The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.

The CpxAR two-component signal transduction system in Escherichia coli and other pathogens senses diverse envelope stresses and promotes the transcription of a variety of genes that remedy these stresses. An important member of the CpxAR regulon is cpxP. The CpxA-dependent transcription of cpxP has been linked to stresses such as misfolded proteins and alkaline pH. It also has been proposed that acetyl phosphate, the intermediate of the phosphotransacetylase (Pta)-acetate kinase (AckA) pathway, can activate the transcription of cpxP in a CpxA-independent manner by donating its phosphoryl group to CpxR. We tested this hypothesis by measuring the transcription of cpxP using mutants with mutations in the CpxAR pathway, mutants with mutations in the Pta-AckA pathway, and mutants with a combination of both types of mutations. From this epistasis analysis, we learned that CpxR integrates diverse stimuli. The stimuli that originate in the envelope depend on CpxA, while those associated with growth and central metabolism depend on the Pta-AckA pathway. While CpxR could receive a phosphoryl group from acetyl phosphate, this global signal was not the primary trigger for CpxR activation associated with the Pta-AckA pathway. On the strength of these results, we contend that the interactions between central metabolism and signal transduction can be quite complex and that successful investigations of such interactions must include a complete epistatic analysis.

SummaryProtein acetylation has historically been considered a predominantly eukaryotic phenomenon. Recent evidence, however, supports the hypothesis that acetylation broadly impacts bacterial physiology. To explore more rapidly the impact of protein acetylation in bacteria, microbiologists can benefit from the strong foundation established by investigators of protein acetylation in eukaryotes. To help advance this learning process, we will summarize the current understanding of protein acetylation in eukaryotes, discuss the emerging link between acetylation and metabolism and highlight the best-studied examples of protein acetylation in bacteria.

Bacterial biofilm infections remain prevalent reasons for implant failure. Dental implant placement occurs in the oral environment, which harbors a plethora of biofilm-forming bacteria. Due to its trans-mucosal placement, part of the implant structure is exposed to oral cavity and there is no effective measure to prevent bacterial attachment to implant materials. Here, we demonstrated that UV treatment of titanium immediately prior to use (photofunctionalization) affects the ability of human polymicrobial oral biofilm communities to colonize in the presence of salivary and blood components. UV-treatment of machined titanium transformed the surface from hydrophobic to superhydrophilic. UV-treated surfaces exhibited a significant reduction in bacterial attachment as well as subsequent biofilm formation compared to untreated ones, even though overall bacterial viability was not affected. The function of reducing bacterial colonization was maintained on UV-treated titanium that had been stored in a liquid environment before use. Denaturing gradient gel-electrophoresis (DGGE) and DNA sequencing analyses revealed that while bacterial community profiles appeared different between UV-treated and untreated titanium in the initial attachment phase, this difference vanished as biofilm formation progressed. Our findings confirm that UV-photofunctionalization of titanium has a strong potential to improve outcome of implant placement by creating and maintaining antimicrobial surfaces.

Summary In eukaryotes, lysine acetylation is a well-established posttranslational modification that has been implicated in virtually all aspects of eukaryotic physiology. Although homologs of the enzymes that catalyze protein acetylation are widely conserved and distributed amongst bacterial species, not much is known about the impact of protein acetylation on bacterial physiology. Here, we present evidence that the Gcn5-like acetyltransferase YfiQ and the sirtuin deacetylase CobB play crucial roles in the transcription regulation of the periplasmic stress-responsive promoter cpxP when cells of Escherichia coli grow in the presence of glucose, an environment that induces protein acetylation. Under this growth condition, several acetylation sites were detected on three of the RNA polymerase (RNAP) subunits: β, β’ and α. We focused on acetylations of the carboxy-terminal domain (CTD) of α because of its relative small size and its limited acetylation. We determined that K298 of α is acetylated in a glucose and YfiQ-dependent manner and that K298 is specifically required for glucose-induced cpxP transcription. Since the αCTD aids in promoter recognition by RNA polymerase, we propose its acetylation may influence bacterial physiology through effects on gene expression.