An archaeal histone is required for transformation of Thermococcus kodakarensis

Abstract

Archaeal histones wrap DNA into complexes, designated archaeal nucleosomes, that resemble the tetrasome core of a eukaryotic nucleosome. Therefore, all DNA interactions in vivo in Thermococcus kodakarensis, the most genetically versatile model species for archaeal research, must occur in the context of a histone-bound genome. Here we report the construction and properties of T. kodakarensis strains that have TK1413 or TK2289 deleted, the genes that encode HTkA and HTkB, respectively, the two archaeal histones present in this archaeon. All attempts to generate a strain with both TK1413 and TK2289 deleted were unsuccessful, arguing that a histone-mediated event(s) in T. kodakarensis is essential. The HTkA and HTkB amino acid sequences are 84% identical (56 of 67 residues) and 94% similar (63 of 67 residues), but despite this homology and their apparent redundancy in terms of supporting viability, the absence of HTkA and HTkB resulted in differences in growth and in quantitative and qualitative differences in genome transcription. A most surprising result was that the deletion of TK1413 (ΔhtkA) resulted in a T. kodakarensis strain that was no longer amenable to transformation, whereas the deletion of TK2289 (ΔhtkB) had no detrimental effects on transformation. Potential roles for the archaeal histones in regulating gene expression and for HTkA in DNA uptake and recombination are discussed.

Fig 1

Alignments of the T. kodakarensis histone sequences (6) using clustalW. Shown are alignments of the amino acid (A) and the encoding nucleic acid (B) sequences of HTkA (TK1413) and HTkB (TK2289). Between the sequences, identical amino acids and nucleotides are indicated by asterisks, and similar amino acids are indicated by colons. The codon changes introduced into TK1413 in plasmid pTS706 to generate plasmids pTS707 and pTS708 are indicated by underlining. An oligonucleotide with the sequence double underlined in TK1413 was synthesized, labeled with ³²P, and used as the probe for Southern blotting (Fig. 2E).

Fig 2

Construction and confirmation of T. kodakarensis LC124 (ΔTK1413). (A) Plasmid pLC113 was generated from pTS535 (Table 1) by cloning amplicons from T. kodakarensis genomic DNA upstream and downstream of the TK0254-TK0664 cassette. Plasmid pLC124, generated by deletion of TK1413 from pLC113, was used as the donor DNA to transform T. kodakarensis TS517. The genome structure of the intermediate strain generated was confirmed, as illustrated, and recombination between the duplicated sequences deleted the cassette to yield T. kodakarensis LC124 with the genome as shown. The positions of primers (primers a to g [see Table S1 in the supplemental material for sequences]) used to generate diagnostic amplicons (C) and probes for Southern blotting (D to F) are indicated by arrows above the T. kodakarensis TS517 genome. The 2,905-bp PvuII restriction fragment to which the probes hybridized is indicated. (B) Expanded illustration of the primer locations (heavy arrows) and the amplicons generated. (C) Ethidium bromide-stained agarose gel for electrophoretic separation of the amplicons generated using the primer pairs a-b and c-d from T. kodakarensis LC124, LC125, and TS517 genomic DNAs. (D to F) Southern blots of PvuII-digested T. kodakarensis TS517, LC124, and LC125 DNAs probed with DIG-labeled amplicons (primer pairs c-d and f-g) or ³²P-labeled oligonucleotide primer e. In panel D, the 2,905-bp PvuII fragment that contains TK1413 in T. kodakarensis TS517 is indicated by an arrow. This fragment is not present in T. kodakarensis LC124. The PvuII fragment present in both genomic DNAs which contains TK2289 and thus cross-hybridizes with the probe is noted by an asterisk. In panel F, as the probe includes sequences immediately adjacent to TK1413, it hybridized to the PvuII fragment in T. kodakarensis LC124 DNA that contains the ΔTK1413 deletion (gray arrow). As the probe contains the entire TK1413 sequence, it cross-hybridized to the ∼1-kbp PvuII fragment present which contains TK2289 (asterisk) in both T. kodakarensis TS517 and LC124 DNAs. M, molecular size marker (in base pairs).

Fig 3

Construction and confirmation of T. kodakarensis LC125 (ΔTK2289). (A) Plasmid pLC114 was constructed by cloning amplicons from T. kodakarensis genomic DNA upstream and downstream of the TK0254-TK0664 cassette. Plasmid pLC125, generated by deletion of TK2289 from pLC114, was used as the donor DNA to transform T. kodakarensis TS517. The genome structure of the intermediate strain generated was confirmed, as illustrated, and recombination between the duplicated sequences deleted the cassette to yield T. kodakarensis LC125 with the genome as shown. The positions of primers (primers h to m [see Table S1 in the supplemental material]) used to generate the diagnostic amplicons (C and D) and the probe used for Southern blotting (E) are indicated by arrows above the T. kodakarensis TS517 genome. The 5,534- and 5,330-bp EcoRI restriction fragments to which the probe hybridized in digests of T. kodakarensis TS517 and LC125 genomic DNAs are indicated. (B) Expanded illustration of the primer locations (heavy arrows) and the amplicons generated. (C and D) Ethidium bromide-stained agarose gel electrophoretic separations of the amplicons generated with primer pairs h-i and j-I (see Table S1 in the supplemental material) from T. kodakarensis LC124, LC125, and TS517 genomic DNAs. (E) Southern blot of EcoRI-digested T. kodakarensis TS517 and LC125 DNAs. The amplicon generated by primer pair k-m was DIG labeled and used as the probe. The 5,534-bp EcoRI fragment present in T. kodakarensis TS517 DNA (black arrow) is shortened to 5,330-bp by the ΔTK2289 mutation, as indicated for T. kodakarensis LC125 DNA (gray arrow). The probe also cross-hybridized to an ∼11-kbp EcoRI fragment (*) present in both genomes that contains TK1413.

Fig 4

Comparison of the growth profiles of T. kodakarensis TS517, LC124, and LC125 cultures in ASW-YT-S⁰ medium at 85°C monitored by measurements of the increases in the optical density at 600 nm. The curves show the average values, with errors, obtained in three independent experiments from a total of 9 cultures of each strain.