Mutations in interaction surfaces differentially impact E. coli Hfq association with small RNAs and their mRNA targets

Abstract

The RNA chaperone protein Hfq is required for the function of all small RNAs (sRNAs) that regulate mRNA stability or translation by limited base pairing in Escherichia coli. While there have been numerous in vitro studies to characterize Hfq activity and the importance of specific residues, there has been only limited characterization of Hfq mutants in vivo. Here, we use a set of reporters as well as co-immunoprecipitation to examine 14 Hfq mutants expressed from the E. coli chromosome. The majority of the proximal face residues, as expected, were important for the function of sRNAs. The failure of sRNAs to regulate target mRNAs in these mutants can be explained by reduced sRNA accumulation. Two of the proximal mutants, D9A and F39A, acted differently from the others in that they had mixed effects on different sRNA/mRNA pairs and, in the case of F39A, showed differential sRNA accumulation. Mutations of charged residues at the rim of Hfq interfered with positive regulation and gave mixed effects for negative regulation. Some, but not all, sRNAs accumulated to lower levels in rim mutants, suggesting qualitative differences in how individual sRNAs are affected by Hfq. The distal face mutants were expected to disrupt binding of ARN motifs found in mRNAs. They were more defective for positive regulation than negative regulation at low mRNA expression, but the defects could be suppressed by higher levels of mRNA expression. We discuss the implications of these observations for Hfq binding to RNA and mechanisms of action.

Keywords: ArcZ; BSA; ChiX; DsrA; LB; Luria–Bertani; McaS; NSWB; RyhB; bovine serum albumin; co-IP; co-immunoprecipitation; non-stringent wash buffer; sRNA; small RNA; wild type; wt.

Fig. 1

Chromosomal Hfq mutants. (a, c, e) Space-filling representation of the E. coli Hfq crystal structure (PDB 1HK9) showing the locations of amino acids mutated viewed from the proximal face (Q8, D9, F39, F42, K56 and H57), the rim (R16, R17 and R19), or distal face (Y25, G29, I30 and K31). (b, d, f) Hfq protein levels in mutant strains. Extracts were prepared from derivatives of SG30200 (P_BAD-rpoS-lacZ) carrying wild-type and mutant hfq alleles (see Table S5); cells were grown in LB medium at 37 °C to early stationary phase (OD₆₀₀ ~ 1.0). The levels of Hfq protein were determined by immunoblot analysis using anti-Hfq serum and ECL Western Blotting System. Ponceau S staining of the immunoblots showed that equal total protein amounts were loaded.

Fig. 2

Effects of hfq mutations on sRNA-dependent activation of rpoS and flhD. (a, c) Derivatives of SG30200 (P_BAD-rpoS-lacZ) and (e) derivatives of NRD688 (P_BAD-flhD-lacZ) carrying the indicated sRNA overexpression plasmids were grown on lactose MacConkey plates containing 50 µg/ml ampicillin and arabinose (at 0.0001% for (a) and (c) and at 0.0002% for (e)) at 37 °C for 16 h. (b, d, f) β-galactosidase activity measured in wild-type and a subset of the mutant cells shown in (a, c, e). Cells were grown in LB medium containing 100 µg/ml ampicillin, 100 µM IPTG, 0.0002% arabinose at 37 °C to early stationary phase (OD₆₀₀ ~ 1.0) and assayed. Data is an average of three assays and brackets denote the standard deviation of the mean.

Fig. 3

Effects of hfq mutations on sRNA-dependent repression of flhD, chiP, sodB and sdhC. (a) Derivatives of NRD688 (P_BAD-flhD-lacZ) carrying a plasmid expressing McaS were grown on a lactose MacConkey plate containing 50 µg/ml ampicillin and 0.001% arabinose for 24 h. (c) Derivatives of DJS2677 (P_BAD-chiP-lacZ) were grown on lactose MacConkey plates with 0.0005% arabinose for 11 h at 37 °C. (e) Derivatives of DJS2546 (fur::kan) were grown in MOPS EZ rich defined medium with 0.4% glycerol to late exponential phase at 37 °C, and RNA was extracted and sodB RNA levels were analyzed as described in Materials and Methods. (g) The same strains as for (e) were grown in minimal succinate medium at 37° C overnight and growth determined by OD₆₀₀ normalized to growth in minimal glucose medium. (b, d, f, h) β-galactosidase activity measured in wild-type and a subset of hfq mutants shown in (a, c, e, g). Strains in (a, c), derivatives of DJS2676 (P_BAD-sodB-lacZ) overexpressing RyhB, and derivatives of DJS2729 (P_BAD-sdhC-lacZ) overexpressing RyhB were grown in LB medium containing 100 µg/ml ampicillin, 100 µM IPTG, 0.0002% arabinose at 37 °C to early stationary phase (OD₆₀₀ ~ 1.0) and assayed for β-galactosidase activity. Data is an average of three assays and brackets denote the standard deviation of the mean.

Fig. 4

Effects of hfq mutations on sRNA levels under specific growth conditions. Extracts were prepared from wild-type and mutant derivatives of SG30200 (P_BAD-rpoS-lacZ) grown in LB medium at 37 °C to early stationary phase (OD₆₀₀ ~ 1.0). ArcZ levels were analyzed by Northern blots while the levels of all other sRNAs were analyzed by primer extension. In both cases, 5 µg of total RNA and primers specific to the indicated RNAs were used.

Fig. 5

Effects of hfq mutations on Hfq association with select (a) sRNAs and (b) mRNAs. Extracts were prepared from wild-type and hfq mutant derivatives of SG30200 (P_BAD-rpoS-lacZ) cells grown in LB medium at 37 °C to early stationary phase (OD₆₀₀ ~ 1.0). co-IP was carried out with anti-Hfq antiserum. ArcZ levels were analyzed by Northern blots while the levels of all other RNAs were analyzed by primer extension. In both cases, 5 µg of total RNA or 0.5 µg of co-IP RNA and primers specific to the indicated RNAs were used.