Figures and data
Phenotypes of ΔahcobB and ΔahcobQ strains. (A) Construction of ahcobB- and ahcobQ-defective strains. M: Marker; Lanes 1 and 3: PCR products of WT using the P7/P8 primer pairs of ahcobB and ahcobQ, respectively; Lanes 2 and 6: PCR products of ΔahcobB and ΔahcobQ, respectively, using P7/P8 primer pairs; Lanes 5 and 7: PCR products of WT using P5/P6 primer pairs of ahcobB and ahcobQ, respectively; Lanes 4 and 8: PCR products of ΔahcobB and ΔahcobQ, respectively, using the P5/P6 primer pairs. (B) Bacterial migration ability; (C) hemolytic activity; (D) histogram of the biofilm formation ability (OD 595 nm). **P < 0.005.
Homology comparison and deacetylase activity assay of AhCobQ and AhCobB.
(A) and (B) Homologous and conserved domain analysis of AhCobQ and AhCobB family proteins. (C) and (D) Dot blot and western blot verified the whole cell protein Kac level among WT, ΔahcobB, and ΔahcobQ strains. (E–H) Effect of NAD+, NAM, Zn2+, and ATP on KDAC enzymatic activity of AhCobQ.
Validation of the KDAC activity of AhCobQ by LC-MS/MS. (A) Retention time of synthetic peptides YNGVFQECCQAEDKGACLLPK (STD) and YNGVFQECCQAEDKacGACL-L-PK (STD*Kac) with or without AhCobB and AhCoQ treatment; (B) MS1 spectrum of synthetic peptides STD and STD*Kac with or without AhCobB and AhCobQ treatment; (C) and (D) MS2 spectrum of acetylated and unmodified peptides.
Lysine deacetylase activity of AhCobQ truncated proteins. Western blot analysis of the KDAC activity of (A) AhCobQ1–179, AhCobQ179–264, AhCobQ1–264, AhCobQ189–264, AhCobQ199–264, AhCobQ195–264, AhCobQ195–255, and AhCobQ200–255; (B) AhCobQ179–264, AhCobQ189–264, AhCobQ189–255, AhCobQ189–250, AhCobQ189–245, and AhCobQ189–240 truncated proteins treated with Kac-BSA. The first lane represents Kac-BSA without AhCobQ truncated proteins. The upper part of the figure shows the WB results of the Kac level of truncated proteins with Kac-BSA, and the lower part shows the PVDF membrane R350 staining for the loading amount control. (C) Design of the series of truncated proteins and the summary of the WB results in this experiment. Blue indicates the AAA_31 domain (1–179 aa), and red indicates the unknown range (180–264 aa) in AhCobQ. Black and white indicate the positive and negative WB results, respectively, in this study.
Phylogenetic tree of the AhCobQ KDAC activity domain (195– 245 aa). Different colors indicate different branches, and Aeromonas hydrophila spp. is highlighted in red.
Proteomics analysis of upregulated proteins in ΔahcobB, ΔahcobQ, ΔahacuC, and WT strains.
(A) Venn diagrams visualized the overlapped upregulated Kac proteins and Kac peptides among the ΔahcobB, ΔahcobQ, ΔahacuC, and WT strains. The number of acetylated proteins and peptides (sites) are shown in the figure. (B) KEGG metabolic pathway enrichment analysis of upregulated Kac proteins and peptides by the three KDACs. Unique and overlapped Kac protein and peptides (sites) are presented in different colors.
Western blot validation of the site-specific Kac protein substrates regulated by the three KDACs. (A–C) Kac specific sites were regulated by AhCobQ alone, AhCobQ with AhCobB, and all three KDACs together. The overlapping proteomic features of the target Kac proteins regulated by the three KDACs are shown on the left side of the figure. The detected proteins are in the pink area of the Venn diagram; (D) different Kac sites in one protein substrate were regulated by different KDACs. The predicted 3D structure of the target proteins with different Kac sites is shown on the left side of the figure. The middle section shows the WB results of the deacetylation effect of the three KDACs on site-specific Kac protein substrates, and the right shows the PVDF membrane R350 staining for the loading amount control.
AhCobQ positively regulates the ICD enzymatic activity through deacetylation of K388 on ICD. (A) western blot verified the deacetylation effect of AhCobQ on ICD and ICD-K388; (B) western blot verified the deacetylation effect of AhCobQ, AhCobB and AhAcuC on ICD-K388. The PVDF membrane R350 staining for the loading amount control was displayed under the WB results; (C) Enzymatic activities of ICD (yellow), ICD-Kac388(blue), ICD-Kac388 treated with AhCobQ (red), ICD-Kac388 treated with AhCobB (green), ICD-Kac388 treated with AhAcuC (brown); (D) The histogram showed the effect of AhCobQ/AhcobB on ICD or ICD-Kac388 enzymatic activities at 5min. ***P < 0.001.
