Tomonori Nakai

Control of expression by the cellulose synthase (bcsA) promoter region from Acetobacter xylinum BPR 2001

The 5 upstream region (about 3.1kb) of the cellulose synthase operon (bcs operon) has been isolated by cloning from Acetobacter xylinum strain BPR 2001. The expression level of the upstream region was determined using sucrose synthase cDNA as a reporter gene in the shuttle vector pSA19. The expression occurred with the 1.1-kb upstream sequence from the ATG start codon of the bcs operon but not with the 241-bp upstream sequence in A. xylinum, although neither the 1.1-kb nor the 241-bp upstream sequence caused any expression as a promoter in Escherichia coli. The level of expression with the 1. 1-kb upstream sequence in A. aceti was 75% of that in A. xylinum. These results suggest that the upstream region functions as a specific promoter for the Acetobacter genus. The expression was reduced by the introduction of the 241-bp upstream region between the lac promoter and the reporter gene in E. coli and was not detected in A. xylinum. This suggests that the short upstream region composed of 241bp contains the site(s) which causes a negative regulation on the transcription for bcs operon. The production of recombinant protein with the ribosome-binding site (RBS) of A. xylinum obtained from the bcs operon, was reduced to about half in E. coli, and that with the site of the lac promoter was also reduced to about half in A. xylinum. This shows that a species-specific predominance occurs during interaction between mRNA and 16S rRNA in the RBS between A. xylinum and E. coli.

Formation of callose from sucrose in cotton fiber microsomal membranes

Callose is formed from exogenous sucrose by cotton fiber microsomal membranes that contain both sucrose synthase and callose synthase activity. Although the coupled reaction between sucrose and callose synthases occurs predominantly to channel glucose from sucrosederived uridine diphosphate (UDP)-glucose into callose in the membranes, there is no difference in the UDP-glucose-forming/sucrose-forming activity ratios between the soluble and membrane-bound forms of sucrose synthase. The consumption of UDP-glucose from sucrose into callose probably leads to UDP-glucose formation rather than sucrose formation despite the lower affinity of sucrose synthase for sucrose than for UDP-glucose. Callose formation is markedly stimulated by the addition of either recombinant Glu11 (S11E) or in vitro phosphorylated Ser11 mung bean sucrose synthase but not by the wild-type nonphosphorylated Ser11 enzyme. We propose that a negative charge (by phosphorylation or mutagenesis) at Ser11 in sucrose synthase causes the enzyme to promote a coupled callose-forming reaction.