Targeted disruption of the alpha-amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus.
Sulfolobus solfataricus secretes an acid-resistant alpha-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 alpha-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal alpha-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-alpha-amylase antibodies raised against the purified protein immunoprecipitated secreted alpha-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 alpha-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome.
Worthington
Penny
P
Beadle Center for Genetics, University of Nebraska, Lincoln 68588, USA.
Hoang
Viet
V
Perez-Pomares
Francisco
F
Blum
Paul
P
D000595:Amino Acid Sequence; D003470:Culture Media; D019848:Gene Expression Regulation, Archaeal; D018390:Gene Targeting; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic; D021241:Spectrometry, Mass, Electrospray Ionization; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D003470:Culture Media; D000516:alpha-Amylases