Diguanylate Cyclases AdrA and STM1987 Regulate Salmonella enterica Exopolysaccharide Production during Plant Colonization in an Environment-Dependent Manner

Abstract

Increasing evidence indicates that despite exposure to harsh environmental stresses, Salmonella enterica successfully persists on plants, utilizing fresh produce as a vector to animal hosts. Among the important S. enterica plant colonization factors are those involved in biofilm formation. S. enterica biofilm formation is controlled by the signaling molecule cyclic di-GMP and represents a sessile lifestyle on surfaces that protects the bacterium from environmental factors. Thus, the transition from a motile, planktonic lifestyle to a sessile lifestyle may represent a vital step in bacterial success. This study examined the mechanisms of S. enterica plant colonization, including the role of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), the enzymes involved in cyclic di-GMP metabolism. We found that two biofilm components, cellulose and curli, are differentially required at distinct stages in root colonization and that the DGC STM1987 regulates cellulose production in this environment independent of AdrA, the DGC that controls the majority of in vitro cellulose production. In addition, we identified a new function for AdrA in the transcriptional regulation of colanic acid and demonstrated that adrA and colanic acid biosynthesis are associated with S. enterica desiccation tolerance on the leaf surface. Finally, two PDEs with known roles in motility, STM1344 and STM1697, had competitive defects in the phyllosphere, suggesting that regulation of motility is crucial for S. enterica survival in this niche. Our results indicate that specific conditions influence the contribution of individual DGCs and PDEs to bacterial success, perhaps reflective of differential responses to environmental stimuli.

FIG 1

Cellulose and curli are temporally important for root colonization, and cellulose production is independent of adrA in this environment. (A) Mutant strains were compared with the wild type for colonization of alfalfa roots after coinoculation with the wild type at a 1:1 ratio. Gray and white bars indicate the proportion of the population (% total population) that is composed of the mutant strain at 24 and 48 hpi, respectively. (B) Cellulose production was monitored by measuring calcofluor fluorescence and is presented as logarithmic relative fluorescence units (log₁₀ RFU) for wild-type and mutant strains at 24 and 48 hpi. Each experiment was repeated three times, and the results of representative experiments are presented here. Error bars indicate standard deviations, while asterisks indicate significant differences either from 50% (A; dashed line), a percentage that represents equal proportions of the wild type and mutant, or from the wild type (B) (P < 0.05).

FIG 2

The DGC STM1987 contributes to root colonization and in planta cellulose production. (A) The 12 DGC mutants were compared to the wild type for colonization of alfalfa roots after coinoculation at a 1:1 ratio. (B) Complementation experiments for the adrA, STM1987, STM3375, and STM3615 mutants. Each respective gene was provided in trans, and competition experiments were performed as described. Gray and white bars indicate the proportion of the population (% total population) that is composed of the mutant strain at 24 and 48 hpi, respectively. (C) Cellulose production was monitored by measuring calcofluor fluorescence and is presented as logarithmic relative fluorescence units (log₁₀ RFU) for wild-type and mutant strains at 24 and 48 hpi. Each experiment was repeated three times, and the results of representative experiments are presented here. Error bars indicate standard deviations while asterisks indicate significant differences from 50% (dashed line), a percentage that represents equal proportions of wild type and mutant (P < 0.05), and letters indicate significant differences between strains within a single time point (uppercase letters for 24 hpi and lowercase letters for 48 hpi; P < 0.01).

FIG 3

c-di-GMP metabolism and exopolysaccharide production impact S. enterica persistence on leaves. (A) DGC (yellow) and PDE (blue) mutants and (B) cellulose, curli, and colanic acid mutants were compared to the wild type for colonization of alfalfa leaves after coinoculation at a 1:1 ratio. The results of representative experiments showing persistence of each mutant as a proportion of the total population (% total population) at 48 hpi are shown here. Experiments were performed three times, error bars indicate the standard deviations, and asterisks indicate significant differences from 50% (dashed line; P < 0.05).

FIG 4

AdrA and colanic acid production are important for desiccation tolerance on leaves. Leaf persistence competition assays were repeated under two relative humidity conditions: 85% humidity (black squares) and 15% humidity (red triangles). All data points from three independent experiments were combined and are presented as the proportion of the population (% total population) that is composed of the mutant strain (adrA [A] or wcaB [B]) at 24, 48, and 72 hpi. Lines (dashed black, humid conditions; solid red, dry conditions) correspond to a linear regression model, and shaded areas correspond to the respective 95% confidence interval.

FIG 5

The regulation and importance of exopolysaccharide biofilm components in S. enterica plant colonization depends on the environment. The regulation presented here is modeled after data collected from in vitro studies (dashed lines) (14, 60, 61) but also highlights the results from plant experiments (solid lines). Colors indicate factors with plant colonization defects in the rhizosphere (tan) or phyllosphere (green). Putative DGCs and PDEs are shown as rectangles and ovals, respectively. The asterisk after STM2123 denotes that the root colonization defect observed in this mutant occurs specifically during single inoculation experiments and not competition assays.