CONTRIBUTION OF A NEOPULLULANASE, A PULLULANASE, AND AN ALPHA-GLUCOSIDASE TO GROWTH OF BACTEROIDES-THETAIOTAOMICRON ON STARCH

Bacteroides thetaiotaomicron, a gram-negative colonic anaerobe, can ut ilize three forms of starch: amylose, amylopectin, and pullulan. Previ ously, a neopullulanase, a pullulanase, and an cu-glucosidase from B. thetaiotaomicron had been purified and characterized biochemically, Th e neopullulanase and alpha-glucosidase appeared to be the main enzymes involved in the breakdown of starch, because they were responsible fo r most of the starch-degrading activity detected in B. thetaiotaomicro n cell extracts. To determine the importance of these enzymes in the s tarch utilization pathway, we cloned the genes encoding the neopullula nase and ol-glucosidase. The gene encoding the neopullulanase (susA) w as located upstream of the gene encoding the ol-glucosidase (susB), Bo th genes were closely linked to another starch utilization gene, susC, which encodes a 115-kDa outer membrane protein that is essential for growth on starch, The gene encoding the pullulanase, pull, was not loc ated in this region in the chromosome, Disruption of the neopullulanas e gene, susA, reduced the rate of growth on starch by about 30%, Elimi nation of susA in this strain allowed us to detect a low residual leve l of enzyme activity, which was localized to the membrane fraction. Pr eviously, we had shown that a disruption in the pull gene did not affe ct the rate of growth on pullulan, We have now shown that a double mut ant, with a disruption in susA and in the pullulanase gene, pull, was also able to grow on pullulan, Thus, there is at least one other starc h-degrading enzyme besides the neopullulanase and the pullulanase. Dis ruption of the cx-glucosidase gene, susB, reduced the rate of growth o n starch only slightly. No residual cx-glucosidase activity was detect able in extracts from this strain, Since this strain could still grow on maltose, maltotriose, and starch, there must be at least one other enzyme capable of degrading the small oligomers produced by the starch degrading enzymes, Our results show that the starch utilization syste m of B. thetaiotaomicron is quite complex and contains a number of app arently redundant degradative enzymes.