Publication: Isolation and purification of a Campylobacter upsaliensis autolysin
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Issued Date
1999
Resource Type
File Type
application/pdf
ISSN
84166
Other identifier(s)
2-s2.0-0033053368
Rights Holder(s)
มหาวิทยาลัยศรีนครินทรวิโรฒ
Bibliographic Citation
Canadian Journal of Microbiology. Vol 45, No.1 (1999), p.23-30
Suggested Citation
Santiwatanakul S., Krieg N.R. Isolation and purification of a Campylobacter upsaliensis autolysin. Canadian Journal of Microbiology. Vol 45, No.1 (1999), p.23-30. doi:10.1139/cjm-45-1-23 Retrieved from: https://hdl.handle.net/20.500.14740/7132
Author(s)
Abstract
Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of Campylobacter upsaliensis could not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS) - PAGE gels containing either purified Escherichia coli peptidoglycan or whole cells of Micrococcus luteus (Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained Escherichia coli peptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells of M. luteus, only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containing Escherichia coli peptidoglycan. This common autolysin was isolated by adsorbing it from Campylobacter upsaliensis soluble fractions onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing Escherichia coli peptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to the M. luteus cells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.
Subject(s)
Peptidoglycan
Adsorption
Amino acid sequence
Antibody production
Article
Autolysis
Bacterium isolation
Campylobacter upsaliensis
Cell lysate
Culture medium
Nonhuman
Polyacrylamide gel electrophoresis
Priority journal
Protein purification
Sequence analysis
Zymography
Amino Acid Sequence
Animals
Blotting, Western
Campylobacter
Electrophoresis, Polyacrylamide Gel
Molecular Sequence Data
N-Acetylmuramoyl-L-alanine Amidase
Rabbits
Adsorption
Amino acid sequence
Antibody production
Article
Autolysis
Bacterium isolation
Campylobacter upsaliensis
Cell lysate
Culture medium
Nonhuman
Polyacrylamide gel electrophoresis
Priority journal
Protein purification
Sequence analysis
Zymography
Amino Acid Sequence
Animals
Blotting, Western
Campylobacter
Electrophoresis, Polyacrylamide Gel
Molecular Sequence Data
N-Acetylmuramoyl-L-alanine Amidase
Rabbits