Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae.
Iron limitation, glucose deprivation, and growth under low oxygen supply (environmental stress) increased the expression of several proteins of Neisseria gonorrhoeae, including a 63-kilodalton protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This gonococcal stress protein (GSP63) was detected in the cytosol and copurified with lithium acetate-derived outer membranes. Successful purification of the protein was achieved by sucrose density gradient centrifugation and by chromatography on phenyl-Sepharose. Gel filtration of the purified protein revealed a molecular weight of approximately 450,000, suggesting that in its native state, the protein consists of a multimer of six to eight subunits. Isoelectric focusing indicated a pI of 5.2. Immunoblotting experiments using a polyclonal antiserum raised against the purified protein demonstrated cross-reactivity with a protein of the same electrophoretic mobility as GSP63 in all eight gonococcal isolates tested. N-terminal amino acid sequencing of the protein revealed up to 65% homology with members of the Hsp60 heat shock protein family, suggesting that GSP63 is related to this group of proteins. This relationship was further substantiated by the immunological cross-reactivity of GSP63 with mycobacterial Hsp60 and the ATP-binding activity of the gonococcal stress protein.
Pannekoek
Y
Y
Department of Medical Microbiology, University of Amsterdam, The Netherlands.
van Putten
J P
JP
Dankert
J
J
D000085:Acetates; D019342:Acetic Acid; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D000942:Antigens, Bacterial / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D005947:Glucose / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D007501:Iron / Q000378:metabolism; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009344:Neisseria gonorrhoeae / Q000737:chemistry*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000737:chemistry
D000085:Acetates; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; C077560:GSP63 stress protein, Neisseria gonorrhoeae; D006360:Heat-Shock Proteins; D000255:Adenosine Triphosphate; D007501:Iron; D005947:Glucose; D019342:Acetic Acid