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Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis   

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A brief version of this protocol appeared in:
Molecular Microbiology
Jun 2016

Abstract

One of the most successful fluorescent proteins, used as a reporter of gene expression in many bacterial, plant and animals, is green fluorescent protein and its modified forms, which also function well in cyanobacteria. However, these fluorescent proteins do not allow rapid and economical quantitation of the reporter gene product, as does the popular reporter gene lacZ, encoding the enzyme β-galactosidase. We provide here a protocol for the in situ localization of β-galactosidase activity in cyanobacterial cells. This allows the same strain to be used for both a simple, quantitative, colorimetric assay with the substrate ortho-nitrophenyl-β-galactoside (ONPG) and for sensitive, fluorescence-based, cell-type localization of gene expression using 5-dodecanolyaminofluorescein di-β-D-galactopyranoside (C12-FDG).

Keywords: β-galactosidase, in situ localization, Heterocysts, Cyanobacteria, lacZ reporter

Background

Anabaena variabilis is a filamentous cyanobacterium that differentiates specialized cells called heterocysts that function specifically for nitrogen fixation (Kumar et al., 2010; Maldener and Muro-Pastor, 2010). We use the lacZ gene of Escherichia coli as a transcriptional reporter of cyanobacterial gene expression because of the ease of a quantitative, enzymatic, colorimetric, β-galactosidase assay in 96-well plates (Griffith and Wolf, 2002) and the ability to use the same strain for in situ localization of gene expression using the fluorescent substrate 5-dodecanolyaminofluorescein di-β-D-galactopyranoside (C12-FDG) (Thiel et al., 1995; Ma et al., 2016). One of the earliest reports of lacZ as a reporter was the fusion of malF, encoding the maltose transporter, to lacZ, which resulted in localization of β-galactosidase activity to the cytoplasmic membrane in E. coli (Silhavy et al., 1976). Since then lacZ has been used as a reporter in bacterial, plant and animal systems; e.g., the stable transfection of mouse tumor cells with lacZ allowed single cell histochemical staining using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (Arlt et al., 2012). In fact, most cellular localization of expression of lacZ has used X-gal, which is relatively inexpensive, easy to use and provides an easy visual screen. Our initial attempts to use X-gal and other chromogenic substrates in Anabaena were unsuccessful because the colored products were toxic to cyanobacteria and often resulted in cell lysis. In addition, the cyanobacterial pigments, including chlorophyll, phycocyanin, and carotenoids, made color detection difficult. We also attempted to use the fluorescent substrate, 4-methylumbelliferone β-D-galactopyranoside, whose product, 4-methylumbelliferone, emits in the blue range; however, we were not able to detect fluorescence over the background fluorescence of the cells. Finally we tried fluorescein β-D-galactopyranoside (FDG), a very sensitive fluorogenic substrate for β-galactosidase. FDG, which is not fluorescent, is hydrolyzed in two steps by β-galactosidase, first to fluorescein monogalactoside and then to fluorescein. We modified the method developed to visualize lacZ expression during sporulation in Bacillus subtilis (Bylund et al., 1994; Chung et al., 1995). That protocol specified 5-octanolyaminofluorescein di-β-D-galactopyranoside (C8-FDG); however we had poor results with C8-FDG, so we tried the more lipophilic 5-dodecanolyaminofluorescein di-β-D-galactopyranoside (C12-FDG) (Miao et al., 1993; Plovins et al., 1994; Zhang et al., 1991), which has 12 carbons added to the fluorescein in FDG. C12-FDG proved to function well in cyanobacteria. Using C12-FDG we have been able to easily visualize heterocyst-specific expression of genes, such as cnfR1, the activator of the nitrogenase genes in heterocysts (Pratte and Thiel, 2016), fused to lacZ (Figure 1).

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Pratte, B. S. and Thiel, T. (2017). Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis. Bio-protocol 7(1): e2084. DOI: 10.21769/BioProtoc.2084.
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