Genetic and biochemical characterization of a Pseudomonas solanacearum gene cluster required for extracellular polysaccharide production and for virulence

Abstract

Infection of host plants by Pseudomonas solanacearum results in wilting, which is thought to be due largely to the occlusion of xylem vessels by the P. solanacearum extracellular polysaccharide (EPS) that primarily consists of N-acetylK gld me (GaINAc). By means of Ta3 mutagenesis, we identified a 6.5-kb gene cluster that contains five complementation units required for EPS production and virulence in this bacterium. There was positive correlation between the amount of EPS produced in culture and (i) in planta growth and (ii)virulence. Based on analysis of 0-glucuronidase-gene fusions, these genes are expressed both in broth cultures and in planta and may be constitutive. Both wild-type and mutant strains contained similar amounts of UDP-GaINAc, the predicted primary substrate for EPS synthesis. Thus, the EPS mutants we obtained should be useful in the analysis of steps in the assembly of the polysaccharide and how this process is related to virulence.Virulence in Pseudomonas solanacearum (E.F.Sm.) is a complex and multifaceted phenomenon. Most of the known or presumed virulence factors in P. solanacearum are extracellular products, including the plant cell wall-degrading enzymes polygalacturonase (3, 27) and endoglucanase (26) and the plant hormones auxin, cytokinin, and ethylene (9). Although this array of extracellular products would seem to constitute a formidable arsenal for virulence, the most intensively studied virulence factor from P. solanacearum is a relatively inert product, extracellular polysaccharide (EPS) (1,11,13,19,31,36,38). This high-molecular-weight polymer is thought to be responsible for induction of the wilt symptoms that characterize the disease caused by P. solanacearum.Wild-type virulent strains of P. solanacearum produce EPS that forms a loosely associated slime both in culture and in planta. Since the pathogen is limited primarily to plant xylem vessels, EPS is thought to cause wilt symptoms by obstructing water flow. This was first suggested by Hussain and Kelman (19) (1, 11, 13), but the presence of these sugars is probably the result of incomplete purification. Instead, two amino sugars, bacillosamine (2,4-diamino-2,4,6-trideoxyglucose) and galactosaminuronic acid, probably are integral components of the P. solanacearum EPS. The latter sugar is apparently responsible for the acidic nature of the molecule (33a).Most of the early work on the relationship of EPS to virulence in P. solanacearum was based on spontaneous EPS-mutants. However, interpretation of results obtained with these mutants is confounded by the fact that such mutants are pleiotropic and are affected in other characteristics that also are likely to be important for virulence (9,25

A complex network regulates expression of eps and other virulence genes of Pseudomonas solanacearum

“…Virulence (i.e., rate of wilting and killing) or growth of P. solanacearum in planta was assessed three times by stem inoculation of at least four tomato plants with 10 4 cells of each strain as described previously (11). Analysis of EPS I and EXPs.…”

“…Production of EPS I requires the 18-kb eps gene cluster, which encodes several membrane-associated and soluble polypeptides involved in its biosynthesis and export (11,22,46). Synthesis of EPS I and O antigen of P. solanacearum also involves portions of the ops gene cluster, which may encode synthesis of a common sugar precursor (10,29,30).…”

Twitching motility of Ralstonia solanacearum requires a type IV pilus system

“…The obvious candidates are the lipopolysaccharide and pili (fimbriae). However, biosynthesis of lipopolysaccharides is complex, requiring multiple enzymic steps, and the R. solanacearum LPS-deficient mutants described to date also have defects in EPS1 synthesis or membrane function (Cook & Sequeira, 1991 ;Titarenko et al, 1997). In contrast, pili consist primarily of a single structural protein (pilin), so they are more amenable to reverse genetics to evaluate their role during pathogenesis.…”

Extracellular polysaccharide is required for wild-type virulence of Pseudomonas solanacearum

“…Most EPS-defective mutants were found to be affected in virulence (7,9,10,13,16). At least three clusters of genes participating in EPS production have been identified.…”

“…At least three clusters of genes participating in EPS production have been identified. The ops gene cluster (7,13,19) contains at least seven distinct complementation units within a 6.5-kb sequence in strain K60. Mutations in each complementation unit affect EPS production (7) as well as LPS structure (13).…”