Development of SNAP-tag-mediated live cell labeling as an alternative to GFP inPorphyromonas gingivalis

Abstract

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.

Oral Porphyromonas gingivalis translocates to the liver and regulates hepatic glycogen synthesis through the Akt/GSK-3β signaling pathway

“…SNAP-P.g. was constructed by transforming P. gingivalis ATCC33277 with a shuttle vector containing the SNAP26b gene cloned under the control of the P. gingivalis endogenous trxB promoter into P. gingivalis ATCC33277, as described previously [22] with modifications. The schema of SNAP-P.g.…”

Generalized Schemes for High-Throughput Manipulation of the Desulfovibrio vulgaris Genome

“…Conventional green fluorescent protein derivatives require molecular oxygen for proper chromophore formation and hence cannot be utilized under anaerobic culturing conditions. We therefore explored the use of a modified SNAP tag that has a dead-end reaction with a modified O 6 -benzylguanine (BG) derivative (32,35). To validate the use of the AGT-tagbased method for subcellular localization in anaerobic bacteria, we first compared the SNAP labelings of three AGTtagged proteins from D. vulgaris, DsrC (DVU2776), MreB (DVU0789) (data not shown), and FtsZ (DVU2499), from the respective engineered strains to the unmodified wild-type strain.…”

Syntheses and characterizations of novel pyrrolocoumarin probes for SNAP-tag labeling technology

“…FT-IR spectra were recorded on an AVATAR-360 spectrophotometer. 1 H and 13 C NMR spectra were recorded on Bruker DRX-400, or Bruker DRX-300 spectrometers with TMS as the internal standard. HRMS were measured by using either FTMS-7 or IonSpec 4.7 spectrometers.…”

“…1 In the last decade, the green fluorescent protein (GFP) and its variants have emerged as the most powerful tool in the optical research of protein functions due to their bright clear fluorescence, which is detectable even in single cells. 2 However, the monotone fluorescent properties of GFPs hinders them from further application in some cases.…”