Visualization Of Bacteria On Plants Available Tools

26.2.1 Labeling of Bacteria with Fluorescent Proteins

The recent renaissance in the application of microscopy to the study of bacterial behavior on plants is largely attributable to the discovery of GFP. The popularity of GFP as a fluorophore lies in its bright and relatively stable fluorescence, the expression of gfp in most bacterial species, and the ability to use it as an intrinsic label without the need for a substrate. The latter property means that samples can be mounted without prior processing for visualization under the microscope, and thus disruption of bacterial cells in the environment under study is minimal. This is in sharp contrast with the potential artifacts generated during sample preparation for immunofluorescence or electron microscopy. Additionally, mutants of GFP that have enhanced fluorescence intensity and emit at various wavelengths of the visible spectrum have provided investigators with versatile tools to circumvent problems with detecting the GFP signal against autofluorescent backgrounds. This problem is especially prevalent in plant tissues, which emit with various intensities in different regions of the visible spectrum [1]. It is exacerbated also by contamination of the image with stray fluorescence, but may be minimized by the use of confocal microscopy. Because the confocal laser scanning microscope (CLSM) collects the fluorescent signal solely from the focal plane by rejecting scattered light with its pinhole, the CLSM can improve greatly the detection of fluorescently tagged bacterial cells on plants.

Despite the availability of GFP mutants with enhanced fluorescence intensity, such as the widely used S65T mutant [2], the detection of GFP fluorescence at low levels of expression in bacterial cells remains difficult. Therefore, intrinsic labeling with GFP is commonly achieved by transforming the bacterial strain of interest with a plasmid that is maintained stably at a moderate copy number per cell and that harbors gfp expressed from strong promoters. Adverse effects of GFP on the fitness of bacteria that were transformed with high copy number plasmids encoding GFP have been reported [3], presumably because the resultant high concentrations of GFP overload bacterial metabolism or disrupt cellular functions, and thus use of such plasmids should be avoided.

GFP production from single insertion of gfp into the bacterial chromosome may circumvent problems associated with excessive GFP concentrations or plasmid instability, but may still decrease the competitiveness of the tagged strain compared to its parental strain under stressful conditions such as carbon-substrate limitation [4]. In addition, gfp expression from a single location on the bacterial chromosome may yield a signal-to-noise ratio that approaches the lower limit of detection for GFP in a strong fluorescent background. In such cases, GFP fluorescence may be imaged only by high signal gain, which results in poor definition of the bacterial cell profile and in grainy images [5]. This is particularly true for imaging of dim GFP-tagged bacteria on plant tissue that emits in the green range, e.g., the roots of certain plant species.

Expression of gfp from plasmids requires that plasmid stability in the transformed bacterial strain be assessed in the plant environment under study. By comparison of population sizes of E. coli O157:H7 cells that showed green fluorescence to those that were detected by immunolabeling, Takeuchi and Frank demonstrated that E. coli O157:H7 pEGFP retained pEGFP, or GFP per se, at higher frequency on lettuce leaves and cauliflower florets than on tomato [6]. Since few broad-host-range plasmids are completely stable in any bacterial species, it is expected that the frequency at which a GFP-encoding plasmid is lost in a cell population will increase as the bacterial growth rate increases, or as physiological stress impacts that population. Therefore, the retention of a GFP plasmid among a bacterial population may vary greatly depending on the experimental conditions and on the plant host tissue or species studied.

In addition to GFP and its color variants, intrinsic fluorophores emitting in the near red (DsRed) and far red (HcRed) have been cloned from Discoma and Heteractis crispa, respectively [7,8]. The availability of fluorophores that span the visible range allow for multispectral imaging of bacteria within a same sample. The excitation and emission spectra of the red fluorescent proteins and of the most widely used variants of GFP are shown in Figure 26.1. The peaks of their excitation and emission spectra are listed in Table 26.1.

Wavelength (nm)

FIGURE 26.1 Excitation (A) and emission (B) spectra of blue (BFP), cyan (CFP), green (GFP), and red (DsRed and HcRed) fluorescent proteins suitable for intrinsic labeling of bacteria. The Y-axis represents the normalized fluorescence intensity. (Fluorescence data courtesy of BD Biosciences Clontech, Palo Alto, CA.)

Wavelength (nm)

FIGURE 26.1 Excitation (A) and emission (B) spectra of blue (BFP), cyan (CFP), green (GFP), and red (DsRed and HcRed) fluorescent proteins suitable for intrinsic labeling of bacteria. The Y-axis represents the normalized fluorescence intensity. (Fluorescence data courtesy of BD Biosciences Clontech, Palo Alto, CA.)

26.2.2 Labeling of Bacteria With Dyes and Fluorescent Conjugates

In addition to fluorescent proteins, a wide range of fluorescent dyes and bioconjugates are available for the visualization of bacteria on plants. Exhaustive lists of fluorescent probes and their applications in biological microscopy are beyond the scope of this chapter, but can be found in an excellent review by Kasten [9]. The DNA-intercalating stain acridine orange (AO) and the newer SYTO® dyes (Molecular Probes, Eugene, OR), which are available in a wide range of the visible spectrum, are particularly useful to visualize bacteria against the autofluorescent plant background. 4',6-Diamidino-2-phenylindole (DAPI), also a nucleic acid stain, requires ultraviolet (UV) illumination, and thus may cause extensive damage to plant cells. Other dyes, such as Sypro® Orange, a general protein stain, and Nile Red (Molecular Probes), which stains lipid-hydrophobic sites , have been applied to the study of bacterial biofilms [10].

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