Yfmk Is an Nε-Lysine Acetyltransferase That Directly Acetylates the Histone

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 combinations 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 organization, 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 nucleoids 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) Schematic displaying the seven acetylation sites identified on The corresponding acetylated mimic, hbsK41Q, had a re- HBsu. (B) Wild-type (BD630), hbsK41Q (BD8147), and hbsK41R (BD8148) cells producible decrease in DAPI staining intensity (Fig. 1B), which were grown in minimal glucose medium to exponential phase and their → nucleoids were stained with DAPI (pseudocolored green). Representative was true for most of the K Q mutants (SI Appendix, Fig. S3). microscopic images are displayed. DIC, differential interference contrast. This observed decrease in DAPI staining intensity makes accurate and reliable determination of nucleoid areas difficult. For a subset (C) Wild-type, GFP-expressing cells (BD8012) were mixed with the hbsK41R → mutant, and their nucleoids were stained with DAPI. Nucleoid areas of at of the K Q mutants, this was likely due to a decrease in DNA least 4,500 cells for each strain were determined as described in Materials content, as determined by flow cytometry (SI Appendix, Fig. S4). and Methods. Cumulative distribution plots are displayed, where the 50th In this asynchronous population, the hbsK3Q, hbsK41Q,and percentile represents the median of the population distribution. The hbsK86Q mutants showed a reproducible distribution shift toward hbsK41R mutant nucleoid distribution was significantly different from the less TOTO-3 staining intensity, signifying a lower average DNA wild type (P < 0.0001), as determined using the Kolmogorov–Smirnov test. content. The remaining Q mutants and all of the R mutants were nearly identical to wild type in their DNA content (SI Appendix, Fig. S4 B and C). the unacetylated and acetylated forms of HBsu. Using fluo- HBsu contains lysines distributed throughout the protein, and rescence microscopy, we show that R substitutions for key it is likely that some of these positively charged residues are HBsu lysines lead to a more compacted nucleoid structure. critical for binding the negatively charged DNA backbone. BLAST analysis identified more than 50 proteins encoded by B. Acetylation of lysine would neutralize the positive charge, and subtilis that contain a conserved GNAT domain and are would be predicted to decrease DNA binding affinity. Inspection therefore potential acetyltransferases. In a microscopic screen, of the HBsu structure, modeled using the structure from Bacillus the inactivation of five of these putative protein acetyl- stearothermophilus (SI Appendix, Fig. S5), suggests that at least transferases increased nucleoid compaction. Epistasis experi- K41, K80, K86, and perhaps K3 may directly contact the bound ments showed that specific K→Q mutations on HBsu partially DNA. In accordance with this, mutation of K80 or K86 to ala- bypassed the effects of inactivation of two of these, YfmK and nine resulted in a severe reduction in DNA binding (20), both which were identified as acetylation sites in vivo (15). To test the YdgE. Enzymatic acetyltransferase reactions with radiolabeled hypothesis that acetylation impairs DNA binding, we performed acetyl-CoA determined that purified YfmK acetylated HBsu DNA binding studies with acetylated and unacetylated mimic mu- in vitro, and that acetylation was most prominent in the C tants. We purified wild-type HBsu, and HBsuK41Q and HBsuK41R terminus of HBsu. YfmK therefore represents the second variants, and analyzed their DNA binding ability by electrophoretic protein acetyltransferase identified in B. subtilis. We propose mobility-shift assay (Fig. 2). Wild-type HBsu completely shifted that at least one biological role of HBsu acetylation is to reg- the probe with only 8 pmol of protein, as previously observed (20). ulate nucleoid compaction. More work is needed to elucidate if It should be noted that shifted smears were observed rather than these acetylations coordinate with other PTMs in a bacterial discrete bands, perhaps because HBsu binds nonspecifically. The “histone-like code.” HBsuK41R mutant shifted nearly all of the probe with only 2 pmol

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1815511116 Carabetta et al. Downloaded by guest on September 28, 2021 Wild type HBsuK41R HBsuK41Q mutants, with the exception of hbsK37Q, which did not display HBsu (pmol)- 2 4 6 8 24 6 8 2481216 nucleoid compaction (SI Appendix,Fig.S2A). Bypass was further confirmed with the measurement of nucleoid areas. As shown in Fig. 3A and SI Appendix,Fig.S7, the compaction phenotypes of both the yfmK and ydgE deletions are partially bypassed by the hbsK41Q allele (P < 0.0001). This partial bypass may be explained by the action of more than one acetyltransferase at K41 (Fig. 3B) or that YfmK and YdgE act at multiple sites. YfmK also acts at Free probe K3, K18, and K80, while YdgE may also act at K86 (Fig. 3B and SI Appendix,Figs.S7–S11). As expected, in no case did an R mutant affect the compaction due to eliminationofanacetyltransferase. Although these data are most simply explained by the direct action Fig. 2. HBsuK41Q leads to a decrease in DNA binding affinity. EMSAs were carried out as described in Materials and Methods. Purified HBsu, HBsuK41R, or of YfmK and YdgE on HBsu, it is conceivable that their effects on HBsuK41Q was incubated in the indicated amounts with 50 ng of probe DNA in compaction are indirect. We therefore tested whether YfmK is in- 10-μL reaction volumes. Bands were visualized by staining with SYBR Green. deed a protein acetyltransferase that can act specifically on HBsu.

YfmK Is an Ne-Lysine Protein Acetyltransferase. To investigate whether of protein, demonstrating stronger binding affinity than the wild YfmK and YdgE act as bona fide HBsu acetyltransferases, these type. Conversely, the HBsuK41Q showed a clear reduction in proteins, and HBsu, were cloned into expression vectors and puri- DNA binding affinity compared with the wild type, with the fied from E. coli (SI Appendix,Fig.S12). Acetyltransferase assays 14 unshifted probe mostly disappearing with the addition of 16 rather were carried out in the presence of C-labeled acetyl-CoA (Fig. than 8 pmol of protein. 4A). AcuA, the known B. subtilis acetyltransferase, and its primary substrate AcsA were included as a positive control for the reaction Identification of Uncharacterized Protein Acetyltransferases. We next (lane 2). In in vitro reactions with the AcsA substrate protein pre- examined if HBsu was acetylated via an enzymatic mechanism. sumed degradation products were frequently observed, suggesting it Although at present B. subtilis possesses only one characterized is unstable under these reaction conditions. AcuA did not acetylate protein GNAT, AcuA, a BLAST search against the B. subtilis HBsu (lane 7), as expected because HBsu lacks the AcuA motif proteome using the highly conserved GNAT domain of Saccha- (12). YdgE appeared to self-acetylate (lane 3) and did not acetylate romyces cerevisiae Gcn5p (residues 95 to 253) revealed more than HBsu to detectable levels in vitro (lane 8), so we did not study it MICROBIOLOGY 50 potential protein acetyltransferases (SI Appendix, Table S1). further. HBsu was acetylated in the presence of YfmK (compare Deletion mutants for the 50 cognate genes were obtained from the lanes 6 and 9), which did not acetylate itself (lane 4). YfmK point Bacillus Stock Genetic Center and confirmed by DNA sequencing. mutants predicted to eliminate or reduce acetyltransferase activity Each mutant was grown to exponential phase, and examined by (27) (SI Appendix,Fig.S13A) were constructed, and mutant pro- microscopy after DAPI staining. Five of the 50 mutants displayed teins (G88A, Y89A, F124A, and F129A) were tested for a nucleoid a nucleoid compaction phenotype, similar to those observed with compaction phenotype as described above. All four led to different HBsu R mutants (SI Appendix,Fig.S6). These five deletion alleles extents of nucleoid compaction. yfmKG88A was found to pheno- were transformed into the BD630 background for further study copy the yfmK mutant most closely in vivo (SI Appendix,Fig.S13B). (SI Appendix,TableS2). If one or more of these acetyltransferases Furthermore, hbsK41Q was able to bypass the yfmKG88A pheno- directly acetylates HBsu, in its absence HBsu may be relatively less type (SI Appendix,Fig.S14), suggesting that the cognate protein is acetylated, thus recapitulating an R mutant phenotype. If so, the catalytically inactive and that the wild-type enzyme acetylates K41 introduction of a Q mutation at a target site would be expected to in vivo. YfmKG88A and F124A, a less severely affected mutant bypass the effect of the acetyltransferase deletion, at least par- protein, were selected for in vitro acetyltransferase experiments. tially. All allelic pairwise combinations were tested visually for As expected, the acetylation of HBsu increased with increasing each of the five putative protein acetyltransferases and the hbs Q wild-type YfmK, while the two YfmK point mutants no longer

Fig. 3. hbsK41Q partially bypasses the yfmK nucleoid compaction phenotype. (A) After growth to exponential phase, wild-type (BD8012) cells were m2 m2 individually mixed with yfmK, yfmK hbsK41Q,or yfmK hbsK41R (BD8347) cells, and nucleoids were B stained with DAPI. Nucleoid areas of at least 4,500 cells for each strain were determined as described in Ma- terials and Methods. Cumulative distribution plots are displayed, where the 50th percentile represents the median of the population distribution. All distribu- tions were significantly different from wild type (P < 0.0001), as determined using the Kolmogorov– Smirnov test. (B) Summary of YdgE and YfmK putative interactions with HBsu, based on in vivo genetic bypass experiments (SI Appendix, Figs. S7–S11).

Carabetta et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 28, 2021 A B

Fig. 4. YfmK directly acetylates HBsu in vitro. (A)In vitro acetylation assays were performed as described in Materials and Methods. In a reaction mixture of 100 μL, 3 μg of AcuA, YdgE, and YfmK were incubated alone or with 5 μg HBsu or AcsA for 2 h at CD37 °C in the presence of [14C]acetyl-CoA. A mixture of AcuA and AcsA was included as a positive control. (B) YfmK was incubated with additional substrates, including BSA, a soluble domain of ComEC, AcsA, and the V. cholera protein CqsR. The catalytically inactive mutant YfmKG88A was included for comparison. (C) Relative quantification of HBsu lysine acetylation by tandem mass spectrometry following incubation with YfmK compared with control. *, not quantified. (D) Relative quantification of HBsu lysine acetylation by tandem mass spectrometry following incubation with YfmK for 1, 2, and 4 h compared with a time- matched control.

acetylated HBsu (Fig. 4B and SI Appendix,Fig.S13C). Next, K80, the acetylation level increased throughout the time course, the specificity of YfmK was explored (Fig. 4B). YfmK was perhaps indicating greater accessibility. We were able to estimate incubated with several lysine-rich proteins including BSA, acetylation stoichiometry for K80, because both the modified Vibrio cholera protein CqsR, a soluble domain of B. subtilis and unmodified tryptic peptide are represented by the same ComEC, and AcsA. YfmK failed to acetylate any of these sequence (VPAFKPGK). In the absence of enzyme, about 0.3% proteins, with the exception of AcsA. Although YfmK exhibits of purified HBsu was acetylated at K80, which is likely due to considerable specificity, it is possible that AcsA contains a site nonenzymatic acetylation (SI Appendix, Fig. S19), while, in the that resembles its target on HBsu. presence of YfmK, 4.1% of HBsu was acetylated, a 13.7-fold To identify and quantify the specific HBsu lysine residues increase. This measured stoichiometry is representative of levels acetylated by YfmK in vitro, HBsu was incubated with non- found endogenously in E. coli (28, 29). However, it is certainly radioactive acetyl-CoA in the presence or absence of YfmK and possible that additional cofactors or proteins may be involved then subjected to trypsin digestion followed by label-free quan- in vivo in modulating the catalytic efficiency and specificity of the titative mass spectrometry. The overall sequence coverage of reaction. Next, we performed an experiment to measure endoge- tryptic peptides was 90%, which allowed for the theoretical de- nous levels of HBsu acetylation from exponentially growing wild- tection of all lysine residues, except for K23, which was not found type and yfmK mutant cells using the mass spectrometry method to be acetylated in our previous unbiased acetylome analysis (15) of parallel reaction monitoring (PRM). PRM is a sensitive tar- (Fig. 1A). In vitro acetylation of HBsu was localized to seven geted mass spectrometry method for selective quantification of lysine residues by MS/MS fragmentation (SI Appendix, Fig. S15; proteins or peptides from a complex biological sample (30). Using representative spectra are shown in SI Appendix, Fig. S16), the this approach, we were able to detect three acetylation sites (K80, majority of which were also detected in vivo (Fig. 1A). Acetyla- K83, and K86), thereby confirming their presence on the endog- tion of the HBsu N-terminal tryptic peptide (containing K3) was enous protein. Of these, K80 acetylation was present at a sufficient detected, but it was ambiguous whether acetylation occurred on the level for its reliable quantification. We found that acetylation of NterminusoronK3(SI Appendix,Fig.S16), so reliable quantifi- K80 was reduced 15-fold in a yfmK mutant compared with wild cation could not be performed. The only previously identified type (SI Appendix,Fig.S20). Altogether, both the in vivo and in vivo site (Fig. 1A) that was not detected by tandem MS in this in vitro data strongly suggest that YfmK represents a protein analysis was K37, which does not appear to be important for HBsu- acetyltransferase in B. subtilis that acts directly on HBsu. dependent nucleoid compaction (SI Appendix,Fig.S2A). In- terestingly, all detected sites showed a YfmK-dependent increase Discussion in acetylation, ranging from 2- to 100-fold (Fig. 4C). It should be The HU family of NAPs have been suggested to be functional noted that while K75 acetylation could be detected, in this exper- homologs of histones, despite the absence of sequence or structural imental design, it could not be independently quantified, since K80 homology (31). Histone acetylation has a well-established role in and K75 are within the same tryptic peptide. Moreover, in an in- regulating DNA transactions including gene expression, replica- dependent experiment, incubation of HBsu with YfmKG88A tion, repair, and recombination (reviewed in refs. 32 and 33). confirmed the severely impaired enzymaticactivityofthismutant, Previously, we discovered that the essential, histone-like protein resulting in a decrease in acetylation at all sites to levels similar to HBsu contains seven novel acetylation sites in vivo (3). Interest- the no-enzyme control (SI Appendix,Fig.S17). ingly, HBsu was also reported to contain phosphorylated threonine We also performed a time course acetylation reaction in the (T4) and arginine (R61) residues (34, 35). It is tempting to spec- presence or absence of YfmK. For most sites, under these ex- ulate that these modifications are part of a bacterial histone-like perimental conditions, acetylation levels were nearly constant code. For these modifications to play a regulatory role, they must after an hour (Fig. 4D and SI Appendix, Fig. S18). However, for have biological consequences. Here, we used substitutions that

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1815511116 Carabetta et al. Downloaded by guest on September 28, 2021 mimic the acetylated (Q) and unacetylated (R) states to probe the can act in vitro to acetylate most of the known in vivo sites in physiological consequences of acetylation. These substitutions are HBsu, it seems to have preferential activity, namely greater ac- frequently employed to study acetylation (3, 22–26), although they cessibility to the C-terminal residues (Fig. 4C and SI Appendix, are chemically distinct from the acetylated and unacetylated side Fig. S18). This result seems partially incongruent with the in vivo chains. Thus, although these substitutions provide a useful tool, genetic bypass experiments, which suggested YfmK acetylation of results from their use must ultimately be corroborated using ad- K3, K18, and K41, in addition to K80, are functionally relevant. ditional approaches, such as phenotypic analysis of acetyltransfer- One possible explanation is that YfmK has limited access to some ase mutants, as in the present study. of the C-terminal lysine residues in vivo because they are steri- We have provided evidence that one role for the acetylation of cally blocked by DNA binding. Alternatively, other nearby PTMs HBsu is to regulate nucleoid compaction and hence the orga- (succinylation, phosphorylation, etc.) may be present on HBsu nization of the B. subtilis chromosome (Fig. 1 and SI Appendix, that influence YfmK’s ability to access the other C-terminal resi- Figs. S2 and S3). As acetylation of lysine residues neutralizes its dues. Additional studies are required to understand the regulation positive charge, and because many of these residues on HBsu are of yfmK itself and to identify other substrates of YfmK. It is cur- located in regions that directly contact DNA (SI Appendix, Fig. rently unknown if HBsu acetylation is reversible and if AcuC, S5), it is likely that the acetylation of key lysine residues weakens SrtN, or an unknown deacetylase is responsible. DNA binding affinity. In agreement, we showed that the acetylation mimic mutant HBsuK41Q led to a decrease in DNA Materials and Methods binding (Fig. 2). These data, combined with the nucleoid com- Bacterial Strains, Media, and Growth Conditions. All bacterial strains were paction phenotype of the hbs R and acetyltransferase deletion constructed in the BD630 background (his leu met) and are listed in SI Ap- mutants, suggest that a possible role of acetylation is to decon- pendix, Table S2. Plasmid construction for allelic replacement using the dense the chromosome, either locally or globally. Interestingly, pMiniMAD2 strategy (51) is described in SI Appendix. Deletion alleles of the Q and R mutants of HBsu had other observable phenotypes: putative acetyltransferases were acquired from the Bacillus Genetic Stock hbsK3Q, K41Q, and K86Q may cause a reduction in DNA con- Center, and were confirmed by DNA sequencing performed by Eton Biosci- tent, suggesting a role for acetylation in DNA replication or in ences. All B. subtilis strains were constructed by transformation (52). Minimal the coordination of DNA replication and cell division (SI Ap- glucose liquid and solid media were prepared as described previously (53), with the addition of 50 μg/mL each of histidine, leucine, and methionine, or pendix hbsK3Q K80Q , Fig. S4), and and strains make an un- tryptophan when appropriate. Bacteria were grown at 37 °C with aeration, identified yellow pigment when growing in minimal media, and growth was monitored in a Klett colorimeter. Antibiotics were added as suggesting a change in gene expression. Although these changes appropriate, and used at the following concentrations: 5 μg/mL erythro-

may be indirect consequences of altered compaction, their site mycin, 25 μg/mL tetracycline, 5 μg/mL kanamycin, 100 μg/mL spectinomycin, MICROBIOLOGY specificity suggests additional complexity. and 100 μg/mL ampicillin. HU family members are acetylated in diverse bacteria (36–49). Some specific acetylation events were even conserved between B. Quantitative Microscopy Data Analysis. Cells were grown, harvested, and subtilis and distant bacteria. For example, K3 is also acetylated in prepared for microscopy as described in SI Appendix. Nucleoid length, width, Mycobacterium tuberculosis, Geobacillus kaustophilus, and Pseu- and area were measured using the NIS-Elements AR (version 4.40; Nikon) domonas aeruginosa, and K18 is acetylated in E. coli HUβ and G. automated General Analysis feature. Thresholds were set up to identify the kaustophilus. Recently, in M. tuberculosis, it was demonstrated GFP-expressing cells and the nucleoids of each cell, which were applied to that acetylated mtHU reduced its interaction with DNA and each image separately. Using the Combine feature, the program automati- altered its DNA compaction properties (50). An acetyltransfer- cally sorted the nucleoids based on GFP expression. The program then ase was identified that was able to acetylate mtHU in vitro. measured the nucleoid length, width, and area. At least 4,500 cells per strain Additionally, in Acinetobacter baumannii, acetylation of K13, were counted, collected from three independent experiments. To determine which was conserved among some bacteria, alters the thermo- boundaries for length and width of the nucleoids of exponentially growing stability and DNA binding kinetics of the protein (49). Together, cells, 100 nucleoids were manually measured. The data generated were these data suggest that acetylation of key residues in HU family exported to Microsoft Excel for further processing. For each strain, lengths and widths were manually inspected, and sizes outside of 0.25 to 1.5 μmfor proteins may be an evolutionarily conserved mechanism. widths or 0.5 to 3.2 μm for lengths were removed. For each strain, cumu- In B. subtilis, AcuA is the only known protein acetyltransferase lative distribution plots were made by sequentially organizing the measured (12), which inactivates AcsA at a conserved active-site lysine. As nucleoid areas from low to high, and plotting those values against their expected, AcuA did not acetylate HBsu, which does not contain cumulative fraction. The 50th percentile represents the median of the the AcsA active-site motif (Fig. 4). We have now identified five population. The Kolmogorov–Smirnov test (54) was performed using JMP potential novel acetyltransferases that may directly acetylate HBsu Pro 13 (SAS Institute). (SI Appendix,Fig.S6). Using genetic bypass experiments, we found suggestive evidence that YfmK acetylates HBsu at K3, K18, K41, Electrophoretic Mobility-Shift Assays. EMSAs were carried out as described and K80 in vivo, while YdgE may acetylate HBsu at K41 and K86 previously (55), with modifications. A 500-bp fragment was amplified from (Fig. 3 and SI Appendix,Figs.S7–S11). When characterizing the the B. subtilis chromosome using the primers RicAfor and RicArev (SI Ap- Bacillus acetylome, we found that negatively charged residues were pendix, Table S3). Increasing amounts of HBsu (2 to 8 pmol of wild type and enriched in the immediate area surrounding an acetylation site, HBsuK41R, and 2 to 16 pmol of HBsuK41Q) were incubated with 50 ng of while positively charged residues were statistically underrepre- probe in a 10-μL reaction containing binding buffer (50 mM Tris, pH 8.0, sented (15). In contrast, most of the lysine acetylation sites in HBsu 1 mM EDTA, 50 mM NaCl) for 10 min at 37 °C. Following incubation, 0.1 contain positively charged residues in their neighborhoods. We volume of sample buffer (10 mM Tris, pH 7.5, 30% glycerol, 0.1% bromo- suspect that the strong enrichment for aspartate and glutamate phenol blue) was added to each reaction, and half of each sample was × residues proximal to many acetylation sites is part of an auto- loaded on a vertical, 4.5% polyacrylamide gel in 0.5 TBE (Tris-borate-EDTA). catalytic mechanism of lysine acetylation, while HBsu, which is After separation for 1 h at 10 mA, the gels were removed and stained with SYBR Green (EMSA Kit; Thermo Fisher Scientific), as per the manufacturer’s rich in basic amino acids, is a substrate for enzymatic acetylation, instructions. Bands were visualized with a ChemiDoc MP imager (Bio-Rad). as is AcsA. YfmK directly acetylates HBsu and AcsA in vitro (Fig. 4 and In Vitro Acetylation Assays. HBsu, YdgE, YfmK, AcuA, and AcsA were purified SI Appendix, Fig. S13C) and, in the absence of yfmK, at least one as described in SI Appendix. In vitro acetylation assays were carried out as in vivo site of acetylation, HBsu K80, is reduced (SI Appendix, described (13), with minor modifications as described in SI Appendix. Briefly, Fig. S20). Although it is a small protein that largely consists of a 3 μg of enzyme (AcuA, YdgE, or YfmK) was incubated with 5 μg of substrate single GNAT domain (SI Appendix, Fig. S13), YfmK possesses (AcsA or HBsu) and 25 μM[1-14C]acetyl-CoA (specific activity, 47 mCi/mmol; substrate specificity, as it was not capable of acetylating BSA, Moravek Biochemicals) for 2 h at 37 °C. Proteins were precipitated and re- ComEC, or the V. cholera protein CqsR, all of which have solved by SDS/PAGE. Radiolabeled bands were detected using a Typhoon multiple K residues (Fig. 4B).WhileweconfirmedthatYfmK FLA 7000 (GE Healthcare) phosphoimager.

Carabetta et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 28, 2021 Liquid Chromatography-Tandem Mass Spectrometry. Nanoliquid chromatography- making strains BD8012 and BD8822, respectively. We thank Felix Adusei- tandem mass spectrometry was used for quantification of HBsu acetylation from Danso for purifying AcuA, Matthew Neiditch for providing Ulp1, and (i) in vitro acetyltransferase reactions using MS1-based quantification, and (ii) Evan Waldron and Atul Khataokar for guidance and assistance with purify- wild-type and yfmK deletion strains using PRM-based quantification, as described ing other proteins. We thank Atul Khataokar for generating the HBsu in SI Appendix. structural model. We thank Andrew Tanner for statistical discussions and guidance. We thank Riccardo Arrigucci for assistance with flow cytometry. ACKNOWLEDGMENTS. We thank Jorge Escalante for providing strains and This work was supported by Grant GM43756 (to D.D.), GM114141 purified AcsA. We thank Christine Diethmaier and Jeanie Dubnau for (to I.M.C.), and startup support from Rowan University (to V.J.C.).

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