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Journal of Bacteriology, July 2003, p. 3948-3957, Vol. 185, No. 13
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.13.3948-3957.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization and Functional Complementation of a Nonlethal Deletion in the Chromosome of a ß-Glycosidase Mutant of Sulfolobus solfataricus

Simonetta Bartolucci,1 Mosè Rossi,2 and Raffaele Cannio2*

Istituto di Biochimica delle Proteine, CNR,2 Dipartimento di Chimica Biologica, Università degli Studi di Napoli Federico II, Naples, Italy1

Received 4 November 2002/ Accepted 18 March 2003

LacS- mutants of Sulfolobus solfataricus defective in ß-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 ß-glycosidase gene (lacS). Fine mapping performed in comparison to the genomic sequence of S. solfataricus P2 indicated an extended deletion of ~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 ß-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside).

* Corresponding author. Mailing address: Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via Pietro Castellino 111, 80131, Naples, Italy. Phone: (39)0816132285. Fax: (39)0816132248. E-mail: cannio{at}dafne.ibpe.na.cnr.it.

Journal of Bacteriology, July 2003, p. 3948-3957, Vol. 185, No. 13
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.13.3948-3957.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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  • Erauso, G., Stedman, K. M., van de Werken, H. J. G., Zillig, W., van der Oost, J. (2006). Two novel conjugative plasmids from a single strain of Sulfolobus. Microbiology 152: 1951-1968 [Abstract] [Full Text]  


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