Global Profiling of Lysine Acetylation in Borrelia burgdorferi B31 Reveals Its Role in Central Metabolism

Abstract

The post-translational modification of proteins has been shown to be extremely important in prokaryotes. Using a highly sensitive mass spectrometry-based proteomics approach, we have characterized the acetylome of B. burgdorferi. As previously reported for other bacteria, a relatively low number (5%) of the potential genome-encoded proteins of B. burgdorferi were acetylated. Of these, the vast majority were involved in central metabolism and cellular information processing (transcription, translation, etc.). Interestingly, these critical cell functions were targeted during both ML (mid-log) and S (stationary) phases of growth. However, acetylation of target proteins in ML phase was limited to single lysine residues while these same proteins were acetylated at multiple sites during S phase. To determine the acetyl donor in B. burgdorferi, we used mutants that targeted the sole acetate metabolic/anabolic pathway in B. burgdorferi (lipid I synthesis). B. burgdorferi strains B31-A3, B31-A3 ΔackA (acetyl-P- and acetyl-CoA-) and B31-A3 Δpta (acetyl-P+ and acetyl-CoA-) were grown to S phase and the acetylation profiles were analyzed. While only two proteins were acetylated in the ΔackA mutant, 140 proteins were acetylated in the Δpta mutant suggesting that acetyl-P was the primary acetyl donor in B. burgdorferi. Using specific enzymatic assays, we were able to demonstrate that hyperacetylation of proteins in S phase appeared to play a role in decreasing the enzymatic activity of at least two glycolytic proteins. Currently, we hypothesize that acetylation is used to modulate enzyme activities during different stages of growth. This strategy would allow the bacteria to post-translationally stimulate the activity of key glycolytic enzymes by deacetylation rather than expending excessive energy synthesizing new proteins. This would be an appealing, low-energy strategy for a bacterium with limited metabolic capabilities. Future work focuses on identifying potential protein deacetylase(s) to complete our understanding of this important biological process.

The Functional and Molecular Effects of Doxycycline Treatment on Borrelia burgdorferi Phenotype

“…The mechanisms governing the development of persister cells are not easily discerned by these RNASeq analyses, perhaps because the spirochetes stochastically enter dormancy (Caskey and Embers, 2015) and regrowth is determined by non-heritable traits. One supposition may be that instead, post-transcriptional, or even post-translational events such as lysine acetylation (Fisher et al, 2017; Bontemps-Gallo et al, 2018) govern the entry and exit from dormancy in the antibiotic environment. For example, in the toxin-mediated growth reduction within S. typhimurium , the toxins (TacT) add a post-translational modification (acetylation) to tRNA, which is reversed by a peptidyl tRNA hydrolase (Cheverton et al, 2016).…”

Post-translational Protein Acetylation: An Elegant Mechanism for Bacteria to Dynamically Regulate Metabolic Functions

“…It is now clear that the extent of acetylation, the mechanisms by which acetylation occurs, the identities of acetylated proteins, and the locations of acetylation on those proteins can vary between organisms; yet, in most cases, there is great conservation in core cellular processes (Nakayasu et al, 2017). Such approaches have shown that acetylation occurs on proteins involved in a wide diversity of cellular process, especially central metabolism and translation (Wang et al, 2010; Weinert et al, 2013a; Baeza et al, 2014; Kuhn et al, 2014; Kosono et al, 2015; Schilling et al, 2015; Kentache et al, 2016; Birhanu et al, 2017; Jers et al, 2017; Nagano-Shoji et al, 2017; Post et al, 2017; Bontemps-Gallo et al, 2018; Gaviard et al, 2018; Sun et al, 2018; Vasileva et al, 2018; Yoshida et al, 2019). As stated above, many of these acetylations are conserved.…”

“…Furthermore, attempts to reduce the levels of AcCoA, such as by draining acetyl groups into an acetyl group polymer such as polyhydroxybutyrate, do not avoid any contribution by KATs (Hu, 2013). Work in an organism where AcCoA can be non-essential, such as the reduced genome bacterium Borrelia burgdorferi that solely synthesizes AcCoA to generate mevalonate, a precursor of cell wall lipid I, might be a useful reductionist system to determine whether this truly occurs in vivo (Bontemps-Gallo et al, 2018). Without knowing all possible KATs and eliminating them, it is hard to truly ever say whether an AcCoA-dependent acetylation is truly non-enzymatic in vivo .…”

Mechanisms, Detection, and Relevance of Protein Acetylation in Prokaryotes

“…In a few cases, we know that acetylation of a lysine can affect protein function (1, 2, 20, 137, 186–193). However, there remain thousands of acetyllysines without functional assessment.…”