High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by transposable elements

Abstract

We have isolated uracil-auxotrophic mutants of the hyperthermophilic archaeon Sulfolobus solfataricus in order to explore the genomic stability and mutational frequencies of this organism and to identify complementable recipients for a selectable genetic transformation system. Positive selection of spontaneous mutants resistant to 5-fluoroorotate yielded uracil auxotrophs with frequencies of between 10(-4) and 10(-5) per sensitive, viable cell. Four different, nonhomologous insertion sequences (ISs) were identified at different positions within the chromosomal pyrEF locus of these mutants. They ranged in size from 1,058 to 1,439 bp and possessed properties typical of known transposable elements, i.e., terminal inverted repeats, flanking duplicated target sequences, and putative transposase genes encoding motifs that are indicative of the IS4-IS5 IS element families. Between 12 and 25 copies of each IS element were found in chromosomal DNAs by Southern analyses. While characteristic fingerprint patterns created by IS element-specific probes were observed with genomic DNA of different S. solfataricus strains, no homologous sequences were identified in DNA of other well-characterized strains of the order Sulfolobales.

FIG. 1

Growth of S. solfataricus P1 wt (open circles) and of 5-FOA-resistant mutants (filled symbols; triangles, P1-18; squares, P1-17; diamonds, PH1-15) on medium containing 0.2% d-arabinose and 0.1% tryptone as carbon sources with (10 μg/ml) or without uracil. OD, optical density.

FIG. 2

Genomic region of pyrimidine biosynthesis genes in S. solfataricus P2. The positions of the primers pyr-R and pyr-L, used to amplify the pyrEF genes from wt and mutant DNA, are indicated, as well as the sites of insertion of the four different IS elements found in seven different mutants. The two AflIII sites within this region are also shown. pyrE and pyrF are genes coding for putative orotidine-5′-monophosphate pyrophosphorylase (PyrE) or orotidine-5′-monophosphate decarboxylase (PyrF) as identified by homology to known protein sequences from the database.

FIG. 3

Agarose gel electrophoresis (a) and corresponding Southern hybridization (b) of AflIII-digested genomic DNAs identifying the pyrEF loci in S. solfataricus P1 (wt) and in uracil-auxotrophic mutants. The pyrEF probe was obtained by PCR amplification of a 1,099-bp fragment (position 394 through 1493 in Fig. 2). M, markers.

FIG. 4

Sequences of structural features of the four IS elements. Dotted arrows, duplicated target sequences (direct repeats) in the pyrEF genes flanking the IS element in each mutant; solid arrows, inverted repeats at termini of IS elements (lowercase letters represent mismatches within the inverted repeat).

FIG. 5

Sequence similarity of the deduced amino acid sequences of the largest ORFs to representative transposases of the IS4-IS5 family of insertion sequences. The two conserved signatures, D-N-G/A-Y/F and Y-N₂-R-N₃-E-N₆-K, of the N3 and the C1 region, respectively, as described by Rezsöhazy et al. (23), were found in the putative transposases of the S. solfataricus elements. Identical amino acids are shaded in black; similar amino acids are shaded in grey (with a 40% cutoff). Sources of known IS elements (accession numbers are given in brackets) from archaea: ISH27-1, H. salinarum PHH1 (X54432); ISH8, H. salinarum SD108 (M58557). Those for bacteria: IS10, Salmonella enterica serovar Typhimurium Tn10 (J01829); IS186A, E. coli RR1 (M11300); IS4Sa, Synechocystis sp. strain PCC6803 (U38915); IS1151, Clostridium perfringens NCTC2062 (Z18246); IS231A, Bacillus thuringiensis (X03397); IS4, E. coli K-12 (J01733); IS5377, Bacillus stearothermophilus CU21 (X67862); IS903, E. coli (J01839). IS elements from this study are underlined.

FIG. 6

Southern hybridization using IS element-specific probes and AflIII-digested chromosomal DNA of S. solfataricus P1 (wt) and uracil-auxotrophic mutants derived from P1 ( and 18) or PH1 (, , , , and ; β-galactosidase mutant with a copy of ISC1217 in lacS [26]). Each hybridizing fragment corresponds to at least one IS element copy, because AflIII does not cut within ISC1058, ISC1217, and ISC1359 and only close to the terminus of ISC1439 (Fig. 3b). Large arrows indicate the positions of fragments containing the IS element in the pyrEF region (as in Fig. 3, detected with the pyrEF probe). The respective mutants containing the IS element in this region are boxed. Note additional fragments in several mutants caused by multiple transposition events.

FIG. 7

Southern hybridization with pyrEF (a)- and IS element (b)-specific probe of ISC1058 to AflIII-digested chromosomal DNAs of S. solfataricus P1 (lanes 1), the β-galactosidase mutant PH1 (lanes 2), strain P2 (lanes 3), S. shibatae (lanes 4), S. acidocaldarius (lanes 5), A. ambivalens (lanes 6), isolate PIT3 (lanes 7), and isolate KAW2 (lanes 8).