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. 2019 Oct 9;8:e51603. doi: 10.7554/eLife.51603

Figure 5. The discovery of acetylation of K134 in MsNrtR.

(A) Use of Western blot to probe the acetylation of recombinant MsNrtR protein in both E. coli and M. smegmatis. The two forms of recombinant NrtR protein were purified from E. coli BL21 and M. smegmatis, and analyzed by western blotting using both anti-acetyl-lysine antibody (α-Acetyl) and poly anti-MsNrtR rabbit serum. The bigger version of MsNrtR is produced by the pET28 expression plasmid in E. coli, whose N-terminus is fused to the 6xHis-containing tag of 23 residues (Supplementary file 1). By contrast, the smaller version of MsNrtR is generated by pMV261 in M. smegmatis, which is only tagged with C-terminal 6xHis. The altered molecular mass (~2 kDa) is the reason why the migration rate of protein electrophoresis differs slightly for the two MsNrtR versions. A representative result is given from three independent trials. (B) The discovery of a unique Lys134 acetylation site in MsNrtR. A LC/MS spectrum reveals that a charged peptide (LVAkLSYTNIGFALAPK) of MsNrtR bears an acetylated lysine (K134Ac). The sequence depicted in the yellow box illustrates the K134 site of acetylation in the context of the modeled structure of MsNrtR-DNA. (C) The mutation of K134A results in reduced acetylation of MsNrtR in M. smegmatis MC2 155 (Magni et al., 2004). A representative result from three independent experiments is given.

Figure 5.

Figure 5—figure supplement 1. Dependence on K134 acetylation in the binding of MsNrtR to a cognate DNA target.

Figure 5—figure supplement 1.

(A) Conservation analysis of the NrtR K134 residue of M. smegmatis through multiple sequence alignment. An arrow denotes the conserved lysine residue. The amino acid sequences are from M. smegmatis MC2 155 (Magni et al., 2004) (Acc. no.: YP_887512), M. avium (Acc. no.: WP_011725315), M. tuberculosis H37Rv (Acc. no.: NP_216109), Pseudomonas aeruginosa (Acc. no.: PTC34113.1), Streptomyces avermitilis-1 (Acc. no.: WP_010987913), Streptomyces avermitilis-2 (Acc. no.: WP_010983884), Hahella chejuensis (Acc. no.: WP_011400661), Acinetobacter baumannii (Acc. no.: WP_000155308), Acinetobacter baylyi (Acc. no.: WP_011182178), Delftia acidovorans (Acc. no.: WP_012207554), Rhizobium etli (Acc. no.: WP_011428339.1), Cyanothece sp. (Acc. no.: WP_009544558), Corynebacterium glutamicum (Acc. no.: WP_011014097), Beutenbergia cavernae (Acc. no.: YP_002882784), and Brachybacterium faecium (Acc. no.: YP_003153838). (B) Size exclusion chromatographic profile and inset SDS-PAGE gel of M. smegmatis NrtR and its three mutants K134A, K134Q, and K134R. This work was carried out as described in Figure 3—figure supplement 1. Designations: OD280, optical density at 280 nm; mAU, milli-absorbance units. (C) Binding of wild-type NrtR to DNA containing the MsNrtR palindrome evaluated by EMSA. (D) Binding of the NrtR K134R mutant to DNA containing the MsNrtR palindrome evaluated by EMSA. (E) Binding of the NrtR K134A mutant to DNA containing the MsNrtR palindrome evaluated by EMSA. (F) Binding of the NrtR K134Q mutant to DNA containing the MsNrtR palindrome evaluated by EMSA. The amount of NrtR added per lane is increased from left to right (0, 5, 10, and 20 pmol), with the minus sign denoting the absence of NrtR protein. (G) Comparative analyses of the relative DNA-binding affinity of MsNrtR and its three K134 mutants (K134R, K134A and K134Q).
Figure 5—figure supplement 2. Acetylation is ubiquitous in three bacterial NrtR proteins.

Figure 5—figure supplement 2.

Western blot was routinely applied to detect the acetylation of the NrtR homologs MsNrtR (MSMEG_3198, 27.35 kDa), VcNrtR (VCA0097, 29 kDa) of Vibrio cholerae, and SsNrtR (SSU05_1971, 32 kDa) of Streptococcus suis. These recombinant NrtR proteins were overexpressed, purified from E. coli BL21, and analyzed by Western blot using both anti-acetyl-lysine antibody (α-Acetyl) and anti-6 ×His antibody (α−6 × His). A representative result is given from three independent trials. The molecular weights of the NrtR homologs were predicted by an online server (web.expasy.org).
Figure 5—figure supplement 3. Construction and identification of NrtR K134 point mutants on the M. smegmatis chromosome.

Figure 5—figure supplement 3.

(A) A schematic for the construction of an in-frame deletion of nrtR and its K134 point mutants on the chromosome. The M. smegmatis lines carrying the point-mutants of K134 in NrtR are constructed by re-introducing the mutated version of nrtR after the removal of full-length nrtR-encoding sequence from M. smegmatis. Primers 1 and 2, abbreviated as P1 and P2, represent the primer pair nrtR-U (PacI)-F and nrtR-D-(NheI)-R (Supplementary file 2). (B) PCR confirmation of the in-frame nrtR deletion mutant of M. smegmatis, ΔnrtR. (C) Direct DNA sequencing of the three point-mutants (K134A, K134Q, and K134R) of nrtR on the chromosome of M. smegmatis. (D) Western blot to evaluate the expression of NrtR in wild-type M. smegmatis and its derivatives (ΔnrtR and the three point-mutants [K134A, K134R, and K134Q]). Anti-MsNrtR rabbit serum refers to a primary antibody.