CHARACTERIZATION OF A GENE-CLUSTER FOR GLYCOGEN BIOSYNTHESIS AND A HETEROTETRAMERIC ADP-GLUCOSE PYROPHOSPHORYLASE FROM BACILLUS-STEAROTHERMOPHILUS

A chromosomal region of Bacillus stearothermophilus TRBE14 which conta ins genes for glycogen synthesis was cloned and sequenced. This region includes five open reading frames (glgBCDAP). It has already been dem onstrated that glgB encodes branching enzyme (EC 2.4.1.18 [H. Takata e t al., Appl. Environ. Microbiol. 60:3096-3104, 1994]). The putative Gl gC (387 amino acids [aa]) and GlgD (343 aa) proteins are homologous to bacterial ADP-glucose pyrophosphorylase (AGP [EC 2.7.7.27]): the sequ ences share 42 to 70% and 20 to 30% identities with AGP, respectively. Purification of GlgC and GlgD indicated that AGP is an alpha(2) beta( 2)-type heterotetrameric enzyme consisting of these two proteins. AGP did not seem to be an allosteric enzyme, although the activities of mo st bacterial AGPs are known to be allosterically controlled. GlgC prot ein had AGP activity without GlgD protein, but its activity was lower than that of the heterotetrameric enzyme. The GlgA (485 aa) and GlgP ( 798 aa) proteins were shown to be glycogen synthase (EC 2.4.1.21) and glycogen phosphorylase (EC 2.4.1.1), respectively. We constructed plas mids harboring these five genes (glgBCDAP) and assayed glycogen produc tion by a strain carrying each of the derivative plasmids on which the genes were mutated one by one. Glycogen metabolism in B. stearothermo philus is discussed on the basis of these results.