Quantitative Immunofluorescence of Regulated eps Gene Expression in Single Cells of Ralstonia solanacearum

Abstract

Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>107 cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. ForR. solanacearum, QIF was used to determine the amount of β-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis ofR. solanacearum cells recovered from stems of infected tomato plants showed that expression of epsduring pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed inR. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.

A Single Regulator Mediates Strategic Switching between Attachment/Spread and Growth/Virulence in the Plant Pathogen Ralstonia solanacearum

“…Most traits putatively regulated by PhcA have been experimentally verified only in vitro , with the important exception of EPS. Immunofluorescence microscopy of R. solanacearum -infected tomato plants using anti-EPS antibodies demonstrated that EPS production is dependent on cell density in planta ( 23 , 24 ). Accumulating evidence suggests that in vitro studies are not reliable for understanding R. solanacearum regulation during plant pathogenesis.…”

Fastidian gum: theXylella fastidiosaexopolysaccharide possibly involved in bacterial pathogenicity

“…The formation of biofilms is an important factor in the pathogenicity of several organisms [3]. Examples include the biofilm produced by Pseudomonas aeruginosa , an infectious bacterium in humans [4], and those produced by Ralstonia solanacearum , the ESP of which is important for the rapid systemic colonization of tomato plants and the subsequent symptoms caused by bacterial infection [5]. The EPS produced by Xanthomonas campestris may be required for this species’ virulence [6].…”

Strain characterization and 16S‐23S probe development for differentiating geographically dispersed isolates of the phytopathogen Ralstonia solanacearum

“…On entry, bacterial cells penetrate the xylem vessels and spread throughout the plant establishing foci of infection. The colonizing bacteria cause rot and tissue disintegration through the action of lytic enzymes, extracellular polysaccharides (EPS), endoglucanase (EG) and endopolygalacturonase (PG), which are the major virulence factors (Denny, 1995;Kang et al, 1999). In particular, EPS production contributes to the biomass of colonizing bacteria, resulting in the rapid wilting of infected plants (Saile et al, 1997;Araud-Razou et al, 1998).…”