Riboswitch and small RNAs modulate btuB translation initiation in Escherichia coli and trigger distinct mRNA regulatory mechanisms

Abstract Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.

agar plates, BYE (LB without salt) agar plates supplemented with 6% (w/v) sucrose (BS plates) or liquid and solid minimal medium A. Acid LB (pH 4.7) was made by addition of HCl.When needed, media were supplemented with an appropriate antibiotic.Unless otherwise stated, kanamycin (Kan) at 50 μg/mL, chloramphenicol (Cam) at 20 μg/mL, ampicillin (Amp) at 150 μg/mL or tetracycline (Tet) at 10 μg/mL were utilized for plasmid maintenance.For selection of strains carrying a single copy of a drug resistance marker the concentrations were twice as low.During the P1 transduction, selectable plates also contained 5 mM sodium citrate.

Designing an E. coli strain for construction of chromosomal mScarlet fluorescent reporter fusions
The strain OK510 was engineered for constructing mScarlet fusions.It is a DJ624 (MG1655 ΔlacX74 mal::lacIq) derivative that carries a mini-λ-Tet prophage (3), which provides λ Red recombination functions that are induced at 42°C, and an mScarlet locus placed into the 6 bp intergenic region argG-yhbX.This locus (see Supplementary Figure S15) comprises a synthetic bidirectional transcriptional terminator L3S2P21 (TT1) (4), an inactive PLtetO-1 promoter (5) that is deprived of the -10 region, the Pcat-cat-sacB cassette expressed in the opposite direction to that of following 'mScarlet region (mScarlet-I ORF missing the translation initiation codon), and the nptII ORF, which is transcriptionally coupled to 'mScarlet and flanked by two FRT sites.The FRT sites allow for optional elimination of the nptII ORF via Flp-FRT recombination.The last element of the locus is a natural bidirectional transcriptional terminator, ECK120026481 (TT2) (4).Hence, the strain features Tet R (due to the presence of mini-λ-Tet), Sucr S , Cam R and Kan S as both 'mScarlet and nptII genes are not expressed.
The construction of mScarlet fusions was made with PCR cassette containing ~40 bp homologies on both ends that allow for recombining with its 5'-end within the inactive PLtetO-1 restoring the -10 region, and with its 3'-end within the 'mScarlet (restoring the translation initiation codon), so that it replaces the Pcat-cat-sacB sequences.This generates a translational fusion to mScarlet, which is expressed from a strong constitutive (in the absence of the Tet repressor) promoter PLtetO-1 and provides expression of the downstream nptII ORF that renders the recombinants resistant to kanamycin.Thus, the recombinants can be selected for both resistance to sucrose and kanamycin, which greatly reduces occurrence of false-positive clones as compared to selection on sucrose alone.Selection at 37°C also facilitates elimination the mini-λ-Tet prophage in the majority of recombinants.This OK510 strain was made through multiple steps of recombineering, summarized in Supplementary Figure S15.First, a cat-sacB cassette was amplified in 3 steps in order to add upstream the inactive PLtetO-1 promoter lacking the -10 region, as well as upstream and downstream transcription terminators (TT1 and TT2, respectively) and homology regions to the argG/yhbX locus.The first step PCR template was the genomic DNA of a strain carrying a cat-sacB cassette and the primers used are (i) Ptetno-10-cat-for and Ter-catsacRev (1st step), (ii) Ter-Ptet-for and yhbX-Ter-rev (2nd step = reamplification of PCR of 1st step) and (iii) argG-Ter-for and yhbX-Ter-rev (3rd step = reamplification of PCR of 2nd step).This last PCR product was recombined in strain MG1432 and recombinants were selected on LB-Cam at 30°C, and then checked for sucrose sensitivity and Tet resistance (to ensure that they kept the mini-l Tet for further recombineering).The structure of the locus was checked by sequencing and the resulting strain was called MG2346.In a second recombineering step, the nptII gene followed by an FRT site (PCR product amplified from genomic DNA with primers sacB-KanR-For and FRT-sacB-Rev) was introduced downstream of the sacB gene, giving rise to the strain MG2348 after selection on Kan and sequencing of the locus.The third step consisted in replacing the cat-sacB cassette by the mScarlet gene devoid of the start codon, and followed by an FRT site.This was again done by recombineering, into strain MG2348 this time, using a PCR product amplified from the pNF02-mScI plasmid (a gift from N. Fraikin and L. Van Melderen, (6)) with primers Ptetno-10-mSC-For and mSc-FRT-Rev, and then reamplified with primers Ptet-55-12For and postFRT-KanR-Rev to increase the length of the homology regions.Recombinants were selected at 30°C on BS plates, and checked for CamS and TetR.Clones whose structure of this TT1-Ptet(no-10)-'mScarlet(no AUG)-FRT-nptII-FRT-TT2 region was confirmed by sequencing were found to be KanS; out of those, strain MG2352 was used for the subsequent step.
At the last step, the Pcat-cat-sacB cassette, made with oligos AK411 and AK412 using chromosomal DNA of NC397 strain (7) as a template, was recombined into MG2352 with subsequent selection of recombinants on LB-Cam plates at 30°C giving rise to the strain OK509.To avoid mutations that could have accumulated upon multiple recombineering passages during construction of the OK509 strain, the 'mScarlet locus of OK509 was transferred into the initial strain, MG1432, selecting transductants on LB-Cam plates at 30°C.The resulting strain, OK510, was used for construction of the mScarlet fusions used in this study.

lacZ fusions construction
Construction of lacZ fusions was done by replacing a cat-sacB cassette upstream of lacZ by recombineering into strain PM1205 ((8), for construction of PBAD driven fusions) or MG1508 ((9), for construction of PLtetO-1 driven fusions) and OK868 (an MG1508 variant with the ∆lacY::FRT deletion).At low IPTG concentrations, its intracellular levels are largely dependent on the LacY-mediated uptake.As lacZ and lacY are co-expressed, the LacY levels (and IPTG uptake activity) vary according to the lacZ fusion activity.To uncouple the intracellular IPTG levels from the activity of a lacZ fusion, i.e. to provide equal induction conditions in different lacZ fusion strains, ∆lacY::FRT background was used (for Supplementary Figure S12).To this end, the ∆lacY::FRT-nptII-FRT allele was PCR amplified from JW0334 chromosome using AK51 and AK52 primers, and the resulting PCR product was recombined into MG1508, selecting on Kan (generating OK865).The nptII was eliminated from OK865 via Flp-FRT recombination (leaving ∆lacY::FRT scar) using pCP20 plasmid giving rise to OK866.Then, the PLtetO-1-cat-sacB-lacZ-∆lacY::FRT region was transferred into MG1432 via P1 transduction selecting on Cam (at 30°C, to preserve the mini-λ-Tet), and the obtained ∆lacY::FRT version of MG1508 was referred to as OK868.
In general, the PCR products were obtained using MG1655 genomic DNA as a template, and, when needed, the length of the homology regions was extended by re-amplification with primers Ptet-55-12For or lacZ28-66rev as necessary.The mutations EP1, EP2, EP3, M9, M11 and mutH1 were present on the primers, and the same was true for the lowPLtetO-1 mutant that changes the -35 region consensus of PLtetO-1 from TTGACA into TGGACA.For the construction of the PBAD-btuB-lacZ transcriptional fusions, successive PCR reactions introduced, downstream of the selected btuB region, an in-frame sequence encoding a DPAF peptide terminated by a stop codon, followed by lacZ sequence starting To produce ∆lacY::FRT versions of the lacZ fusions, the fusion regions were PCR amplified with primers Ptet-55-12For and lacZ28-66rev using corresponding WT lacY fusion strains chromosome as a template.The PCR products were recombineered into OK868.
PLtetO-1-driven versions of the transcriptional fusions were obtained by recombineering in MG1508.In each case, a relevant PCR product for recombineering was made in two steps: 1) amplification of the fusion region with primers 5'PtetBtuB-240 and lacZ28-66rev using a corresponding PBAD-driven fusion strain chromosome as a template, and 2) reamplification of the step 1 PCR product with Ptet-55-12For and lacZ28-66rev.
Recombinants were selected on BS plates supplemented with 0.002% X-gal at 37°C, picking blue colonies and checking them for sensitivity to chloramphenicol (loss of the cat-sacB cassette) and tetracycline (loss of the mini-λ-Tet).The lac loci of resulting strains were verified by sequencing.

Elimination of the nptII ORF from FRT-nptII-FRT cassettes
Unnecessary nptII ORFs originating from the OK510 derivatives and from Keio collection knockouts (11) used in this study were eliminated via Flp-FRT recombination.To this end, cells were transformed with the pCP20 plasmid (12).The plasmid provides in trans synthesis of flippase (Flp) and possesses a thermo-sensitive replication origin.
Transformants were selected at 30°C on LB-Cam (10 μg/mL) plates supplemented with chloramphenicol and purified once on the same medium.Then, an individual transformant colony was grown overnight in 10 mL of LB medium at 42°C and plated on LB at ~10 2 cfu per plate 37°C.After such passage vast majority of clones were both KanS and CamS.

Constructing Hfq variant strains carrying the mScarlet fusions under study
The construction of strains carrying different hfq mutations is based on (13), with minor modifications to combine these different alleles with mScarlet fusions.First, the ΔargG::FRT-nptII-FRT allele was transferred from JW3140 (from the Keio collection (11)) to DJ624 by P1 transduction to generate strain OK523.This OK523 strain was then cured of nptII as described above generating OK530.The Δhfq::cat-sacB allele was cotransduced with nearby purA::FRT-nptII-FRT from DJS2604 (from D. Schu, NIH) to OK530 selecting on LB-Cam, thus giving rise to OK564.The hfq alleles were transferred Bastet et al.
Then, each fusion was transduced into the five hfq variant strains.Arginine prototroph transductants were selected at 37°C on minimal A-agar plates supplemented with 0.2% glucose (mScarlet locus is 100% co-transduced with the wild type argG allele).The resulting strains, OK586 to OK605, are listed in the Supplementary Table S1.

Cloning Hfq under an IPTG-inducible promoter
A pCAS18 plasmid was given by M. Springer.It carries E. coli rpsR gene that was cloned in a pCA24N (14) using BseRI and HindIII restriction sites (SalI site is present between the rpsR stop codon and HindIII site).A unique NdeI restriction site was made at the rpsR start codon in pCAS18 by PCR with primers AK74 and AK75 giving rise to pCAS18n.
Then, a 233 bp NdeI-SalI region of pCAS18n containing rpsR was replaced by a 354 bp Hfq-containing fragment of the pHfq (15).The resulting plasmid that provides IPTGinduced expression of Hfq was referred to as pNK139.Thus, the pNK139 allows IPTGinducible synthesis of Hfq from the PT5-lac promoter, using host RNA polymerase.This provides an opportunity to observe the dose-dependent effect of Hfq on cellular processes such as model fusion expression.
Absorbance at 600 nm and fluorescence (excitation at 560±15 nm, emission at 600±15 nm with 580 nm dichroic filter) was measured every 12 minutes for 16 h.Each experiment was made in triplicate (starting from three independent transformant colonies).Experimental  OK581

E. coli hfq under PT5-lac control in pCAS18n
This study; used in Fig. S12 (pCA-Hfq) FRT is the Flp recombinase target site.mini-λ-Tet is a λ prophage lacking replication and lytic functions.It provides Red functions required for recombination, and resistance to tetracycline (3).mScarlet is a fluorescent reporter (28); the mScarlet-I gene was optimized for codon usage in E. coli (6).nptI and nptII are kanamycin resistance ORFs originating from Tn903 and Tn5, respectively.PLtetO-1 and PLlacO-1 are hybrid tetracycline and IPTG-inducible promoters, respectively (5).

Templates for T7 in vitro transcription of radiolabeled probes for Northern blots (used in Fig. 1 )
T7btuBprobe-for TAATACGACTCACTATAGCGTACGCCATCAATTAACACCAAC in vitro transcription of the antisense of btuB (