Papers in the Biological Sciences
Document Type
Article
Date of this Version
2003
Citation
Journal of Bacteriology, Jan. 2003, p. 482–488 Vol. 185, No. 2
Abstract
Sulfolobus solfataricus secretes an acid-resistant _-amylase (amyA) during growth on starch as the sole
carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand
_-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant
was characterized. Internal _-amylase peptide sequences obtained by tandem mass spectroscopy were used to
identify the amyA coding sequence. Anti-_-amylase antibodies raised against the purified protein immunoprecipitated
secreted _-amylase activity and verified the enzymatic identity of the sequenced protein. A new
gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the
S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in
the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole
carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the
mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or
Western blot analysis. These results clarify the biological role of the α-amylase and provide additional methods
for the directed genetic manipulation of the S. solfataricus genome.
Comments
Copyright © 2003, American Society for Microbiology. All Rights Reserved.