Evaluation of cell wall binding domain of Staphylococcus aureus autolysin as affinity reagent for bacteria and its application to bacterial detection.
We evaluated the cell wall binding (CWB) domain of Staphylococcus aureus autolysin as an affinity reagent for bacteria. A fusion of CWB domain and green fluorescent protein (CWB-GFP) bound to S. aureus with a dissociation constant of 15 nM. CWB-GFP bound to a wide range of gram-positive bacteria, but not to most gram-negative bacteria. We suspected that the outer membrane of gram-negative bacteria inhibits the access of CWB-GFP to peptidoglycan layer. Indeed, CWB-GFP bound to gram-negative bacteria when they were treated with benzalkonium chloride. Because CWB-GFP bound to the bacterial peptidoglycan layer, it appeared to be an effective affinity reagent for bacteria and CWB fusion with reporter proteins could be applied to detect bacteria. We also constructed a fusion of CWB and luciferase, which can be used for the rapid detection of bacteria.
Ahmed
Abo Bakr F
AB
Department of Molecular Biotechnology, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima, Japan.
Noguchi
Kanako
K
Asami
Yasuo
Y
Nomura
Kazutaka
K
Fujii
Hiroya
H
Sakata
Minoru
M
Tokita
Akihiko
A
Noda
Kenichi
K
Kuroda
Akio
A
D001419:Bacteria / Q000737:chemistry / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods*; D002473:Cell Wall / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D007778:Lactobacillus / Q000737:chemistry / Q000302:isolation & purification; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D013171:Spores, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D009238:N-Acetylmuramoyl-L-alanine Amidase