The Role of Two Linear ß-Glucans Activated by c-di-GMP in Rhizobium etli CFN42.

Bacterial exopolysaccharides (EPS) have been implicated in a variety of functions that assist in bacterial survival, colonization, and host-microbe interactions. Among them, bacterial linear ß-glucans are polysaccharides formed by D-glucose units linked by ß-glycosidic bonds, which include curdlan, cellulose, and the new described Mixed Linkage ß-Glucan (MLG). Bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a universal bacterial second messenger that usually promote EPS production. Here, we report Rhizobium etli as the first bacterium capable of producing cellulose and MLG. Significant amounts of these two ß-glucans are not produced under free-living laboratory conditions, but their production is triggered upon elevation of intracellular c-di-GMP levels, both contributing to Congo red (CR+) and Calcofluor (CF+) phenotypes. Cellulose turned out to be more relevant for free-living phenotypes promoting flocculation and biofilm formation under high c-di-GMP conditions. None of these two EPS are essential for attachment to roots of Phaseolus vulgaris, neither for nodulation nor for symbiotic nitrogen fixation. However, both ß-glucans separately contribute to the fitness of interaction between R. etli and its host. Overproduction of these ß-glucans, particularly cellulose, appears detrimental for symbiosis. This indicates that their activation by c-di-GMP must be strictly regulated in time and space and should be controlled by different, yet unknown, regulatory pathways.

Screening of c-di-GMP-Regulated Exopolysaccharides in Host Interacting Bacteria.

Bacterial exopolysaccharides (EPS) often confer a survival advantage by protecting the cell against abiotic and biotic stresses, including host defensive factors. They are also main components of the extracellular matrix involved in cell-cell recognition, surface adhesion and biofilm formation. Biosynthesis of a growing number of EPS has been reported to be regulated by the ubiquitous second messenger c-di-GMP, which promotes the transition to a biofilm mode of growth in an intimate association with the eukaryotic host. Here we describe a strategy based on the combination of an approach to artificially increase the intracellular level of c-di-GMP in virtually any gram-negative bacteria with a high throughput screening (HTS) for the identification of monosaccharide composition and carbohydrate fingerprinting of novel EPS, or modified variants, that can be involved in host-bacteria interactions.

Mini-Tn7 vectors for stable expression of diguanylate cyclase PleD* in Gram-negative bacteria.

BACKGROUND: The cyclic diguanylate (c-di-GMP) is currently considered an ubiquitous second messenger in bacteria that influences a wide range of cellular processes. One of the methodological approaches to unravel c-di-GMP regulatory networks involves raising the c-di-GMP intracellular levels, e.g. by expressing a diguanylate cyclase (DGC), to provoke phenotypic changes. RESULTS: We have constructed mini-Tn7 delivery vectors for the integration and stable expression of the pleD* gene encoding a highly active DGC, which can be used to artificially increase the intracellular levels of c-di-GMP in Gram negative bacteria. The functionality of these new vectors has been validated in several plant-interacting α- and γ-proteobacteria. Similarly to vector plasmid-borne pleD*, the genome-borne mini-Tn7pleD* constructs provide significant increases in intracellular c-di-GMP, provoking expected phenotypic changes such as enhanced polysaccharide production, biofilm formation and reduced motility. However, the mini-Tn7pleD* constructs resulted far more stable in the absence of antibiotics than the plasmid-based pleD* constructs. Furthermore, we have also implemented an inducible system to modulate pleD* expression and intracellular c-di-GMP rises "on demand". CONCLUSIONS: mini-Tn7pleD* constructs are very stable and are maintained during bacterial free-living growth as well as during interaction with eukaryotic hosts, in the absence of selective pressure. This high stability ensures experimental homogeneity in time and space with regard to enhancing c-di-GMP intracellular levels in bacteria of interest.

Novel mixed-linkage ß-glucan activated by c-di-GMP in Sinorhizobium meliloti.

An artificial increase of cyclic diguanylate (c-di-GMP) levels in Sinorhizobium meliloti 8530, a bacterium that does not carry known cellulose synthesis genes, leads to overproduction of a substance that binds the dyes Congo red and calcofluor. Sugar composition and methylation analyses and NMR studies identified this compound as a linear mixed-linkage (1 → 3)(1 → 4)-ß-D-glucan (ML ß-glucan), not previously described in bacteria but resembling ML ß-glucans found in plants and lichens. This unique polymer is hydrolyzed by the specific endoglucanase lichenase, but, unlike lichenan and barley glucan, it generates a disaccharidic → 4)-ß-D-Glcp-(1 → 3)-ß-D-Glcp-(1 → repeating unit. A two-gene operon bgsBA required for production of this ML ß-glucan is conserved among several genera within the order Rhizobiales, where bgsA encodes a glycosyl transferase with domain resemblance and phylogenetic relationship to curdlan synthases and to bacterial cellulose synthases. ML ß-glucan synthesis is subjected to both transcriptional and posttranslational regulation. bgsBA transcription is dependent on the exopolysaccharide/quorum sensing ExpR/SinI regulatory system, and posttranslational regulation seems to involve allosteric activation of the ML ß-glucan synthase BgsA by c-di-GMP binding to its C-terminal domain. To our knowledge, this is the first report on a linear mixed-linkage (1 → 3)(1 → 4)-ß-glucan produced by a bacterium. The S. meliloti ML ß-glucan participates in bacterial aggregation and biofilm formation and is required for efficient attachment to the roots of a host plant, resembling the biological role of cellulose in other bacteria.

Responses to elevated c-di-GMP levels in mutualistic and pathogenic plant-interacting bacteria.

Despite a recent burst of research, knowledge on c-di-GMP signaling pathways remains largely fragmentary and molecular mechanisms of regulation and even c-di-GMP targets are yet unknown for most bacteria. Besides genomics or bioinformatics, accompanying alternative approaches are necessary to reveal c-di-GMP regulation in bacteria with complex lifestyles. We have approached this study by artificially altering the c-di-GMP economy of diverse pathogenic and mutualistic plant-interacting bacteria and examining the effects on the interaction with their respective host plants. Phytopathogenic Pseudomonas and symbiotic Rhizobium strains with enhanced levels of intracellular c-di-GMP displayed common free-living responses: reduction of motility, increased production of extracellular polysaccharides and enhanced biofilm formation. Regarding the interaction with the host plants, P. savastanoi pv. savastanoi cells containing high c-di-GMP levels formed larger knots on olive plants which, however, displayed reduced necrosis. In contrast, development of disease symptoms in P. syringae-tomato or P. syringae-bean interactions did not seem significantly affected by high c-di-GMP. On the other hand, increasing c-di-GMP levels in symbiotic R. etli and R. leguminosarum strains favoured the early stages of the interaction since enhanced adhesion to plant roots, but decreased symbiotic efficiency as plant growth and nitrogen contents were reduced. Our results remark the importance of c-di-GMP economy for plant-interacting bacteria and show the usefulness of our approach to reveal particular stages during plant-bacteria associations which are sensitive to changes in c-di-GMP levels.