Role of Archaeal HerA Protein in the Biology of the Bacterium Thermus thermophilus

Abstract

Intense gene flux between prokaryotes result in high percentage of archaeal genes in the genome of the thermophilic bacteria Thermus spp. Among these archaeal genes a homolog to the Sulfolobus spp. HerA protein appears in all of the Thermus spp. strains so far sequenced (HepA). The role of HepA in Thermus thermophilus HB27 has been analyzed using deletion mutants, and its structure resolved at low resolution by electron microscopy. Recombinant HepA shows DNA-dependent ATPase activity and its structure revealed a double ring, conically-shaped hexamer with an upper diameter of 150 Å and a bottom module of 95 Å. A central pore was detected in the structure that ranges from 13 Å at one extreme, to 30 Å at the other. Mutants lacking HepA show defective natural competence and DNA donation capability in a conjugation-like process termed "transjugation", and also high sensitivity to UV and dramatic sensitivity to high temperatures. These data support that acquisition of an ancestral archaeal HerA has been fundamental for the adaptation of Thermus spp. to high temperatures.

Keywords: DNA repair; HerA; Thermus; hexameric ATPase; lateral gene transfer; transformation; transjugation.

Figure 1

Genomic context of hepA homologs. Synteny of the cluster in which the common homolog of HepA is found in T. thermophilus HB27 (ttc0147), T. thermophilus HB8 (ttha0522), T. thermophilus SG0.5JP16-17 (tthe16_0521), T. thermophilus JL18 (TtJL18_1554) and T. scotoductus (Tsco_c06870), Deinoccoccus radiodurans (DR_0837), Oceanithermus profundus (Ocepr_1235), and the archaea Aeropyrum pernix (APE_0107), Acidolobus saccharovorans (ASAC_055), Thermofilum pendens (Tpen_1571), and Methanobacteirum lacus (metl_2080).

Figure 2

HepA is a cytoplasmic protein. Western blot with an anti-HepA antiserum on whole cells of exponential culture of T. thermophilus HB8 (T, lane 1), and its soluble (S, lane 3) and membrane fractions (M, lane 2). Purified His-tagged HepA was employed as the control (P, lane 4).

Figure 3

Localization of HepA. HepA-sYFP fusion expressed from a single copy in the chromosome under its natural promoter (a–c) or ectopically from a multicopy plasmid in a constitutive way (d). Panel (a) corresponds to untreated cells. Panels (b) and (c) correspond to different magnifications of UV-treated cells as described in the Materials and Methods Section.

Figure 4

Mutants defective in HepA grow at low temperatures. Growth curves in TB medium at 60 °C of the wild-type strain (black circles), and its ΔhepA mutant (empty circle). Error bars represent the deviation of the mean value of three independent samples.

Figure 5

Sensitivity of hepA mutants to UV. Figure shows the ratios between viable cells of UV treated and untreated cells at different cell densities (OD₆₀₀) along the growth curves of T. thermophilus HB27 gdh:.kat (wt) and T. thermophilus HB27 ΔhepA::kat mutant.

Figure 6

Effects of the absence of HepA in transformation and transjugation. (a) Parallel cultures of T. thermophilus HB27 gdh::kat (wt, black bars) and the ΔhepA::kat mutant (gray bars) were transformed with 150 ng of plasmid pMH118 or with 15 ng of genomic DNA from an isogenic strain labelled with Hyg resistance in the chromosome; and (b) transjugation assays between the indicated mates. (1) ΔhepA::kat × wild-type::hyg; (2) ΔhepA::kat × ΔpilA::hyg; (3) ΔhepA::kat-ΔpilA double mutant × wild-type::hyg; (4) ΔhepA::kat-ΔpilA double mutant containing a plasmid expressing HepA ectopically × wild-type resistant to Cm. (p-value < 0.005 is shown as **).

Figure 7

Purification and ATPase activity of HepA. (a) Coomassie blue-stained SDS-PAGE gel showing purification of HepA as described in materials and methods. (M) Proteins of the indicated size (kDa) used as markers; (1) IMAC column flow-through; (2–6) fractions eluted with imidazole. (b) ATPase activity expressed as the ATP consumed (%) after incubation for 1 h at 65 °C with the indicated concentrations of HepA. The initial concentration of ATP was 10⁻⁴ M.

Figure 8

HepA single-particle electron microscopy reconstruction. (a) Representative electron micrograph of a negatively-stained HepA sample. Bar, 50 nm; (b) six two-dimensional averaged classes of the oligomeric HepA; (c) three-dimensional reconstruction of the hexameric HepA with C6 symmetry; and (d) a semitransparent model of the hexameric HepA with a docked atomic model of the hexameric HerA (pink).