Development of SNAP-tag-mediated live cell labeling as an alternative to GFP in Porphyromonas gingivalis.
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.
Nicolle
Ophélie
O
Equipe de Microbiologie, UPRES-EA 1254, Université Européenne de Bretagne, Université de Rennes I, Campus Médecine, Pharmacie, UFR Odontologie, Rennes, France.
Rouillon
Astrid
A
Guyodo
Helene
H
Tamanai-Shacoori
Zohreh
Z
Chandad
Fatiha
F
Meuric
Vincent
V
Bonnaure-Mallet
Martine
M
D000693:Anaerobiosis; D001431:Bacteriological Techniques / Q000379:methods*; D018441:Biofilms; D005434:Flow Cytometry; D005453:Fluorescence; D015870:Gene Expression; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D008967:Molecular Biology / Q000379:methods*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D054773:Streptococcus gordonii / Q000235:genetics / Q000254:growth & development
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase