Switching fatty acid metabolism by an RNA-controlled feed forward loop

Abstract

Hfq (host factor for phage Q beta) is key for posttranscriptional gene regulation in many bacteria. Hfq's function is to stabilize sRNAs and to facilitate base-pairing with trans-encoded target mRNAs. Loss of Hfq typically results in pleiotropic phenotypes, and, in the major human pathogen Vibrio cholerae, Hfq inactivation has been linked to reduced virulence, failure to produce biofilms, and impaired intercellular communication. However, the RNA ligands of Hfq in V. cholerae are currently unknown. Here, we used RIP-seq (RNA immunoprecipitation followed by high-throughput sequencing) analysis to identify Hfq-bound RNAs in V. cholerae Our work revealed 603 coding and 85 noncoding transcripts associated with Hfq, including 44 sRNAs originating from the 3' end of mRNAs. Detailed investigation of one of these latter transcripts, named FarS (fatty acid regulated sRNA), showed that this sRNA is produced by RNase E-mediated maturation of the fabB 3'UTR, and, together with Hfq, inhibits the expression of two paralogous fadE mRNAs. The fabB and fadE genes are antagonistically regulated by the major fatty acid transcription factor, FadR, and we show that, together, FadR, FarS, and FadE constitute a mixed feed-forward loop regulating the transition between fatty acid biosynthesis and degradation in V. cholerae Our results provide the molecular basis for studies on Hfq in V. cholerae and highlight the importance of a previously unrecognized sRNA for fatty acid metabolism in this major human pathogen.

Keywords: RNase E; Vibrio cholerae; fatty acid metabolism; feed-forward loop; small RNA.

Fig. 1.

RIP-seq analysis of Hfq-binding sRNAs. (A) V. cholerae wild-type cells (control) and cells carrying a 3XFLAG epitope at the C-terminal end of the chromosomal hfq gene were cultivated in LB medium to low (OD₆₀₀ of 0.2) and high cell densities (OD₆₀₀ of 2.0) and subjected to coimmunoprecipitation. Protein samples were collected at different steps of the IP procedure and analyzed by Western blots. Culture refers to total protein before treatment, lysate refers to total protein after cell lysis, supernatant refers to remaining protein after incubation with anti-FLAG antibody and protein G Sepharose, wash refers to remaining protein in the lysis buffer after five washing steps, and co-IP indicates coimmunoprecipitated protein sample. The relative amount of cells loaded (OD₆₀₀ units) is indicated. RNAP served as loading control. (B) RNA samples of co-IP and total RNA (lysate) fractions were loaded on a Northern blot and analyzed for Qrr4 levels. 5S rRNA served as loading control. (C) Pie charts of control and Hfq co-IP samples showing the relative fractions of the different RNA classes. The relative amount of total cDNA reads from each class in the control and Hfq co-IP libraries are shown. (D) Distribution of reads of significantly enriched sRNAs (fold enrichment > 2, P value ≤ 0.05) in Hfq co-IP libraries obtained from low (OD₆₀₀ of 0.2) and high cell densities (OD₆₀₀ of 2.0). Reads matching to a given sRNA were compared to all enriched sRNAs in the cDNA libraries. Shown are all sRNAs corresponding to at least 0.1% of the mapped reads. The relative amount of reads and enrichment factors for each sRNA are listed in SI Appendix, Table S2. (E) Co-IP and total RNA (lysate) fractions were obtained from V. cholerae wild-type and hfq::3XFLAG-tagged strains cultivated in LB medium to low (OD₆₀₀ of 0.2) and high cell densities (OD₆₀₀ of 2.0). The RNA was loaded on Northern blots and probed for the indicated sRNAs. 5S rRNA served as a loading control.

Fig. 2.

Identification and expression of the FarS sRNA. (A) Classification of Hfq-binding sRNAs according to their genomic location. The pie chart shows the relative fractions of Hfq-binding sRNAs (fold enrichment > 2, P value ≤ 0.05) originating from 3′UTRs, intergenic regions (IGRs), 5′UTRs, and coding sequences (CDSs). (B) Schematic representation of the fabB-farS genomic organization. Scissors indicate the processing site. Numbers correspond to the fabB promoter truncations tested in E. (C) Alignment of farS sequences in different Vibrio species. The sequences were aligned using the Multalign tool (76). The start of the sRNA and the Rho-independent terminator are indicated. The stop codon of fabB in V. cholerae is marked with a black box. Vch, Vibrio cholerae; Vfu, Vibrio furnissii; Van, Vibrio anguillarum; Vco, Vibrio coralliilyticus; Vca, Vibrio campbellii; Vha, Vibrio harveyi; Vpa, Vibrio parahaemolyticus; Vvu, Vibrio vulnificus. (D) V. cholerae wild-type and ΔfadR cells harboring either a control plasmid (pBAD-ctr) or a plasmid containing the fadR gene and its native promotor (p-PfadR) were cultivated in LB medium. Total RNA samples were collected at different stages of growth, and expression of FarS was analyzed on Northern blot. 5S rRNA was used as loading control. (E) V. cholerae wild-type and ΔfarS strains harboring different plasmids containing fabB-farS gene fragments (as indicated in B) were grown to stationary phase (OD₆₀₀ of 2.0) in LB medium. Northern blot analysis was performed to determine FarS levels. Probing for 5S rRNA served as a loading control.

Fig. 3.

RNase E is required for FarS production. (A) V. cholerae ΔfarS carrying either a wild-type (rne WT) or temperature-sensitive RNase E (rne TS) allele and the pBAD-fabB-farS plasmid were cultivated at 30 °C in LB medium. When cells reached an OD₆₀₀ of 1.0, cultures were split and kept at permissive temperature (30 °C) or shifted to nonpermissive temperature (44 °C) and incubated for 30 min. Next, expression of fabB-farS was induced using L-arabinose (0.2% final concentration, 30 min), and FarS levels were monitored by Northern blot. Probing for 5S rRNA served as loading control. (B) V. cholerae wild-type or ΔfarS cells harboring either a control plasmid (pBAD-ctr), a plasmid containing the fabB-farS gene locus and the fabB promotor, or a version of the plasmid where the first three base pairs of farS were mutated (TTT to GGG) were cultivated in LB medium. Total RNA samples were collected when cells reached an OD₆₀₀ of 1.0. The Northern blot was probed for FarS, and 5S rRNA was used as a loading control.

Fig. 4.

Structure of FarS and base-pairing to fadE target mRNAs. (A) Secondary structure of FarS. The secondary structure was derived from chemical and enzymatic structure probing experiments (SI Appendix, Fig. S4B). The base-pairing site is marked in red, and the Hfq binding site is indicated. (B) Predicted base-pairing of FarS with vc1740. Arrows indicate the single nucleotide mutations tested in D. (C) Predicted base-pairing of FarS with vc2231. Arrows indicate the single nucleotide mutations tested in E. (D) Discoordinate regulation of FLAG::VC1741 and VC1740::GFP. E. coli cells carrying a reporter plasmid for FLAG::VC1741 and VC1740::GFP or VC1740*::GFP (C10 to G) were cotransformed with a control plasmid (p-ctr), p-farS, or p-farS* (G54 to C). Cells were grown in LB medium to stationary phase (OD₆₀₀ of 2.0). GFP and FLAG levels were measured by Western blot, and FarS levels were determined by Northern blot. RNAP and 5S rRNA served as loading controls for the Western and Northern blots, respectively. (E) E. coli harboring a reporter plasmid for VC2231::GFP or VC2231*::GFP (C17 to G) and either a control plasmid (p-ctr), p-farS, or p-farS* (G54 to C) were grown in LB medium to OD₆₀₀ of 2.0. GFP and FarS levels were monitored as in D.

Fig. 5.

FarS inhibits FadE protein production. (A and B) V. cholerae wild-type and ΔfarS strains carrying a chromosomal 3XFLAG epitope either at the vc1740 (A) or at the vc2231 (B) gene and harboring the indicated plasmids were cultivated in M9 minimal medium. Protein and total RNA samples were collected at the indicated OD₆₀₀ readings. FadE::3XFLAG protein production (A, VC1740::3XFLAG; B, VC2231::3XFLAG) was analyzed on Western blots, and expression of FarS was monitored on Northern blots. RNAP and 5S rRNA served as loading controls for the Western and Northern blots, respectively. Percentages indicate the amount of protein relative to the wild-type level at the corresponding growth phase. A quantification of data obtained from three independent biological replicates is shown in SI Appendix, Fig. S6 A and B. (C) V. cholerae wild-type and Δvc1740/Δvc2231 strains carrying the indicated plasmids were cultivated for 10 h in M9 minimal medium containing fatty acid (sodium oleate) as sole carbon source. Serial dilutions were prepared and recovered on agar plates, and colony-forming units (CFU) per milliliter were determined. Dots represent individual replicates (n = 4), and lines indicate the mean CFU.

Fig. 6.

FarS is part of a mixed feed-forward loop. (A) Schematic display of a mixed type 3 coherent feed-forward loop involving the transcription factor FadR, the fabB mRNA, FarS, and the two fadE mRNAs. (B and C) V. cholerae wild-type and ΔfarS strains carrying a chromosomal 3XFLAG epitope either at the vc1740 (B) or at the vc2231 (C) gene were cultivated in M9 minimal medium to stationary phase (OD₆₀₀ of 2.0). Total protein and RNA samples were collected before and after addition of fatty acids (+FA; sodium oleate, 0.005% final concentration) at the indicated time points. Expression patterns of the VC1740 (B) and VC2231 (C) proteins were analyzed on Western blots, and expression of FarS was determined using Northern blot analysis. RNAP and 5S rRNA served as loading controls for the Western and Northern blots, respectively. (D and E) V. cholerae wild-type and ΔfarS strains carrying a chromosomal 3XFLAG epitope either at the vc1740 (D) or at the vc2231 (E) gene were cultivated in M9 minimal medium containing sodium oleate (0.005% final concentration) to an OD₆₀₀ of 2.0. Cells were washed with PBS and resuspended in M9 minimal medium lacking fatty acids (-FA). Total protein and RNA samples were collected before and after removal of fatty acids at the indicated time points. Western and Northern blots show VC1740 (D) and VC2231 (E) protein and FarS levels, respectively. RNAP was used as loading control for Western blots; 5S rRNA for Northern blots.

Fig. 7.

Regulatory model for FarS-mediated fatty acid metabolism in V. cholerae. The fabB gene is part of the fatty acid biosynthesis regulon. It encodes β-ketoacyl-ACP synthase catalyzing the rate-limiting step in the synthesis of unsaturated fatty acid. The downstream reactions are catalyzed by FabG (3‐ketoacyl‐ACP reductase), FabZ (3‐hydroxyacyl‐ACP dehydratase), and FabV (enoyl‐ACP reductase). FarS is produced from the 3′UTR of fabB and posttranscriptionally inhibits the expression of two paralogous fadE mRNAs. The fadE genes encode acyl-CoA dehydrogenase catalyzing the initial step in fatty acid β-oxidation. Here, long‐chain fatty acids are transported across the outer and inner membranes by FadL and FadD, respectively. Following FadE activity, the remaining steps in fatty acid degradation are performed by a complex consisting of FadB and FadA.