Staphylococcal sRNA IsrR downregulates methylthiotransferase MiaB under iron-deficient conditions

ABSTRACT Staphylococcus aureus is a major contributor to bacterial-associated mortality, owing to its exceptional adaptability across diverse environments. Iron is vital to most organisms but can be toxic in excess. To manage its intracellular iron, S. aureus, like many pathogens, employs intricate systems. We have recently identified IsrR as a key regulatory RNA induced during iron starvation. Its role is to reduce the synthesis of non-essential iron-containing proteins under iron-depleted conditions. In this study, we unveil IsrR’s regulatory action on MiaB, an enzyme responsible for methylthio group addition to specific sites on transfer RNAs (tRNAs). We use predictive tools and reporter fusion assays to demonstrate IsrR’s binding to the Shine-Dalgarno sequence of miaB RNA, thereby impeding its translation. The effectiveness of IsrR hinges on the integrity of a specific C-rich region. As MiaB is non-essential and has iron-sulfur clusters, IsrR induction spares iron by downregulating miaB. This may improve S. aureus fitness and aid in navigating the host’s nutritional immune defenses. IMPORTANCE In many biotopes, including those found within an infected host, bacteria confront the challenge of iron deficiency. They employ various strategies to adapt to this scarcity of nutrients, one of which involves regulating iron-containing proteins through the action of small regulatory RNAs. Our study shows how IsrR, a small RNA from S. aureus, prevents the production of MiaB, a tRNA-modifying enzyme containing iron-sulfur clusters. With this illustration, we propose a new substrate for an iron-sparing small RNA, which, when downregulated, should reduce the need for iron and save it to essential functions.


INTRODUCTION
Staphylococcus aureus is the leading cause of bacteria-associated mortality worldwide (1).
Its pathogenicity is due to its adaptability across various biotopes and the expression of multiple virulence determinants.Iron is a trace element universally required for growth, but toxic in excess.S. aureus, akin to other pathogens, has elaborate systems for maintaining its intracellular iron homeostasis to match environmental conditions (2).
We recently identified and characterized IsrR, a small regulatory RNA (sRNA) of S. aureus, contributing to an adaptive response to iron scarcity (3).Most sRNAs exert their activity by pairing with target mRNAs whose stability and/or translation they affect.They are generally expressed conditionally and contribute to adaptation to various growth conditions.Their estimated number in S. aureus varies from around 50 to a few hundred according to studies.
They are major regulators or contributors to fine-tune processes including metabolism, virulence, and antibiotic resistance; however, the function of most of them remains unknown (4)(5)(6).
IsrR, by restricting the production of non-essential iron-containing proteins, is expected to spare iron for indispensable functions.isrR expression is repressed by the ferric uptake regulator (Fur) which is active in iron-rich environments.Induced during iron deficiency, IsrR targets Shine-Dalgarno motifs on mRNAs coding for iron-containing proteins, notably restraining translation of specific mRNAs (fdhA, narG, nasD, and gltB2) linked to nitrate respiration and encoding [Fe-S] cluster-dependent enzymes (3).Given limited iron requirements, fermentative pathways are favored under iron-limited conditions.IsrR is a functional analog of RyhB from Escherichia coli (7) and of other sRNAs found in diverse bacterial species (reviewed in (8,9)) sharing targets and iron regulation.However, IsrR does not share sequence similarities or the same enzymatic requirements for its activity with RyhB and its reported functional analogs.IsrR, conserved within the Staphylococcus genus, is likely not an ortholog of these sRNAs, pointing to a convergent evolutionary solution for adaptation to low-iron conditions (3).
Hosts prevent pathogen growth through an arsenal of defense mechanisms, one of which, known as nutritional immunity, is the sequestration of nutrients such as iron (10).We reported that IsrR deficiency results in reduced virulence of S. aureus in a murine infection model, highlighting the role of IsrR in facilitating optimal spread of S. aureus in the host environment, probably critical for evasion of nutritional immunity (3).
Here, we demonstrate that IsrR downregulates miaB encoding a non-essential protein containing [Fe-S] clusters.This new example shows that IsrR is a central player in the maintenance of iron homeostasis in S. aureus.
S. aureus strains were grown aerobically in Brain Heart Infusion (BHI) broth at 37°C.E. coli strains were grown aerobically in Luria-Bertani (LB) broth at 37°C.Antibiotics were added to media as needed: ampicillin 100 μg/ml and chloramphenicol 20 μg/ml for E. coli; chloramphenicol 5 μg/ml and tetracycline 2.5 μg/ml for S. aureus.Iron-depleted media was obtained by the addition of DIP (2,2'-dipyridyl) 0.5 mM incubated for 30 min before any use.

Biocomputing analysis
Pairing predictions between IsrR and the mRNA targets were made using IntaRNA (15) set with default parameters except for suboptimal interaction overlap set to "can overlap in both".
The sequences used for IsrR and mRNA targets were extracted from the S. aureus NCTC8325 strain (NCBI RefSeq NC_007795.1).For the mRNA targets, the sequences used started at the TSS when known (e.g., EMOTES (16) or were arbitrarily made to start at nucleotide -60 with respect to the +1 of translation.The sequences contain the 5' UTR and the 17 first codons of the CDS.

Statistical tests
For Western-blots and fluorescence experiments, statistical analyses between two groups were made by testing the equality of variances between the groups using a Fisher test and, if and only if the two variances are equal, comparing them using a t-test.Error bars represent the standard deviation of the results.

Western blot analysis
The chromosomal miaB-flag reporter gene was constructed as described (Table 1).Strains were grown overnight with and without DIP.Protein extracts were prepared as follows: 4 mL of the bacterial culture were harvested by centrifugation at 4°C and 3800 g for 5 min.The resulting pellet was resuspended in 400 µL of 50 mM Tris-HCl.Cell disruption was achieved using a Fast-Prep homogenizer (MP Biomedicals).After disruption, crude protein extracts were obtained by centrifugation at 4°C and 21000 g for 15 min.The protein concentration of each extract was determined using the Bradford protein assay method.Ten µg of each protein extract were loaded onto a NuPAGE 4%-12% Bis-Tris gel (Invitrogen).
Electrophoresis was carried out at 150V for 45 min.Proteins were transferred from the gel to a PVDF membrane using the iBind system (Invitrogen).Immunolabeling was performed overnight using a rabbit anti-Flag antibody (Invitrogen) as primary antibody and an anti-rabbit IgG HRP-conjugated antibody (Promega) as secondary antibody.The iBlot system (Invitrogen) was employed for antibody incubation.Images of the immunolabeled membrane were acquired using a ChemiDoc MP imaging system (Bio-Rad).Bands were quantified using the Image Lab software (Bio-Rad).

Quantitative reverse transcriptase PCR
Overnight cultures HG003 and HG003 ΔisrR (biological triplicates, N=3) were diluted to an OD 600 = 0,005 and incubated in BHI supplemented with DIP (0.5 mM) at 37°C.At OD 600 = 1, rifampicin (concentration 200 µg/ml) was added to the growth medium.Bacterial cultures were harvested before rifampicin addition (time 0) and at 2, 5, and 10 min after rifampicin addition.Total RNAs were extracted as described (17) and treated with DNase (Qiagen) according to manufacturer's instructions.The last purification step was performed with the of the RNAs was verified using the Agilent 2100 bioanalyzer with the RNA 6000 Nano kit (Agilent Technologies).qRT-PCR experiments and data analysis were performed as described (18).The Ct values of rrsA were used to normalize the data and the determination of the gene expression ratio was achieved using the ∆∆Ct method.Primers used for qRT-PCRs are indicated in Table 1.

MiaB is a putative IsrR-target
We used CopraRNA (19), a software designed for predicting targets of bacterial sRNAs across diverse species, to unveil potential targets of IsrR.Given isrR conservation throughout the Staphylococcus genus, CopraRNA emerged as an efficient tool for IsrR target prediction (20), resulting in the successful validation of four of its targets as reported (3).
Among the predicted targets was miaB mRNA (3,21).The latter encodes MiaB, a methylthiotransferase catalyzing the addition of a methylthio group (ms 2 ) to isopentenyl adenine (i 6 A) at position 37 (ms 2 i 6 A-37) of transfer RNA (tRNA) anticodons ( 22).This modification is present in most tRNAs reading codons starting with a U and is conserved from bacteria to human (23); it contributes to stabilizing the codon-anticodon interaction (24) and affects translational accuracy (25).
To substantiate the proposed interaction between IsrR and miaB mRNA, we used the IntaRNA software (15).The input was the complete IsrR sequence and the miaB 5' untranslated region (UTR) with 25 codons.IsrR is characterized by three single-stranded Crich regions, denoted as CRR1, CCR2, and CRR3 (3).The CRRs are common sequences among Firmicutes' regulatory RNAs where they are presumably seed-binding motifs to G-rich sequences (26).IntaRNA analysis suggests a potential pairing of IsrR with the Shine-Dalgarno sequence of miaB RNA (Figure 1A).The pairing involves 55 nucleotides, an unusually high number of pairs.The energy of sRNA-mRNA pairing is low (-22.33 kcal/mol) supporting its existence.It involves the CCR3 GCCCG sequence, which could serve as a seed motif of the interaction.Of note, the isrR/ miaB mRNA pairing also is conserved within the Staphylococci genus (Figure 1B).

IsrR-dependent reduction of MiaB expression
To explore the potential influence of IsrR on miaB expression, a gene fusion was constructed wherein the native miaB gene was replaced by a copy harboring the complete miaB open reading frames extended with a C-terminal Flag sequence (Figure 2A).The resulting MiaB-Flag fusion protein was detectable through anti-Flag antibodies, thereby serving as a quantifiable surrogate for wild-type gene expression levels.This reporter fusion was integrated into the chromosomes of both HG003 and its corresponding ΔisrR isogenic derivative.Western blot analyses conducted on HG003 cultures cultivated under nutrient-rich conditions show an unaltered MiaB-Flag abundance, irrespective of the presence or absence of isrR.This status quo was in line with the known Fur-mediated repression of isrR under iron-replete conditions.We previously reported that introducing the iron chelator 2,2'-dipyridyl (DIP) into the growth medium elicits a release from Fur repression, consequently inducing IsrR expression (3).Remarkably, under DIP-induced conditions, no MiaB-Flag was detected regardless of the isrR background (Figure 2B).This result is interpreted as iron depletion leading to apo-MiaB being unstable; indeed, the stability of proteins with Fe-S clusters often depends on cluster integrity (27).For example, the glutamine phosphoribosylpyrophosphate amidotransferase of Bacillus subtilis has a [4Fe-4S] cluster which when degraded by O 2 favors enzyme degradation (28).
To overcome this issue, a fur::tet allele (29) was introduced by phage-mediated transduction in the miaB-flag and ΔisrR miaB-flag strains rendering isrR constitutively expressed, as previously reported (3).Expectedly, in this genetic background, MiaB-Flag was barely detected in the fur strain as opposed to its fur ΔisrR derivative (Figure 2C-D).

IsrR-mediated post-transcriptional regulation of miaB expression
In the absence of Fur, isrR is expressed and miaB is downregulated; the observation is explained by predicted base pairing interaction between IsrR and miaB RNA.In many cases, sRNAs targeting mRNAs affect their stability.We therefore considered that IsrR might decrease miaB mRNA stability.This question was addressed by comparing miaB mRNA stability in wild-type and ΔisrR strains under iron-starved condition.At OD 600 =1, RNA synthesis was inhibited by rifampicin addition to the growth medium, and miaB mRNA turnover was determined by qRT-PCR quantification at different time points.No significant differences were observed, suggesting that IsrR does not affect miaB mRNA stability (Figure 2E).We, therefore, considered that the regulation of miaB by IsrR occurred primarily at the translational level, a feature already observed for the regulation of fdhA and gltB2 by IsrR (3).
To examine this hypothesis, a genetic reporter of miaB translation was constructed.It is based on pRN112, a replication thermosensitive plasmid for chromosomal integration carrying the mAmetrine gene (mAm) under the transcriptional control of the P1 sarA promoter (P1 sarA ) (14).The 5' untranslated region (5'UTR) of miaB, along with an additional 54 nucleotides, was inserted within pRN112 under the transcriptional control of P1 sarA .The first 18 miaB codons were positioned upstream and in frame mAm (Figure 3A).With this construct, the mAmetrine fluorescence served as a proxy for miaB expression levels, and as the reporter gene is under P1 sarA transcriptional regulation, its expression is expected to be constitutive.To mitigate concerns associated with multi-copy reporter plasmids, the engineered sequence was integrated into the chromosome of both HG003 strain and its ΔisrR derivative.As for the endogenous miaB, the 5'UTRmiaB-mAm reporter exhibited significant downregulation under iron-starved conditions in an IsrR-dependent manner, while its transcription was driven from P1 sarA (Figure 3B).
Our experimental findings support that IsrR downregulates the expression of the reporter fusion by a post-transcriptional event.
The C-rich region 3 (CRR3) of IsrR plays a pivotal role in the downregulation of miaB mRNA.
Pairing interactions between sRNAs and their targets are initiated by a kissing complex between single-stranded regions, which then propagates within both partners (30).sRNAs can have multiple seed regions, usually C-rich in S. aureus (26), specific to given targets as exemplified by RNAIII (review in ( 4)) or with redundant activities as in RsaE (31).To determine specific regions of IsrR responsible for its regulatory activity against miaB mRNA, experiments using the HG003 ΔisrR strain, harboring the 5'UTR miaB fluorescent reporter were conducted.Plasmids expressing various IsrR derivatives, including wild-type IsrR, IsrR lacking CRR1, CRR2, CRR3, or all three CRRs were used.isrR was placed under the control of the tet promoter (P tet ) in the absence of the Tet repressor (TetR), although it should be noted that a Fur binding site is present within the transcribed sequence (3).Consequently, we supplemented the growth media with DIP to prevent Fur repression on isrR expression.
The presence of a plasmid expressing IsrR markedly diminished the fluorescence of the reporter fusion when compared to a control plasmid or a plasmid expressing IsrR with no CRR motif.In contrast, the deletion of CRR3 resulted in a significant increase in mAmetrine fluorescence indicating a loss of activity against the reporter fusion.Note that the same construct producing IsrRΔC1 is expressed and active against gltB2 and fdhA mRNAs (3).
However, the deletion of CRR1 and CRR2 in isolation had no substantial impact on IsrR's activity against the miaB 5'UTR reporter (Figure 3C).This observation suggests that these two motifs may either be dispensable for the regulatory activity or possess redundant functions with respect to the miaB 5'UTR.
To discern between these possibilities, we introduced into the ΔisrR::tag135 strain plasmids expressing different isrR derivatives with deletions of two CRRs.As expected, strains carrying alleles with the CRR3 deletion (isrRΔC1C3 and isrRΔC2C3) failed to complement the ΔisrR allele.In contrast, isrRΔC1C2 was still capable of downregulating the fluorescence of the P sarA miaB_5'UTRmAm reporter (Figure 3C).These findings align with the results obtained from IntaRNA analysis, which indicates that the IsrR/miaB mRNA pairing energy remains substantial as long as CRR3 and its surrounding region remain intact (Figure 1).To further support the involvement of CRR3 in IsrR/miaB mRNA pairing, the reporter system was tested with point mutations altering the predicted pairing.First, the CRR3 motif was changed from CCC to ATA (Figure 3D).Expectedly, mutated IsrR prevented the miaB reporter fusion downregulation (Figure 3D).A compensatory mutation restoring the pairing with the mutated IsrR was introduced within the miaB sequence by changing a GGG sequence upstream miaB Shine Dalgarno sequence to TAT.These mutations altering the 5'UTR region led to a higher level of reporter gene expression.However, while wild-type IsrR did not affect the expression of the mutated reporter fusion, mutated-IsrR downregulated its expression.These experiments support the conclusion that CRR3/5'UTR miaB RNA pairing is needed for the IsrR-dependent downregulation of miaB.
It is worth noting that the requirement for CRRs in IsrR activity appears to be contingent upon its target mRNA (Figure 4).For instance, the absence of CRR3 does not impede the downregulation of gltB2 and fdhA mRNAs (3).Conversely, for the latter mRNAs, the integrity of both CRR1 and CRR2 is indispensable, presenting a contrast to miaB mRNA regulation.This observation suggests that the acquisition of multiple CRRs, together with their adjacent regions, confers on regulatory RNAs the ability to control a wider range of target mRNAs.sRNAs likely evolve by acquiring new domains to interact with different targets (e.g.miaB RNA vs gltB2 mRNA).At the same time, mRNAs may evolve to adapt to the available sRNA motifs (e.g.gltB2 RNA vs fdhA mRNA).

DISCUSSION
MiaB is a conserved enzyme that modifies tRNAs acting after MiaA on an adenine adjacent to the anticodon site (22)(23)(24)(25).In some species, the modified adenosine is then hydroxylated by MiaE, but this enzyme is not present in S. aureus (32,33).RNA modifications optimize codon/anticodon interactions and improve translation fidelity; for example, the absence of MiaB was shown to affect the efficiency of a suppressor tRNA (22).
The relationship between iron metabolism and the MiaB enzyme has been a subject of scientific interest for several decades.As far back as the 1960s, researchers noted the absence of certain tRNA modifications in E. coli cultured in iron-free media, underscoring the connection between iron availability and tRNA modifications (34).Subsequent investigations revealed that the miaB gene encodes the tRNA methylthiotransferase responsible for these modifications, and mutations in miaB were found to reduce decoding efficiencies under specific conditions (22).Notably, MiaB was initially thought to contain two [4Fe-4S] clusters (35), but a more recent study concludes that MiaB has one [4Fe-4S] and one [3Fe-4S] (36).
Under iron-starved conditions, clusters depletion renders MiaB inactive.Furthermore, as shown here, in iron-starved condition, MiaB-Flag was not detectable suggesting that in the absence of iron cluster, MiaB is degraded as observed for other proteins with [Fe-S] clusters (27).
In E. coli, the influence of MiaB extends to the regulation of Fur through a distinctive mechanism (37).The fur gene is preceded by uof, an open reading frame located "upstream of fur", and the translation of fur is tightly coupled to that of uof.An intriguing aspect of this regulatory relationship is the presence of a codon UCA, decoded by a MiaB-dependent tRNA Ser .Consequently, efficient uof/fur translation relies on functional MiaB.When ironstarvation conditions prevail, MiaB becomes inactive, impairing tRNA methylthiolation.This, in turn, leads to reduced uof/fur translation, ultimately favoring the expression of iron uptake genes.However, this regulation is not conserved in S. aureus since no uof-like sequence was observed.
In standard laboratory conditions, Enterobacteria appear to be resilient to the absence of MiaB (38).In the Gram-positive bacterium B. subtilis, miaB (aka ymcB) is also non-essential under the tested conditions (39) while its inactivation is associated with the disappearance of ms 2 i 6 A post-transcriptional modifications (40).There is a 68% amino acid identity between B. subtilis and S. aureus MiaBs, suggesting that Staphylococcal MiaB indeed methylthiolates tRNAs.miaB in Staphylococcus aureus is also not essential (41).Consequently, preventing MiaB activity is likely to have minimal or no discernible consequences in various growth conditions.Since MiaB needs [Fe-S] clusters as cofactors, suppressing its synthesis in iron-processes.
It is noteworthy that the inhibition of miaB RNA translation by a sRNA associated with the iron-sparing response is a feature observed thus far only in S. aureus.Surprisingly, in E. coli, RyhB does not target miaB mRNA (42), presumably because MiaB function is more critical to some cellular functions in this organism.The absence of MiaB can result in reduced translation accuracy, impacting gene expression.Therefore, modulating miaB expression to spare iron represents a viable alternative for bacteria, provided the fitness benefits outweigh the costs.* Plasmids were constructed by isothermal assembly with the indicated PCR product(s) (11).Numbers (#/#) are primer pairs used for PCR amplifications on the indicated substrate.For primer sequences, see Table 1 bottom part.

Figure 1 :
Figure 1: Computer prediction of IsrR and mutated IsrR pairing with miaB mRNA A) IntaRNA (15) pairing prediction of IsrR and IsrR derivatives deleted for CRR1, CRR2 or CRR3 with miaB mRNA.Blue sequences, SD; red sequences, CRRs; bold underline characters, GUG start codon; E, energy; HE, hybridization energy.B) IntaRNA pairing prediction of IsrR and miaB mRNA in different species from the Staphylococcus genus.IsrR sequences from the indicated species were obtained with GLASSgo (44).For legends, see Figure 1A.

Figure 2 :
Figure 2: MiaB-Flag reporter detection A) Schematic representation of the MiaB-Flag reporter fusion.Blue, 3' end of miaB ORF; Orange, flag sequence; Yellow, native stop codon; Grey, first nts of miaB 3' UTR.B) Western blot experiment with anti-Flag antibodies in presence or absence of iron chelators (N=4).Genotypes and growth conditions are indicated.75 kDa, non-specific signal present in all samples including Flag-less strain.60 kDa, signals corresponding to MiaB-Flag.C) Western blot experiment with anti-Flag antibodies with fur and fur::tet strains (N=3) as explained in Figure 2B.D) Histograms.MiaB-Flag signals from Western blot (Figure 2C) were normalized to the 75 kDa signals from their corresponding lane.Histogram y-axis indicates the values of normalized MiaB-Flag signals of indicated strains divided by the normalized MiaB-Flag signal from the wild-type background strain (fur + ).Error bars indicate the standard deviation of three biological replicates.***, P-value < 0,001 for Student test (N=3).E) The stability of miaB mRNA is not significantly affected by IsrR.HG003 and its isogenic ΔisrR derivative were grown in rich medium supplemented with DIP.At t0, rifampicin (200 µg/ml) was added to the growth medium.Cultures were sampled at t0, 2, 5, and 10 min after the addition of rifampicin, total RNA was extracted, and the amounts of miaB mRNA, rrsA rRNA (control), and IsrR were determined by qRT-PCR.Histograms Y-axis show the quantification of miaB mRNA, rrsA sRNA, and IsrR normalized to t0.Error bars indicate the standard deviation of biological triplicates (N=3).

Figure 3 :
Figure 3: Translational repression of miaB reporter fusion by IsrR A) Chromosomal reporter fusion for detection of IsrR activity.Red nt; transcription start site, blue bold nts; RBS, black bold nts; start codon, underlined nts; nts in interaction with IsrR as predicted by IntaRNA.B) The translational activity of the miaB-mAm reporter fusion was quantified by growing strains HG003 and ΔisrR HG003 harboring the fusion on BHI and BHI supplemented with DIP.Results (N=3) are normalized to OD 600 =1.C) The ΔisrR strain harboring the miaB-mAm reporter fusion was transformed with plasmids expressing different

Figure 4 :
Figure 4: Contribution of CRRs to downregulate different mRNA targets Negative arrows starting for either CRR1, 2 or 3 and pointing to mRNA targets indicate CRRs required for IsrR activity toward the corresponding targets.Data from in vivo results with fluorescent reporters in (3) and this work.

AATGATACGGGCAAATAGAAAGGATTTTGTAAA
fur::tet ∆isrR miaB-flag fur::tet miaB-flag D Y K D D D D K GAG CCG GAA ATG GTG ATT CAA GAC TAC AAA GAC GAT GAC GAC AAG TAA ATGTATAATA GTG AAC GAA GAA CAA AGA AAA GCA AGT TCT GTA GAT GTT TTA GCT GAG AGA GAT

Table 1 ;
Strains, plasmids, and DNA primers used in this study.