Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella

Abstract

Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.

Keywords: Hfq; RprA; feedforward control; plasmid conjugation; sRNA.

Fig. 1.

Multiple target regulation by RprA in Salmonella. (A) Alignment of the rprA gene from selected enterobacterial species (ECA, Erwinia carotovora; ECO, Escherichia coli K12; KPN, Klebsiella pneumoniae; PLU, Photorhabdus luminescens; SFL, Shigella flexneri; STM, Salmonella enterica sv. Typhimurium). Transcription control regions −10 and −35 are boxed, the transcription initiation site is marked by an arrow. Scissors indicate the RprA processing site, and inverted arrows refer to the rho-independent terminator. (B) Northern blot analysis of RprA in Salmonella. Samples were collected at several stages of growth (OD₆₀₀ of 0.5, 1.0, and 2.0, at 3 and 6 h after cells had reached OD₆₀₀ of 2.0, and after 24 h of cultivation). 5S rRNA served as loading control. (C) Microarray analysis of RprA full-length and RprA processed pulse expression. Expression profiles of pulse-induced full-length and processed RprA were compared with samples carrying control plasmids. A heat map of genes regulated by full-length RprA (more than twofold) is shown and compared with regulation by processed RprA. (D) Western and Northern blot analyses of σ^S, RicI::3×FLAG, and RprA production after pulse expression of full-length and processed RprA. Wild-type and ΔrprA carrying the indicated plasmids were grown to early stationary phase (OD₆₀₀ of 1.5) and induced for pBAD expression. 5S rRNA (Northern blot) and GroEL (Western blot) served as loading controls.

Fig. 2.

Anti-antisense activation of ricI. (A) Graphical presentation of the ricI 5′-UTR alone (Top) or in complex with RprA (Bottom). Numbering of ricI and RprA is relative to their transcription start site. ricI base pairs with the 5′ terminal end of the processed RprA form. Arrows denote mutations introduced in ricI::gfp and RprA, respectively. (B) Western blot analysis of ricI::gfp variants (as indicated in A, Top) expressed in Salmonella ΔrprA cells. (C) Western blot analysis of ΔrprA Salmonella harboring plasmid pRprA or mutant plasmid, pRprAC63, in combination with either wild-type ricI::gfp or mutant ricIG45::gfp fusion plasmids. GroEL served as loading control.

Fig. S1.

Posttranscriptional control of ricI. (A) Secondary structure of the ricI 5′-UTR. Truncations and mutations tested in B are indicated by arrows. The SD sequence and the start codon are underlined. (B) Salmonella ΔrprA cells carrying the indicated ricI::gfp variants were tested for GFP production by Western blot. (C) Salmonella carrying a GFP control plasmid (pXG-1) were cotransformed with a control plasmid (pctr.) or a RprA overexpression plasmid (pRprA) and tested by Western blot for GFP production. (D) Salmonella carrying the truncated ricI::gfp plasmid (pKP130; A) were cotransformed with a control plasmid (pctr.) or a RprA overexpression plasmid (pRprA) and tested by Western blot for GFP production. (E) Salmonella carrying the mutated ricI::gfp plasmid (pKP138; A) were cotransformed with a control plasmid (pctr.) or a RprA overexpression plasmid (pRprA) and tested by Western blot for GFP production. (B–E) GroEL served as loading control.

Fig. 3.

Bile-induced expression of RicI. (A) Western blot analysis of RicI::3×FLAG expression. Wild-type and ΔrprA cells were grown to late exponential phase (OD₆₀₀ of 1.0) and treated with bile salts (3% final concentration). Whole-protein samples were collected at the indicated time points and probed for RicI::3×FLAG production. (B) Wild-type and the indicated Salmonella mutants (ΔrprA, ΔrcsF, ΔrcsC, ΔrcsB, and ΔrpoS) were cultivated in LB media (with or without 3% bile salts) to OD₆₀₀ of 1.0 and probed for bile-mediated activation of RicI::3×FLAG (Western blot) and RprA (Northern blot). (C) RicI::3×FLAG production in the context of RprA overexpression. Wild-type, ΔrprA, ΔrcsB, and ΔrpoS cells transformed with a control plasmid (pctr.) or the RprA overexpression plasmid (pRprA) were tested for RicI::3×FLAG expression on Western blot.

Fig. S2.

Gene synteny analysis of ricI. The genomic regions upstream and downstream of ricI (STM4242) from related enterobacteria were inspected for sequence conservation (ECO, Escherichia coli K12; KPN, Klebsiella pneumoniae; SFL, Shigella flexneri; SL1344, Salmonella enterica sv. Typhimurium SL1344; STM, Salmonella enterica sv. Typhimurium LT2; STY, Salmonella typhi CT18). Coloring refers to the GC content of the individual genes.

Fig. S3.

Role of σ^S for ricI expression. (A) Same as Fig. 2C (lanes 1 and 2), but RicI::GFP expression was monitored in a Salmonella ΔrpoS strain. (B) 5′-RACE analysis of the ricI transcriptional start site. C, control (genomic DNA); M, DNA size marker; T−, no TAP treatment; T+, TAP-treated samples.

Fig. 4.

σ^S activates ricI transcription. (A) Alignment of the ricI promoter sequence from Salmonellae and Enterobacter species (ESP, Enterobacter sp. 638; SAG, S. enterica sv. Agona; SAR, S. enterica sv. arizonae; SBO, Salmonella bongori; SEB, S. enterica sv. Bovismorbificans; SSG, S. enterica sv. Schwarzengrund; STM, S. enterica sv. Typhimurium). Transcription control regions −10 and −35 are boxed, and the transcription initiation (+1) site is marked by an arrow. Residue C-13 is shown in bold. (B) Wild-type, ΔrprA, and ΔrpoS cells carrying the transcriptional ricI::lacZ reporter were monitored for β-galactosidase production at the indicated stages of growth. (C) Wild-type and ΔrpoS cells transformed with either a wild-type (pricI::gfp) or the mutant [pricI::gfp (G13)] reporter were cultivated to early stationary phase (OD₆₀₀ of 2.0) and assayed for GFP production.

Fig. 5.

An FFL with AND-gate logic controls RicI production. (A) Schematic display of the FFL regulating RicI production. Both RprA and σ^S are required for RicI expression. Dashed lines indicated posttranscriptional regulation, and solid lines denote control at the transcriptional level. (B) Secondary structure of RprA. Mutations tested in C are indicated by arrows. Scissors mark the RprA processing site. (C) Salmonella carrying the translational ricI::lacZ reporter were transformed with the indicated plasmids and tested for β-galactosidase production upon induction of pBAD expression. (D) The indicated strains (wild type, ΔrpoS, ΔrprA, ΔricI*, ΔrpoS/ricI*, and ΔrprA/ricI*) carrying the translational ricI::lacZ reporter were assayed for β-galactosidase production at the indicated time points of growth. (E) Analyses of σ^S, RicI::3×FLAG, and RprA expression after A22-mediated induction of the Rcs pathways. Samples were collected at the indicated time points and probed for σ^S and RicI::3×FLAG (Western blot) as well as RprA (Northern blot) production. GroEL and 5S rRNA served as loading controls.

Fig. S4.

Dual control of ricI expression. (A) Salmonella ΔrprA cells carrying the ricI::gfp reporter and cotransformed with the indicated plasmids (Fig. 5B) were grown to early stationary phase and tested for RicI::GFP and σ^S production by Western blot. GroEL served as loading control. (B) Wild-type and ΔrprA cells carrying the translational ricI::lacZ reporter were cultivated to early stationary phase and tested for β-galactosidase production in the presence of bile salts or A22. (C) Western blot analyses of σ^S and RicI::3×FLAG production in ΔrprA cells (transformed with a low-copy plasmid carrying either the rprA, rprAC37, or rprAC63 allele) after A22 treatment. GroEL served as loading controls. (D) Analyses of σ^S, RicI::3×FLAG, and RprA production following activation and deactivation of the Rcs pathway using A22. Samples were collected at the indicated time points and probed for σ^S and RicI::3×FLAG (Western blot) as well as RprA (Northern blot) production. GroEL and 5S rRNA served as loading controls.

Fig. S5.

Alignment of RicI protein sequences. The protein sequences of homologs of the RicI protein from various bacterial species were aligned (E22, Escherichia coli E22; ECA, Erwinia carotovora; ESP, Enterobacter sp. 638; ESA, Enterobacter sakazakii; PIN, Psychromonas ingrahamii; R64, Escherichia coli O26:H11; SEN, Salmonella enterica sv. Newport; SPU, Shewanella putrefaciens; STM, Salmonella enterica sv. Typhimurium LT2; SWO, Shewanella woodyi; VCO, Vibrio cholerae; VFI, Vibrio fischeri; VSH, Vibrio shiloni; VSP, Vibrio splendidus).

Fig. 6.

RicI inhibits pSLT conjugation in Salmonella. (A) Conjugation rates of the pSLT plasmid in the indicated donor strains. (B) Same as A but conjugation was tested in the presence of 4% bile salts. (C, Top) Alexa 488-labeled R17 bacteriophage was used to visualize the pSLT conjugation pili in wild-type and ΔricI cells. (Bottom) Quantification of labeled wild-type and ΔricI cells using FACS analysis. Flow cytometry analysis of Alexa 488 fluorescence intensity and percentages of cells that do not show R17 binding (blue) and cells exhibiting R17 binding (red). Histograms represent the percentages of fluorescent (R17-bound) and nonfluorescent cells. (D) Yeast two-hybrid assays of RicI–TraV interaction. Combination of RicI and TraV fusion proteins restores growth of yeast cells on selective medium, whereas expression of the individual fusion proteins (in combination with the control plasmids pGBKT7 or pGADT7) is insufficient. SlrP-Trx provided a positive control (74), and the negative controls were RicI/TraC and RicI/TrbE.

Fig. S6.

Role of RicI in pSLT transfer. (A) Production of β-galactosidase from a traJ::lacZ reporter in wild-type, ΔricI, and Δdam cells. (B) Same as A, but activity of a traB::lacZ reporter was tested. (C) Fractionation of Salmonella cells producing the RicI::3×FLAG protein. RicI::3×FLAG, Lon, Dam, and TraT proteins were detected by Western blot using specific antibodies. (D) Silver staining of coimmunoprecipitated proteins using RicI::3×FLAG protein as bait. The PtetO-ricI::3×FLAG strain expresses the RicI::3×FLAG protein from the constitutive ptetO promoter. TraV and three additional proteins (STM14_0317, YhjJ, and StbA) coimmunoprecitated with RicI are indicated by arrows. Untagged (no FLAG epitope) Salmonella wild-type cells served as negative control. M, protein size marker.

Fig. 7.

Model of RicI-mediated conjugation inhibition in Salmonella. (Left) Under regular growth conditions (no membrane stress), the Rcs system is inactive and RprA is not produced. Therefore, RprA cannot activate rpoS and ricI will not be expressed. Expression and assembly of the pSLT conjugation apparatus is permitted. (Right) When the Rcs system is activated (e.g., by bile salts or A22), full-length RprA will activate the rpoS mRNA leading to σ^S production. σ^S activates the transcription of ricI, and the ricI mRNA can be activated by the processed RprA variant. Finally, RicI interacts with TraV to inhibit assembly of the pSLT conjugation pilus.