牙龈卟啉单胞菌
格氏链球菌
生物膜
生物
绿色荧光蛋白
微生物学
荧光显微镜
牙周病原体
质粒
厌氧菌
细菌
DNA
生物化学
荧光
基因
物理
量子力学
遗传学
作者
Ophélie Nicolle,Astrid Rouillon,Hélène Guyodo,Zohreh Tamanai-Shacoori,Fatiha Chandad,Vincent Meuric,Martine Bonnaure‐Mallet
出处
期刊:Fems Immunology and Medical Microbiology
[Oxford University Press]
日期:2010-08-01
卷期号:59 (3): 357-363
被引量:22
标识
DOI:10.1111/j.1574-695x.2010.00681.x
摘要
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.
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