Reprogramming of anaerobic metabolism by the FnrS small RNA

Abstract

Small RNAs (sRNAs) that act by base pairing with trans-encoded mRNAs modulate metabolism in response to a variety of environmental stimuli. Here, we describe an Hfq-binding sRNA (FnrS) whose expression is induced upon a shift from aerobic to anaerobic conditions and which acts to downregulate the levels of a variety of mRNAs encoding metabolic enzymes. Anaerobic induction in minimal medium depends strongly on FNR but is also affected by the ArcA and CRP transcription regulators. Whole genome expression analysis showed that the levels of at least 32 mRNAs are downregulated upon FnrS overexpression, 15 of which are predicted to base pair with FnrS by TargetRNA. The sRNA is highly conserved across its entire length in numerous Enterobacteria, and mutational analysis revealed that two separate regions of FnrS base pair with different sets of target mRNAs. The majority of the target genes were previously reported to be downregulated in an FNR-dependent manner but lack recognizable FNR binding sites. We thus suggest that FnrS extends the FNR regulon and increases the efficiency of anaerobic metabolism by repressing the synthesis of enzymes that are not needed under these conditions.

Fig. 1. FnrS RNA encoded in the ydaN-dbpA intergenic region

A. Map of ydaN-dbpA region. B. Sequence of the first 217 nucleotides of the ydaN-dbpA intergenic region. +1 indicates the mapped start of the FnrS RNA, the stop codon of ydaN is in bold, the probable FnrS Rho-independent terminator is underlined, and putative binding sites for FNR/CRP and ArcA are boxed in continuous and dotted lines, respectively. C. Alignment of the region encompassing FnrS created using the CLC sequence viewer (www.clcbio.com). Sequences used for this alignment are from Escherichia coli K12, Shigella dysenteriae Sd197, Salmonella thyphimurium LT2, Klebsiella pneumoniae subsp. pneumoniae MGH 78578, Yersinia pseudotuberculosis PB1/+, Enterobacter sp. 368, Citrobacter koseri ATCC BAA-895, Erwinia carotovora subsp. atroseptica SCRI1043, Serratia proteamaculans 568, Sodalis glossinidius str. ‘morsitans’.

Fig. 2. FnrS expression under different growth conditions and in various mutant strains

A. MG1655 cells were grown in M63 with 0.2% glucose to OD₆₀₀ ≈ 0.4 under aerobic conditions, the culture was split into multiple aliquots, the cells were collected and resuspended in the indicated medium (M63 with 0.2% glucose or 0.4% glycerol and 20 mM nitrate or 40 mM fumarate) and incubated aerobically or anaerobically for 20 min. B. MG1655, MG1655Δcrp, MG1655 Δfnr and MG1655 ΔarcA were grown in M63 with 0.2% glucose to OD₆₀₀ ≈ 0.4 under aerobic conditions, the cultures were split into two aliquots, the cells were collected and resuspended in M63 with 0.4% glycerol and 40 mM fumarate and incubated aerobically or anaerobically for 20 min. C. MG1655 and MG1655_fnr- strain were grown in M63 with 0.2% glucose to OD₆₀₀ ≈ 0.4 under aerobic conditions, the cultures were split into four aliquots, cells were collected and resuspended in M63 with either 0.2% glucose or 0.4% glycerol and incubated aerobically or anaerobically for 20 min. D. MG1655_fnr- cells were grown and treated as for (A). E. MG1655_fnr-, MG1655_fnr- Δcrp, MG1655_fnr- Δfnr or MG1655_fnr- ΔarcA were grown and treated as for (B). For all samples, total RNA (5 μg) was separated on an acrylamide gels, transferred to nitrocellulose and probed with a ³²P-labelled oligonucleotide specific to FnrS. For all panels the position of the band corresponding to a 100-nucleotide marker RNA is indicated on the left. The Northern blots in (A), (B) and (C) were exposed overnight, while the Northern blots in (D) and (E) were exposed for one week.

Fig. 3. FnrS structure

A. FnrS structure predicted by Mfold and supported by dimethylsulfate (DMS) modification data. The sequence complementary to the oligonucleotide used in the reverse transcription reaction is in bold. Dots indicate residues that reacted with DMS and boxes denote residues modified in mutants I, II and III (see Fig. 5A). B. In vivo probing of the FnrS RNA structure. Cells were grown anaerobically in M63 with 0.2% glucose and 40 mM fumarate and half of the culture was treated with dimethylsulfate for four min. Total RNA extracted from these cultures was analyzed by primer extension reactions.

Fig. 4. FnrS repression of maeA, gpmA, sodB, folE and folX

Cultures of MG1655 carrying pAZ3 or pAZ3-FnrS were grown in LB to OD₆₀₀ ≈ 0.4 and treated with 0.2% arabinose. After 15 min, cells were washed two times in LB + 0.2% glucose and grown an additional 15 min. The time of incubation (min) with arabinose (ara) and glucose (glu) before RNA extraction are indicated on top. For all samples, total RNA (5 μg) was separated on an agarose gels, transferred to nitrocellulose and probed with a ³²P-labelled oligonucleotides specific the genes indicated on the right.

Fig. 5. Base pairing between FnrS and target mRNAs

A. Predicted base pairing interactions. Black arrows indicate the promoters mapped by 5′ RACE PCR, and the numbers correspond to the number of nucleotides between the transcriptional and the translational start sites. The regions of base pairing between FnrS and its targets as predicted by the TargetRNA program are symbolized by short bars on the left and are given on the right. FnrS mutations I, II and III are also indicated. The ribosome binding sites are italicized and the start codons are in bold. The sequences of the compensatory mutations are also given. B. Repression of maeA, gpmA, sodB, folE expression by FnrS and FnrS mutants. Total RNA was extracted from MG1655 before and 30 min after the induction of FnrS (from pBR-FnrS) or FnrS mutant I, II or III (pBR-FnrS-I, II or III) with 100 μM IPTG. Genes probed are indicated on the left. The last panel shows the levels of the wild-type and mutant FnrS transcripts. The Northern blots were carried out as in Fig. 2 and 4.

Fig. 6. Mutational analysis of FnrS base pairing

A. β-galactosidase assays of FnrS target mRNA-lacZ fusions in presence of pBR (empty plasmid), pBR-FnrS, pBR-FnrS-I, pBR-FnrS-II and pBR-FnrS-III. B. β-galactosidase assays of FnrS target mRNA-lacZ fusions carrying complementary mutations. For both (A) and (B), expression of the lacZ fusions was pre-induced for 5 min by the addition of 0.2% arabinose, after which cells were treated with 100 μM IPTG to induce the P_lac promoter on the pBR plasmids. The levels of β-galactosidase activity were assayed 30 min later. The averages for the activity in Miller units determined in three independent experiments are shown together with the standard deviation.

Fig. 7. Effect of RydD on gpmA and sodB expression upon shifts between aerobic and anaerobic conditions

MG1655 and MG1655 ΔfnrS strains were grown in M63 with 0.2% glucose and 40 mM fumarate to OD₆₀₀ ≈ 0.4 under aerobic conditions. Cells were collected and resuspended in the same medium and grown anaerobically. After 20 min, cells were again harvested, resuspended in the same medium and incubated aerobically for 60 min. Total RNA was extracted at several times (min) during this cycle. Northern blots were probed as in Fig. 2 and 4 for the genes indicated on the right.