Identification and Characterization of one fru locus and two aga loci of Bacillus megaterium

• Main Authors: Chiou Chih Yung, 邱志勇
• Other Authors: 邵國銓
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/00456745410907448175

博士 === 國立陽明大學 === 生物化學研究所 === 91 === Bacillus megaterium ATCC 14581 could utilize sucrose, melibiose, and raffinose as a sole carbon source for growth. A genetic locus that is adjacent to the gene encoding the small acid-soluble protein SASP C-4 of B. megaterium has been defined. This locus, designated fru, contains a b-fructosidase gene (fruA), a gene coding for a hydrophobic protein that is closely related to non-PTS sugar permeases of the proton symport type (fruP), and a gene coding for a transcriptional regulator of the LacI/GalR family (fruR). The FruA protein can hydrolyze sucrose and raffinose, but not maltose, isomaltose, trehalose, melibiose, or lactose. The transcription initiation site of fruP has been mapped and the fruP promoter identified. Gel mobility shift assays showed that the FruR protein could specifically bind to a DNA fragment containing the fruP promoter region. DNase I footprinting analysis has defined the FruR binding site. Disruption of fruR led to high-level constitutive expression of fruPA, but had no effect on expression from the fruR promoter itself, indicating that FruR acts as a repressor of fruPA expression, but does not autoregulate its own synthesis. Interestingly, expression of fruPA in B. megaterium was not induced by sucrose, raffinose, fructose, or inulin, whereas the constitutive expression of fruPA in a fruR mutant was repressed by both glucose and sucrose. Possible physiological implications of these findings are discussed. To investigate the raffinose utilization system of B. megaterium, two a-galactosidase genes from B. megaterium were cloned. These genes, designated agaA and agaB, were located at different loci. The locus containing agaA also includes a gene coding for a putative sugar-binding protein (msmE), and two genes coding for membrane proteins (msmF, msmG). AgaA is similar to a-galactosidases of family 4 in the classification of glycosyl hydrolases, and exhibits unique requirement for NAD+ and Mn2+ for activity. Partially purified AgaA could hydrolyze melibiose and p-nitrophenyl-a-D-galactopyranoside (pNP-a-gal), but not o-nitrophenyl-b-D-galactopyranoside (oNP-a-gal), p-nitrophenyl-a-D-glucopyranoside (pNP-a-glc), lactose, sucrose, and maltose. Expression of agaA in B. megaterium could not be induced by melibiose, raffinose, lactose, and galactose. Sugar binding assays revealed that MsmE could bind lactose, melibiose, and maltose. Disruption of msmE led to slower growth of B. megaterium cells on lactose, melibiose, and raffinose, indicating that the locus might play a role in sugar utilization in B. megaterium. AgaB is similar to a-galactosidases of family 36 in the classification of glycosyl hydrolases. AgaB could also hydrolyze pNP-a-gal and melibiose. Insertional inactivation of agaB resulted in no detectable a-galactosidase activity in the absence of additional NAD+ and Mn2+ in the crude extract of B. megaterium. In contrast with agaA, expression of agaB in B. megaterium could be induced by melibiose and lactose, but not by raffinose. Moreover, agaB expression was found to be subject to catabolite repression by glucose, sucrose and raffinose. This phenomenon was rarely observed in other bacteria. The regulatory mechanisms for expression of these two a-galactosidase genes of B. megaterium remain to be further investigated.