GadY, a small-RNA regulator of acid response genes in Escherichia coli

Abstract

A previous bioinformatics-based search for small RNAs in Escherichia coli identified a novel RNA named IS183. The gene encoding this small RNA is located between and on the opposite strand of genes encoding two transcriptional regulators of the acid response, gadX (yhiX) and gadW (yhiW). Given that IS183 is encoded in the gad gene cluster and because of its role in regulating acid response genes reported here, this RNA has been renamed GadY. We show that GadY exists in three forms, a long form consisting of 105 nucleotides and two processed forms, consisting of 90 and 59 nucleotides. The expression of this small RNA is highly induced during stationary phase in a manner that is dependent on the alternative sigma factor sigmaS. Overexpression of the three GadY RNA forms resulted in increased levels of the mRNA encoding the GadX transcriptional activator, which in turn caused increased levels of the GadA and GadB glutamate decarboxylases. A promoter mutation which abolished gadY expression resulted in a reduction in the amount of gadX mRNA during stationary phase. The gadY gene was shown to overlap the 3' end of the gadX gene, and this overlap region was found to be necessary for the GadY-dependent accumulation of gadX mRNA. We suggest that during stationary phase, GadY forms base pairs with the 3'-untranslated region of the gadX mRNA and confers increased stability, allowing for gadX mRNA accumulation and the increased expression of downstream acid resistance genes.

FIG. 1.

GadY exists as three forms. (A) Map of the gadAXYW acid response gene cluster. The gadY gene is shaded in black. (B) Northern blot detection of GadY. The total RNA (5 μg) from MG1655 cells grown to stationary phase (6 h) in LB medium was separated in an 8% polyacrylamide gel and then transferred to a nylon membrane. GadY RNA was detected with an oligonucleotide (JK138) that was complementary to GadY. Samples were run alongside Decade RNA markers (Ambion) with the indicated sizes. (C) Primer extension analysis of GadY RNA. Primer extension assays were performed with RNA isolated from MG1655 cells grown to stationary phase (4 h) in LB medium and with oligonucleotide IS183-A4, which is complementary to the 3′ end of GadY. (D) Sequence of the gadY gene in E. coli K-12. The −10 and −35 promoter sequences are underlined, and bold letters denote the gadY coding sequence. A promoter mutant (GSO109) was constructed in which the −10 sequence was changed to six guanine nucleotides, as indicated by lowercase letters. The 5′ ends of the three forms of the GadY RNA are displayed in larger letters and are labeled 105, 90, and 59, representing the sizes of these RNAs. The inverted repeat corresponding to the stem-loop of the transcription terminator is indicated by solid arrows. A putative single-stranded Hfq binding site (UUUAU) is adjacent to the putative stem-loop indicated by dashed arrows.

FIG. 2.

GadY expression is rpoS dependent. (A) GadY expression at different stages of growth. The total RNA was harvested at the indicated time points from MG1655 cells grown in LB medium. Samples (5 μg) were run alongside Decade RNA markers with the indicated sizes. (B) Level of GadY RNAs in wild-type and rpoS mutant strains. Total RNAs were harvested from MG1655 wild-type and rpoS::Tn10 mutant strains after 2 and 6 h of growth. For both panels, total RNAs were separated, transferred, and probed as described in the legend to Fig. 1B.

FIG. 3.

Hfq binding to GadY. Cell extracts were prepared from MC4100 and hfq-1 mutant cells grown for 16 h in LB medium. Immunoprecipitations were then carried out with the MC4100 extracts, using an Hfq antiserum or preimmune serum, and were compared to total RNAs isolated from 1/10 extract equivalents of the wild-type and hfq-1 mutant cells. The RNAs were separated, transferred, and probed as described in the legend to Fig. 1B. As observed in Fig. 2A, GadY(90) was much more abundant than GadY(105) or GadY(59) after 16 h. In addition, the GadY(105) or GadY(59) RNA species may have decreased stability under the conditions used to isolate the RNAs for this experiment.

FIG. 4.

Effect of GadY overproduction on GadA and GadB levels. (A) Protein expression patterns in cells overexpressing GadY. Equal amounts of total protein from MG1655 cells carrying vector pRI, pRI-GadY, or pRI-YdaG and grown in LB medium for 2 h were separated in SDS-10% polyacrylamide gels. The indicated bands were identified by in-gel trypsin digestion and MALDI-TOF mass spectrometry. (B) gadA mRNA levels in cells overexpressing GadY. Total RNAs (5 μg each) from MG1655, MG1655/pRI, MG1655/pRI-GadY, and MG1655 ΔgadX::kan/pRI-GadY grown in LB medium for 4 h were separated in a 1% agarose-0.05 M MOPS-1 mM EDTA-3.3% formaldehyde gel and then transferred to a nylon membrane. gadA mRNA was detected with an oligonucleotide (GadAB-A1) that was complementary to gadA. Samples were run alongside Millenium RNA size markers (Ambion), which were probed separately. The gadA mRNA migrated in the gel at the expected size of 1.4 kb. The two longer transcripts observed with GadY overproduction may correspond to gadAX and gadBC transcripts, and the shorter transcript is likely a degradation product.

FIG. 5.

Effects of increased and decreased GadY expression on gadX mRNA levels. (A) Map of overlap between GadY small RNA and gadX 3′ UTR. (B) gadX mRNA levels in cells overexpressing GadY. Total RNAs (5 μg each) from wild-type MG1655, MG1655/pRI-GadY, MG1655/pRI, and MG1655 ΔgadX::kan/pRI-GadY(105) grown in LB medium for 4 h were separated, transferred, and probed as described in the legend to Fig. 4B, except that the gadX mRNA was detected with an oligonucleotide (GadX-A1) that was complementary to gadX. (C) GadY RNA levels in gadY −10 promoter mutant strain. Total RNAs (5 μg each) from MG1655 and the gadY −10 mutant strain (GSO109) grown in LB medium for 6 h were separated, transferred, and probed as described in the legend to Fig. 1B. (D) gadX mRNA levels in cells with decreased GadY expression. Total RNAs (5 μg each) from MG1655 and the gadY −10 mutant grown in LB medium for 6 h were separated, transferred, and probed as described in the legend to Fig. 4B. The samples were run alongside Millenium RNA size markers, which were probed separately. The gadX mRNA migrated at the expected size of approximately 1 kb. The Northern blot in panel D was overexposed compared to that in panel B to allow detection of the gadX transcript from MG1655. The nonspecific hybridization to the 16S rRNA detected with the long exposure of panel D served as a loading control.

FIG. 6.

Effect of deleting gadX 3′ UTR. (A) Maps of placZ-gadX_{gadY −10} and placZ-gadX_ΔgadY constructs. (B) gadX mRNA levels from lacZ promoter fusion constructs in cells overexpressing GadY. Total RNAs (5 μg) from MG1655/placZ-gadX_{gadY −10 mutant} pRI, MG1655/placZ-gadX_{gadY −10 mutant} pRI-GadY, MG1655/placZ-gadX_ΔgadY pRI, and MG1655/placZ-gadX_ΔgadY pRI-GadY grown in LB medium for 4 h were separated, transferred, and probed as described in the legend to Fig. 4B. The reason for the lower gadX mRNA levels in MG1655/placZ-gadX_ΔgadY carrying pRI-GadY than in the same background strain carrying pRI is unknown.