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(A) Top line, SDS-PAGE and Western blot analysis of purified PykF and its acetylated variants from BL21 (DE3) cells. The acetylated variants with mutation of a single lysine site in PykF to glutamine. Lane 1, wild-type PykF; Lane 2, PykF-K13Q; Lane 3, PykF-K19Q; Lane 4, PykF-K52Q; Lane 5, PykF-K59Q; Lane 6, PykF-K68Q; Lane 7, PykF-145Q; Lane 8, PykF-K317Q; Lane 9, PykF-K319Q; Lane 10, PykF-K340Q; Lane 11, PykF-K368Q; Lane 12, PykF-K382Q. The same amount of protein was loaded. Anti-AcK: anti-acetyl lysine antibody. Bottom line, pyruvate kinase activity of PykF and its acetylated variants. Pyruvate kinase activity of the wild-type PykF was set as 1. Mean values and standard deviations were calculated based on three replicates (n = 3). (B) Top line, SDS-PAGE and Western blot analysis of purified PykF and its deacetylated variants from BL21 (DE3) cells. The deacetylated variants with mutation of a single lysine site in PykF to arginine. Lane 1, wild-type PykF; Lane 2, PykF-K13R; Lane 3, PykF-K19R; Lane 4, PykF-K52R; Lane 5, PykF-K59R; Lane 6, PykF-K68R; Lane 7, PykF-145R; Lane 8, PykF-K317R; Lane 9, PykF-K319R; Lane 10, PykF-K340R; Lane 11, PykF-K368R; Lane 12, PykF-K382R. The same amount of protein was loaded. Anti-AcK, anti-acetyl lysine antibody. Bottom line, pyruvate kinase activity of PykF and its deacetylated variants. Pyruvate kinase activity of the wild-type PykF was set as 1. Mean values and standard deviations were calculated based on three replicates (n = 3). Two-tailed P-values were determined by the t-test, and the significance level is 0.05. *p < 0.05.

(A) Construction of complemented and overexpression strains. Lane 1, wild-type (WT); Lane 2, WT:pykF overexpression strain; Lane 3, deletion of cobB gene strain (ΔcobB); Lane 4, ΔcobB:pykF complemented strain; Lane 5, deletion of pykF gene strain (ΔpykF); Lane 6, ΔpykF:pykF complemented strain; Lane 7, ΔpykF:K52R complemented strain; Lane 8, ΔpykF:K68R complemented strain; Lane 9, ΔpykF:K317R complemented strain. Anti-His: His Tag Mouse Monoclonal Antibody. (B) Analysis of pyruvate kinase activity throughout the V. alginolyticus ΔpykF strain and WT:pykF overexpression strain. Pyruvate kinase activity of V. alginolyticus WT was set as 1. (C) Analysis pyruvate kinase activity of site-directed mutagenesis complemented strains. Pyruvate kinase activity of V. alginolyticus ΔpykF:pykF was set as 1. Mean values and standard deviations were calculated based on three replicates (n = 3). Two-tailed P-values were determined by the t-test, and the significance level is 0.05. *p < 0.05.

(A) CobB deacetylation PykF in vitro. Top line, Western blot was performed on PykF treated with CobB for 1 h. The same amount of protein was loaded in all lanes. Bottom line, pyruvate kinase activity was measured, and the enzyme activity of lane 1 sample was set as 1. Mean values and standard deviations were calculated based on three replicates (n = 3). Two-tailed P-values were determined by the t-test, and the significance level is 0.05. (B) CobB deacetylates PykF in vivo. Top line, Western blot analysis of purified PykF from V. alginolyticus WT:pykF and ΔcobB:pykF. Lane 1, purified PykF from V. alginolyticus WT:pykF; Lane 2, purified PykF from V. alginolyticus ΔcobB:pykF. The same amount of protein was loaded in all lanes. Anti-His, His Tag Mouse Monoclonal Antibody. Anti-AcK: anti-acetyl lysine antibody. Bottom line, pyruvate kinase activity of V. alginolyticus WT and ΔcobB. Pyruvate kinase activity of V. alginolyticus WT was set as 1. Mean values and standard deviations were calculated based on three replicates (n = 3). Two-tailed P-values were determined by the t-test, and the significance level is 0.05.

Top line, acetylation of PykF. SDS-PAGE and Western blot analysis were performed on purified PykF treated with AcP in different concentrations and incubation time. The same amount of protein was loaded. Anti-AcK, acetyl lysine antibody. Bottom line, pyruvate kinase activity was measured, and the enzyme activity of lane 1 sample was set as 1. Mean values and standard deviations were calculated based on three replicates (n = 3). Two-tailed P-values were determined by the t-test, and the significance level is 0.05.

(A) Growth of V. alginolyticus. Analysis of growth throughout the V. alginolyticus ΔpykF strain and WT:pykF overexpression strain at 24 h. (B) Analysis of growth throughout V. alginolyticus ΔpykF:pykF, ΔpykF:K52R, ΔpykF: K68R and ΔpykF:K317R complemented strains at 24 h. (C) Analysis of extracellular protease activity throughout the V. alginolyticus ΔpykF mutant strain and WT:pykF overexpression strain. Extracellular protease activity of V. alginolyticus WT was set as 1. (D) Analysis site-directed mutagenesis complemented strains on extracellular protease activity. Extracellular protease activity of V. alginolyticus ΔpykF:pykF was set as 1. Mean values and standard deviations were calculated based on three replicates (n = 3). Two-tailed P-values were determined by the t-test, and the significance level is 0.05. *p < 0.05.