Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum.
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 Hind III fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the Hind III 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. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.
Saxena
I M
IM
Department of Botany, University of Texas, Austin 78713-7640.
Lin
F C
FC
Brown
R M
RM
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D005798:Genes, Bacterial; D041822:Gluconacetobacter xylinus / Q000201:enzymology / Q000235:genetics*; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012689:Sequence Homology, Nucleic Acid
D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D005964:Glucosyltransferases; C457769:PRC1 protein, Arabidopsis; C028634:cellulose synthase (UDP-forming)