Integrating anaerobic/aerobic sensing and the general stress response through the ArcZ small RNA

Abstract

The alternative sigma factor RpoS responds to multiple stresses and activates a large number of genes that allow bacteria to adapt to changing environments. The accumulation of RpoS is regulated at multiple levels, including the regulation of its translation by small regulatory RNAs (sRNAs). A library of plasmids expressing each of 26 Escherichia coli sRNAs that bind Hfq was created to globally and rapidly analyse regulation of an rpoS-lacZ translational fusion. The approach can be easily applied to any gene of interest. When overexpressed, four sRNAs, including OxyS, previously shown to repress rpoS, were observed to repress the expression of the rpoS-lacZ fusion. Along with DsrA and RprA, two previously defined activators of rpoS translation, a third new sRNA activator, ArcZ, was identified. The expression of arcZ is repressed by the aerobic/anaerobic-sensing ArcA-ArcB two-component system under anaerobic conditions and adds translational regulation to the ArcA-ArcB regulon. ArcZ directly represses, and is repressed by, arcB transcription, providing a negative feedback loop that may affect functioning of the ArcA-ArcB regulon.

Figure 1

Use of a dedicated library of sRNAs to study regulation of an rpoS–lacZ translational fusion. (A) Screening of the sRNA library on the P_BAD–rpoS–lacZ fusion (PM1409). The effect of the overexpression of each sRNA on the rpoS–lacZ fusion was plotted as a function of the fold change compared with the basal activity of PM1409 containing a pBR-plac control vector. Fold changes greater than two were considered significant. Grey bars represent sRNAs for which effects were not considered significant; black and light grey bars indicate sRNAs having an activating or a repressing effect, respectively. (B) sRNA overexpression effect on the P_BAD–rpoS–lacZ fusion independent of the known positively acting sRNAs. As above, but with PM1417, a dsrA rprA arcZ triple deletion mutant derivative of PM1409.

Figure 2

ArcZ direct pairing with the rpoS leader. (A) The rpoS mRNA hairpin, adapted from Soper and Woodson (2008), is shown. The region of the rpoS leader involved in base-pairing with the sRNAs is shaded in grey in this panel and (B). Nucleotides are numbered from the +1 of the rpoS mRNA. The AUG translation start codon is boxed, as are the nucleotides mutated in the experiments in Figure 2C. RBS, ribosome binding site. (B) Predicted base-pairings of each of three sRNAs with the shaded portion of the rpoS leader are shown. Mutated nucleotides are boxed and the changes made in them are shown; positions are numbered according to the transcriptional start site. The sequence in the striped box in ArcZ is not present in the short processed form of the RNA (see text). (C) Effect of the wild-type sRNAs or the C-to-G mutations in each of the sRNAs were tested on the wild-type P_BAD–rpoS–lacZ fusion (PM1409), rpoS(G463C)–lacZ fusion (PM1433), rpoS(C561G)–lacZ fusion (PM1438), and rpoS(G463C-C561G)–lacZ fusion (PM1439). Note the difference in the y-axis in the left- and right-hand panels.

Figure 3

Role of DsrA, RprA, and ArcZ in expression of rpoS. (A) Isogenic P_BAD–rpoS–lacZ strains deleted either for dsrA (PM1411), rprA (PM1412), arcZ (PM1413), or carrying combinations of double or triple mutations in each of the three sRNAs (see Supplementary Table S1) were grown at 37°C in LB containing 0.002% arabinose to stationary phase. Samples were taken and β-galactosidase activity was measured as described previously (Majdalani et al, 1998). An hfq mutant (PM1419) was also tested. The wild-type strain (PM1409) is shown in black; all mutants are dark grey. (B) A subset of the strains in (A), carrying either single mutations or the triple mutation, were grown in minimal medium containing 0.2% arabinose and assayed for rpoS expression.

Figure 4

ArcZ sRNA. (A) Alignment of arcZ in various enterobacterial strains. Flanking genes are shown; the start codon for elbB and stop codon for arcB are boxed. Sequence identities are shaded in grey. The transcription start site (+1) and the deduced arcZ promoter elements are labelled. A putative ArcA binding site overlapping the −35 element is indicated and residues close to the ArcA box consensus, as defined by McGuire et al (1999), are shown with stars. The boxed sequence in the sRNA is the sequence predicted to be involved in pairing with the rpoS leader. The ^* and ^** show minor and major sRNA cleavage sites, respectively; cleavage is between A and T for the major site of processing. The 5′ and 3′ probes used for the northern blot in Figure 4B detect the underlined sequences. Arrows indicate the position of 3′ RACE clones for arcB mRNA, and the number of isolates for each position. The 3′-end was determined in a mutant not expressing ArcZ (PM1520); no clones could be obtained from the wild-type cells. (B) Northern blot experiments showing the pattern of expression of ArcZ sRNA. An overnight culture of the WT strain MG1655 was diluted 500-fold in LB and grown at 37°C; samples were collected at the indicated OD₆₀₀. RNA was extracted and northern blots were performed with either a probe directed against the 5′-end (ArcZNB1, left panel) or the 3′-end (ArcZ NB4, right panel) of the sRNA. (C) β-galactosidase activity of the ParcZ (-100)–lacZ fusion as a function of growth. Overnight cultures of PM1450, containing a ParcZ(-100)–lacZ fusion (indicated by black diamonds) and PM1451, containing the same promoter fusion with a TG-to-CC mutation at position −14/15 of the promoter (empty squares) were diluted 500-fold in LB medium at 37°C. The t0 sample was taken after 2 h of growth; then samples were taken at regular intervals and β-galactosidase activity determined (solid lines, y-axis on the left) and OD_600nm measured (dashed lines, y-axis on the right).

Figure 5

Effect of ArcA on expression of ArcZ and RpoS. (A) Expression of the ParcZ–lacZ transcriptional fusion as a function of oxygen. Wild-type cells containing the ParcZ(-100)–lacZ fusion (PM1450) and isogenic derivatives deleted for arcA (PM1453) or arcB (PM1456) were grown in minimal medium overnight in presence (dark bars) or absence (light bars) of oxygen before samples were removed and assayed for β-galactosidase activity. (B) Northern blot showing expression of ArcZ as a function of oxygen. Strain NM525 and its arcA∷kan derivative PM1493 were grown overnight at 37°C in minimal medium before samples were removed and the RNA extracted. Northern blots were subsequently performed and probed with the ArcZ-NB4 probe (see Supplementary Table S2). (C) Expression of the P_BAD–rpoS–lacZ fusion as a function of arcZ and arcA. Left panel: Experiments are as in (A), but using cells carrying the rpoS–lacZ translational fusion (PM1409) and its arcA (PM1480), arcZ (PM1413), and arcA, arcZ (PM1481) mutant derivatives grown in minimal medium containing 0.2% arabinose. As in Figures 1, 2 and 3, the promoter of this fusion is P_BAD and the fusion protein is not subject to RpoS-specific degradation. Right panel: Assay of a control P_BAD–lacZ fusion (lacZ under control of the lacZ RBS) (PM1051) under anaerobic and aerobic conditions as for the left panel. (D) Strain SG30013, carrying an rpoS–lacZ translational fusion containing the promoter region of rpoS and enough of the rpoS gene to be subject to RssB-dependent degradation, and its arcA∷kan (PM1620), arcZ∷tet (PM1621), and arcA∷kan, arcZ∷tet double mutant (PM1622) derivatives were grown in LB at 37°C until the indicated OD. Cells were then lysed and β-galactosidase activity was measured as described in the Materials and methods section.

Figure 6

An ArcZ and ArcA/ArcB regulatory loop. (A) Diagram of overlap of the arcB and arcZ transcripts, and expression of arcB under the control of a P_BAD promoter. On the right side, mutations in the arcZ promoter inactivate its expression without disrupting the overlap region. (B) Four isogenic strains were examined for the expression of arcB mRNA and expression of ArcZ. Lanes 1–5: PM1560, in which the arcB promoter has been replaced by an arabinose-inducible P_BAD promoter; lanes 6–10: PM1561, a derivative of PM1560 in which the −10 element of the arcZ promoter was inactivated; lanes 11–15, PM1562, an arcA∷kan derivative of PM1560; lanes 16–20, PM1563, an arcA∷kan derivative of PM1561. All strains were grown in LB to OD₆₀₀=1 and samples were collected (Time 0); cultures were split and 0.2% arabinose was added to one of two parallel cultures. Samples were collected after 15 and 60 min of growth, RNA extracted, and northern blot analyses performed using probes against arcB (upper panels) and ArcZ (second row). Northern blot signals were quantified relative to the SsrA control, and variation in intensity during the course of the experiment was compared by setting the Time 0 point to 1. (C) Strains PM1560 (P_BAD–arcB arcZ⁺) and PM1561 (P_BAD–arcB arcZ⁻) were grown as in Figure 6B. After 15 min of induction (Time: 0; lanes 2, 8, and 14), samples were removed and rifampicin was added at a final concentration of 250 μM. Samples were collected at the described intervals and RNA prepared for northern blots. SsrA levels were used to normalize as in (B).

Figure 7

Regulatory circuits for ArcA, ArcB, and ArcZ. See text.