Characterization and functional complementation of a nonlethal deletion in the chromosome of a beta-glycosidase mutant of Sulfolobus solfataricus.
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).
Bartolucci
Simonetta
S
Dipartimento di Chimica Biologica, Università degli Studi di Napoli Federico II, Naples, Italy.
Rossi
Mosè
M
Cannio
Raffaele
R
D001483:Base Sequence; D002874:Chromosome Mapping; D019847:Chromosomes, Archaeal / Q000235:genetics*; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D005959:Glucosidases / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D027981:Symporters; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase