Identification and Characterization of CAMP Cohemolysin as a Potential Virulence Factor of Riemerella anatipestifer
| Autor: | Sumathi Subramaniam, Hai-Meng Tan, Hilda Loh, Kim Lee Chua, Joachim Frey, Karen Crasta |
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| Rok vydání: | 2002 |
| Předmět: |
DNA
Bacterial Sequence analysis Molecular Sequence Data Gene Expression Biology medicine.disease_cause Flavobacterium Microbiology Riemerella law.invention Hemolysin Proteins Plasmid Bacterial Proteins law Endopeptidases medicine Amino Acid Sequence Cloning Molecular Molecular Biology Escherichia coli Immunoassay Gel electrophoresis pBluescript Sequence Homology Amino Acid Virulence Gene Amplification Riemerella anatipestifer Sequence Analysis DNA Enzymes and Proteins Molecular biology Recombinant DNA Genome Bacterial |
| Zdroj: | Journal of Bacteriology. 184:1932-1939 |
| ISSN: | 1098-5530 0021-9193 |
| DOI: | 10.1128/jb.184.7.1932-1939.2002 |
| Popis: | Riemerella anatipestifer is responsible for exudative septicemia in ducks. The genetic determinant of the CAMP cohemolysin, cam , from a strain of R. anatipestifer was cloned and expressed in Escherichia coli. Chromosomal DNA from serotype 19 strain 30/90 was used to construct a gene library in pBluescript II SK(−) vector in E. coli XL-1-Blue strain. The clones containing recombinant plasmids were screened for the CAMP reaction with Staphylococcus aureus . Those that showed cohemolysis were chosen for further analysis by sequencing. One of these clones, JFRA8, was subcloned to identify the smallest possible DNA fragment containing the CAMP cohemolysin determinant, which was located on a 3,566-bp Bam HI- Bst XI fragment which specified a 1,026-bp open reading frame. Clones containing recombinant plasmids carrying cam obtained by PCR cloning into E. coli M15 strain secreted an active CAMP cohemolysin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses confirmed that the recombinant strain expressed a protein with a molecular mass of 37 kDa and that strains from serotypes 1, 2, 3, 5, 6, and 19 expressed the cohemolysin. The deduced amino acid sequence showed high homology to those of O -sialoglycoprotein endopeptidases. Hydrolysis of radioiodinated glycophorin A confirmed that Cam is a sialoglycoprotease. |
| Databáze: | OpenAIRE |
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