Here, we present a protocol for visualizing gene expression in the filamentous cyanobacterium Trichodesmium and the single-celled species Synechocystis and Cyanothece using the catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) technique. We describe steps for fixation, agarose coating, enzymatic permeabilization, and sample handling. This protocol is broadly applicable to cyanobacteria, and the detection of rbcL mRNA in Trichodesmium, used as an example, supports its use to study the heterogeneity of physiological processes at single-cell level.

Department of Fisheries Oceanography and Marine Ecology National Marine Fisheries Research Institute Hugo Kołłątaja 1 81 332 Gdynia Poland

Laboratory of Photosynthesis Institute of Microbiology of the Czech Academy of Sciences Centre Algatech Novohradská 237 379 01 Třeboň Czech Republic

Laboratory of Photosynthesis Institute of Microbiology of the Czech Academy of Sciences Centre Algatech Novohradská 237 379 01 Třeboň Czech Republic; Faculty of Science University of South Bohemia in České Budějovice Branišovská 1645 31a 370 05 České Budějovice Czech Republic

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Amann R., Fuchs B.M. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat. Rev. Microbiol. 2008;6:339–348. doi: 10.1038/nrmicro1888. PubMed DOI

Amann R.I., Binder B.J., Olson R.J., Chisholm S.W., Devereux R., Stahl D.A. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 1990;56:1919–1925. doi: 10.1128/aem.56.6.1919-1925.1990. PubMed DOI PMC

Amann R.I., Ludwig W., Schleifer K.H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 1995;59:143–169. doi: 10.1128/mr.59.1.143-169.1995. PubMed DOI PMC

Wagner M., Horn M., Daims H. Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. Curr. Opin. Microbiol. 2003;6:302–309. doi: 10.1016/S1369-5274(03)00054-7. PubMed DOI

Mahbub M., Hemm L., Yang Y., Kaur R., Carmen H., Engl C., Huokko T., Riediger M., Watanabe S., Liu L.-N., et al. mRNA localization, reaction centre biogenesis and thylakoid membrane targeting in cyanobacteria. Nat. Plants. 2020;6:1179–1191. doi: 10.1038/s41477-020-00764-2. PubMed DOI

Mahbub M., Mullineaux C.W. Locations of membrane protein production in a cyanobacterium. J. Bacteriol. 2023;205:e0020923. doi: 10.1128/jb.00209-23. PubMed DOI PMC

Wang K., Mahbub M., Mastroianni G., Valladares A., Mullineaux C.W. mRNA localization and thylakoid protein biogenesis in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. J. Bacteriol. 2024;206:e00328-24. doi: 10.1128/jb.00328-24. PubMed DOI PMC

Pilhofer M., Pavlekovic M., Lee N.M., Ludwig W., Schleifer K.H. Fluorescence in situ hybridization for intracellular localization of nifH mRNA. Syst. Appl. Microbiol. 2009;32:186–192. doi: 10.1016/j.syapm.2008.12.007. PubMed DOI

Mota C.R., So M.J., de los Reyes F.L. Identification of nitrite-reducing bacteria using sequential mRNA fluorescence in situ hybridization and fluorescence-assisted cell sorting. Microb. Ecol. 2012;64:256–267. doi: 10.1007/s00248-012-0018-x. PubMed DOI

Zeller P., Méjean A., Biegala I., Contremoulins V., Ploux O. Fluorescence in situ hybridization of Microcystis strains producing microcystin using specific mRNA probes. Lett. Appl. Microbiol. 2016;63:376–383. doi: 10.1111/lam.12634. PubMed DOI

Brient L., Ben Gamra N., Periot M., Roumagnac M., Zeller P., Bormans M., Méjean A., Ploux O., Biegala I.C. Rapid characterization of microcystin-producing cyanobacteria in freshwater lakes by TSA-FISH (tyramid signal amplification-fluorescent in situ hybridization) Front. Environ. Sci. 2017;5:43. doi: 10.3389/fenvs.2017.00043. DOI

Lee N., Nielsen P.H., Andreasen K.H., Juretschko S., Nielsen J.L., Schleifer K.H., Wagner M. Combination of fluorescent in situ hybridization and microautoradiography—a new tool for structure-function analyses in microbial ecology. Appl. Environ. Microbiol. 1999;65:1289–1297. doi: 10.1128/AEM.65.3.1289-1297.1999. PubMed DOI PMC

Schönhuber W., Fuchs B., Juretschko S., Amann R. Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification. Appl. Environ. Microbiol. 1997;63:3268–3273. doi: 10.1128/aem.63.8.3268-3273.1997. PubMed DOI PMC

Pernthaler A., Pernthaler J., Amann R. Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl. Environ. Microbiol. 2002;68:3094–3101. doi: 10.1128/AEM.68.6.3094-3101.2002. PubMed DOI PMC

Lim E.L., Amaral L.A., Caron D.A., DeLong E.F. Application of rRNA-based probes for observing marine nanoplanktonic protists. Appl. Environ. Microbiol. 1993;59:1647–1655. doi: 10.1128/aem.59.5.1647-1655.1993. PubMed DOI PMC

Bakermans C., Madsen E.L. Detection in coal tar waste-contaminated groundwater of mRNA transcripts related to naphthalene dioxygenase by fluorescent in situ hybridization with tyramide signal amplification. J. Microbiol. Methods. 2002;50:75–84. doi: 10.1016/S0167-7012(02)00015-5. PubMed DOI

Pernthaler A., Amann R. Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria. Appl. Environ. Microbiol. 2004;70:5426–5433. doi: 10.1128/AEM.70.9.5426-5433.2004. PubMed DOI PMC

Masuda T., Majerová D., Piwosz K., Tsurumaki T., Fujita Y., Prášil O. Immunocytochemical visualization of proteins from cyanobacterial cells with high autofluorescence of phycoerythrin and phycourobilin. J. Vis. Exp. 2023;199 doi: 10.3791/65168. PubMed DOI

Mullineaux C.W., Wang K., Mahbub M. RNA-FISH as a probe for heterogeneity at the cellular and subcellular levels in cyanobacteria. Front. Photobiol. 2024;2 doi: 10.3389/fphbi.2024.1511953. DOI

Vijayan V., Jain I.H., O'Shea E.K. A high resolution map of a cyanobacterial transcriptome. Genome Biol. 2011;12:R47. doi: 10.1186/gb-2011-12-5-r47. PubMed DOI PMC

Moran M.A., Satinsky B., Gifford S.M., Luo H., Rivers A., Chan L.-K., Meng J., Durham B.P., Shen C., Varaljay V.A., et al. Sizing up metatranscriptomics. ISME J. 2013;7:237–243. doi: 10.1038/ismej.2012.94. PubMed DOI PMC

Zhang J.Y., Hess W.R., Zhang C.C. Life is short, and art is long: RNA degradation in cyanobacteria and model bacteria. mLife. 2022;1:21–39. doi: 10.1002/mlf2.12015. PubMed DOI PMC

Grujcic V., Taylor G.T., Foster R.A. One cell at a time: advances in single-cell methods and instrumentation for discovery in aquatic microbiology. Front. Microbiol. 2022;13 doi: 10.3389/fmicb.2022.881018. PubMed DOI PMC

Daims H., Brühl A., Amann R., Schleifer K.H., Wagner M. The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 1999;22:434–444. doi: 10.1016/S0723-2020(99)80053-8. PubMed DOI

Wallner G., Amann R., Beisker W. Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry. 1993;14:136–143. doi: 10.1002/cyto.990140205. PubMed DOI

Levitan O., Sudhaus S., LaRoche J., Berman-Frank I. The influence of pCO2 and temperature on gene expression of carbon and nitrogen pathways in Trichodesmium IMS101. PLoS One. 2010;5 doi: 10.1371/journal.pone.0015104. PubMed DOI PMC

Koedooder C., Landou E., Zhang F., Wang S., Basu S., Berman-Frank I., Shaked Y., Rubin-Blum M. Metagenomes of Red Sea subpopulations challenge the use of marker genes and morphology to assess Trichodesmium diversity. Front. Microbiol. 2022;13 doi: 10.3389/fmicb.2022.879970. PubMed DOI PMC

Grujcic V., Nuy J.K., Salcher M.M., Shabarova T., Kasalický V., Boenigk J., Jensen M., Šimek K. Cryptophyta as major bacterivores in freshwater summer plankton. ISME J. 2018;12:1668–1681. doi: 10.1038/s41396-018-0057-5. PubMed DOI PMC

Pernthaler A., Pernthaler J., Amann R. In: Molecular Microbial Ecology Manual. 2nd edition. Kowalchuk G., de Bruijn F.J., Head I.M., Akkermans A.D., Elsas J.D., editors. Kluwer Academic Publishers; 2004. Sensitive multicolor fluorescence in situ hybridization for the identification of environmental microorganisms; pp. 711–726.

Piwosz K., Mukherjee I., Salcher M.M., Grujčić V., Šimek K. CARD-FISH in the sequencing era: Opening a new universe of protistan ecology. Front. Microbiol. 2021;12 doi: 10.3389/fmicb.2021.640066. PubMed DOI PMC

Schneider C.A., Rasband W.S., Eliceiri K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods. 2012;9:671–675. doi: 10.1038/nmeth.2089. PubMed DOI PMC

Chen Y.B., Zehr J.P., Mellon M. Growth and nitrogen fixation of the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp IMS 101 in defined media: evidence for a circadian rhythm. J. Phycol. 1996;32:916–923. doi: 10.1111/j.0022-3646.1996.00916.x. DOI

Rippka R. Isolation and purification of cyanobacteria. Methods Enzymol. 1988;167:3–27. doi: 10.1016/0076-6879(88)67004-2. PubMed DOI

Wang S., Koedooder C., Zhang F., Kessler N., Eichner M., Shi D., Shaked Y. Colonies of the marine cyanobacterium Trichodesmium optimize dust utilization by selective collection and retention of nutrient-rich particles. iScience. 2022;25 doi: 10.1016/j.isci.2021.103587. PubMed DOI PMC

Zhang F., Wang S., Visser A.-N., Koedooder C., Eichner M., Anderson O.R., Dyhrman S.T., Shaked Y. Recurrent association between Trichodesmium colonies and calcifying amoebae. ISME Commun. 2024;4:ycae137. doi: 10.1093/ismeco/ycae137. PubMed DOI PMC

Berman-Frank I., Lundgren P., Chen Y.B., Küpper H., Kolber Z., Bergman B., Falkowski P. Segregation of nitrogen fixation and oxygenic photosynthesis in the marine cyanobacterium Trichodesmium. Science. 2001;294:1534–1537. doi: 10.1126/science.1064082. PubMed DOI

El-Shehawy R., Lugomela C., Ernst A., Bergman B. Diurnal expression of hetR and diazocyte development in the filamentous non-heterocystous cyanobacterium Trichodesmium erythraeum. Microbiol. 2003;149:1139–1146. doi: 10.1099/mic.0.26170-0. PubMed DOI

Sandh G., Xu L., Bergman B. Diazocyte development in the marine diazotrophic cyanobacterium Trichodesmium. Microbiol. 2012;158:345–352. doi: 10.1099/mic.0.051268-0. PubMed DOI

Hania A., López-Adams R., Prášil O., Eichner M. Protection of nitrogenase from photosynthetic O2 evolution in Trichodesmium: Methodological pitfalls and advances over 30 years of research. Photosynthetica. 2023;61:58–72. doi: 10.32615/ps.2023.007. PubMed DOI PMC

Siddiqui P.J.A., Carpenter E.J., Bergman B. Ultrastructure and immunolocalization of phycobiliproteins and ribulose-1,5-bisphosphate carboxylase/oxygenase in the marine cyanobacterium Trichodesmium thiebautii. J. Phycol. 1992;28:320–327. doi: 10.1111/j.0022-3646.1992.00320.x. DOI

Lin S., Henze S., Lundgren P., Bergman B., Carpenter E.J. Whole-cell immunolocalization of nitrogenase in marine diazotrophic cyanobacteria, Trichodesmium spp. Appl. Environ. Microbiol. 1998;64:3052–3058. doi: 10.1128/AEM.64.8.3052-3058.1998. PubMed DOI PMC