title: Codon-optimized fluorescent mTFP and mCherry for microscopic visualization and genetic counterselection of streptococci and enterococci.
abstract: Despite the powerful potential of fluorescent proteins for labeling bacteria, their use has been limited in multi-species oral biofilm models. Fermentative metabolism by streptococcal species that initiate biofilm colonization results in an acidic, reduced microenvironment that may limit the activities of some fluorescent proteins which are influenced by pH and oxygen availability. The need to reliably distinguish morphologically similar strains within biofilms was the impetus for this work. Teal fluorescent protein (mTFP1) and red fluorescent protein (mCherry) were chosen because their fluorescent properties made them promising candidates. Since tRNA availability has been implicated in efficient translation of sufficient quantities of protein for maximum fluorescence, a streptococcal codon optimization approach was used. DNA was synthesized to encode either protein using codons most frequently used in streptococci; each coding region was preceded by an engineered ribosomal binding site and restriction sites for cloning a promoter. Plasmids carrying this synthesized DNA under control of the Streptococcus mutans lactate dehydrogenase promoter conferred fluorescence to nine representative streptococcal and two Enterococcus faecalis strains. Further characterization in Streptococcus gordonii showed that mTFP1 and mCherry expressions could be detected in cells grown planktonically, in biofilms, or in colonies on agar when expressed on an extrachromosomal plasmid or in single copy integrated into the chromosome. This latter property facilitated counterselection of chromosomal mutations demonstrating value for bacterial strain construction. Fluorescent and non-fluorescent bacteria were distinguishable at acidic pH. These codon-optimized versions of mTFP1 and mCherry have promising potential for use in multiple experimental applications.
authors.affiliation: Department of Periodontics and Endodontics, University at Buffalo School of Dental Medicine, Buffalo, NY, USA; Department of Oral Biology, University at Buffalo School of Dental Medicine, Buffalo, NY, USA. Electronic address: mmv4@buffalo.edu.
authors.forename: M Margaret
authors.initials: MM
authors.lastname: Vickerman
authors.affiliation: Department of Periodontics and Endodontics, University at Buffalo School of Dental Medicine, Buffalo, NY, USA; Department of Oral Biology, University at Buffalo School of Dental Medicine, Buffalo, NY, USA.
authors.forename: Jillian M
authors.initials: JM
authors.lastname: Mansfield
authors.affiliation: Dows Institute for Research, University of Iowa College of Dentistry, Iowa City, IA, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Zhu
authors.affiliation: Central Microscopy Research Facility, University of Iowa, Iowa City, IA, USA.
authors.forename: Katherine S
authors.initials: KS
authors.lastname: Walters
authors.affiliation: Dows Institute for Research, University of Iowa College of Dentistry, Iowa City, IA, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Banas
mesh_terms: D001483:Base Sequence; D018441:Biofilms / Q000254:growth & development; D003062:Codon; D016983:Enterococcus / Q000166:cytology / Q000235:genetics*; D005456:Fluorescent Dyes; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D049408:Luminescent Agents; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009154:Mutation; D011401:Promoter Regions, Genetic; D013291:Streptococcus / Q000166:cytology / Q000235:genetics*; D054773:Streptococcus gordonii / Q000166:cytology / Q000235:genetics / Q000254:growth & development; D000097573:Red Fluorescent Protein
chemical_list: D003062:Codon; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D049408:Luminescent Agents; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C485182:Cyan Fluorescent Protein
keywords: Biofilms; Codon-optimized; Fluorescent proteins; Oral streptococci; mCherry; mTFP