SUMMARY Acylation of biomolecules (e.g., proteins and small molecules) is a process that occurs in cells of all domains of life and has emerged as a critical mechanism for the control of many aspects of cellular physiology, including chromatin maintenance, transcriptional regulation, primary metabolism, cell structure, and likely other cellular processes. Although this review focuses on the use of acetyl moieties to modify a protein or small molecule, it is clear that cells can use many weak organic acids (e.g., short-, medium-, and long-chain mono- and dicarboxylic aliphatics and aromatics) to modify a large suite of targets. Acetylation of biomolecules has been studied for decades within the context of histone-dependent regulation of gene expression and antibiotic resistance. It was not until the early 2000s that the connection between metabolism, physiology, and protein acetylation was reported. This was the first instance of a metabolic enzyme (acetyl coenzyme A [acetyl-CoA] synthetase) whose activity was controlled by acetylation via a regulatory system responsive to physiological cues. The above-mentioned system was comprised of an acyltransferase and a partner deacylase. Given the reversibility of the acylation process, this system is also referred to as r eversible l ysine a cylation (RLA). A wealth of information has been obtained since the discovery of RLA in prokaryotes, and we are just beginning to visualize the extent of the impact that this regulatory system has on cell function.

Acetyl phosphate (AcP) is a small-molecule metabolite that can act as a phosphoryl group donor for response regulators of two-component systems (TCSs). The serious human respiratory pathogen Streptococcus pneumoniae (pneumococcus) synthesizes AcP by the conventional pathway involving phosphotransacetylase and acetate kinase, encoded by pta and ackA, respectively. In addition, pneumococcus synthesizes copious amounts of AcP and hydrogen peroxide (H 2 O 2 ) by pyruvate oxidase, which is encoded by spxB. To assess possible roles of AcP in pneumococcal TCS regulation and metabolism, we constructed strains with combinations of spxB, pta, and ackA mutations and determined their effects on ATP, AcP, and H 2 O 2 production. Unexpectedly, ⌬ackA mutants were unstable and readily accumulated primary suppressor mutations in spxB or its positive regulator, spxR, thereby reducing H 2 O 2 and AcP levels, and secondary capsule mutations in cps2E or cps2C. ⌬ackA ⌬spxB mutants contained half the cellular amount of ATP as a ⌬spxB or spxB ؉ strain. Acetate addition and anaerobic growth experiments suggested decreased ATP, rather than increased AcP, as a reason that ⌬ackA mutants accumulated spxB or spxR suppressors, although experimental manipulation of the AcP amount was limited. This finding and other considerations suggest that coping with endogenously produced H 2 O 2 may require energy. Starting with a ⌬spxB mutant, we constructed ⌬pta, ⌬ackA, and ⌬pta ⌬ackA mutants. Epistasis and microarray experiment results were consistent with a role for the SpxB-Pta-AckA pathway in expression of the regulons controlled by the WalRK Spn , CiaRH Spn , and LiaSR Spn TCSs involved in sensing cell wall status. However, AcP likely does not play a physiological role in TCS sensing in S. pneumoniae.

Adaptation to endogenous oxidative stress is an integral aspect of Streptococcus pneumoniae colonization and virulence. In this work, we identify key transcriptomic and proteomic features of the pneumococcal endogenous oxidative stress response. The thiol peroxidase TpxD plays a critical role in adaptation to endogenous H2O2 and serves to limit protein sulfenylation of glycolytic, capsule, and nucleotide biosynthesis enzymes in S. pneumoniae.

Bacterial genomes evolve in complex ecosystems and are best understood in this natural context, but replicating such conditions in the lab is challenging. We used transposon sequencing to define the fitness consequences of gene disruption in the bacterium Caulobacter crescentus grown in natural freshwater, compared with axenic growth in common laboratory media. Gene disruptions in amino-acid and nucleotide sugar biosynthesis pathways and in metabolic substrate transport machinery impaired fitness in both lake water and defined minimal medium relative to complex peptone broth. Fitness in lake water was enhanced by insertions in genes required for flagellum biosynthesis and reduced by insertions in genes involved in biosynthesis of the holdfast surface adhesin. We further uncovered numerous hypothetical and uncharacterized genes for which disruption impaired fitness in lake water, defined minimal medium, or both. At the genome scale, the fitness profile of mutants cultivated in lake water was more similar to that in complex peptone broth than in defined minimal medium. Microfiltration of lake water did not significantly affect the terminal cell density or the fitness profile of the transposon mutant pool, suggesting that Caulobacter does not strongly interact with other microbes in this ecosystem on the measured timescale. Fitness of select mutants with defects in cell surface biosynthesis and environmental sensing were significantly more variable across days in lake water than in defined medium, presumably owing to day-to-day heterogeneity in the lake environment. This study reveals genetic interactions between Caulobacter and a natural freshwater environment, and provides a new avenue to study gene function in complex ecosystems.

Protein and small-molecule acylation reactions are widespread in nature. Many of the enzymes catalyzing acylation reactions belong to the Gcn5-related N-acetyltransferase (GNAT; PF00583) family, named after the yeast Gcn5 protein. The genome of Salmonella enterica serovar Typhimurium LT2 encodes 26 GNATs, 11 of which have no known physiological role. Here, we provide in vivo and in vitro evidence for the role of the MddA (methionine derivative detoxifier; formerly YncA) GNAT in the detoxification of oxidized forms of methionine, including methionine sulfoximine (MSX) and methionine sulfone (MSO). MSX and MSO inhibited the growth of an S. enterica ⌬mddA strain unless glutamine or methionine was present in the medium. We used an in vitro spectrophotometric assay and mass spectrometry to show that MddA acetylated MSX and MSO. An mddA ؉ strain displayed biphasic growth kinetics in the presence of MSX and glutamine. Deletion of two amino acid transporters (GlnHPQ and MetNIQ) in a ⌬mddA strain restored growth in the presence of MSX. Notably, MSO was transported by GlnHPQ but not by MetNIQ. In summary, MddA is the mechanism used by S. enterica to respond to oxidized forms of methionine, which MddA detoxifies by acetyl coenzyme A-dependent acetylation.T he Gcn5-related N-acetyltransferase (GNAT; PF00583) superfamily of proteins (Ͼ10,000 members) is present in all domains of life. GNATs transfer the acetyl group from acetyl coenzyme A (acetyl-CoA) to proteins or small molecules (for reviews, see references 1 and 2). The acetylation targets of GNATs include the N termini of proteins (3, 4), aminoglycoside antibiotics (5), glutamate (6), spermidine (7), aminoalkylphosphonic acid (8), dTDP-fucosamine (9), and tRNAs (10). Some of the first bacterial GNATs characterized were the aminoglycoside N-acetyltransferases from Enterococcus faecium (5) and Serratia marcescens (11), demonstrating GNAT-dependent acetylation and inactivation of antibiotics.GNATs provide protection against a myriad of cellular stressors (5,11,12), and it is possible that the diversity of stressors controlled by GNATs correlates with the environment encountered by the cell. Therefore, the relevance of GNAT function to cell physiology varies among organisms, with respect to not only cellular stressors but metabolic pathways as well. For example, Salmonella enterica (13) and Escherichia coli (14) each contain ϳ26 GNATs, yet actinomycetes, such as Streptomyces lividans (15), encode up to ϳ70 putative GNATs. This suggests that S. lividans occupies a more diverse environment while also maintaining a more complex metabolism.At present, there is limited to no information available on the cellular processes several putative S. enterica GNATs may affect. Not surprisingly, the signals that trigger the synthesis of GNATs, the transcription factors involved in sensing such signals, and the determinants of GNAT substrate specificity remain unknown.In S. enterica, MddA (methionine derivative detoxifier A; formerly YncA [STM1590]) is a putative GNAT with no characterized...