Abstract
Here we describe protocols for using the red fluorescent protein mCherryOpt in Clostridium difficile. The protocols can be readily adapted to similar fluorescent proteins (FPs), such as green fluorescent protein (GFP) and cyan fluorescent protein (CFP). There are three critical considerations for using FPs in C. difficile. (1) Choosing the right color: Blue and (especially) red are preferred because C. difficile exhibits considerable yellow-green autofluorescence. (2) Codon optimization: Most FP genes in general circulation have a GC content of ~60 %, so they are not well expressed in low-GC bacteria. (3) Fixing anaerobically grown cells prior to exposure to O2: The FPs under consideration here are non-fluorescent when produced anaerobically because O2 is required to introduce double bonds into the chromophore. Fixation prevents C. difficile cells from becoming degraded during the several hours required for chromophore maturation after cells are exposed to air. Fixation can probably be omitted for studies in which maintaining cellular architecture is not important, such as using mCherryOpt to monitor gene expression.
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References
Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544. doi:10.1146/annurev.biochem.67.1.509
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909. doi:10.1038/nmeth819
Remington SJ (2011) Green fluorescent protein: a perspective. Protein Sci 20(9):1509–1519. doi:10.1002/pro.684
Margolin W (2012) The price of tags in protein localization studies. J Bacteriol 194(23):6369–6371. doi:10.1128/JB.01640-12
Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci U S A 91(26):12501–12504
Buckley AM, Petersen J, Roe AJ, Douce GR, Christie JM (2015) LOV-based reporters for fluorescence imaging. Curr Opin Chem Biol 27:39–45. doi:10.1016/j.cbpa.2015.05.011
Drepper T, Huber R, Heck A, Circolone F, Hillmer AK, Buchs J, Jaeger KE (2010) Flavin mononucleotide-based fluorescent reporter proteins outperform green fluorescent protein-like proteins as quantitative in vivo real-time reporters. Appl Environ Microbiol 76(17):5990–5994. doi:10.1128/AEM.00701-10
Ransom EM, Ellermeier CD, Weiss DS (2015) Use of mCherry Red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile. Appl Environ Microbiol 81(5):1652–1660. doi:10.1128/AEM.03446-14
Zhang C, Xing XH, Lou K (2005) Rapid detection of a gfp-marked Enterobacter aerogenes under anaerobic conditions by aerobic fluorescence recovery. FEMS Microbiol Lett 249(2):211–218. doi:10.1016/j.femsle.2005.05.051
Ransom EM, Williams KB, Weiss DS, Ellermeier CD (2014) Identification and characterization of a gene cluster required for proper rod shape, cell division, and pathogenesis in Clostridium difficile. J Bacteriol 196(12):2290–2300. doi:10.1128/JB.00038-14
Sastalla I, Chim K, Cheung GY, Pomerantsev AP, Leppla SH (2009) Codon-optimized fluorescent proteins designed for expression in low-GC gram-positive bacteria. Appl Environ Microbiol 75(7):2099–2110. doi:10.1128/AEM.02066-08
Fagan RP, Fairweather NF (2011) Clostridium difficile has two parallel and essential Sec secretion systems. J Biol Chem 286(31):27483–27493. doi:10.1074/jbc.M111.263889
Trieu-Cuot P, Arthur M, Courvalin P (1987) Origin, evolution and dissemination of antibiotic resistance genes. Microbiol Sci 4(9):263–266
Ausubel FM (2002) Short protocols in molecular biology: a compendium of methods from Current protocols in molecular biology, 5th edn. Wiley, New York
Gibson DG (2009) Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Res 37(20):6984–6990. doi:10.1093/nar/gkp687
Heap JT, Pennington OJ, Cartman ST, Minton NP (2009) A modular system for Clostridium shuttle plasmids. J Microbiol Methods 78(1):79–85. doi:10.1016/j.mimet.2009.05.004
Barra-Carrasco J, Olguin-Araneda V, Plaza-Garrido A, Miranda-Cardenas C, Cofre-Araneda G, Pizarro-Guajardo M, Sarker MR, Paredes-Sabja D (2013) The Clostridium difficile exosporium cysteine (CdeC)-rich protein is required for exosporium morphogenesis and coat assembly. J Bacteriol 195(17):3863–3875. doi:10.1128/JB.00369-13
Pereira FC, Saujet L, Tome AR, Serrano M, Monot M, Couture-Tosi E, Martin-Verstraete I, Dupuy B, Henriques AO (2013) The spore differentiation pathway in the enteric pathogen Clostridium difficile. PLoS Genet 9(10):e1003782. doi:10.1371/journal.pgen.1003782
Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60(5):523–533. doi:10.1007/s00253-002-1158-6
Acknowledgments
The work described in this chapter was funded by National Institutes of Health grants GM-083975 to D.S.W., AI-087834 to C.D.E., and the Department of Microbiology at The University of Iowa.
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Ransom, E.M., Weiss, D.S., Ellermeier, C.D. (2016). Use of mCherryOpt Fluorescent Protein in Clostridium difficile . In: Roberts, A., Mullany, P. (eds) Clostridium difficile. Methods in Molecular Biology, vol 1476. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6361-4_5
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DOI: https://doi.org/10.1007/978-1-4939-6361-4_5
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