Random mutagenesis of a hyperthermophilic archaeon identified tRNA modifications associated with cellular hyperthermotolerance

Abstract

Random mutagenesis for the hyperthermophilic archaeon Thermococcus kodakarensis was established by the insertion of an artificial transposon designed to allow easy identification of the transposon-inserted locus. The phenotypic screening was applied for the isolation of thermosensitive mutants of T. kodakarensis, which resulted in the isolation of 16 mutants showing defective growth at the supraoptimal temperature 93°C. The high occurrence of the mutants suggested that the high thermotolerance of hyperthermophiles was achieved by a combination of diverse gene functions. The transposon insertion sites in two-thirds of the mutants were identified in a group of genes responsible for tRNA modifications including 7-formamidino-7-deaza-guanosine (archaeosine), N1-methyladenosine/N1-methylinosine, N4-acetylcytidine, and N2-dimethylguanosine/N2,N2-dimethylguanosine. LC-MS/MS analyses of tRNA nucleosides and fragments exhibited disappearance of the corresponding modifications in the mutants. The melting temperature of total tRNA fraction isolated from the mutants lacking archaeosine or N1-methyladenosine/N1-methylinosine decreased significantly, suggesting that the thermosensitive phenotype of these mutants was attributed to low stability of the hypomodified tRNAs. Genes for metabolism, transporters, and hypothetical proteins were also identified in the thermosensitive mutants. The present results demonstrated the usefulness of random mutagenesis for the studies on the hyperthermophile, as well as crucial roles of tRNA modifications in cellular thermotolerance.

Figure 1.

Overview of the strategy for construction of T. kodakarensis randomly mutagenized library based on in vitro transposition and double crossover homologous recombination.

Figure 2.

A PCR analysis of the transposon-inserted gDNA library. The plasmid extracted from randomly selected eight E. coli clones in the library was used as a template. PCR was carried out with a primer pair of (a) M13P4/M13Rv, (b) M13P4/tk0149-r, (c) tk0149-f/M13Rv, (d) M13P4/tk0149-f and (e) tk0149-r/M13Rv. (A) The primer annealing sites are shown by arrowheads. (B) Southern blot analysis of T. kodakarensis transposon-inserted clones with the pdaD probe. Lanes: ΔpdaD, genomic DNA from the strain ΔpdaD; KOD1, genomic DNA from the wild strain KOD1; 1–14, genomic DNA from randomly selected 14 clones in the transposon-inserted mutant library of T. kodakarensis; M, molecular size marker.

Figure 3.

Growth properties of T. kodakarensis thermosensitive mutants (left) and the complemented strains (right) isolated from the transposon (Tn)-inserted random mutant library. The cells were cultivated in 8 ml MA-YT-Pyr medium in a test tube (n = 3). Left: Growth of tRNA modification mutants at 85°C (A) and 93°C (B), and growth of the complemented strains at 93°C (C). (○) KD2239 control strain; (✦) FFH05 (queE::Tn); (▴) FFH18 (tgtA::Tn); (•); FFH20 (trmI::Tn); (▪)FFH32 (tmcA::Tn); (×) FFH35 (trm11::Tn). Right: Growth of other mutants at 85°C (D) and 93°C (E), and growth of the complemented strains at 93°C (F). (○) KD2239 control strain; (✦) FFH15 (tk0672::Tn); (▴) FFH08 (tk1402::Tn); (•) FFH12 (tk1803::Tn); (▪) FFH27 (tk0647::Tn); (×) FFH22 (tk2145::Tn). In the panels (C) and (F), growth of the strains transformed with the corresponding complementation vector are shown, except for the control strain KD2239.

Figure 4.

LC–MS/MS analysis of total tRNA nucleosides prepared from T. kodakarensis thermosensitive mutants having transposon insertion within a predicted tRNA-modification gene. The panels represent mass chromatograms that detect monovalent positive ions of the modified nucleosides of G⁺ (A), ac⁴C (B), m¹A, m¹I, m⁵s²U and m⁵U (C) and m^2,2G and monomethyl guanosines (D), respectively. The vertical axes show relative abundance (%) of each the modified nucleoside normalized by 5-methylcytidine. The horizontal axes show retention time (min).