YfmK is an Ne-lysine acetyltransferase that directly acetylates the histone-like protein HBsu in Bacillus subtilis Valerie J. Carabettaa,1, Todd M. Grecob, Ileana M. Cristeab, and David Dubnauc,1 aDepartment of Biomedical Sciences, Cooper Medical School of Rowan University, Camden, NJ 08103; bDepartment of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08544; and cPublic Health Research Institute Center of New Jersey Medical School, Rutgers University, Newark, NJ 07103 Edited by Graham C. Walker, Massachusetts Institute of Technology, Cambridge, MA, and approved January 11, 2019 (received for review September 7, 2018) e N -lysine acetylation is an abundant and dynamic regulatory post- high, AcuA inactivates AcsA by acetylating a conserved lysine translational modification that remains poorly characterized in residue in its active site. Two deacetylases, the KDAC AcuC (13) bacteria. In bacteria, hundreds of proteins are known to be acet- and the sirtuin SrtN (14), lead to the reactivation of AcsA when ylated, but the biological significance of the majority of these acetyl-CoA is deficient. The KAT AcuA is highly specific for Acs- events remains unclear. Previously, we characterized the Bacillus like enzymes, as its inactivation has only minor effects on global subtilis acetylome and found that the essential histone-like pro- protein acetylation (9, 15). B. subtilis contains hundreds of acety- tein HBsu contains seven previously unknown acetylation sites lated proteins (9, 15, 16), suggesting that other protein acetyl- in vivo. Here, we investigate whether acetylation is a regulatory transferases may exist. component of the function of HBsu in nucleoid compaction. Using While characterizing the B. subtilis acetylome (15), we dis- mutations that mimic the acetylated and unacetylated forms of covered that the histone-like protein HBsu is modified by acet- the protein, we show that the inability to acetylate key HBsu ly- ylation at seven sites in vivo (Fig. 1A). In eukaryotic cells, the sine residues results in a more compacted nucleoid. We further histone proteins are responsible for DNA compaction and for investigated the mechanism of HBsu acetylation. We screened de- forming appropriate chromatin structures. Histones contain un- letions of the ∼50 putative GNAT domain-encoding genes in B. structured, highly basic N-terminal tails that can be modified by MICROBIOLOGY subtilis for their effects on DNA compaction, and identified five several different PTMs, including lysine acetylation. The com- binations of these PTMs on the tails are regulatory and can alter candidates that may encode acetyltransferases acting on HBsu. “ Genetic bypass experiments demonstrated that two of these, gene expression. They are commonly referred to as the histone code.” In bacteria, the nucleoid-associated proteins [NAPs (17)] YfmK and YdgE, can acetylate Hbsu, and their potential sites of are largely responsible for chromosome compaction. HBsu be- action on HBsu were identified. Additionally, purified YfmK was longs to the highly conserved HU family of NAPs and is essential able to directly acetylate HBsu in vitro, suggesting that it is the B. subtilis for viability in B. subtilis (18, 19). It binds to curved DNA without second identified protein acetyltransferase in . We pro- apparent sequence specificity (20) and condenses the bacterial pose that at least one physiological function of the acetylation of chromosome (21). However, the potential role of lysine acety- HBsu at key lysine residues is to regulate nucleoid compaction, lation in regulating HBsu function is unknown. In the current analogous to the role of histone acetylation in eukaryotes. study, we investigated the role of HBsu acetylation in modulating bacterial chromosome (nucleoid) compaction. We generated acetylation | histone | nucleoid compaction | acetylase | GNAT K→R and K→Q substitution mutations that respectively mimic e -lysine acetylation is an important and ubiquitous regula- Significance Ntory posttranslational modification (PTM), conserved among all three domains of life (1, 2). Within the past decade, it Ne-lysine acetylation is a dynamic regulatory posttranslational has been appreciated that protein acetylation is widespread in modification that affects hundreds of proteins in all three do- many different bacteria and may regulate hundreds of proteins mains of life. In bacteria, acetylated proteins can be found in with diverse cellular functions (3, 4). Although the physiological many essential pathways, and may also be important for viru- significance of the majority of bacterial protein acetylation events lence in pathogenic species. However, the biological relevance of remains unknown, an impact of acetylation on the functions of the overwhelming majority of these acetylation events remains several bacterial proteins (3) has been shown. poorly characterized. Here, we discover that acetylation of the In bacteria, there are two known mechanisms for acetylation histone-like protein HBsu regulates its ability to control nucleoid of lysine residues, enzymatic and nonenzymatic. Nonenzymatic compaction, and we identify the second protein acetyltransfer- acetylation occurs via an autocatalytic mechanism using acetyl Bacillus subtilis phosphate as the primary acetyl donor (5–9). In Escherichia coli,it ase in . Moving forward, the targeting of bacte- was demonstrated that the majority of global protein acetylation rial protein acetylation may be exploited to aid in the design of occurs at a low stoichiometry and nonenzymatically (10). Enzy- novel therapeutics, as it has been successful in the treatment of matic acetylation is carried out by acetyltransferases (KATs). The certain cancers and latent viral and fungal infections. highly conserved GCN5-like N-acetyltransferases (GNATs) cata- lyze the transfer of an acetyl group from acetyl-CoA (CoA) to a Author contributions: V.J.C., T.M.G., I.M.C., and D.D. designed research; V.J.C. performed target primary amine, either on a lysine residue or an N-terminal research; V.J.C. and T.M.G. analyzed data; and V.J.C., T.M.G., I.M.C., and D.D. wrote + the paper. amino group. Deacetylation is carried out by the Zn -dependent + lysine deacetylase (KDAC) family or NAD -dependent sirtuins The authors declare no conflict of interest. (reviewed in refs. 3 and 11). The first class of bacterial enzymes This article is a PNAS Direct Submission. found to be regulated by reversible acetylation were acetyl-CoA Published under the PNAS license. synthetases (Acs’s), the enzyme responsible for converting acetate 1To whom correspondence may be addressed. Email: [email protected] or into acetyl-CoA, and other AMP intermediate-forming enzymes [email protected]. (reviewed in ref. 11). In Bacillus subtilis, an Acs ortholog, AcsA, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. is regulated by AcuA, the only characterized protein acetyl- 1073/pnas.1815511116/-/DCSupplemental. transferase in this organism (12). When acetyl-CoA levels are www.pnas.org/cgi/doi/10.1073/pnas.1815511116 PNAS Latest Articles | 1of6 Downloaded by guest on September 28, 2021 A Results Acetylation of HBsu Regulates Nucleoid Compaction. It has been established previously that HBsu is essential, and is important for chromosomal compaction (19, 20). To confirm these observations, a depletion strain was constructed in the BD630 background (his leu met; SI Appendix,TableS2), in which hbs was expressed from the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible Phyperspank B promoter, and the native copy was replaced with a tetracycline resistance cassette. This strain will form colonies on agar plates only in the presence of IPTG. When depleted for HBsu in liquid cultures the cells were filamented and, as expected, contained ex- panded and irregularly spaced nucleoids (SI Appendix,Fig.S1). Additionally, anucleate cells formed frequently, suggesting a DNA segregation defect (SI Appendix,Fig.S1, white arrows). Effects of Mutants That Mimic Acetylated and Unacetylated Lysine Residues. After confirming the role of HBsu in nucleoid organi- zation, we examined the effects of protein acetylation. To eval- uate the potential impact of acetylation on nucleoid compaction, glutamine (acetylated mimic) and arginine (unacetylated mimic) point mutant substitutions are commonly used (3, 22–26). Based on our previous identification of seven in vivo lysine acetylation C sites of HBsu, we introduced Q and R substitutions individually into the native hbs locus (BD630; Fig. 1A), using the pMini- MAD2 cloning strategy, as described in SI Appendix. Exponen- tially growing cells were sampled from minimal glucose media, and their nucleoids were stained with DAPI. Cells from the hbsK41R mutant contained more compacted and circular nucle- oids compared with the wild type (Fig. 1B). Interestingly, a similar phenotype was observed for six of the seven R substitutions, with hbsK37R being the lone exception (SI Appendix,Fig.S2). Quan- tification of the nucleoid area from fluorescence microscopy of over 4,500 cells per strain revealed that the population medians, represented as the 50th percentile of cumulative distribution plots of the K→R mutants, were left-shifted relative to the wild-type distribution (Fig. 1C and SI Appendix, Fig. S2B; P < 0.0001), confirming that the measured nucleoids are more compacted, as Fig. 1. HBsu mutants containing unacetylated mimics (R) lead to compacted observed visually (Fig. 1B). nucleoids. (A)