Gene knockout of the intracellular amylase gene by homologous recombination in Streptococcus bovis

被引:13
作者
Brooker, JD
McCarthy, JM
机构
[1] Department of Animal Science,
[2] University of Adelaide Waite Campus,undefined
[3] Private Bag Glen Osmond,undefined
[4] South Australia 5064,undefined
[5] Australia ,undefined
关键词
D O I
10.1007/s002849900226
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Streytococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular a-amylase gene from Streptococcus bovis WI-I was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15-20% of The wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium.
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页码:133 / 138
页数:6
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