Background

Ca²⁺ is an emerging intracellular messenger of bacteria that impacts a wide array of cellular processes such as the maintenance of cell integrity, cell division (Dominguez et al., 2015), motility (Tisa and Alder, 1995; Gode-Potratz et al., 2010; Cruz et al., 2012; Guragain et al., 2013; Parker et al., 2015; Fishman et al., 2018), type III secretion (DeBord et al., 2003; Dasgupta et al., 2006; Gode-Potratz et al., 2010; Fishman et al., 2018), gene expression (Dominguez et al., 2015), quorum sensing (Werthén and Lundgren, 2001), biofilm formation (Patrauchan et al., 2005; Sarkisova et al., 2005; Rinaudi et al., 2006; Cruz et al., 2012; Das et al., 2014; Zhou et al., 2014; Parker et al., 2016) or biofilm suppression (Bilecen and Yildiz, 2009; Shukla and Rao, 2013). Recently, it was demonstrated that the intracellular Ca²⁺ concentration controls virulence of Pseudomonas savastanoi pv. savastanoi (Psav) (Moretti et al., 2019). Furthermore, several known virulence genes were upregulated in the presence of increasing Ca²⁺ concentrations in Pseudomonas syringae pv. tomato (Pto) DC3000 (Fishman et al., 2018), and Xylella fastidiosa (Parker et al., 2016). Measurement of the intracellular free-calcium concentration, [Ca²⁺]_i, in prokaryotes has, therefore, become of great interest to study its role as intracellular messenger of bacteria in response to environmental cues. Monitoring the [Ca²⁺]_i within bacterial cells, which is indispensable for understanding the correlations between the transport of Ca²⁺ across the plasma membrane and cellular processes was thus far difficult, as it was hampered by the small size of the bacterial cells, the semi-selective nature of the bacterial cell wall, the low membrane permeability, and the toxicity of many Ca²⁺ chelators used (Gangola and Rosen, 1987; Knight et al., 1991; Futsaether and Johnsson, 1994; Norris et al., 1996; Herbaud et al., 1998; Jones et al.,1999; Torrecilla et al., 2001). A well-established method to determine changes in the [Ca²⁺]_i in prokaryotes is based on the heterologous expression of Aequorin (a calcium-activated photoprotein) bacterial cells (Watkins et al., 1995). This method employs the expression of recombinant Aequorin (from a plasmid or integrated in the bacterial genome), which emits light upon Ca²⁺ binding. This method is rather time-consuming, requires the availability of molecular biology tools, and is technically challenging. In fact, the method is better suited for eukaryotic cells even if it was successfully used to monitor [Ca²⁺]_i in several bacterial species (Naseem et al., 2007; Guragain et al., 2016). To overcome these limitations of Aequorin, we developed an alternative and complementary spectrofluorometric method based on the chemical probe Fura 2-AM {1-[2-(5-carboxyoxazol-2-yl)-6-amino-benzofuran-5-oxy]-2-(2’-amino-5’-methylphenoxy) ethan-N,N,N’,N’-tetraacetic acid}. Importantly, both the assay solution used and the Fura 2-AM do not compromise cell viability at the concentrations here used (Gangola and Rosen, 1987; Futsaether and Johnsson, 1994; Tisa and Alder, 1995; Norris et al., 1996; Jones et al., 1999), meaning that this method allows quantification of [Ca²⁺]_i in response to external cues and different conditions without the need of advanced equipment (Moretti et al., 2019). It must be pointed out that Fura 2-AM is a probe that diffuses across the cell membrane of viable bacterial cells and its subsequent rapid de-esterification by cellular esterases yields Fura 2, which retains the ability to bind the cytosolic Ca²⁺ while it losses the ability to diffuse across the cell membrane (Figure 1). When Fura 2 forms a complex with Ca²⁺, the intensity of the fluorescence at λ=510 nm increases with increasing Ca²⁺ concentration (Grynkiewicz et al., 1985) (Figure 2). In addition, Fura 2 is unable to permeate bacterial cells itself due to the selective permeability of the cell walls and membranes (Grynkiewicz et al., 1985). Of notice, since the measurements make use of a fluorescence signal that only becomes apparent inside cells (when Fura 2-AM is converted to Fura 2), it is not necessary to use unloaded viable cells as negative control. In fact, if the cells are not viable, the cell membrane loses its integrity and the probe would not be trapped in the cells but rather remain dispersed in the incubation medium.

Figure 1. The fate of Fura 2-AM in cells. Fura 2-AM is de-esterified by cellular esterases and transformed in Fura 2, which is able to form a complex with cytosolic calcium (Ca²⁺) and cannot passively cross the cell membrane.

Figure 2. Excitation spectra of Fura 2. Excitation spectra of Fura 2 in solution containing 0 to 39.8 µM of free calcium (Ca²⁺). Modified from Grynkiewicz et al. (1985).

We find that the fluorescent probe Fura 2-AM is highly sensitive allowing us to determine changes in cytosolic Ca²⁺ levels in Pseudomonas savastanoi pv. savastanoi DAPP-PG 722 (Moretti et al., 2019) and Pseudomonas syringae pv. tomato DC3000 (Trabalza et al., in preparation) cells by using a spectrofluorometer equipped with a stirred semi-micro cuvette.