Cell-wall synthesis and ribosome maturation are co-regulated by an RNA switch in Mycobacterium tuberculosis

Abstract The success of Mycobacterium tuberculosis relies on the ability to switch between active growth and non-replicating persistence, associated with latent TB infection. Resuscitation promoting factors (Rpfs) are essential for the transition between these states. Rpf expression is tightly regulated as these enzymes are able to degrade the cell wall, and hence potentially lethal to the bacterium itself. We have identified a regulatory element in the 5′ untranslated region (UTR) of rpfB. We demonstrate that this element is a transcriptionally regulated RNA switch/riboswitch candidate, which appears to be restricted to pathogenic mycobacteria, suggesting a role in virulence. We have used translation start site mapping to re-annotate the RpfB start codon and identified and validated a ribosome binding site that is likely to be targeted by an rpfB antisense RNA. Finally, we show that rpfB is co-transcribed with ksgA and ispE downstream. ksgA encodes a universally conserved methyltransferase involved in ribosome maturation and ispE encodes an essential kinase involved in cell wall synthesis. This arrangement implies co-regulation of resuscitation, cell wall synthesis and ribosome maturation via the RNA switch.


In vitro transcription
Using mfold (4) we found that adding GUAAA to the 5' end of the RpfB 5' UTR did not alter the predicted structures. This enabled halting the RNA polymerase at position 11. Templates were generated by PCR amplification of the pGAMrrnX vector, agarose gel extraction of the amplicon which itself was then used as template in a second PCR. Amplified template was ethanol precipitated and re-suspended in DEPC treated H2O (Ambion) at 200 nM. The 302 basepair transcription template contained a -80 to -8 rrnB promoter fragment from pKA303 (5) in which the -10 box (acgTAACTT) was modified to an extended -10 box (TGnTAACTT) to enhance factor independent initiation. This was followed by a SpeI site and the sequence 'GTAAA' fused with the rpfB riboswitch sequence spanning from the P1 TSS +1 to +176 bp followed by the synB synthetic terminator for termination at 211 nt (3).
In vitro transcription reactions were carried out using E. coli RNA polymerase holoenzyme Transcription reactions were separated using denaturing 7 M urea PAGE 10% (v/v) 19:1 acrylamide:bis-acrylamide (MP Bio Science), exposed to a phosphorimager screen and developed using Typhoon FLA 9500 (GE), sizing transcripts using radiolabelled Decade and Century RNA markers (Ambion).

b-galactosidase assay for integrated reported and target expression (pIRATE)
Constructs utilised for assaying asRNA influence on riboswitch/RpfB expression in Figure S2 correspond to the following in Table S1:

Alignment of the tatD-rpfB intergenic regions
The multiple alignment of tatD-rpfB intergenic regions was built in stages following the rationale that the "switch" part of the intergenic region would be aligned optimally using a structure-guided alignment whereas the rest of the intergenic region should be aligned using a sequence-based alignment, aligning the most similar sequences first. Hence, the predicted "switch" regions of species with P1 promoters and predicted terminators (M. When aligning intergenic regions, approximately 10 codons (30 nucleotides) were added on either end to anchor the alignment. The use of additional nucleotides beyond the annotated intergenic region for each species are required in this case because the promoter regions and putative "switch" in M. tuberculosis overlap the coding region of tatD and the annotated CDS for rpfB are not consistent across mycobacterial species. Moreover, the start of the rpfB coding region of M. avium is annotated as 1090233 but it is clear from alignment to the M. tuberculosis rpfB that this could be extended 22 codons further upstream, hence the intergenic region plus buffer that was used for our alignment ends at nucleotide 1090196 instead. The three additional species whose intergenic regions appear more diverged are: Mycobacterium leprae TN (AL450380.1:314669-315016); Mycobacterium abscessus (NC_010397.1:1141551-1141702); Mycobacterium smegmatis str. MC2 155 (CP000480.1:c5523574-5523378). In M. abscessus, tatD appears to end prematurely compared with tatD from M. tuberculosis, hence the region used in the alignment did not include a tatD buffer "anchor" in this case.
MAFFT-produced alignments were saved in clustal format and reloaded into Jalview (8) to produce the relevant figures.

Prediction of terminators in intergenic regions
The program TransTermHP (v2.09) (9) was used to predict putative terminators in the intergenic regions between tatD and rpfB. Default parameter values were used for all searches (windows of DNA of length 6 containing at least 3 thymines were treated as potential terminator tails; each side of the putative stem had to be at least 4 nucleotides long; the length of the loop had to be between 3 and 13 nucleotides long; the total length of the stem+loop had to be less than 59 nucleotides). Putative terminators were returned if both the hairpin score and the tail score were less than the default cut-off values of -2 and -2.5 respectively. Where no terminators were predicted, the program 2ndscore from the same software suite (TransTermHP) was used (with default parameters) to predict the best hairpins starting at every nucleotide in the sequence. The best scoring, non-overlapping hairpins in the region of the M.tuberculosis predicted terminator were selected. Actinobacteria tatD rpfB ksgA metS ispE Figure S1: Conservation of the rpfB locus. The image shows the rpfB neighbourhood, according to STRING (https://string-db.org). The five genes, metS, tatD, rpfB, ksgA and ispE reside in the same locus in a range of species. Small, white arrows indicate non-conserved genes. UTR and first codons (from +1 to ATG at +215 in Fig. 1) was cloned between a constitutive promoter and lacZ in pIRATE-P1RpfB (Table S1). The asRNA was fused to another, divergently expressed heterologous promoter. Results indicate mean and SD of three biological replicates.