Abstract

   Lysine acetylation is a major post-translational modification that
   plays an important regulatory role in almost every aspects in both
   eukaryotes and prokaryotes. Bacillus amyloliquefaciens, a Gram-positive
   bacterium, is very effective for the control of plant pathogens.
   However, very little is known about the function of lysine acetylation
   in this organism. Here, we conducted the first lysine acetylome in B.
   amyloliquefaciens through a combination of highly sensitive
   immune-affinity purification and high-resolution LC−MS/MS. Overall, we
   identified 3268 lysine acetylation sites in 1254 proteins, which
   account for 32.9% of the total proteins in this bacterium. Till date,
   this is the highest ratio of acetylated proteins that have been
   identified in bacteria. Acetylated proteins are associated with a
   variety of biological processes and a large fraction of these proteins
   are involved in metabolism. Interestingly, for the first time, we found
   that about 71.1% (27/38) and 78.6% (22/28) of all the proteins tightly
   related to the synthesis of three types of pepketides and five families
   of lipopeptides were acetylated, respectively. These findings suggest
   that lysine acetylation plays a critical role in the regulation of
   antibiotics biosynthesis. These data serves as an important resource
   for further elucidation of the physiological role of lysine acetylation
   in B. amyloliquefaciens.
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   Nowadays the intensive use of agrochemicals has led to the emergence of
   pathogen resistance and severe negative environmental impacts[32]^1.
   With the increased demand for pesticide-free food, biological control
   through the use of natural antagonistic microorganisms has emerged as a
   promising alternative to chemical pesticides for more rational and safe
   crop management. Bacillus amyloliquefaciens, a Gram-positive bacterium,
   is highly effective for the biocontrol of multiple plant diseases
   caused by soilborne or post-harvest
   pathogens[33]^2,[34]^3,[35]^4,[36]^5,[37]^6. Especially, the strain
   FZB42 of B. amyloliquefaciens has been commercially used due to its
   high efficacy against fungal and bacterial pathogens[38]^7. Antibiotics
   of this organism play an important role in its biocontrol
   activity[39]^8. Lipopeptides (LPs) and polyketides, generated by
   complex enzymes known as non-ribosomal peptide synthetases (NRPS) and
   polyketide synthases (PKS), respectively[40]^9,[41]^10, are the best
   known antibiotics produced by B. amyloliquefaciens. These secondary
   metabolites not only suppress disease pressure in plants by
   antimicrobial activities and activating plant defense, but also are
   important for biofilm formation and root colonization of bacteria on
   crop plants[42]^11,[43]^12,[44]^13,[45]^14.

   Protein acetylation is an evolutionarily conserved post-translational
   modification (PTM) in both eukaryotes and prokaryotes[46]^15. Through
   reversible addition of an acetyl group to lysine residues, protein
   acetylation regulates protein localization, activity, protein-protein
   and protein-nucleic acid interactions[47]^16. Advancements in mass
   spectrometry (MS) and high affinity purification of acetylated peptides
   have made it possible to study lysine acetylation on a proteomic scale.
   As a result, there is increasing evidence indicating that lysine
   acetylation is involved in diverse cellular processes, especially in
   regulating central metabolic
   pathways[48]^17,[49]^18,[50]^19,[51]^20,[52]^21,[53]^22,[54]^23,[55]^24
   . It was shown that in Salmonella the relative activities of key
   enzymes controlling the direction of glycolysis versus gluconeogenesis
   and the branching between citrate cycle and glyoxylate bypass were all
   regulated by acetylation[56]^25. However, to date, there is little
   information regarding the relationship between lysine acetylation and
   biologically active secondary metabolites production. The recent
   discoveries that several enzymes involved in the biosynthesis of
   secondary metabolites were found to be modified by acetyl groups
   implies a role of lysine acetylation in these processes[57]^26,[58]^27.
   In Saccharopolyspora erythraea, TDP-4-keto-6-deoxyhexose 2, 3-reductase
   and nonribosomal peptide synthetas, which are involved in the
   biosynthesis of erythromycin and siderophore, respectively, were
   identified as acetylated proteins. One NRPS involved in the production
   of unknown secondary metabolites in Streptomyces roseosporus was found
   to be acetylated on Lys703, a highly conserved residue in all NRPS
   adenylation domains[59]^27. Based on these observations, we speculate
   that lysine acetylation plays an important role in the regulation of
   the production of LPs and polyketides in B. amyloliquefaciens.

   To test this hypothesis, we performed investigation on the
   acetylproteome of B. amyloliquefaciens by high-resolution LC-MS/MS
   coupled with highly sensitive immune-affinity purification. In total,
   we identified 3268 lysine acetylation sites in 1254 proteins, which
   account for 32.9% of the total proteins in the cell. The identified
   acetylated proteins are involved in diverse biological functions and
   cellular processes. Many enzymes associated with central carbon
   metabolism, protein metabolism and secondary metabolism are acetylated.
   Lysine acetylation is notably observed in the conserved domains of many
   key enzymes involved in the biosynthesis of polyketiedes and LPs,
   indicating that the biosynthesis of secondary metabolites could be
   regulated by acetylation. These results provide the first comprehensive
   view of the acetylome of B. amyloliquefaciens.

Results

Identification and analysis of lysine-acetylated proteins in B.
amyloliquefaciens

   To determine the protein acetylome of B. amyloliquefaciens, a proteomic
   method based on sensitive immune-affinity purification and
   high-resolution LC-MS/MS was applied to identify acetylated proteins
   and their modification sites in B. amyloliquefaciens ([60]Fig. 1). A
   total of 3268 lysine acetylation sites distributed in 1254 acetylated
   proteins was identified ([61]Supplementary Table S1 online). The
   identified proteins account for 32.9% (1254/3811) of the total proteins
   in B. amyloliquefaciens, which is much higher than the percentage of
   acetylation reported in other bacteria ([62]Table 1). To confirm the
   validation of MS data, the mass error of all the identified peptides
   were checked. The distribution of mass error is near zero and most of
   them are less than 0.02 Da which means the mass accuracy of the MS data
   fits the requirement ([63]Supplementary Fig. S1a online). The length of
   most peptides was distributed between 8 and 20, which agrees with the
   property of tryptic peptides ([64]Supplementary Fig. S1b online) and
   means sample preparation reaches the standard. The mass spectrometry
   proteomics data have been deposited to the ProteomeXchange Consortium
   via the PRIDE partner repository with the dataset identifier PXD003339.

Figure 1. Overview of experimental procedures used in this study.

   Figure 1
   [65]Open in a new tab

Table 1. Comparison of B. amyloliquefaciens acetylome with other published
bacteria acetylomes.

   Species Total Proteins Acetylated proteins Percentage of acetylated
   proteins (%) References