Genetic evidence for the importance of protein acetylation and protein deacetylation in the halophilic archaeon Haloferax volcanii

Abstract

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 genome of the haloarchaeon Haloferax volcanii led to the identification of three putative protein acetyltransferases belonging to the Gcn5 family, Pat1, Pat2, and Elp3, and two deacetylases, Sir2 and HdaI. Intriguingly, the gene that encodes HdaI shares an operon with an archaeal histone homolog. We performed gene knockouts to determine whether the genes encoding these putative acetyltransferases and deacetylases are essential. A sir2 deletion mutant was able to grow normally, whereas an hdaI deletion mutant was nonviable. The latter is consistent with the finding that trichostatin A, a specific inhibitor of HdaI, inhibits cell growth in a concentration-dependent manner. We also showed that each of the acetyltransferases by itself is dispensable for growth 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.

FIG. 1.

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

FIG. 2.

HdaI alignment. A partial alignment of the halophilic H. volcanii HdaI and H. marismortui HdaI versus the amino-terminal part of the S. cerevisiae HdaI and the Homo sapiens HDAC1. Known conserved domains are marked by black lines. Amino acid residues that were proven to 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 an operon with the archaeal “double-histone” homolog. The hdaI start codon overlaps with the histone stop codon. The 3′ region of the HdaI open reading frame overlaps the 3′ region of the essential gene that encodes CCA tRNA nucleotidyltransferase, which is transcribed on the opposite DNA strand.

FIG. 4.

Pat alignment. The S. solfataricus Pat and the carboxy-terminal 95 amino acid residues of S. enterica Pat were aligned against H. volcanii Pat1 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 highly conserved R/QXXGXG/A motif, which is important for acetyl-CoA recognition and binding (38, 54), is marked by arrows. Black and gray shading represent 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 highly conserved R/QXXGXG/A motif is marked by black arrows. Two tyrosine residues that were shown to be highly important for Elp3 acetylation activity 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 and similarity, respectively.

FIG. 6.

Growth curve of the H. volcanii wild-type (wt) strain in the presence of TSA. H. volcanii was grown on rich HY medium without TSA or at different concentrations of TSA, as indicated. The culture turbidity was measured as the OD₆₀₀.

FIG. 7.

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