Overproduction of the Brucella melitensis heat shock protein DnaK in Escherichia coli and its localization by use of specific monoclonal antibodies in B. melitensis cells and fractions.
The Brucella melitensis dnaK gene was amplified by the polymerase chain reaction using primers chosen according to the published sequence of B. ovis and cloned in multiple copy plasmids enabling expression under the control of the Plac promoter. Monoclonal antibodies (mAb) obtained by immunizing mice with B. melitensis B115 cell wall (CW) fraction or by infecting mice with virulent B. melitensis strain H38 and recognizing a 73-kDa band in immunoblotting of the B. melitensis CW fraction reacted with the cloned dnaK gene product and were thus shown to be specific for the heat shock protein DnaK. The anti-Dnak protein mAbs did not react with Escherichia coli control cells or cell lysates and could therefore be specific to Brucella DnaK protein epitopes. These mAbs were further used to study overproduction of the DnaK protein. B. melitensis DnaK overproduction in E. coli resulted in a defect in cell septation and formation of cell filaments. Immunogold labelling with the mAbs and electron microscopy localized the DnaK protein inside as well as outside the E. coli cells, probably resulting from lysis due to toxicity of the overproduced DnaK protein. These results indicated that overproduction of the B. melitensis DnaK protein in E. coli had similar physiological consequences as that of E. coli overproduced in E. coli. The DnaK protein localization in B. melitensis cells was essentially cytoplasmic, as shown by immunoelectron microscopy. Heat shock treatment of these cells resulted in increased binding of mAbs and labelling in the cytoplasm. However, in subcellular fractions the DnaK protein was predominantly found in the cell envelope fraction of B. melitensis, which could perhaps be due to interaction of the DnaK protein with membrane proteins.
Cloeckaert
A
A
Laboratoire de Pathologie infectieuse et Immunologie, Institut National de la Recherche Agronomique, Nouzilly, France.
Grépinet
O
O
Salih-Alj Debbarh
H
H
Zygmunt
M S
MS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D017347:Brucella melitensis / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000302:isolation & purification; D015151:Immunoblotting; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D051379:Mice; D008854:Microscopy, Electron; D016133:Polymerase Chain Reaction / Q000379:methods*
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; C063842:dnaK protein, E coli