genetic evidence for the importance of protein acetylation...

JOURNAL OF BACTERIOLOGY, Mar. 2009, p. 1610–1617 Vol. 191, No. 50021-9193/09/$08.00�0 doi:10.1128/JB.01252-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Genetic Evidence for the Importance of Protein Acetylation andProtein Deacetylation in the Halophilic Archaeon Haloferax volcanii�†

Neta Altman-Price and Moshe Mevarech*Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences,

Tel Aviv University, Tel Aviv 69978, Israel

Received 8 September 2008/Accepted 16 December 2008

Protein acetylation and deacetylation reactions are involved in many regulatory processes in eukaryotes.Recently, it was found that similar processes occur in bacteria and archaea. Sequence analysis of the genomeof the haloarchaeon Haloferax volcanii led to the identification of three putative protein acetyltransferasesbelonging to the Gcn5 family, Pat1, Pat2, and Elp3, and two deacetylases, Sir2 and HdaI. Intriguingly, the genethat encodes HdaI shares an operon with an archaeal histone homolog. We performed gene knockouts todetermine whether the genes encoding these putative acetyltransferases and deacetylases are essential. A sir2deletion mutant was able to grow normally, whereas an hdaI deletion mutant was nonviable. The latter isconsistent with the finding that trichostatin A, a specific inhibitor of HdaI, inhibits cell growth in a concen-tration-dependent manner. We also showed that each of the acetyltransferases by itself is dispensable forgrowth but that deletion of both pat2 and elp3 could not be achieved. The corresponding genes are therefore“synthetic lethals,” and the protein acetyltransferases probably have a common and essential substrate.

Protein acetylation is one of several classes of posttranslationalregulatory processes that occur in living cells. In contrast to thoseof other protein modifications, such as phosphorylation, whichhas been studied for over 50 years, the role of protein acetylationin cellular events is less well understood (23, 35). Two types ofprotein acetylation take place in the cell. N acetylation is theacetylation of the amino termini of newly synthesized proteinsthat mostly follows the removal of the first methionine residue.This type of acetylation is common in eukaryotes but also occursto some extent in archaea (1, 14). The second type of acetylationis a reversible modification that occurs on lysine residues of ma-ture proteins, resulting in charge neutralization of these residues.The latter type of protein acetylation is catalyzed by a family ofhistone acetyltransferases (HATs) that function to transfer anacetyl group from acetyl-coenzyme A (acetyl-CoA) to theε-amino group of certain lysine side chains. The HAT family iscomposed of five enzyme subfamilies, of which the Gcn5-relatedN-acetyltransferase (GNAT) subfamily is the best characterized(reviewed in references 11, 17, 26, 29, and 46). Acetylation reac-tions catalyzed by HATs can be reversed by a family of histonedeacetylases (HDACs). HDACs are divided into three classes:class I HDACs are related to yeast Rpd3, class II HDACs arerelated to yeast HdaI, and class III HDACs are related to theyeast transcriptional repressor Sir2. Class I and class II HDACsshare some homology in their catalytic domains and hydrolyze theacetamide bond in similar manners, whereas class III HDACsshare no homology with class I and II HDACs and employ adifferent enzymatic mechanism (reviewed in references 7, 10, 20,39, and 47).

The reversible posttranslational protein acetylation is notrestricted to eukaryotes and has been demonstrated to takeplace in bacteria and archaea. A metabolic role for the Sir2homolog CobB was reported in the bacterium Salmonellaenterica. It was shown that CobB activates the acetyl-CoAsynthetase (ACS) via deacetylation (42), while Pat, an acetyl-transferase that exhibits homology in its C-terminal 95-amino-acid-residue region to the eukaryotic Gcn5 acetyltransferases,deactivates it (43). Recent studies of the archaeon Sulfolobussolfataricus revealed that the archaeal homolog of Sir2 forms astable complex with Alba, one of the most abundant archaealchromatin proteins. In vitro, Sir2 is responsible for deacetyla-tion of Alba, causing an increase in its affinity to DNA andthereby repressing transcription (5, 57). Subsequent studiesshowed that Alba acetylation is carried out by a homolog of S.enterica Pat (31). Sir2 homologs are not the only HDACs inarchaea and are not even the most common. All archaea (withthe exception of Nanoarchaeum equitans) possess homologs ofHdaI. However, no genetic analysis has been performed toestablish the significance of HDACs and HATs in archaea.

Haloferax volcanii is an obligate halophilic and aerobic ar-chaeon of the euryarchaeota lineage. It has become a modelorganism for molecular genetic studies of archaea due to thewide range of available genetic tools (2). Also, the completegenome nucleotide sequence of the organism was recently de-termined and annotated by The Institute for Genomic Re-search. In this communication, we present bioinformatic evi-dence for the existence in H. volcanii of genes encoding thereversible protein acetylation/deacetylation reactions and pro-vide genetic evidence for their essentiality.

MATERIALS AND METHODS

Strains and culture conditions. The properties of the various H. volcaniistrains used in this work are given in Table 1.

H. volcanii was routinely grown in rich (HY) medium containing (per liter):150 g of NaCl, 36.9 g of MgSO4 · 7H2O, 5 ml of a 1 M KCl solution, 1.8 ml ofa 75-mg/liter MnCl2 solution, 5 g yeast extract (Difco), and Tris-HCl (pH 7.2) at

* Corresponding author. Mailing address: Department of MolecularMicrobiology and Biotechnology, George S. Wise Faculty of Life Sci-ences, Tel Aviv University, Tel Aviv 69978, Israel. Phone: 972-3-6408715. Fax: 972-3-6409407. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 29 December 2008.

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a final concentration of 50 mM. After autoclaving and cooling of the medium, 5ml of 10% (wt/vol) CaCl2 was added. Agar plates contained 18 g of Bacto Agar(Difco) per liter. Casamino Acids (CA) medium contained the same componentsas the HY medium except that the yeast extract was replaced by 5 g/liter of CA(Difco). Minimal medium contained (per liter) 150 g of NaCl, 36.9 g of MgSO4

· 7H2O, 5 ml of a 1 M KCl solution, 50 ml of 1 M NH4Cl, 45 ml of 10% (vol/vol)glycerol, 5 ml of 10% (wt/vol) sodium succinate, 2 ml of 0.5 M K2HPO4, andTris-HCl (pH 7.2) at a final concentration of 50 mM. After autoclaving andcooling of the medium, the following materials were added: 5 ml of 10% (wt/vol)CaCl2, 1 ml trace element solution, 0.8 ml of 1-mg/ml thiamine, and 0.1 ml of1-mg/ml biotin.

For counterselection for uracil auxotrophs, 5-fluoroorotic acid (U.S. Biologi-cal) was added to the medium at a final concentration of 100 �g/ml. Whenneeded, novobiocin (Sigma-Aldrich) was added to the medium at a final con-centration of 2 �g/ml. When required, thymidine was added to a final concen-tration of 40 �g/ml, and leucine, tryptophan, and uracil were added to a finalconcentration of 50 �g/ml. Trichostatin A (TSA) (Sigma-Aldrich) was added tothe growth media at the concentrations indicated in the text.

Gene knockouts and gene replacements. Gene knockouts and gene replace-ments were performed according to the “pop-in pop-out” methodology as de-scribed previously (3, 6). In this methodology, the upstream and downstreamflanking regions of the genes to be deleted are PCR amplified and clonedtogether into the “suicide plasmid” pGB70 or pTA131, which carries the pyrEselectable genetic marker but cannot replicate autonomously in H. volcanii. Theplasmids are transformed into an H. volcanii �pyrE mutant, and transformants inwhich the plasmids have been integrated into the chromosome are selected onplates that lack uracil. Upon counterselection on plates containing uracil and5-fluoroorotic acid, the only cells that survive are those in which the integratedplasmids have been excised by spontaneous intrachromosomal homologous re-combination, either restoring the wild-type gene or resulting in its deletion. Genereplacements were performed according to the method of Allers et al. (3).

HY medium was used as a thymidine-minus medium for hdrB cassette selec-tion. CA medium was used as a uracil- and tryptophan-minus medium for trpAcassette selection. Minimal medium was used as a leucine-minus medium forleuB cassette selection.

The pop-out strains were screened using pairs of external “short up” and“short down” primers located approximately 100 bp upstream and 100 bp down-stream of the entire flanking construct. All the deletions were also verified by theinability to PCR amplify the coding region of the deleted genes in mutantsrunning in parallel control reactions with the “wild-type” strains.

A list of all integrative plasmids, shuttle vectors, and other vectors that wereused for this study is given in Table 2. A list of all primers used in this study isgiven in Table S1 in the supplemental material.

Transformation procedures. Transformation of H. volcanii was carried outusing the polyethylene glycol method as described previously (6).

Determination of growth rates and TSA inhibition. To determine growthrates, cells were grown in HY medium at 42°C to the stationary phase, diluted(1:50), grown to an optical density at 600 nm (OD600) of 0.6 to 0.7, and thendiluted again to an OD600 of 0.05 in fresh medium. When the effect of TSA onthe growth rate was determined, the fresh medium was supplemented with theindicated amount of TSA. OD600 measurements were taken at 2- to 4-h intervalsfollowing an overnight lag phase.

Genomic-data analyses. H. volcanii genome sequence data were obtained fromThe UCSC Genome Browser (http://archaea.ucsc.edu/cgi-bin/hgGateway?db�haloVolc1.) Multiple-alignment analysis was performed using Multalin software(12; http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html). Multiple-alignment figures were created using BoxShade 3.21 software (http://www.ch.embnet.org/software/BOX_form.html).

RESULTS

H. volcanii contains genes predicted to encode putative pro-tein acetylases and deacetylases. To identify putative compo-nents of the H. volcanii posttranslational protein acetylation/deacetylation machinery, its genome database (see Materialsand Methods) was screened using sequences of known HATsand HDACs as BLAST queries. Several putative open readingframes that showed significant homology to known HATs andHDACs were identified. The H. volcanii genome was found tocontain genes belonging to two HDAC families: one gene is ahomolog of the Sir2 family (HVO_2194) (40% identity and57% similarly to S. solfataricus Sir2), and another is a homologof the HdaI family (HVO_0522) (37% identity and 55% sim-ilarity to Saccharomyces cerevisiae HdaI). Figures 1 and 2present alignments of the yeast and archaeal Sir2 homologsand the yeast and archaeal HdaI homologs, respectively, andshow that the proteins are closely related and conserve impor-tant functional domains.

The hdaI gene encoding HdaI occurs in an operon contain-ing a histone gene (Fig. 3). The putative histone is predicted tohave a tandem H3-H4 core domain similar to that present inthe Methanopyrus kandleri HMk histone, which specifically

TABLE 1. Strains used in this study

Strain Description Derivation orreference

H133 DS70 �pyrE2 �trpA �leuB �hdrB 3WR580 H133 �sir2 This studyWR643 H133 �pat1::trpA This studyWR644 H133 �pat2::hdrB This studyWR645 H133 �pat2::hdrB �pat1::trpA This studyWR660 H133 �elp3::leuB This studyWR669 H133 sir2::pMM915 This studyWR671 H133 hdaI::pMM1028 This studyWR709 H133 �pat1::trpA �elp3::leuB This studyWR718 H133 �hdaI 71-920 (pMM1142) This study

TABLE 2. Plasmids used in this study

Plasmid Relevant properties Source orreference

pGB70 pUC19 containing the H. volcanii pyrE2 gene 6pTA105 pUC19 containing the leuB cassette (H. volcanii leuB gene expressed from the ferredoxin promoter) Thorsten AllerspTA106 pUC19 containing the trpA cassette (H. volcanii trpA gene expressed from the ferredoxin promoter) Thorsten AllerspTA131 pBluescript II containing the H. volcanii pyrE2 gene 3pTA187 pUC19 containing the hdrB cassette (H. volcanii hdrB gene expressed from the ferredoxin promoter) Thorsten AllerspMM915 H. volcanii sir2-flanking regions cloned into pTA131 This studypMM956 trpA cassette cut from pTA106 and cloned into pMM951 between the pat1-flanking regions This studypMM1028 Plasmid designed to allow the introduction of an in-frame deletion of nucleotides 71–920 of H.

volcanii hdaIThis study

pMM1033 hdrB cassette cut from pTA187 and cloned into pMM961 between the pat2-flanking regions This studypMM1108 leuB cassette cut from pTA105 and cloned into pMM952 between the elp3-flanking regions This studypMM1142 H. volcanii hdaI gene under the control of the tryptophanase promoter; cloned between the NdeI

and EcoRI sites28

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binds DNA (13, 33, 41). Interestingly, hdaI overlaps with theCCA-adding tRNA nucleotidyltransferase gene, which is onthe opposite DNA strand. The same genome arrangement isfound in other halophilic archaea whose genomes have beensequenced but not in any other archaea.

Three homologs of known HATs were identified in the H.volcanii genome. The first two, Pat1 (HVO_1756) and Pat2(HVO_1821), belong to the Gcn5 family. They are related to S.solfataricus Pat, and the C-terminal 95-amino-acid regionshares similarity with the S. enterica enzyme (26% identity and45% similarity to S. solfataricus Pat and 34% identity and 45%similarity to the C terminus of S. enterica Pat, respectively)(Fig. 4). The third HAT (HVO_2888) is a homolog of yeastElp3, which also belongs to the Gcn5 family (Fig. 5) (37%identity and 58% similarity to S. cerevisiae Elp3). Elp3 is asubunit of the “elongator” complex possessing acetyltrans-ferase activity (51, 53).

The H. volcanii sir2 homolog is dispensable, but hdaI isessential. To determine the essentiality of the two HDACs, weemployed the “pop-in pop-out” strategy for constructing geneknockouts previously developed for H. volcanii (3, 6) (see Ma-

terials and Methods). In this procedure, if the deletion of thetarget gene has no effect on the growth properties of the cells,it is expected that in about half of the cells excision of thechromosomally integrated plasmid leaves behind the wild-typeallele of the target gene and in about half of the cells theexcision creates the desired deletion. The sir2 genomic dele-tion plasmid pMM915 was transformed into H. volcanii strainH133 and integrated into its chromosome to create the sir2“pop-in” strain WR669. Following “pop-out” counterselection,it was found that in about half of the cells in which the pyrE-containing plasmid was excised, deletion of sir2 had occurred(to give strain WR580), as determined by PCR analysis (seeFig. S1 in the supplemental material). The H. volcanii sir2homolog is therefore not essential. The growth rate of themutant strain in rich HY medium was similar to that of theparental strain, H133, grown under the same conditions. Un-like the S. enterica cobB knockout mutant (45, 48), the H.volcanii sir2 deletion mutant grew normally on low concentra-tions of acetate as the sole energy source. The H. volcanii �sir2mutant also showed no apparent growth impairment comparedto the wild-type strain when cultured at salt concentrations

FIG. 1. Alignment of the Sir2 homologs of different organisms versus the Sir2 homolog of H. volcanii. Several amino acid residues that wereproven to be essential for deacetylation activity are marked by gray arrows. Highly conserved amino acids that interact with NAD� are markedwith black arrows (4). The CXXC(15–20X)CXXC zinc finger motif is marked by black lines. Black and gray shading represent amino acid identityand similarity, respectively. A. fulgidus, Archaeoglobus fulgidus.

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ranging from 4 M to 1.5 M NaCl and at temperatures rangingfrom 37°C to 45°C (data not shown).

The same “pop-in pop-out” strategy was employed in at-tempts to inactivate hdaI. Since the hdaI gene partially over-laps the essential CCA-adding transferase gene that is in-

volved in tRNA maturation (Fig. 3), attempts were made todelete nucleotides 71 to 920 of the nonoverlapping region ofthe hdaI gene. Plasmid pMM1028 was used to create the “pop-in”strain WR671. However, no hdaI deletions were obtained fol-lowing the “pop-out” counterselection, suggesting that hdaI isessential.

To confirm that hdaI is essential, a plasmid carrying thecomplete hdaI gene was cloned into the pRV1-ptna-bgaHplasmid (28) between the NdeI and EcoRI sites, placing thehdaI gene under the control of the tryptophanase promoter(pMM1142). Plasmid pMM1142 transformed into the “pop-in” strain, WR671. In this genetic background, it was pos-sible to knock out the chromosomal gene and create strainWR718 (see Fig. S2 in the supplemental material). Thenovobiocin resistance gene present on the pMM1142 plas-mid was used to confirm the presence of the plasmid in the

FIG. 2. HdaI alignment. A partial alignment of the halophilic H. volcanii HdaI and H. marismortui HdaI versus the amino-terminal part of theS. cerevisiae HdaI and the Homo sapiens HDAC1. Known conserved domains are marked by black lines. Amino acid residues that were provento be essential for deacetylation activity are marked by black arrows. Black and gray shading represent amino acid identity and similarity,respectively.

FIG. 3. Halophilic hdaI operon. The halophilic hdaI genes share anoperon with the archaeal “double-histone” homolog. The hdaI startcodon overlaps with the histone stop codon. The 3� region of the HdaIopen reading frame overlaps the 3� region of the essential gene thatencodes CCA tRNA nucleotidyltransferase, which is transcribed onthe opposite DNA strand.



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cell, together with the genomic knockout background. Otherevidence to support the essentiality of hdaI was obtainedfrom the effect of the HdaI-specific inhibitor TSA (16, 50,55) on the growth rate of H. volcanii. Figure 6 shows thatTSA inhibits the growth of H. volcanii in a concentration-dependent manner.

A pat1 and pat2 double-knockout strain is viable. H. volcaniicontains genes coding for two homologs of the S. solfataricusPat. Plasmid pMM956 was designed to allow replacement ofpat1 by the trpA cassette, and pMM1033 was designed to allowreplacement of pat2 by the hdrB cassette (see Fig. S3 in thesupplemental material). The growth rates in rich HY media ofthe two single-deletion mutants (WR643 for pat1 replacementwith the trpA cassette and WR644 for pat2 replacement withthe hdrB cassette) and the double-deletion mutant (WR645)were comparable to that of the parental strain, H133 (data notshown).

The elp3 knockout is viable, but elp3 and pat2 are “syntheticlethals.” The elp3 gene was replaced by the leuB selectablemarker using the pMM1108 plasmid. It was possible to createan elp3 knockout in the wild-type background (WR660), aswell as in the background of the �pat1 strain (WR709). How-

ever, attempts to create an elp3 null strain in the �pat2 single-mutant or the �pat1 �pat2 double-mutant strain failed. Theseresults imply that elp3 and pat2 are “synthetic lethals,” namely,their products may share the same target(s).

Acetyltransferase knockout strains show no resistance toTSA. The growth rates of all acetyltransferase knockout strains(�pat1, �pat2, �pat1 �pat2, �elp3, and �pat1 �elp3) in rich HYmedium were found to be comparable to those of the parentalH133 strain (data not shown). We also examined the growthproperties of the mutants in rich media containing TSA. How-ever, none of the single-mutant strains were resistant to TSA.Similarly, attempts to knock out hdaI in strains carrying thevarious acetyltransferase gene knockouts also failed.

DISCUSSION

Posttranslational protein modification plays a key role inmany cellular processes. The natures of these modificationscannot, in most cases, be deduced from genomic informationand have to be determined by elaborate procedures. Whilearchaeal genomic data are accumulating rapidly with innova-tions in genome-sequencing techniques, progress regarding ar-

FIG. 4. Pat alignment. The S. solfataricus Pat and the carboxy-terminal 95 amino acid residues of S. enterica Pat were aligned against H. volcaniiPat1 and Pat2. Motifs A and B of the Gcn5 family are marked by black lines (32, 46), with the partial sequence of S. cerevisiae Gcn5. The highlyconserved R/QXXGXG/A motif, which is important for acetyl-CoA recognition and binding (38, 54), is marked by arrows. Black and gray shadingrepresent amino acid identity and similarity, respectively.

FIG. 5. Alignment of archaeal and S. cerevisiae Elp3 sequences. Motifs A and B of the Gcn5 family are marked with black lines. The highlyconserved R/QXXGXG/A motif is marked by black arrows. Two tyrosine residues that were shown to be highly important for Elp3 acetylationactivity are marked by gray arrows. HV, HL, HM, MB, MM, MK, and SC stand for H. volcanii, Halorubrum lacusprofundi, H. marismortui,Methanosarcina barkeri, Methanosarcina mazei, M. kandleri, and S. cerevisiae, respectively. Black and gray shading represent amino acid identity andsimilarity, respectively.



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chaeal protein modification is much slower. The present studydescribes a genetic approach to explore the potential of pro-tein acetylation and deacetylation in H. volcanii by identifyingthe putative protein acetylase and protein deacetylase genes,followed by attempts to delete these genes. The results of thisstudy are summarized in Table 3.

The plethora of acetyltransferases and deacetylases in yeastsis not observed in archaea in general and in H. volcanii inparticular. Unlike many organisms (8, 9), H. volcanii (andother archaea [27, 30]) contains only one putative Sir2 ho-molog. The second H. volcanii histone deacetylase is an HdaIhomolog. Interestingly, Sir2 is widely distributed among bac-teria and eukarya (9, 15), whereas in archaea, HdaI is morecommon. In fact, HdaI is present in all archaeal orders and ismissing only from the Nanoarchaeota genome. Similarly, elp3 iswidely distributed and highly conserved among all archaealgenomes (37, 49).

S. cerevisiae sir2 knockout mutants, as well as those in othereukaryotes, display a wide range of phenotypes, whereas thesingle H. volcanii sir2 knockout mutant had no recognizablephenotype. In contrast, the yeast hdaI knockout mutant had amild phenotype and the hdaI rpd3 double mutant was viable(39). Since addition of TSA to the culture medium causes asevere defect in the growth of H. volcanii and deletion of thehdaI gene is lethal, it seems likely that HdaI is the main proteindeacetylase in H. volcanii.

Single null mutations of S. cerevisiae gcn5 or elp3, homologsof H. volcanii pat2 and elp3, are viable, and the growth prop-erties of the single-mutant strains are comparable to those ofthe wild-type strain under most conditions (53). However, S.cerevisiae elp3 and gcn5 double mutants, though viable, havemore severe growth defects. These defects can be partly re-lieved by an accompanying hdaI deletion (52).

Similarly, single-deletion mutations in each of the three pu-tative H. volcanii acetyltransferase genes had no discernibleeffect on growth or on TSA resistance. Nevertheless, it was notpossible to delete both pat2 and elp3. These results imply thatPat2 and Elp3 share a substrate whose acetylation is essentialand that this substrate cannot be acetylated by Pat1. The factthat hdaI is also essential indicates that indiscriminate acety-lation of lysine residues is harmful and necessitates selectiveremoval by protein deacetylases. So far, we have been unable

to create a strain in which pat2, elp3, and hdaI are all deletedin analogy to the situation described above for S. cerevisiae.

Given that the acetylation/deacetylation machinery is an es-sential cellular process, we can address the issue of the natureof the possible protein targets at which it acts. The archaealchromatin protein Alba was shown to undergo acetylation/deacetylation (31). Alba is found in many archaea but is miss-ing in all halophilic archaea (40, 49). ACS was recently shownin S. enterica to be acetylated on residue K609 by Pat anddeacetylated by CobB (a Sir2 homolog). Residue L641 wasfound to be important for enzymatic acetylation (44). H. vol-canii has four ACS homologs. Two ACS genes (HVO_1585and HVO_0894) are on the chromosome, while the other twogenes (HVO_A0158 and HVO_A0156) are on an extrachro-mosomal megaplasmid (pHV4). The four enzymes show con-siderable similarity to the bacterial ACS, and their acetylationsites are also well preserved. Many other archaea have ACShomologs and have preserved the acetylation site (some exam-ples are given in Fig. 7). Nevertheless, the H. volcanii sir2deletion mutant displays no phenotypic growth impairmentsand, unlike the cobB mutant, can grow on minimal media usinglow concentrations of acetate as the sole carbon source. Theonly haloarchaeal protein known so far to be acetylated in vivois the 2Fe-2S ferredoxin. The amino acid sequences of theHalobacterium salinarum (18) and Haloarcula marismortui (19)ferredoxins were determined and shown to contain a uniqueacetylated lysine close to their carboxyl termini. The conserva-tion of the acetylated lysines in the two distantly related halo-philic archaea might indicate their functional significance.

Among the eukaryotes, the best-studied protein acetylationprocess is that of the amino-terminal histone tails. Their acet-ylation, and other posttranslational modifications, plays an im-portant role in the regulation of gene expression (some modelsfor regulation are reviewed in references 22, 24, 25, 34, and36). H. volcanii histone lacks the tail extension and consists

FIG. 6. Growth curve of the H. volcanii wild-type (wt) strain in thepresence of TSA. H. volcanii was grown on rich HY medium withoutTSA or at different concentrations of TSA, as indicated. The cultureturbidity was measured as the OD600.

TABLE 3. Summary of knockouts of H. volcanii acetylation-deacetylation genes

Gene Status Source

Single knockoutsir2 Viable No selectable marker was

usedhdaI Essential Knockout was obtained only

in the presence of anextrachromosomal copy

pat1 Viable Knockout was obtainedusing a selectable marker

pat2 Viable Knockout was obtainedusing a selectable marker

elp3 Viable Knockout was obtainedusing a selectable marker

Double knockoutpat1 pat2 Viable Knockout was obtained

using a selectable markerfor both genes

pat1 elp3 Viable Knockout was obtainedusing a selectable markerfor both genes

pat2 elp3 Synthetic lethal Knockout could not beobtained



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only of sequences homologous to the core sequence of theeukaryotic H3-H4 histones. Evidently, the potential target ofthe acetylation-deacetylation machinery in H. volcanii cannotbe the histone tail. While acetylation of the histone tail is welldocumented, evidence regarding histone core modifications isgradually accumulating. Recently, a core acetylation modifica-tion was observed in Lys79 of histone H4 in yeasts (21, 56).This lysine residue is conserved in the core H3-H4 histone ofmost archaea and also in H. volcanii histone. This conservation,along with the physical location of the halophilic histone genein an operon with the essential HdaI deacetylase gene, suggeststhat the histone is a possible target for acetylation.

ACKNOWLEDGMENTS

This work was supported by a grant from the Israel Science Foun-dation (ISF-998-07).

We thank Thorsten Allers from the University of Nottingham forthe generous donation of plasmids, Gerald Cohen for critical readingof the manuscript, and Adit Naor for technical assistance.

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FIG. 7. Amino acid alignment of different archaeal ACS enzymes. Lys609 of S. enterica ACS, which is known to undergo acetylation by Pat anddeacetylation by Sir2, is marked with an arrow. Leu641 of S. enterica ACS, which is important for the acetylation/deacetylation process, is alsomarked. Black and gray shading represent amino acid identity and similarity, respectively. H. lacusprofundi, Halorubrum lacusprofundi; N.pharaonis, Natronomonas pharaonis; A. fulgidus, Archaeoglobus fulgidus.



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genetic evidence for the importance of protein acetylation...

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