Localization of c-di-GMP-binding protein with the linear terminal complexes of Acetobacter xylinum.
Specific labeling of a single row of cellulose-synthesizing complexes (terminal complexes, TC subunits, TCs, or TC arrays) in Acetobacter xylinum by antibodies raised against a 93-kDa protein (the cyclic dignanylic acid-binding protein) has been demonstrated by using the sodium dodecyl sulfate (SDS)-freeze-fracture labeling (FRL) technique. The antibodies to the 93-kDa protein specifically recognized the TC subunits on the protoplasmic fracture (PF) face of the outer membrane in A. xylinum; however, nonlabeled TCs were also observed. Two types of TC subunits (particles or pits) are observed on the PF face of the outer membrane: (i) immunogold-labeled TCs showing a line of depressions (pits) with an indistinct particle array and (ii) nonlabeled TC subunits with a distinct single row of particle arrays. The evidence indicates that the labeling patterns differ with respect to the presence or absence of certain TC subunits remaining attached to the replica after SDS treatment. This suggests the presence of at least two TC components, one in the outer membrane and the other in the cytoplasmic membrane. If the TC component in the outer membrane is preferentially fractured and remains attached to the ectoplasmic fracture face (or outer leaflet) of the outer membrane, subsequent replica formation reveals a pit or depression with positive antibody labeling on the PF face of the outer membrane. If the TC component in the outer membrane remains with the PF face (or inner leaflet) of the outer membrane, the innermost TC component is removed during SDS treatment and labeling does not occur. SDS-FRL of TCs in A. xylinum has enabled us to provide the first topological molecular analysis of component proteins in a cellulose-synthesizing TC structure in a prokaryotic organism.
Kimura
S
S
Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Japan.
Chen
H P
HP
Saxena
I M
IM
Brown
R M
RM
Itoh
T
T
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism*; D005614:Freeze Fracturing / Q000379:methods; D041822:Gluconacetobacter xylinus / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D007150:Immunohistochemistry; D008854:Microscopy, Electron
D001426:Bacterial Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D005964:Glucosyltransferases; C076062:cellulose synthase (cyclic diguanylic acid); D006152:Cyclic GMP