Functional expansion of a TCA cycle operon mRNA by a 3' end-derived small RNA

Abstract

Global RNA profiling studies in bacteria have predicted the existence of many of small noncoding RNAs (sRNAs) that are processed off mRNA 3' ends to regulate other mRNAs via the RNA chaperones Hfq and ProQ. Here, we present targets of SdhX (RybD), an Hfq-dependent sRNA that is generated by RNase E mediated 3' processing of the ∼10 000-nt mRNA of the TCA cycle operon sdhCDAB-sucABCD in enteric bacteria. An in silico search predicted ackA mRNA, which encodes acetate kinase, as a conserved primary target of SdhX. Through base pairing, SdhX represses AckA synthesis during growth of Salmonella on acetate. Repression can be achieved by a naturally occurring 38-nucleotide SdhX variant, revealing the shortest functional Hfq-associated sRNA yet. Salmonella SdhX also targets the mRNAs of fumB (anaerobic fumarase) and yfbV, a gene of unknown function adjacent to ackA. Instead, through a slightly different seed sequence, SdhX can repress other targets in Escherichia coli, namely katG (catalase) and fdoG (aerobic formate dehydrogenase). This study illustrates how a key operon from central metabolism is functionally connected to other metabolic pathways through a 3' appended sRNA, and supports the notion that mRNA 3'UTRs are a playground for the evolution of regulatory RNA networks in bacteria.

Figure 1.

(A) Genetic structure of sdhCDAB-sucABCD operon and alignment of sucD 3′UTRs of selected enterobacterial species. Nucleotide sequences were obtained from the following genomes: Eco, E. coli MG1655 (NC_000913); Cko, Citrobacter koseri ATCC BAA-895 (NC_009792); Sal, Salmonella Typhimurium SL1344 (NC_016810); Eae, Enterobacter aerogenes KCTC 2190 (NC_015663); Kpn, Klebsiella pneumoniae 342 (NC_011283); Csa, Cronobacter sakazakii ATCC BAA-894 (NC_009778); Sma, Serratia marcescens FGI94 (NC_020064); Ype, Yersinia pestis CO92 (NC_003143). Red letters indicate conserved nucleotides. The stop codons of the sucD open reading frame are boxed. The conserved seed region and E. coli-specific region complementary to katG are highlighted. The Rho-independent terminator is indicated by inverted arrows. RNase E cleavage sites in Salmonella identified by (26) are indicated by arrow heads, the major and minor sites of which are in black and grey, respectively. (B) Acetate catabolic pathway into TCA cycle. The enzymes encoded on sdhCDAB-sucABCD operon are shown in orange. The enzymes regulated by SdhX in Salmonella are shown in red. (C) Expression profile of TCA cycle proteins and sucD mRNA 3′UTR-derived sRNAs. Salmonella Typhimurium strain SL1344 was aerobically grown in LB medium or minimal medium supplemented with 0.2% glucose (Glu) or 40 mM acetate (Ace). Salmonella cells were collected at the indicated OD₆₀₀ values and whole cell samples and total RNA samples were analyzed on western and northern blots. The size is estimated by DynaMarker RNA Low II ssRNA fragments.

Figure 2.

SdhX is processed by RNase E. (A) Salmonella rne+ (WT: lanes 1–2) and rne3071 (TS: lanes 3–4) strains were grown to OD₆₀₀ of 0.5 at 28°C, split into two flasks, and further incubated at either 28°C (lanes 1, 3) or 44°C (lanes 2, 4) for 30 min. Salmonella rne+ (WT: lane 5) and rneR169K (R169K: lane 6) strains were grown to OD₆₀₀ of 0.5 at 37°C. The size is estimated by pUC19 MspI dsDNA fragments. (B) Predicted secondary structures of SdhX1 and SdhX2. Hfq-bound regions identified by CLIP-seq analysis (12) are indicated by red letters, and the mutations induced by crosslinking to Hfq are highlighted in yellow. See also Supplementary Figure S1.

Figure 3.

The ackA, fumB and katG mRNAs are regulated by Salmonella SdhX. (A) The 5′UTRs or intergenic regions of candidate target mRNAs were cloned into pXG-10sf and pXG-30sf vectors, respectively. Salmonella ΔsdhX strain was transformed by pXG derivative plasmids along with pJV300 control vector (-) or SdhX1 expression plasmid (+). GFP expression on the plate was visualized by LAS4000 imager. (B) Predicted interactions of Salmonella SdhX1 with target mRNAs. Mutated nucleotides (G41C in sdhX and C5G in target mRNAs) were indicated by red letters. The start codon of target mRNAs is underlined. (B) SdhX regulates the target mRNAs by base-pairing mechanism. Salmonella ΔsdhX strain was transformed by combinations of pXG plasmids along with pJV300 control vector (–), SdhX1 expression plasmid (+) or SdhX1 mutant expression plasmid (*) as indicated. GFP expression in the LB liquid medium was quantified by a plate reader and normalized by OD₆₀₀. Since each translational fusion exhibited various GFP intensities, the graphs are shown at different scales. Error bars indicate standard deviations (n = 3).

Figure 4.

Physiological levels of SdhX repress AckA synthesis during aerobic growth on acetate. (A) Salmonella chromosomal sdhX mutants with C-terminal ackA::FLAG fusions (sdhX WT, JVS-11249; sdhXG41C, JVS-11250; sdhXΔ49, MMS-0005) were grown to exponential phase in MOPS minimal medium supplemented with 0.2% glucose (Glu) or 40 mM sodium acetate (Ace). The whole cell samples were analysed by western blot. Expression level of AckA was normalized by that of GroEL, and relative expression levels in sdhX WT and G41C strains to that in Δ49 mutant were plotted. Error bars indicate standard deviations (n = 5). Asterisk indicates P < 0.01. NS indicates not significant (P > 0.05). (B) RNA samples extracted from the cells grown as in (A) were analysed by northern blot using ³²P-labelled oligonucleotides for SdhX2 (MMO-0315) and 5S rRNA (JVO-0322). The G41C mutant exhibited weaker signals when MMO-0317 containing the single mismatch was used (data not shown). The size is estimated by DynaMarker RNA Low II ssRNA fragments. (C) Salmonella strains deleted for transcriptional factors (lane 1, JVS-1574; lane 2, JVS-0673; lane 3, JVS-1227; lane 4, JVS-1626) were grown to exponential phase in LB medium, and analysed by northern blot using MMO-0317 and JVO-0322. A pUC19 MspI dsDNA ladder was used as size marker. (D) Salmonella ackA::FLAG strains (sdhX WT; JVS-11249 and sdhXΔ49; MMS-0005) and those in ΔarcA genetic background carrying either an intact sdhX region (sdhX WT; MMS-0015) or a mutant thereof (sdhXΔ49; MMS-0014) were grown to exponential phase in LB medium, and analysed by western blot with anti-FLAG and anti-GroEL antibodies.

Figure 5.

E. coli fumB and yfbV are not regulated by SdhX due to mutations around their start codons. (A) Difference in fumB-SdhX and yfbV-SdhX interactions between Salmonella and E. coli. The start codon is underlined. Exchanged nucleotides were indicated by red letters. Changes in free energy (ΔG°) upon basepairing are indicated below the interactions. (B) Salmonella ΔsdhX strain was transformed by combinations of mutant pXG plasmids along with pJV300 control vector (null) or SdhX expression plasmid (SdhX). GFP expression was quantified and normalized by OD₆₀₀. Error bars indicate standard deviations (n = 3).

Figure 6.

The fdoG and katG mRNAs are regulated by E. coli SdhX. (A) The 5′UTRs were cloned into pXG-10sf (35). E. coli ΔsdhX strain was transformed by pXG derivative plasmids along with pJV300 control vector (–) or SdhX expression plasmid (+). GFP expression (left) and colony densities (right) were visualized by LAS4000 imager. (B) Predicted interactions of E. coli SdhX with fdoG and katG mRNAs. Mutated nucleotides were indicated by red letters. The start codon of katG is underlined. E. coli ΔsdhX strain was transformed by combinations of pXG plasmids along with pJV300 control vector (–), SdhX (+) or SdhX G62C or C34G mutant expression plasmid (*) as indicated. Since each translational fusion exhibited various GFP intensities, the graphs are shown at different scales. Error bars indicate standard deviations (n = 3).

Figure 7.

Processing of SdhX2 is not necessary for target regulation. Salmonella ΔsdhX mutant with C-terminal ackA::FLAG fusion was transformed by plasmids expressing respective sRNAs (lane 1: pJV300, lane 2: pLM1, lane 3: pLM30, lane 4: pLM34, lane 5: pLM35, lane 6: pJU-19, lane 7: pLM32). The slightly larger size of SdhX2 when expressed as a primary transcript (lane 3) may result from transcription starting at the –1 instead of the +1 position of the constitutive promoter used here. That is, SdhX2 starts with four uridines which are far from ideal for transcription initiation. Indeed, primer extension analysis revealed an extra nucleotide at the 5′ end of SdhX2 expressed from this plasmid (data not shown). The asterisk indicates a putative read-through product, whose transcription terminates at downstream rrnT1 in the vector (34).