139. Abstract
The gene for the catalytic subunit of cellulose
synthase from Acetobacter xylinum has been cloned by using
an oligonucleotide probe designed from the N-terminal amino acid
sequence of the catalytic subunit (an 83 kDa polypeptide) of the
cellulose synthase purified from trypsin-treated membranes of
A. xylinum. The gene was located on a 9.5 kb HindIII fragment
of A. xylinum DNA that was cloned in the plasmid pUC18.
DNA sequencing of approximately 3 kb of the HindIII fragment led
to the identification of an open reading frame of 2169 base pairs
coding for a polypeptide of 80 kDa. Fifteen amino acids in the
N-terminal region (positions 6 to 20) of the amino acid sequence,
deduced from the DNA sequence, match with the N-terminal amino
acid sequence obtained for the 83 kDa polypeptide, confirming
that the DNA sequence cloned codes for the catalytic subunit of
cellulose synthase which transfers glucose from UDP-glucose to
the growing glucan chain. Trypsin treatment of membranes during
purification of the 83 kDa polypeptide cleaved the first 5 amino
acids at the N-terminal end of this polypeptide as observed from
the deduced amino acid sequence, and also from sequencing of the
83 kDa polypeptide purified from membranes that were not treated
with trypsin. Sequence analysis suggests that the cellulose synthase
catalytic subunit is an integral membrane protein with 6 transmembrane
segments. There is no signal sequence and it is postulated that
the protein is anchored in the membrane at the N-terminal end
by a single hydrophobic helix. Two potential N-glycosylation
sites are predicted from the sequence analysis, and this is in
agreement with the earlier observations that the 83 kDa polypeptide
is a glycoprotein [13]. The cloned gene is conserved among a
number of A. xylinum strains, as determined by Southern
hybridization.