6S RNA-Dependent Susceptibility to RNA Polymerase Inhibitors

Abstract

Bacterial small RNAs (sRNAs) contribute to a variety of regulatory mechanisms that modulate a wide range of pathways, including metabolism, virulence, and antibiotic resistance. We investigated the involvement of sRNAs in rifampicin resistance in the opportunistic pathogen Staphylococcus aureus. Using a competition assay with an sRNA mutant library, we identified 6S RNA as being required for protection against low concentrations of rifampicin, an RNA polymerase (RNAP) inhibitor. This effect applied to rifabutin and fidaxomicin, two other RNAP-targeting antibiotics. 6S RNA is highly conserved in bacteria, and its absence in two other major pathogens, Salmonella enterica and Clostridioides difficile, also impaired susceptibility to RNAP inhibitors. In S. aureus, 6S RNA is produced from an autonomous gene and accumulates in stationary phase. In contrast to what was reported for Escherichia coli, S. aureus 6S RNA does not appear to play a critical role in the transition from exponential to stationary phase but affects σ^B-regulated expression in prolonged stationary phase. Nevertheless, its protective effect against rifampicin is independent of alternative sigma factor σ^B activity. Our results suggest that 6S RNA helps maintain RNAP-σ^A integrity in S. aureus, which could in turn help bacteria withstand low concentrations of RNAP inhibitors.

Keywords: 6S RNA; Clostridioides difficile; RNA polymerase; Salmonella enterica; Staphylococcus aureus; antibiotic resistance; fidaxomicin; regulatory RNA; rifampicin; sigma factors.

FIG 1

Fitness loss of the ssrS^Sa mutant in the presence of a sublethal concentration of rifampicin. (A) Scheme of fitness experiment sampling. Three libraries were cultured for 3 days in tryptic soy broth (TSB) with or without rifampicin. Cultures were diluted 1:1,000 at 24 and 48 h. After each dilution step (0, 24, and 48 h), samples were withdrawn for tag counting under both growth conditions, when the OD₆₀₀ reached 1 (samplings 1, 3, and 4) and after the first overnight growth (sampling 2), as indicated. (B) Results of the competition assay between S. aureus sRNA mutants in the presence of 6 μg L⁻¹ rifampicin. Mutant strain names are on the y axis; the x axis shows the proportion of each mutant within the population grown in the presence of rifampicin normalized to the inoculum and to the corresponding sample grown in the absence of rifampicin. For each mutant, four histograms are shown; the color code corresponds to samplings indicated in panel A. Locus 2 and 3 mutants have tag insertions in loci likely not transcribed and not expected to alter the strain fitness. Error bars represent the experimental standard deviations between the three libraries. (Inset) Enlargement of bars for four relevant sRNA mutants: ssrS^Sa, sau60, and the control strain loci 2 and 3.

FIG 2

ssrS deletions confer a conserved rifampicin susceptibility phenotype from S. aureus to S. enterica. Three independent clones were grown overnight for each indicated strain. (A) Serial dilutions of overnight S. aureus (HG003 strain and its derivatives) cultures were spotted on BHI agar with or without 5 μg L⁻¹ rifampicin (RIF). (B) Serial dilutions of overnight S. enterica (LT2 strain and its derivatives) cultures were spotted on LB agar with or without 5 μg mL⁻¹ rifampicin. (C) Growth kinetics of S. aureus strains (HG003 and its derivatives) in BHI with or without 5 μg L⁻¹ rifampicin. OD₆₀₀ is an arbitrary value due to plate reader conditions, not representative of absorbance measurements of S. aureus in flasks. Error bars represent standard deviations from three experiments.

FIG 3

Susceptibility to RNAP inhibitors. (A) Serial dilution of S. aureus overnight cultures plated on solid medium containing rifabutin (RB), fidaxomicin (FDX), aureothricin (AUR), or no antibiotic. The numbers 1, 2, and 3 indicate independent clones. The antibiotic concentrations used were below the MIC. (B) Serial dilutions of C. difficile overnight cultures plated on solid medium containing FDX or no antibiotic. Pictures are representative of four replicates. Thiamphenicol was added in all plates (15 μg mL⁻¹) to maintain the plasmid. p, empty vector pMTL84121; pssrS^Cd, pMTL84121-ssrS^Cd.

FIG 4

ssrS gene expression and 6S RNA sequence in S. aureus. (A) ssrS^Sa expression. Cultures of HG003 grown in BHI were sampled at OD₆₀₀ of 1, 4, and 7 and ON (20 to 24 h incubation). A Northern blot probing for 6S RNA and transfer-messenger RNA (tmRNA) (for normalization) was performed. A quantification of 6S RNA normalized to tmRNA is presented. The standard deviation is based on biological triplicates. (B) Identification of 6S RNA ends by 5′-3′ RACE mapping. Sequences were analyzed separately at different time points (OD of 7, ON [20 to 24 h incubation], and day 4 [D4]) and compiled (mix). Colored letters represent extremities found in analyzed sequences. A color scale indicates the frequency at each 5′ or 3′ end. The highest frequencies are indicated below the corresponding nucleotides.

FIG 5

6S RNA and σ^B interplay in late stationary phase and in rifampicin response in S. aureus. (A) Fluorescence and OD₆₀₀ were monitored simultaneously in three strains (HG003 [parental], HG003 ΔssrS^Sa, and HG002) expressing a fluorescent protein (mAmetrine) under the control of the σ^B promoter of SAOUHSC_00624 from a plasmid (pPsigB-mAmetrine). HG002 (rsbU strain equivalent to the σ^B strain) is a negative control. Error bars represent standard deviations for biological triplicates. (B) Spot test comparing HG003 and HG002 (parental and ΔssrS^Sa strains, respectively) with a sublethal concentration of rifampicin (3.13 μg L⁻¹). Arbitrary values are shown as OD₆₀₀. Experiment was done with independent duplicates.

FIG 6

6S RNA and RNAP holoenzyme interactions in S. aureus. (A) EMSA with 6S [³²P]RNA (6S RNA), RNAP, σ^A, and σ^B. All the proteins were His tagged and purified. [³²P]SprB (SprB) is a control RNA. (B) Immunodetection of σ^A performed by Western blotting of samplings at OD₆₀₀ of 1 and 7, ON, and at day 3 (D3). Quantification of σ^A is relative to the amount of RNAP β/β′ subunits. Experiments were carried out in biological triplicates and analyzed by one-way ANOVA and Tukey’s HSD test [F_OD1(2,6) = 1.54, P = 0.288; F_OD7(2,6) = 5.21, P = 0.049, T_OD7(adjusted P value [P_adj] = 0.045, 95% confidence interval (CI) = 0.026 to 2.00); F_ON(2,6) = 4.50, P = 0.064; F_D3(2,6) = 8.91, P = 0.016, T_D3(P_adj = 0.013, 95% CI = −0.871 to −0.138)]. Significant differences of σ^A/(β/β′) means between strains (P < 0.05) are indicated by a star. W, wild type (HG003); Δ, ssrS^Sa mutant; e, ΔssrS^Sa ecto-ssrS^Sa. (C) Growth curves of HG003, ssrS^Sa mutant (ΔssrS^Sa) and complemented (ΔssrS^Sa ecto-ssrS^Sa) strains in BHI. Strains were cultured in independent triplicates from ON, 2-day, or 3-day precultures. Error bars represent standard deviations.