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Development of SNAP-tag-mediated live cell labeling as an alternative to GFP in Porphyromonas gingivalis
- Source :
- FEMS Immunology and Medical Microbiology, FEMS Immunology and Medical Microbiology, 2010, 59 (3), pp.357-63. ⟨10.1111/j.1574-695X.2010.00681.x⟩, FEMS Immunology and Medical Microbiology, Wiley-Blackwell: No OnlineOpen, 2010, 59 (3), pp.357-63. ⟨10.1111/j.1574-695X.2010.00681.x⟩
- Publication Year :
- 2010
- Publisher :
- HAL CCSD, 2010.
-
Abstract
- International audience; Porphyromonas gingivalis is an anaerobic periodontal pathogen that resides in the complex multispecies microbial biofilm known as dental plaque. Effective reporter tools are increasingly needed to facilitate physiological and pathogenetic studies of dental biofilm. Fluorescent proteins are ideal reporters for conveniently monitoring biofilm growth, but are restricted by several environmental factors, such as a requirement of oxygen to emit fluorescence. We developed a fluorescent reporter plasmid, known as the SNAP-tag, for labeling P. gingivalis cells, which encode an engineered version of the human DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase. Fluorescent substrates containing O(6)-benzylguanine covalently and specifically bind to the enzyme via stable thioether bonds. For the present study, we constructed a replicative plasmid carrying SNAP26b under the control of the P. gingivalis endogenous trxB promoter. The P. gingivalis-expressing SNAP26 protein was successfully labeled with specific fluorophores under anaerobic conditions. Porphyromonas gingivalis biofilm formation was investigated using flow cells and confocal laser scanning microscopy. A specific distribution of a strong fluorescence signal was demonstrated in P. gingivalis-SNAP26 monospecies and bispecies biofilms with Streptococcus gordonii-GFPmut3(*). These findings show that the SNAP-tag can be applied to studies of anaerobic bacteria in biofilm models and is a useful and advantageous alternative to existing labeling strategies.
- Subjects :
- Gene Expression
MESH: Flow Cytometry
MESH: Streptococcus gordonii
MESH: Bacteriological Techniques
biofilm
Green fluorescent protein
MESH: Recombinant Proteins
Genes, Reporter
Immunology and Allergy
MESH: Microscopy, Confocal
Anaerobiosis
Promoter Regions, Genetic
0303 health sciences
Microscopy, Confocal
green fluorescent protein (GFP)
Streptococcus gordonii
General Medicine
MESH: Genetics, Microbial
Flow Cytometry
Recombinant Proteins
SNAP-tag
MESH: Staining and Labeling
Infectious Diseases
[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology
Biochemistry
MESH: Porphyromonas gingivalis
Anaerobic bacteria
Porphyromonas gingivalis
Plasmids
Alkyltransferase
Genetics, Microbial
Microbiology (medical)
MESH: Gene Expression
Green Fluorescent Proteins
Immunology
MESH: Biofilms
Biology
Dental plaque
Microbiology
Fluorescence
O(6)-Methylguanine-DNA Methyltransferase
03 medical and health sciences
MESH: Green Fluorescent Proteins
MESH: Plasmids
MESH: Anaerobiosis
MESH: Promoter Regions, Genetic
medicine
Humans
Molecular Biology
[SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology
030304 developmental biology
Bacteriological Techniques
MESH: Humans
Staining and Labeling
MESH: O(6)-Methylguanine-DNA Methyltransferase
030306 microbiology
MESH: Molecular Biology
MESH: Fluorescence
MESH: Genes, Reporter
Biofilm
biology.organism_classification
medicine.disease
Biofilms
confocal laser scanning microscopy (CLSM)
Subjects
Details
- Language :
- English
- ISSN :
- 09288244 and 1574695X
- Database :
- OpenAIRE
- Journal :
- FEMS Immunology and Medical Microbiology, FEMS Immunology and Medical Microbiology, 2010, 59 (3), pp.357-63. ⟨10.1111/j.1574-695X.2010.00681.x⟩, FEMS Immunology and Medical Microbiology, Wiley-Blackwell: No OnlineOpen, 2010, 59 (3), pp.357-63. ⟨10.1111/j.1574-695X.2010.00681.x⟩
- Accession number :
- edsair.doi.dedup.....feb339c490656c29227d9e1c5d3bbc51