A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii

Abstract

Classical quorum-sensing (autoinduction) regulation, as exemplified by the lux system of Vibrio fischeri, requires N-acyl homoserine lactone (AHL) signals to stimulate cognate transcriptional activators for the cell density-dependent expression of specific target gene systems. For Pantoea stewartii subsp. stewartii, a bacterial pathogen of sweet corn and maize, the extracellular polysaccharide (EPS) stewartan is a major virulence factor, and its production is controlled by quorum sensing in a population density-dependent manner. Two genes, esaI and esaR, encode essential regulatory proteins for quorum sensing. EsaI is the AHL signal synthase, and EsaR is the cognate gene regulator. esaI, DeltaesaR, and DeltaesaI-esaR mutations were constructed to establish the regulatory role of EsaR. We report here that strains containing an esaR mutation produce high levels of EPS independently of cell density and in the absence of the AHL signal. Our data indicate that quorum-sensing regulation in P. s. subsp. stewartii, in contrast to most other described systems, uses EsaR to repress EPS synthesis at low cell density, and that derepression requires micromolar amounts of AHL. In addition, derepressed esaR strains, which synthesize EPS constitutively at low cell densities, were significantly less virulent than the wild-type parent. This finding suggests that quorum sensing in P. s. subsp. stewartii may be a mechanism to delay the expression of EPS during the early stages of infection so that it does not interfere with other mechanisms of pathogenesis.

Figure 1

Restriction map of the esaI/esaR locus (A). The direction of transcription of esaI and esaR is indicated by the arrows. As indicated, a Tn5 insertion in the coding sequence of esaI generates mutant strain ESN51 (B); a HpaI–PstI deletion in the esaR gene is the basis for generating mutant strain ESΔR (C); a KpnI deletion in esaI/esaR becomes the basis for generating the mutant strain ESΔIR (D). The stippled region indicates where the coding segments of the two genes overlap.

Figure 2

(A) Growth-dependent synthesis of EPS. The strains were grown in CPG medium to different cell densities. Cultures of wild-type strain DC283 (—┘—), strain ESN51 (-–-✳-–-), strain ESΔR (---⋄---), and ESΔIR (......) were analyzed for the presence of bound and free EPS. The data points indicated for each strain are the combined data obtained from three separate experiments. The amount of EPS (y axis) is expressed as pg/cell. The number of colony-forming units (CFU), expressed as Log₁₀ cfu/ml, was determined by dilution plating on nutrient agar. (B) Qualitative analysis of EPS production on nutrient agar medium (Left) and CPG medium (Right). The wild-type strain DC283 exhibits a mucoid phenotype only on CPG medium, whereas the ΔesaR and ΔesaIR mutants, ESΔR and ESΔIR, are mucoid on both media. The mutant strain ESN51 is unable to synthesize EPS under either condition.

Figure 3

TLC analysis of AHLs produced during growth. (A) Samples taken from cultures at various optical densities (OD₅₆₀) were chromatographed on C18 reverse-phase thin layer plates in the presence of methanol/water (60:40 vol/vol) and visualized with an A. tumefaciens reporter strain bioassay. Lanes from right to left include: STD, synthetic N-3-oxoacyl HSL standards indicated by their respective acyl side chains C6, C8, and C10; M, an extract of fresh culture medium; lanes indicated as 0.05–0.8 include extracts obtained from cultures grown to these specific optical densities (OD₅₆₀). (B) Graph of the amount of AHL present in cultures grown to different optical densities (OD₅₆₀). The estimated concentration of AHL detected is expressed in nmol/ml culture grown to the corresponding OD₅₆₀.

Figure 4

TLC analysis of AHL samples extracted from cultures of strain DC283 (wild type), and mutant strains ESΔR, ESΔIR, and ESN51. The lane designated STD contains synthetic preparations of N-3-oxoacyl-HSLs identified by their respective acyl side chains. The standards included 0.52 pmol of 3-oxo-C6-HSL, 0.03 pmol of 3-oxo-C8-HSL, 3 pmol of 3-oxo-C10-HSL, and 16 pmol of 3-oxo-C12-HSL. AHLs were detected only in cultures of strains DC283 and ESΔR. All samples applied were extracted from cultures grown to an OD₅₆₀ of 0.6 and concentrated about 20-fold.

Figure 5

Disease progress curves. Sweet corn seedlings (cv. Seneca Horizon) were inoculated with strain DC283 (—□—), ESN51 (...◊...), ESΔR (---○---), and ESΔIR (---▵---) as described in Materials and Methods. Symptoms were rated at 1, 4, 7, 10, and 13 days after inoculation by using the following disease severity scale: 0 = no symptoms, 1 = a few restricted lesions; 2 = moderate water-soaking symptoms; 3 = numerous lesions and slight wilting; 4 = moderately severe wilt; 5 = plant death. Eighteen to 20 plants were inoculated with each strain. The Y-error bars indicate the SD of the individual data points.