Papers in the Biological Sciences


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Journal of Bacteriology, Jan. 2003, p. 482–488 Vol. 185, No. 2


Copyright © 2003, American Society for Microbiology. All Rights Reserved.


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.

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