Research Article: Protein lysine acetylation plays a regulatory role in Bacillus subtilis multicellularity

Date Published: September 28, 2018

Publisher: Public Library of Science

Author(s): Alicyn Reverdy, Yun Chen, Evan Hunter, Kevin Gozzi, Yunrong Chai, Mariola J. Edelmann.


Protein lysine acetylation is a post-translational modification that alters the charge, conformation, and stability of proteins. A number of genome-wide characterizations of lysine-acetylated proteins, or acetylomes, in bacteria have demonstrated that lysine acetylation occurs on proteins with a wide diversity of functions, including central metabolism, transcription, chemotaxis, and cell size regulation. Bacillus subtilis is a model organism for studies of sporulation, motility, cell signaling, and multicellular development (or biofilm formation). In this work, we investigated the role of global protein lysine acetylation in multicellular development in B. subtilis. We analyzed the B. subtilis acetylome under biofilm-inducing conditions and identified acetylated proteins involved in multicellularity, specifically, swarming and biofilm formation. We constructed various single and double mutants of genes known to encode enzymes involved in global protein lysine acetylation in B. subtilis. Some of those mutants showed a defect in swarming motility while others demonstrated altered biofilm phenotypes. Lastly, we picked two acetylated proteins known to be important for biofilm formation, YmcA (also known as RicA), a regulatory protein critical for biofilm induction, and GtaB, an UTP-glucose-1-phosphate uridylyltransferase that synthesizes a nucleotide sugar precursor for biosynthesis of exopolysaccharide, a key biofilm matrix component. We performed site-directed mutagenesis on the acetylated lysine codons in ymcA and gtaB, respectively, and assayed cells bearing those point mutants for biofilm formation. The mutant alleles of ymcA(K64R), gtaB(K89R), and gtaB(K191R) all demonstrated a severe biofilm defect. These results indicate the importance of acetylated lysine residues in both YmcA and GtaB. In summary, we propose that protein lysine acetylation plays a global regulatory role in B. subtilis multicellularity.

Partial Text

Post-translational modification enables bacteria to adapt quickly to changing environments. Instead of spending time and energy to express genes and translate new proteins, cells can quickly modify an existing protein to alter its activity (e.g. from inactive to active) in response to environmental changes [1, 2]. Protein lysine acetylation is a post-translational modification that involves the transfer of an acetyl group from a donor metabolite (e.g. acetyl-CoA or acetyl-phosphate) to a lysine residue of a protein [3]. Protein lysine acetylation can be achieved by two distinct mechanisms: chemical acetylation using acetyl-phosphate as a donor of the acetyl group, and enzymatic acetylation by lysine acetyltransferases using acetyl-CoA as the acetyl group donor [3, 4](Fig 1). During protein lysine acetylation, when a negatively charged acetyl group is covalently attached to a positively charged lysine residue, it can cause a change in charge and/or conformation of the protein, which may subsequently alter the protein activity [1, 4]. Protein lysine acetylation is a reversible process. Lysine deacetyltransferases are responsible for removing the acetyl group from the acetylated lysine residues of a protein. Originally characterized in eukaryotes, such as in the regulation of chromatin organization [3], protein lysine acetylation has now been recognized as an important post-translational modification in bacteria as well (reviewed in [4, 5]). Global acetylome analyses in bacteria have shown that protein lysine acetylation can occur on proteins involved in a variety of different functions such as central metabolism, transcription, DNA binding, motility, and cell size regulation [4, 6–10].

In this study, we investigated the role of one of the post-translational modification mechanisms, protein lysine acetylation, in B. subtilis multicellular development. We analyzed global acetylomes of wild type B. subtilis and two global acetylation mutants grown under biofilm-inducing conditions. We characterized the motifs and diverse categories of proteins modified by lysine acetylation, in particular, a number of acetylated proteins known to be involved in swarming motility and biofilm formation. We investigated the impact of global lysine acetylation on the two multicellular processes by constructing mutants altered in one of the two acetylation mechanisms, chemical or enzymatic acetylation pathways. Many of those mutants showed a clear defect in swarming motility while some also showed a defect in biofilm formation (Figs 3 and 4). In the second half of this study, we picked two individual proteins, YmcA and GtaB, shown to be acetylated in our acetylome analysis and whose acetylation levels significantly decreased in both the Δpta and ΔacuA global acetylation mutants. We conducted further investigations on the role of these acetylated lysine residues in their protein function. The regulatory protein YmcA was previously shown to be essential for biofilm formation [51, 54] while the UTP-glucose-1-phosphate uridylyltransferase GtaB was first shown to be essential for biofilm formation in B. subtilis. By application of site-directed mutagenesis of selected acetylated lysine residues, we demonstrated that some of these modified lysine residues are indeed important for the protein function. Collectively, our results support the idea that protein lysine acetylation plays a global role in bacterial multicellularity in B. subtilis.




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