Characterization and functional complementation of a nonlethal deletion in the chromosome of a beta-glycosidase mutant of Sulfolobus solfataricus

Abstract

LacS(-) mutants of Sulfolobus solfataricus defective in beta-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation. One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the beta-glycosidase gene (lacS). Fine mapping performed in comparison to the genomic sequence of S. solfataricus P2 indicated an extended deletion of approximately 13 kb. The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC1058. In order to complement the LacS(-) phenotype, an expression vector was constructed by inserting the lacS coding sequence with its 5' and 3' flanking regions into the pEXSs plasmid. Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the lacTr (lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily. Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the beta-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside).

FIG. 1.

Growth rate and PCR amplification analysis of the lacS gene in S. solfataricus Gθ and LacS⁻ mutant derivatives. (A) Cell density was monitored at 600 nm in mineral medium supplemented with 0.1% yeast extract, 0.1% Casamino Acids, and 0.1% glucose for cultures of the wild-type Gθ (▵) and mutant GθW (•). Growth trends for the mutants Gθwb1 and Gθwb2 and the parental Gθ matched perfectly. (B) Amplicons were obtained with two pairs of primers, IVS1-IVS2 and lacFw-lacRe, designed on the wild-type DNA sequence to amplify a 248-bp internal region (lane A) or the entire 1,470-bp coding sequence (lane E). Corresponding amplification products from mutant GθW (lanes B and F), as well as Gθwb1 (lanes C and G) and Gθwb2 (lanes D and H), genomic DNAs were analyzed by agarose gel electrophoresis with reference to a mixture of pBR328 DNA cleaved with BglI and pBR328 DNA cleaved with HinfI used as a molecular weight marker (lane M). OD₆₀₀, optical density at 600 nm.

FIG. 2.

Sequence analysis of Gθ-GθW rDNAs. The identical rDNA sequences amplified from both the wild-type and deletion mutant chromosomes were analyzed for distance evaluation in comparison with the five closest Sulfolobus type strains as indicated. The distance matrix of weighted neighbors and the derived phylogenetic tree were produced in the analysis performed with the Ribosomal Database Project Phylip interface program. In the matrix, values above and below the diagonal are similarity and distance indices, respectively. A similarity of 1.000 and a distance of 0.000 correspond to 100% sequence identity.

FIG. 3.

Comparative Southern analysis of the wild-type and mutant lacS loci. Chromosomal DNAs digested with EcoRI (two left lanes of each gel) and XbaI (two right lanes of each gel), identifying the lacS locus in S. solfataricus Gθ (P) and in the LacS⁻ mutant GθW (M), were hybridized with DNA fragments progressively covering the upstream and downstream regions of the lacS coding sequence in independent experiments. The extended 15-kbp region used as a source of these probes is represented in the middle as a solid bar containing already-identified ORFs (xylS, lacS, and lacTr) or putative protein-encoding sequences (ABC, ABC transporter) in their relative orientations. On the left, two arrows indicate the sizes corresponding to the differential cross-hybridization signals on the EcoRI digests hybridized with the extreme 5′ DNA fragment used in the analysis.

FIG. 4.

Fine mapping of the deletion join end. On top is shown the schematic alignment of the lacS loci, considered in this study, on the P2 (S.so P2; positions are indicated in mega-base pairs) and Gθ (S. so Gθ) Sulfolobus chromosomes. The main difference resulted from the insertion of an ISC element (sso3018) in the 3′ end of ORF sso3017 (the bar and arrow indicate the position of the insertion), namely, the lacTr gene, encoding a putative lactose transporter. The extreme 5′ DNA fragment of the 15-kbp region used for Southern analysis was dissected to produce two probes that showed one-band cross-hybridization patterns (A and B), on both the wild-type (P) and mutant (M) genomic DNAs, digested with EcoRI (two left lanes of each gel) and XbaI (two right lanes of each gel). The EcoRI DNA fragment from the mutant genomic DNA corresponding to the 1,250-bp band was cloned, sequenced (C), and compared with the full sequence of the S. solfataricus P2. The larger region of the fragment was identical to part of ORF sso3012, encoding a putative ABC transporter (uppercase letters), abruptly interrupted by a shorter DNA element (lowercase boldface letters) showing identity with multiple repetitive sequences found on the P2 genome and indicated as transposase-coding ICS1058 elements (sso3023).

FIG. 5.

Plasmid map of the pEXSs expression vector derivatives pEXlacS and pEXlacOP. The lacS gene and a genomic fragment, encompassing both lacS and lacTr genes, were inserted into the polycloning site of the pEXSs plasmid to produce the pEXlacS and pEXlacOP DNA constructs. SsV1ORI indicates the 1,700-bp fragment carrying the autonomous replication sequence of the S. shibatae SSV1 viral genome. AspATPr and AspATTer are the promoter and terminator sequences of the S. solfataricus aspartate aminotransferase gene, respectively. hph is the E. coli randomly mutagenized hygromycin phosphotransferase gene. The E. coli pGEM5Zf(−) plasmid moiety lies between the two lacZ gene fragments and comprises the sequences necessary for propagation (ORI) and transformant selection for ampicillin resistance (Amp^r) in E. coli.

FIG. 6.

lac complementation and growth analysis of transformants. The pEXSs derivative vectors pEXlacS and pEXlacOP were transferred into the mutant GθW cells by electroporation, and transformants were selected on gelrite plates containing only lactose as a nutrient. (A) The LacS⁻ GθW did not produce any colonies when plated onto lactose medium (negative control; plate a), and lacS transformants failed to grow, maintaining the lactose metabolic defect (plate b), whereas transformation with the construct containing both lacS and lacTr restored the wild-type phenotype and sustained growth on minimal medium (plate c). (B) Growth of four independent transformants was monitored in both rich tryptone-lactose (Trp-Lac) and minimal lactose (Lac) media and resulted in nearly identical overlapping curves (solid squares) compared to cultures of the wild-type Gθ (•) and untransformed GθW (▵) strains under the same conditions. OD₆₀₀, optical density at 600 nm.

FIG. 7.

β-Glycosidase activity test of isolated clones transformed with pEXlacOP. (A) Four independent transformants were picked from lactose plates, propagated in hygromycin selective medium, and seeded onto fresh plates (1, 2, 3, and 4). After growth, the colonized areas showed β-glycosidase activity, developing blue color upon being stained with the chromogenic substrate X-Gal. Wild-type Gθ and the deficient mutant GθW were used as positive and negative controls for enzyme detection, respectively. Cultures of the same clones were grown to early stationary phase and reinoculated for a subsequent four-step scaling up. Cells were harvested at the early stationary phase of growth for each step, and extracts were assayed for cytosolic β-galactosidase activity (in enzyme units per milligram of total cytosolic proteins). (B) Results for cultures (1, 2, 3, and 4) in both yeast extract-Casamino Acids-glucose (open bars) and tryptone-lactose (solid bars) media at the first (Y1 and T1) and the fourth (Y2 and T2) step of propagation. Activities in the cell extracts of Gθ and GθW (w) cultures were used as reference points for 100 and 0% activity, respectively, in every set of measures.