA*<sub>L</sub>, as compared to what is already known with *mgrA* mRNA and *RsaA*<sub>S</sub> (30). However, RNA-RNA interaction prediction software did not find any new interactions present in *RsaA*<sub>L</sub> that were not also detected with the short form only (Fig. S7), supporting that for *RsaA* to pair with *mgrA* mRNA, only its short-form transcript is required.

Finally, we examined the levels of expression between *RsaA*<sub>S</sub> and *RsaA*<sub>L</sub>. Results showed that there are far higher levels of the short-form transcript in the WT strain when compared to the long-form transcript (Fig. 5A; Fig. S8). This pattern is also reflected in the *ΔrsaA* mutant when complemented with the long form of the gene (Fig. 5A), corresponding with our previous observation that only *RsaA*<sub>L</sub> restores the WT phenotype. However, when the *ΔrsaA* mutant was complemented with *pRsaA*<sub>S</sub>, minimal expression of *RsaA*<sub>S</sub> was detected (Fig. 5A; Fig. S8), suggesting that while only the short form is required for sRNA function (Fig. S7), the short form cannot be expressed successfully on its own, and the expression of the long-form transcript is still needed. When we probed the same Northern blot membranes to detect only *RsaA*<sub>L</sub> and the long-form region, it was revealed that for the complemented *ΔrsaA* mutant with *pRsaA*<sub>L</sub>, we could also see a clear band corresponding to the long-form region *RsaA*<sub>L</sub>, suggesting a cleavage of the long-form region to generate *RsaA*<sub>S</sub> (Fig. 5B; Fig. S8).

To investigate the possible contribution of two sRNA fragments in the *rsaA*-dependent phenotype, the *ΔrsaA* mutant was transformed with *pRsaA*<sub>S</sub> and *pRsaA*<sub>3'L</sub>, two plasmids expressing *RsaA*<sub>S</sub> and *RsaA*<sub>3'L</sub> (*RsaA* sequence present on *RsaA*<sub>L</sub> but not on *RsaA*<sub>S</sub>), respectively (Fig. S9). Spot tests revealed that although the presence of a single plasmid expressing either *RsaA*<sub>S</sub> (via *pRsaA*<sub>S</sub>) or *RsaA*<sub>3'L</sub> (via *pRsaA*<sub>3'L</sub>) cannot restore the WT phenotype, complementation of *ΔrsaA* was observed when both plasmids were present in the same strain, despite the fact that both fragments (*RsaA*<sub>S</sub> and *RsaA*<sub>3'L</sub>) were not present on the same transcript (Fig. 5C). Northern blot experiments indicate the presence of a single plasmid expressing *RsaA*<sub>S</sub> (via *pRsaA*<sub>S</sub>) leads to a weak quantity of *RsaA*<sub>S</sub> likely insufficient to confer a tolerance phenotype. However, the presence of *pRsaA*<sub>3'L</sub> expressing *RsaA*<sub>3'L</sub> allows the production of an increased quantity of *RsaA*<sub>S</sub> restoring the WT phenotype. Surprisingly, a low amount of *pRsaA*<sub>3'L</sub> fragment seems sufficient to lead to the accumulation of *RsaA*<sub>S</sub> (Fig. 5A and B).

Our data suggest that the *RsaA*<sub>L</sub> transcript is possibly cleaved and that the two products are required for *RsaA* to function as a regulatory RNA.

RNA-RNA interaction prediction software identified an interaction between *RsaA*<sub>S</sub> and *RsaA*<sub>3'L</sub> (Fig. S10), which suggests that both could be involved in complex formation allowing the accumulation and therefore the activity of *RsaA*<sub>S</sub> toward its mRNA targets. Combined, these data demonstrate that the expression of *RsaA*<sub>L</sub> is required for the short form to be generated, likely through a cleavage event in which both cleavage products are required to interact for *RsaA* function.
<figure><img src="image_14.png" /><figcaption>**FIG 5** The long RsaA transcript needs to be expressed for the sRNA to function. (A) Northern blot of WT and ΔrsaA complemented with empty vector (pCN38), RsaA short form (pRsaA~S~), RsaA long form (pRsaA~L~), RsaA 3'-end of the long form not present in RsaA~S~ name here RsaA~3'L~ (pRsaA~3'L~) and RsaA~S~ + RsaA~3'L~ (pRsaA~S~ + pRsaA~3'L~) sampled at an OD~600~ of 6 and probed for RsaA 5'-end probe to detect both short and long forms. tmRNA was probed as a loading control. Probes are DIG-labeled riboprobes generated from PCR products. A Northern blot with WT and ΔrsaA complemented with empty vector, RsaA~S~ and pRsaA~L~, probed for RsaA 5'-end probe using a radioactive labeled DNA probe is presented in Fig. S8A. (B) Northern blot using the same cultures and conditions as for Fig. 5A, probed for RsaA~L~ and RsaA~3'L~ only using an RsaA~L~ 3'-end probe, and tmRNA (loading control). A Northern blot with WT and ΔrsaA complemented with empty vector, RsaA~S~ and pRsaA~L~, probed for RsaA~L~ and RsaA~3'L~ only using an RsaA~L~ 3'-end probe using a radioactive labeled DNA probe is presented in Fig. S8B. (C) Growth of WT and ΔrsaA complemented with short form (pRsaA~S~), long-region only (pRsaA~3'L~), or both short- and long-form regions (pRsaA~S~ + pRsaA~3'L~), serially diluted 10-fold and spotted onto BHI agar ±0.6 µg/mL norfloxacin or 0.3 µg/mL cefazolin (n = 3). BHI, Brain Heart Infusion; WT, wild-type.</figcaption></figure>

RNase Y is not required for *rsaA*-dependent phenotypes

Experimental and comparative sequence data indicate that at least 15 ribonucleases (RNases) in S. aureus are involved in RNA processing and degradation (44). Of these, the main staphylococcal exonucleases are RNase J1/J2 and PNPase, while the main endonucleases are RNase III and RNase Y, both of which influence the stability and processing of RsaA~L~ (29, 45). We sought to assess the effect of RNase III and RNase Y deficiency on the processing of RsaA~L~ into RsaA~S~ and RsaA~3'L~. Attempts to construct an RNase III (*rnc*) mutant in strain HG003 were unsuccessful, potentially due to phage-associated toxin systems, as noted in *Bacillus subtilis* (46). Consequently, only RNase Y was tested. Spot tests comparing plating efficiency on norfloxacin between the WT strain and its RNase Y mutant (*Δrny*) showed no difference, suggesting either that no processing of the long form is necessary for RsaA activity or that RNase Y is not involved in this processing (Fig. 6). However, *Δrny* displayed increased sensitivity to cefazolin compared to either the WT or the ΔrsaA mutant, consistent with previous findings that RNase mutants have disordered peptidoglycan layer leading to a hypersensitivity to various antibiotics, including for β-lactam antibiotics like cefazolin, which target the cell wall (47). Since the cefazolin phenotype of *Δrny* was opposite of that observed for ΔrsaA, these findings support the conclusion that RNase Y is unlikely to contribute to RsaA activity.
<figure><img src="image_15.png" /><figcaption>**FIG 6** RNase Y does not affect sensitivity to norfloxacin. Growth of WT, *ΔrsaA*, and *Δmy*, serially diluted 10-fold and spotted onto BHI agar ±0.6 µg/mL norfloxacin or 0.3 µg/mL cefazolin (n = 3). BHI, Brain Heart Infusion; WT, wild-type.</figcaption></figure>

DISCUSSION

sRNAs play a crucial role in regulating key processes related to antibiotic resistance in gram-negative species (15, 24). These processes include cell envelope modifications (25) and drug efflux pumps (26). However, their impact on antibiotic resistance in gram-positive species has received less attention (15).

In *S. aureus*, the sRNA SprX, also known as RsaOR (20), influences vancomycin and teicoplanin glycopeptide resistance by repressing SpoVG (48). Notably, 6S RNA, which targets RNA polymerase, was shown to contribute to increased growth in low levels of rifampicin and related antibiotics in *S. aureus*, as well as in other distantly related bacteria (28). Antibiotics also influence sRNA gene expression, though connections to antibiotic resistance and/or adaptation are less clear. *S. aureus* sRNAs are differentially expressed after exposure to vancomycin, linezolid, ceftobiprole, and tigecycline (49), and exposure of *S. aureus* to sub-inhibitory concentrations of the β-lactam antibiotic oxacillin enhances bacterial virulence via the sRNA Ssr42 (50).

The data presented here connect the RsaA sRNA to antibiotic resistance mechanisms regarding two antibiotic classes: fluoroquinolones (norfloxacin and ciprofloxacin) and β-lactams (cefazolin, cefalexin, cloxacillin, and penicillin G). Although fluoroquinolones act on DNA replication and β-lactams on the cell wall, mechanisms of resistance to both classes can involve the action of efflux pumps. Our results show that RsaA acts through the master regulator MgrA to mediate the sensitivity of *S. aureus* to antibiotics, linking RsaA with the efflux pumps NorA, NorB, NorC, and AbcA. To our knowledge, RsaA is the first example of sRNA linked to fluoroquinolone resistance mechanisms in *S. aureus*. Of note, in *Escherichia coli*, the sRNA SdsR represses the TolC efflux pump and increases sensitivity to fluoroquinolones (26). SdsR activity is dependent on the RNA chaperone Hfq (26), which is likely not the case for RsaA (51).

We found that the effect of RsaA with fluoroquinolones was fully regulated via MgrA, but this regulation was only partial for β-lactams. Since sRNAs often control the expression of multiple mRNA targets to enable a coordinated regulation of cellular processes (12), it is likely that the RsaA-mediated effect on β-lactam sensitivity is mediated by other mRNA targets. A study investigating the RNA targetome of RsaA identified *ssaA_2* and *ssaA2_3* mRNAs as putative RsaA targets and found that these mRNAs form high-affinity complexes with RsaA *in vitro* (52). Since these two genes belong to the family of staphylococcal secretory antigen A (SsaA) proteins involved in peptidoglycan degradation (53), regulation of these substrates by RsaA may affect changes in sensitivity to β-lactam antibiotics, which function by disrupting peptidoglycan cross-linking during cell wall synthesis (54).
Loss of RsxA was complemented only by the long form of the gene, which demonstrates that the short form of the gene is not sufficient for regulating sensitivity to our tested antibiotics. RsxA~L~ was first detected by Northern blot and believed to originate from read-through at the RsxA~S~ transcriptional terminator (29). Both RsxA~L~ and RsxA~S~ share the same 5'-end, as determined by RACE experiments (21). Our study reveals the importance of the long-form *rsaA*~L~ gene being present for the sRNA to function. As no additional interaction sites with *mgrA* mRNA were predicted in RsxA~L~ when compared to RsxA~S~, this correlates with studies on RsxA that have focused only on its short form (29, 30, 52), which is believed to be the functional region. It must be noted that even if RsxA~L~ was not mentioned in these studies, the Δ*rsaA* mutant was complemented using a long stretch of the *S. aureus* genome comprising both *rsaA*~L~ and *rsaA*~S~ genes and the surrounding genomic region (29, 30, 52).

Our data demonstrate that the long form of the *rsaA* gene is needed for regulating staphylococcal sensitivity to norfloxacin and cefazolin, and that for RsxA to function, the long-form transcript requires processing, most likely through cleavage into two halves. This finding aligns with measurements of the half-lives of RsxA short- and long-form transcripts (29), where RsxA~S~ was found to be >2-fold more stable than RsxA~L~ after treatment with rifampicin to inhibit RNA polymerase (29). Based on experimental and homology data, there are believed to be 15 RNases in *S. aureus* that are involved in RNA processing and/or degradation (44). Of these, the main staphylococcal RNases are the exonucleases RNase J1/J2 and PNPase, and the endonucleases RNase III and RNase Y, the latter two of which have been reported to affect the stability and processing of RsxA~L~ (29, 45). We found that RNase Y is unlikely to be the processing enzyme for the cleavage of RsxA~L~ into two halves, though the lack of a viable RNase III mutant in HG003 does not exclude this enzyme from this role.

In *S. aureus*, RsxE is a sRNA possibly processed from a longer transcript to interact more efficiently with some targets, thereby expanding the range of recognised targets. However, this feature is, so far, only reported for its *B. subtilis* and *Staphylococcus epidermidis* orthologs (55, 56). In the case of RsxA, we were able to restore the WT phenotype in a Δ*rsaA* mutant by expressing both RsxA~S~ and the long region of RsxA~L~ from different plasmids (*p*RsxA~S~ and *p*RsxA~3'L*), such that the RsxA~L~ long region is most likely interacting with the short form of RsxA to enable its function, possibly by stabilising the sRNA and/or by promoting interactions with *mgrA* mRNA. We found that the predicted interaction site of RsxA~S~ with the RsxA~L~ long region has minimal overlap with its interaction site with *mgrA* mRNA, suggesting that both interactions could occur at the same time. Complexes forming between RsxA and other mRNAs/sRNAs have been detected by RNase III CLASH, including with *mgrA* mRNA and RNAIII sRNA, both of which interact with the short-form region of RsxA (57). Although an RNase III complex with RsxA~S~ and RsxA~3'L~ was not mentioned, hybrids with high sequence similarity were excluded, and it is possible that the similar sRNA-sRNA interaction may not have been considered.

Additionally, the RsxA~L~ long region was unexpectedly lower in abundance than the short form of RsxA despite being required for its function, suggesting that any interactions may be transient and/or that RsxA~3'L~ may be more susceptible to degradation. We also observed that the abundance of the RsxA~L~ long region from *p*RsxA~3'L~ was much higher when compared to the WT, where it was not detectable. It is possible that these higher levels were due to RsxA~L~ being supplied in multicopy form via a plasmid in the Δ*rsaA* *p*RsxA~L~ strain, which is reflected in the much lower abundance of RsxA~L~ in the WT strain when compared to the Δ*rsaA* mutant expressing *p*RsxA~L~.

sRNAs control many cellular processes and enable rapid adaptation to stress. Here, we show that staphylococcal RsxA acts via the master regulator MgrA to mediate sensitivity to two different antibiotic classes, fluoroquinolones and β-lactams. In addition, we revealed that this regulation requires the full-length version of the *rsaA* gene, the transcript of which is likely processed by the cell to produce two halves that interact to produce functional RsxA. Therefore, exposure of *S. aureus* to sub-inhibitory
fluoroquinolone or β-lactam concentrations can alter the expression of their correspond-
ing efflux pumps, adapting the bacterium to growth in the presence of antibiotics.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions

The bacterial strains used in this study are listed in Table S1. Experiments were performed with HG003, a *S. aureus* model strain widely used for regulation studies (35). Plasmids were engineered by Gibson assembly (58) in *E. coli* IM08B (59) as described (Table S2), using the indicated appropriate primers (Table S3) for PCR amplification. Plasmids were verified by DNA sequencing and transformed into HG003 or its derivatives, as appropriate. HG003 Δ*mgrA* and Δ*rsaA* Δ*mgrA* double mutants were constructed by transferring Δ*mgrA*::tetM (60) into HG003 and HG003 Δ*rsaA*::tag075 using phage-mediated transduction.

S. aureus was routinely cultured in Brain Heart Infusion (BHI) broth at 37°C, with shaking (180 rpm). E. coli was grown in Lysogeny Broth at 37°C with shaking (180 rpm). Media were supplemented with antibiotics as required: ampicillin 100 µg/mL for E. coli; chloramphenicol 5 µg/mL, tetracycline 1 µg/mL, and kanamycin 90 µg/mL for S. aureus.

Fitness competition assay

Mutants with altered fitness in the presence of norfloxacin were identified and analzed
using a reported strategy (27) with three independent libraries containing 48 mutants
(Table S4). The libraries were grown at 37°C in BHI or BHI + 0.25 µg/mL norfloxacin for
3 days. Overnight cultures were diluted 1,000 times into fresh medium for each day.
Samples were withdrawn at an OD₆₀₀ of 1 and after overnight growth as indicated (Fig.
1A).

Spot tests

To assess mutant phenotypes, bacterial growth in the presence of antibiotics was
visualized by spot tests, which enable subtle growth phenotypes to be detected, such
as with sRNA mutants. A total of 5 µL of 10-fold serial dilutions of overnight cultures
(from neat to 10⁻⁷ dilutions) were spotted on agar plates containing either BHI or BHI +
antibiotic at the indicated concentrations. Agar plates were incubated for 24 h at 37°C.

Measurement of bacterial growth in liquid culture

To measure growth of *S. aureus* in liquid culture, bacteria were first grown to stationary phase in BHI at 37°C (180 rpm), then inoculated 1/50 into a flat-bottomed 96-well plate (200 µL total volume) and placed into a CLARIOstar Omega plate reader (BMG Labtech). Antibiotics were added to the media as needed, at the indicated concentrations. Bacteria were grown for 20 h at 37°C (300 rpm), and absorbance at 600 nm was measured every 30 min.

Northern blots

Total RNA preparations and Northern blots were performed as previously described (61). A total of 20 µg of total RNA samples was separated either by agarose (1.3%) gel electrophoresis. Membranes were probed with either primer (Table S3) ³²P-labeled using Terminal Deoxynucleotidyl Transferase (ThermoScientific) and scanned using an Amersham Typhoon imager or T7 generated riboprobes (for PCR matrices, see Table S3) DIG-labeled using the DIG Northern Starter Kit (Sigma-Aldrich) according to the manufacturer's instructions and scanned using a Bio-Rad ChemiDoc imager.
ACKNOWLEDGMENTS

The USA300 LAC ΔmgrA::tetM strain used to transduce into HG003 to generate the HG003 ΔmgrA mutant was a gift from Alexander Horswill. The authors thank our colleagues Mathieu Rougemaille, Nara Figueroa-Bossi, and King Fisher for technical advice and warm support. The research was supported by the "Agence National de la Recherche" (ANR-19-CE12-0006 [sRNA-RRARE]).

AUTHOR AFFILIATION

¹Université Paris-Saclay, CEA, CNRS, Institut de Biologie Intégrative de la Cellule (I2BC), Gif-sur-Yvette, Île-de-France, France

AUTHOR ORCIDs

Kam Pou Ha ID http://orcid.org/0000-0001-7494-3245

Etornam Kofi Kumeko ID http://orcid.org/0009-0002-0506-5906

Philippe Bouloc ID http://orcid.org/0000-0003-4601-3387

FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Agence Nationale de la Recherche</td><td>ANR-19-CE12-0006</td><td>Philippe Bouloc</td></tr></tbody></table>

ADDITIONAL FILES

The following material is available online.

Supplemental Material

Supplemental material (AAC01176-24-s0001.pdf). Tables S1 to S5; Fig. S1 to S10.

REFERENCES

1. Westblade LF, Errington J, Dörr T. 2020. Antibiotic tolerance. PLoS Pathog 16:e1008892. https://doi.org/10.1371/journal.ppat.1008892

2. Foster TJ. 2017. Antibiotic resistance in *Staphylococcus aureus*. Current status and future prospects. FEMS Microbiol Rev 41:430–449. https://doi.org/10.1093/femsre/fux007

3. Darby EM, Trampari E, Siasat P, Gaya MS, Alav I, Webber MA, Blair JMA. 2023. Molecular mechanisms of antibiotic resistance revisited. Nat Rev Microbiol 21:280–295. https://doi.org/10.1038/s41579-022-00820-y

4. Gollan B, Grabe G, Michaux C, Helaine S. 2019. Bacterial persisters and infection: Past, present, and progressing. Annu Rev Microbiol 73:359–385. https://doi.org/10.1146/annurev-micro-020518-115650

5. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. 2004. Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 230:13–18. https://doi.org/10.1016/S0378-1097(03)00856-5

6. Helaine S, Conlon BP, Davis KM, Russell DG. 2024. Host stress drives tolerance and persistence: the bane of anti-microbial therapeutics. Cell Host Microbe 32:852–862. https://doi.org/10.1016/j.chom.2024.04.019

7. Windels EM, Michiels JE, Fauvart M, Wenseleers T, Van den Bergh B, Michiels J. 2019. Bacterial persistence promotes the evolution of antibiotic resistance by increasing survival and mutation rates. ISME J 13:1239–1251. https://doi.org/10.1038/s41396-019-0344-9

8. Levin-Reisman I, Ronin I, Gefen O, Braniss I, Shoresh N, Balaban NQ. 2017. Antibiotic tolerance facilitates the evolution of resistance. Science 355:826–830. https://doi.org/10.1126/science.aaq2191

9. Liu J, Gefen O, Ronin I, Bar-Meir M, Balaban NQ. 2020. Effect of tolerance on the evolution of antibiotic resistance under drug combinations. Science 367:200–204. https://doi.org/10.1126/science.aaq3041

10. Ikuta KS, Swetschinski LR, Aguilar GR, Sharara F, Mestrovic T, Gray AP, Weaver ND, Wool EE, Han C, Hayoon AG. 2022. Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet 400:2221–2248. https://doi.org/10.1016/S0140-6736(22)02185-7

11. Murray C.JL, Ikuta KS, Sharara F, Swetschinski L, Robles Aguilar G, Gray A, Han C, Bisignano C, Rao P, Wool E, et al. 2022. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet 399:629–655. https://doi.org/10.1016/S0140-6736(21)02724-0

12. Waters LS, Storz G. 2009. Regulatory RNAs in bacteria. Cell 136:615–628. https://doi.org/10.1016/j.cell.2009.01.043

13. Chakravarty S, Massé E. 2019. RNA-dependent regulation of virulence in pathogenic bacteria. Front Cell Infect Microbiol 9:337. https://doi.org/10.3389/fcimb.2019.00337

14. Rochat T, Bohn C, Morvan C, Le Lam TN, Razvi F, Pain A, Toffano-Nioche C, Ponien P, Jacq A, Jacquet E, Fey PD, Gautheret D, Bouloc P. 2018. The conserved regulatory RNA RsAE down-regulates the arginine degradation pathway in *Staphylococcus aureus*. Nucleic Acids Res 46:8803–8816. https://doi.org/10.1093/nar/gky584

15. Dersch P, Khan MA, Mühlen S, Görke B. 2017. Roles of regulatory RNAs for antibiotic resistance in bacteria and their potential value as novel drug targets. Front Microbiol 8:803. https://doi.org/10.3389/fmicb.2017.00803

16. Mediati DG, Wu S, Wu W, Tree JJ. 2021. Networks of resistance: small RNA control of antibiotic resistance. Trends Genet 37:35–45. https://doi.org/10.1016/j.tig.2020.08.016

17. Liu W, Rochat T, Toffano-Nioche C, Le Lam TN, Bouloc P, Morvan C. 2018. Assessment of bona fide sRNAs in *Staphylococcus aureus*. Front Microbiol 9:228. https://doi.org/10.3389/fmicb.2018.00228

18. Menard G, Silard C, Suriray M, Rouillon A, Augagneur Y. 2022. Thirty years of sRNA-mediated regulation in *Staphylococcus aureus*: from initial discoveries to *in vivo* biological implications. Int J Mol Sci 23:7346. https://doi.org/10.3390/ijms23137346

19. Bronsky D, Wu Z, Marzi S, Walter P, Geissmann T, Moreau K, Vandenesch F, Caldelari I, Romby P. 2016. *Staphylococcus aureus* RNAIII and its regulon link quorum sensing, stress responses, metabolic adaptation, and regulation of virulence gene expression. Annu Rev Microbiol 70:299–316. https://doi.org/10.1146/annurev-micro-102215-095708
20. Bohn C, Rigoulay C, Chabelskaya S, Sharma CM, Marchais A, Skorski P, Borezée-Durant E, Barbet R, Jacquet E, Jacq A, Gautherset D, Felden B, Vogel J, Bouloc P. 2010. Experimental discovery of small RNAs in *Staphylococcus aureus* reveals a riboregulator of central metabolism. Nucleic Acids Res 38:6620-6636. https://doi.org/10.1093/nar/gkq462

21. Geissmann T, Chevalier C, Cros M-J, Boisset S, Fechter P, Noirot C, Schrenzel J, François P, Vandenesch F, Gaspin C, Romby P. 2009. A search for small noncoding RNAs in *Staphylococcus aureus* reveals a conserved sequence motif for regulation. Nucleic Acids Res 37:7239-7257. https://doi.org/10.1093/nar/gkp668

22. Coronel-Tellez RH, Pospiech M, Barrault M, Liu W, Bordeaux V, Vasnier C, Felden B, Sargueil B, Bouloc P. 2022. sRNA-controlled iron sparing response in staphylococci. Nucleic Acids Res 50:8529-8546. https://doi.org/10.1093/nar/gkac648

23. Barrault M, Chabelskaya S, Coronel-Tellez RH, Toffano-Nioche C, Jacquet E, Bouloc P. 2024. Staphylococcal aconitase expression during iron deficiency is controlled by an sRNA-driven feedforward loop and moonlighting activity. Nucleic Acids Res 52:8241-8253. https://doi.org/10.1093/nar/gkae506

24. Kim T, Bak G, Lee J, Kim K-S. 2015. Systematic analysis of the role of bacterial Hfq-interacting sRNAs in the response to antibiotics. J Antimicrob Chemother 70:1659-1668. https://doi.org/10.1093/jac/dkv042

25. Acuña LG, Barros MJ, Peñaloza D, Rodas PI, Paredes-Sabja D, Fuentes JA, Gil F, Calderón IL. 2016. A feed-forward loop between SroC and MgrR small RNAs modulates the expression of eptB and the susceptibility to polymyxin B in *Salmonella* Typhimurium. Microbiology (Reading) 162:1996-2004. https://doi.org/10.1099/mic.0.000365

26. Parker A, Gottesman S. 2016. Small RNA regulation of TolC, the outer membrane component of bacterial multidrug transporters. J Bacteriol 198:1101-1113. https://doi.org/10.1128/JB.00971-15

27. Le Lam TN, Morvan C, Liu W, Bohn C, Jaszczyszyn Y, Bouloc P. 2017. Finding sRNA-associated phenotypes by competition assays: an example with *Staphylococcus aureus*. Methods 117:21-27. https://doi.org/10.1016/j.ymeth.2016.11.018

28. Esberard M, Hallier M, Liu W, Morvan C, Bossi L, Figueroa-Bossi N, Felden B, Bouloc P. 2022. 6S RNA-dependent susceptibility to RNA polymerase inhibitors. Antimicrob Agents Chemother 66:e0243521. https://doi.org/10.1128/aac.02435-21

29. Lioliou E, Sharma CM, Cadelari I, Helfer A-C, Fechter P, Vandenesch F, Vogel J, Romby P. 2012. Global regulatory functions of the *Staphylococcus aureus* endoribonuclease III in gene expression. PLoS Genet 8:e1002782. https://doi.org/10.1371/journal.pgen.1002782

30. Romilly C, Lays C, Tomasini A, Cadelari I, Benito Y, Hammann P, Geissmann T, Boisset S, Romby P, Vandenesch F. 2014. A non-coding RNA promotes bacterial persistence and decreases virulence by regulating a regulator in *Staphylococcus aureus*. PLoS Pathog 10:e1003979. https://doi.org/10.1371/journal.ppat.1003979

31. Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW. 2013. GenBank. Nucleic Acids Res 41:D36-D42. https://doi.org/10.1093/nar/gks1195

32. Krishnamurthi R, González-Tortuero E, Plahe G, Goodhead IB, Fothergill JL, James CE, Allison HE. 2024. Understanding the impact of temperate bacteriophages on their lysogens through transcriptomics. J Vis Exp 203. https://doi.org/10.3791/64945

33. Matsushiro A, Sato K, Miyamoto H, Yamamura T, Honda T. 1999. Induction of prophages of enterohemorrhagic *Escherichia coli* O157:H7 with norfloxacin. J Bacteriol 181:2257-2260. https://doi.org/10.1128/JB.181.7.2257-2260.1999

34. James CE, Davies EV, Fothergill JL, Walsh MJ, Beale CM, Brockhurst MA, Winstanley C. 2015. Lytic activity by temperate phages of *Pseudomonas aeruginosa* in long-term cystic fibrosis chronic lung infections. ISME J 9:1391-1398. https://doi.org/10.1038/ismej.2014.223

35. Herbert S, Ziebandt AK, Ohlsen K, Schäfer T, Hecker M, Albrecht D, Novick R, Götz F. 2010. Repair of global regulators in *Staphylococcus aureus* 8325 and comparative analysis with other clinical isolates. Infect Immun 78:2877-2889. https://doi.org/10.1128/IAI.00008-10

36. Drlca K. 1999. Mechanism of fluoroquinolone action. Curr Opin Microbiol 2:504-508. https://doi.org/10.1016/s1369-5274(99)00008-9

37. Luong TT, Newell SW, Lee CY. 2003. mgr, a novel global regulator in *Staphylococcus aureus*. J Bacteriol 185:3703-3710. https://doi.org/10.1128/JB.185.13.3703-3710.2003

38. Trotonda MP, Tamber S, Memmi G, Cheung AL. 2008. MgrA represses biofilm formation in *Staphylococcus aureus*. Infect Immun 76:5645-5654. https://doi.org/10.1128/IAI.00735-08

39. Truong-Bolduc QC, Ding Y, Hooper DC. 2008. Posttranslational modification influences the effects of MgrA on norA expression in *Staphylococcus aureus*. J Bacteriol 190:7375-7381. https://doi.org/10.1128/JB.01068-08

40. Truong-Bolduc QC, Hooper DC. 2010. Phosphorylation of MgrA and its effect on expression of the NorA and NorB efflux pumps of *Staphylococcus aureus*. J Bacteriol 192:2525-2534. https://doi.org/10.1128/JB.00018-10

41. Truong-Bolduc QC, Dunman PM, Strahilevitz J, Projan SJ, Hooper DC. 2005. MgrA is a multiple regulator of two new efflux pumps in *Staphylococcus aureus*. J Bacteriol 187:2395-2405. https://doi.org/10.1128/JB.187.7.2395-2405.2005

42. Truong-Bolduc QC, Hooper DC. 2007. The transcriptional regulators NorG and MgrA modulate resistance to both quinolones and beta-lactams in *Staphylococcus aureus*. J Bacteriol 189:2996-3005. https://doi.org/10.1128/JB.01819-06

43. Truong-Bolduc QC, Strahilevitz J, Hooper DC. 2006. NorC, a new efflux pump regulated by MgrA of *Staphylococcus aureus*. Antimicrob Agents Chemother 50:1104-1107. https://doi.org/10.1128/AAC.50.3.1104-1107.2006

44. Bonnin RA, Bouloc P. 2015. RNA degradation in *Staphylococcus aureus*: diversity of ribonucleases and their impact. Int J Genomics 2015:395753. https://doi.org/10.1155/2015/395753

45. Marincola G, Schäfer T, Behler J, Bernhardt J, Ohlsen K, Goerke C, Wolz C. 2012. RNase Y of *Staphylococcus aureus* and its role in the activation of virulence genes. Mol Microbiol 85:817-832. https://doi.org/10.1111/j.1365-2958.2012.08144.x

46. Durand S, Gilet L, Condon C. 2012. The essential function of *B. subtilis* RNase III is to silence foreign toxin genes. PLoS Genet 8:e1003181. https://doi.org/10.1371/journal.pgen.1003181

47. Figaro S, Durand S, Gilet L, Cayet N, Sachse M, Condon C. 2013. *Bacillus subtilis* mutants with knockouts of the genes encoding ribonucleases RNase Y and RNase J1 are viable, with major defects in cell morphology, sporulation, and competence. J Bacteriol 195:2340-2348. https://doi.org/10.1128/JB.00164-13

48. Eyraud A, Tattevin P, Chabelskaya S, Felden B. 2014. A small RNA controls a protein regulator involved in antibiotic resistance in *Staphylococcus aureus*. Nucleic Acids Res 42:4892-4905. https://doi.org/10.1093/nar/gku149

49. Howden BP, Beaume M, Harrison PF, Hernandez D, Schrenzel J, Seemann T, Francois P, Stinear TP. 2013. Analysis of the small RNA transcriptional response in multidrug-resistant *Staphylococcus aureus* after antimicrobial exposure. Antimicrob Agents Chemother 57:3864-3874. https://doi.org/10.1128/AAC.00263-13

50. Horn J, Klepsch M, Manger M, Wolz C, Rudel T, Fraunholz M. 2018. Long noncoding RNA SSR42 controls *Staphylococcus aureus* alpha-toxin transcription in response to environmental stimuli. J Bacteriol 200:e00252-18. https://doi.org/10.1128/JB.00252-18

51. Bohn C, Rigoulay C, Bouloc P. 2007. No detectable effect of RNA-binding protein Hfq absence in *Staphylococcus aureus*. BMC Microbiol 7:1-9. http://doi.org/10.1186/1471-2180-7-10

52. Tomasini A, Moreau K, Chicher J, Geissmann T, Vandenesch F, Romby P, Marzi S, Cadelari I. 2017. The RNA targetome of *Staphylococcus aureus* non-coding RNA RsAA: impact on cell surface properties and defense mechanisms. Nucleic Acids Res 45:6746-6760. https://doi.org/10.1093/nar/gkx219

53. Dubrac S, Boneca IG, Poupel O, Msadek T. 2007. New insights into the Walk/WalR (YycG/YycF) essential signal transduction pathway reveal a major role in controlling cell wall metabolism and biofilm formation in *Staphylococcus aureus*. J Bacteriol 189:8257-8269. https://doi.org/10.1128/JB.00645-07

54. Bush K, Bradford PA. 2016. β-lactams and β-lactamase inhibitors: an overview. Cold Spring Harb Perspect Med 6:a025247. https://doi.org/10.1101/cshperspect.a025247

55. Durand S, Braun F, Lioliou E, Romilly C, Helfer AC, Kuhn L, Quittot N, Nicolas P, Romby P, Condon C. 2015. A nitric oxide regulated small RNA controls expression of genes involved in redox homeostasis in *Bacillus subtilis*. PLoS Genet 11:e1004957. https://doi.org/10.1371/journal.pgen.1004957

56. Schoenfelder SMK, Lange C, Prakash SA, Marincola G, Lerch MF, Wencker FDR, Förstner KU, Sharma CM, Ziebuhr W. 2019. The small non-coding
RNA RsAE influences extracellular matrix composition in *Staphylococcus epidermidis* biofilm communities. PLoS Pathog 15:e1007618. https://doi.org/10.1371/journal.ppat.1007618

57. McKellar SW, Ivanova I, Arede P, Zapf RL, Mercier N, Chu L-C, Mediati DG, Pickering AC, Briaud P, Foster RG, Kudla G, Fitzgerald JR, Caldelari I, Carroll RK, Tree JJ, Granneman S. 2022. RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression. Nat Commun 13:3560. https://doi.org/10.1038/s41467-022-31173-y

58. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343-345. https://doi.org/10.1038/nmeth.1318

59. Monk IR, Tree JJ, Howden BP, Stinear TP, Foster TJ. 2015. Complete bypass of restriction systems for major *Staphylococcus aureus* lineages. MBio 6:e00308-15. https://doi.org/10.1128/mBio.00308-15

60. Crosby HA, Schlievert PM, Merriman JA, King JM, Salgado-Pabón W, Horswill AR. 2016. The *Staphylococcus aureus* global regulator MgrA modulates clumping and virulence by controlling surface protein expression. PLoS Pathog 12:e1005604. https://doi.org/10.1371/journal.ppat.1005604

61. Marchais A, Naville M, Bohn C, Bouloc P, Gautheret D. 2009. Single-pass classification of all noncoding sequences in a bacterial genome using phylogenetic profiles. Genome Res 19:1084-1092. https://doi.org/10.1101/gr.089714.108