Temporal Regulation of the Bacillus subtilis Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth

Abstract

Nε-Lysine acetylation has been recognized as a ubiquitous regulatory posttranslational modification that influences a variety of important biological processes in eukaryotic cells. Recently, it has been realized that acetylation is also prevalent in bacteria. Bacteria contain hundreds of acetylated proteins, with functions affecting diverse cellular pathways. Still, little is known about the regulation or biological relevance of nearly all of these modifications. Here we characterize the cellular growth-associated regulation of the Bacillus subtilis acetylome. Using acetylation enrichment and quantitative mass spectrometry, we investigate the logarithmic and stationary growth phases, identifying over 2,300 unique acetylation sites on proteins that function in essential cellular pathways. We determine an acetylation motif, EK(ac)(D/Y/E), which resembles the eukaryotic mitochondrial acetylation signature, and a distinct stationary-phase-enriched motif. By comparing the changes in acetylation with protein abundances, we discover a subset of critical acetylation events that are temporally regulated during cell growth. We functionally characterize the stationary-phase-enriched acetylation on the essential shape-determining protein MreB. Using bioinformatics, mutational analysis, and fluorescence microscopy, we define a potential role for the temporal acetylation of MreB in restricting cell wall growth and cell diameter.IMPORTANCEThe past decade highlighted Nε-lysine acetylation as a prevalent posttranslational modification in bacteria. However, knowledge regarding the physiological importance and temporal regulation of acetylation has remained limited. To uncover potential regulatory roles for acetylation, we analyzed how acetylation patterns and abundances change between growth phases in B. subtilis. To demonstrate that the identification of cell growth-dependent modifications can point to critical regulatory acetylation events, we further characterized MreB, the cell shape-determining protein. Our findings led us to propose a role for MreB acetylation in controlling cell width by restricting cell wall growth.

Regulation, Function, and Detection of Protein Acetylation in Bacteria

“…Such quantitative data can be used to select likely biologically relevant acetylation events for further study. For example, an acetylation abundance that changes under different growth conditions implies that the modification of that site may provide a regulatory mechanism, as demonstrated for MreB in B. subtilis (42).…”

“…In B. subtilis, the rod shape-determining protein MreB contains three acetylation sites, with K240 acetylation significantly increasing upon entry into stationary phase (42). Wild-type cells get shorter and narrower in stationary phase, and this is also observed during exponential phase in the presence of an MreB K240Q mutant.…”

“…reduce bias for certain acetylation motifs, many studies currently use a mixture of antibodies for acetyl-peptide capture (30,32,34,39,42,43,51). Even so, the isolation efficiency is usually between 25 and 40% (42) and is often not reported.…”

“…Therefore, improvements in the affinity probes could improve the efficiency and accuracy of acetylation site identifications. This is would be especially helpful for detecting lowabundance acetylation events, as the likelihood of identifying an acetylation site increases with increasing protein abundance (42,71). Additional peptide fractionations prior to acetyl-peptide capture can help reduce the peptide complexity or can help focus on a certain cellular compartment, thereby aiding the identification of lowerabundance proteins (32,33,41,44,46).…”

“…The label-free approach is desirable because it is simpler, can be performed in any medium, can be used to compare any number of samples/conditions, and is cost-effective (124). This method was successfully used to monitor the acetylation abundance of E. coli proteins from cells grown in the presence and absence of glucose at various time points (106), as well as to compare acetylation changes in exponential-phase versus stationary-phase growth in B. subtilis (42). Additionally, in E. coli, label-free quantification was used to identify YfiQ-and CobB-sensitive acetylation sites and to show that acetylphosphate is a specific and major determinant of acetylation (59).…”