Correction for Luo et al., An acetyltransferase moonlights as a regulator of the RNA binding repertoire of the RNA chaperone Hfq in Escherichia coli

Correction for “An acetyltransferase moonlights as a regulator of the RNA binding repertoire of the RNA chaperone Hfq in Escherichia coli,” by Xing Luo, Aixia Zhang, Chin-Hsien Tai, Jiandong Chen, Nadim Majdalani, Gisela Storz, and Susan Gottesman, which published November 27, 2024; 10.1073/pnas.2311509120 (Proc. Natl. Acad. Sci. U.S.A. 120, e2311509120).

The authors note: “In Fig. 4F, we tested the effect of deleting the C-terminal domain (CTD) of Hfq (hfq_1-65) on the activity of HqbA expressed from a plasmid and concluded that the Hfq CTD contributed to HqbA function. SI Appendix, Fig. S10F has the growth data associated with this panel. In subsequent experiments, we realized that the strain used for this experiment had a previously undetected point mutation. In reconstructed strains, we found the Hfq CTD is not needed for the activity of HqbA. In following up on the experiments in this publication, the HqbA plasmid was introduced into another strain that carried the hfq_1-65 mutation and, unexpectedly, the HqbA plasmid gave full activity in this strain. This led us to both reconstruct the original set of strains and sequence the full genome of the original strains. The sequencing revealed that, during the construction of the original hfq_1-65 strain, a previously undetected point mutation arose in the translation initiation region of the reporter fusion used in this experiment, leading to reduced expression of the fusion. It was this reduced expression that we interpreted as the need for the Hfq CTD. The error leads to a change in one aspect of the interaction of Hfq and HqbA, changing our previous conclusion. The Hfq CTD is not needed for HqbA function, and it may partially interfere with HqbA access to Hfq. This does not change any other conclusions or data in the paper.”

Fig. 4.

HqbA D98, E102, and Hfq K31 are essential for the interaction with and the modulation of HqbA. (A) HqbA-Hfq₆ complex structure predicted by AlphaFold-Multimer (version 2.1.1). The protein sequence of E. coli HqbA and Hfq hexamer were used as the input. The best scoring model (iptm + ptm score = 0.72) was superposed with the complex structure of HqbA-CoA (PDB: 4SR2) and is displayed in the surface diagram (Top) and ribbon diagram (Bottom). The Hfq hexamer is shown in light gray, the Hfq CTD is shown in dark gray, HqbA is shown in orange and the CoA is shown in lime. Hfq K31 is highlighted in blue, G29 is highlighted in light blue and HqbA D98 and E102 are highlighted in magenta. (B) Western blot analysis of HqbA and Hfq protein levels in total protein and Flag-HqbA pull-down samples. WT hfq (XL290) or hfqK31Q (XL294) strains were transformed with pQE80L, pflagHqbA, or pflagHqbAD98AE102A plasmids and grown and analyzed in for Fig. 2A. (C) HqbA auto-acetylation level measured by in vitro acetylation assay. His-HqbA, His-HqbAY117A, or His-HqbAD98AE102A (2 µg each) were incubated without and with Ac-CoA (0.5 mM) in 50 µL reaction buffer for 5 h at 37 °C. The acetylation signal was measured by western blotting using an antibody against acetylated-lysine. (D) Fluorescence of different reporter strains upon WT HqbA or HqbAD98AE102A overexpression. The fluorescence of each strain was measured as described in Fig. 1. The kinetic OD₆₀₀ and FU for each strain are shown in SI Appendix, Fig. S9. The reporter strains used in this experiment were chiP-mCh (JC1247), sodB-mCh ∆fur (JC1249), and rpoS-mCh (JC1329). (E) Fluorescence of chiP-mCh with different hfq mutants [WT (JC1247), hfqG29A (XL235), hfqK31R (XL237), or hfqK31Q (XL238)] upon HqbA overexpression. The fluorescence of each strain was measured as described in Fig. 1. The kinetic OD₆₀₀ and FU for each strain are shown in SI Appendix, Fig. S10. (F) Fluorescence of chiP-mCh with ∆hqbA (XL290) or hfq_1-65 ∆hqbA (XL871) background upon HqbA overexpression. The fluorescence of each strain was measured as described in Fig. 1. The kinetic OD₆₀₀ and FU for each strain are shown in SI Appendix, Fig. S10F.

As a result, the changes below have been made to the text.

On page 4, right column, second full paragraph, the section title, “Hfq K31 and the Hfq CTD Are Essential for the Modulation by HqbA.” should instead appear as, “Hfq K31 is Essential for the Modulation by HqbA.”

On page 5, left column, first full paragraph, line 11: “While HqbA induction increased the expression of chiP-mCh in the WT hfq strain, the activation effect was abolished in ${\mathit{hfq}}_{1-65}$ (Fig. 4F and SI Appendix, Fig. S10F). These results suggest that the Hfq CTD is also needed for the multicopy HqbA modulation of Hfq.” should instead appear as “HqbA induction increased the expression of chiP-mCh in both the WT hfq strain and in hfq_1-65 (Fig. 4F and SI Appendix, Fig. S10F). These results suggest that the Hfq CTD is not needed for the multicopy HqbA modulation of Hfq.”

On page 5, left column, second full paragraph, line 4: “The Hfq CTD also contributes to the ability of HqbA to suppress Hfq regulation.” should instead appear as “The Hfq CTD is not needed for HqbA to suppress Hfq regulation.”

Fig. 4 and its corresponding legend have also been corrected as a result of this error. Reconstructed strains are used in Fig. 4F, and the legend has been updated to reflect the correct strain names. The corrected figure and its corrected legend appear below.

In addition, SI Appendix, Fig. S10 and its corresponding legend have been corrected. Reconstructed strains are used in SI Appendix, Fig. S10F, and the legend has been updated to reflect the correct strain names.

The authors note that the following statement should be added to the Acknowledgments: “We thank Jemison Yehwah for carrying out the critical experiments leading to our recognition of the mutant in our previous strains.”

The online version of the text, Fig. 4 and the SI Appendix have been corrected.