Analysis of Extracellular Polysaccharide I In Culture and In Planta Using Immunological Methods: New Insights and Implications

McGarvey, Jeff A.; Bell, Colleen; Denny, Timothy P.; Schell, Mark A.

doi:10.1007/978-3-662-03592-4_23

Cited by 18 publications

(12 citation statements)

References 4 publications

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“…This dependence on growth conditions was also observed by the Tetrazolium red exclusion assay and by IFM using the EPS‐specific monoclonal antibody. Related studies have also demonstrated a relationship with cell growth rates, and EPS production was expressed only at cell levels above 10 7 cells ml −1 (McGarvey et al ., 1998). Ralstonia solanacearum is known to regulate the expression of a number of extracellular virulence factors in response to population density and environmental factors (Denny et al ., 1998; McGarvey et al ., 1998).…”

Section: Discussionmentioning

confidence: 99%

“…Related studies have also demonstrated a relationship with cell growth rates, and EPS production was expressed only at cell levels above 10 7 cells ml −1 (McGarvey et al ., 1998). Ralstonia solanacearum is known to regulate the expression of a number of extracellular virulence factors in response to population density and environmental factors (Denny et al ., 1998; McGarvey et al ., 1998). On the other hand, different proteins involved in virulence, such as those encoded by the hrp gene cluster, appear to be induced during the infection of susceptible plant host species.…”

Section: Discussionmentioning

confidence: 99%

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

Timms‐Wilson

Bryant

Bailey³

2001

Environmental Microbiology

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The causative agent of potato brown rot and bacterial wilt, Ralstonia solanacearum, results in serious world-wide economic losses, particularly in the tropics. In the last decade, however, the incidence of bacterial wilt in potatoes grown in Northern Europe has increased, presenting an interesting epidemiological puzzle. Its occurrence may be as a result of changes in agricultural practice or the emergence of a novel bacterial variety, better adapted to cooler conditions. To understand the distribution and genetic diversity of this phytopathogen, we have analysed a collection of 82 isolates from Europe and tropical regions. Both phenotypic [SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) profiling, FAME (fatty acid methyl esters) analysis, growth profiles and EPS (exopolysaccharide) production] and genotypic [16S rRNA RFLP (restriction fragment length polymorphism), ARDRA (amplified ribosomal DNA restriction analysis) and sequence analysis of 16S-23S rRNA ITS and flanking regions] methods were compared. Principal component analysis of FAME profiles clustered isolates into three groups and ARDRA of a 0.85 kb amplified fragment from the 16S-23S ITS region differentiated isolates into four groups. Using sequence analysis, specific primers were designed within the variable region 147-170 of the 23S rRNA. These primers, RsolT2 and RsolT3, respectively, differentiated isolates into two distinct clusters as described previously by Wullings and colleagues (Wullings et al., 1998). The European strains (Biovar 2, race 3) analysed in this study specifically hybridized with RsolT3, and showed considerable genetic homogeneity when compared with strains of other races from 'the rest of the world'. These data indicate the possible selection and proliferation of a 'European'-adapted variant.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Timms‐Wilson

Bryant

Bailey³

2001

Environmental Microbiology

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“…Although secreted plant cell wall-degrading exoenzymes enhance virulence (possibly by facilitating invasion and vascular colonization [23,24,37]), it is exopolysaccharide I (EPS I), a large, nitrogenrich, acidic exopolysaccharide (34), that is the primary virulence factor of R. solanacearum. EPS I is produced in copious amounts and is required for wilting and killing of hosts (8,29). EPS I apparently causes wilting by restricting water flow through xylem vessels (6).…”

mentioning

confidence: 99%

Multicomponent Transcriptional Regulation at the Complex Promoter of the Exopolysaccharide I Biosynthetic Operon of Ralstonia solanacearum

Garg¹,

Huang²,

Yindeeyoungyeon³

et al. 2000

J Bacteriol

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High-level transcription of eps, an operon encoding biosynthesis of an exopolysaccharide virulence factor of the phytopathogen Ralstonia (Pseudomonas) solanacearum, requires the products of at least seven regulatory genes (phcA, phcB, xpsR, vsrA-vsrD, and vsrB-vsrC), which are organized in three converging signal transduction cascades. Because xpsR and the vsrB-vsrC two-component system are the most downstream cascade components required for activation of eps, we explored how these components control transcription from the eps promoter (P eps ). Deletion and PCR mutagenesis identified an upstream region of P eps (nucleotides ؊82 to ؊62) that is critical for transcription activation by VsrB-VsrC and XpsR and also is required for negative control of P eps by the putative eps regulator EpsR. Using PCR mutagenesis we generated the vsrC1 allele that encodes a response regulator that constitutively activates P eps in the absence of its cognate sensor, VsrB. However, activation of P eps by vsrC1 still required xpsR. Unexpectedly, the amino acid substitution conferring the constitutive phenotype on VsrC1 is 12 residues from its C terminus, outside the known functional domains of response regulators. Finally, a modified DNase I footprinting method was used to demonstrate specific binding of both VsrC1 and VsrC to the ؊72 to ؊62 upstream region of P eps .Ralstonia (Pseudomonas) solanacearum, which causes a lethal wilting disease of solanaceous and many other types of other plants (15, 16), enters hosts via natural openings or wounds in roots and then proceeds to extensively colonize xylem vessels of the vascular system (37, 44). Although secreted plant cell wall-degrading exoenzymes enhance virulence (possibly by facilitating invasion and vascular colonization [23,24,37]), it is exopolysaccharide I (EPS I), a large, nitrogenrich, acidic exopolysaccharide (34), that is the primary virulence factor of R. solanacearum. EPS I is produced in copious amounts and is required for wilting and killing of hosts (8,29). EPS I apparently causes wilting by restricting water flow through xylem vessels (6). It also markedly enhances the speed and extent of stem colonization (37).In R. solanacearum, production of EPS I (as well as some exoenzymes) is stringently controlled by a cascading network of more than 10 regulatory genes (5,11,20,41). Inactivation of any of seven genes in this network causes a Ͼ85% reduction in transcription from the eps promoter (P eps ), leading to loss of EPS I production and the ability to wilt and kill. However, inactivation of all but two of these genes (vsrB and vsrC) can be suppressed by constitutive expression of xpsR from a vector promoter (20). These and other data showed that VsrB, VsrC, and XpsR are the most downstream components in the eps regulatory cascade and suggested that they may directly affect interaction of RNA polymerase with P eps .The predicted amino acid sequences of VsrB and VsrC imply that they comprise a two-component system in which VsrB is a sensor kinase and VsrC is its cognate resp...

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“…Thus, eps transcription is low in cultures below 10 7 cells/ml but increases 30-to 50-fold during the next four generations (9). The production of EPS1 in culture follows the same pattern (32).…”

mentioning

confidence: 80%

“…Ralstonia solanacearum, a phytopathogenic bacterium that causes a lethal wilting disease of many plants, produces multiple virulence factors (21,22,43). Among these factors is an acidic high-molecular-mass extracellular polysaccharide (EPS1) (37), which is produced in copious amounts by R. solanacearum both in culture and in planta (2,14,32). EPS1 is important for both the rapid systemic colonization of tomato plants by the pathogen and the subsequent wilt symptoms (2,14,27,42).…”

mentioning

confidence: 99%

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

Kang

Saile

Schell

et al. 1999

Appl Environ Microbiol

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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.

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Analysis of Extracellular Polysaccharide I In Culture and In Planta Using Immunological Methods: New Insights and Implications

Cited by 18 publications

References 4 publications

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

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

Multicomponent Transcriptional Regulation at the Complex Promoter of the Exopolysaccharide I Biosynthetic Operon of Ralstonia solanacearum

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

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