Abstract
BackgroundThe synthesis of cellulose is among the most important but poorly understood biochemical processes, especially in bacteria, due to its complexity and high degree of regulation. In this study, we analyzed both the production of cellulose by all known members of the Rhizobiaceae and the diversity of Rhizobium celABC operon predicted to be involved in cellulose biosynthesis. We also investigated the involvement in cellulose production and biofilm formation of celC gene encoding an endoglucanase (CelC2) that is required for canonical symbiotic root hair infection by Rhizobium leguminosarum bv. trifolii.ResultsANU843 celC mutants lacking (ANU843ΔC2) or overproducing cellulase (ANU843C2+) produced greatly increased or reduced amounts of external cellulose micro fibrils, respectively. Calcofluor-stained cellulose micro fibrils were considerably longer when formed by ANU843ΔC2 bacteria rather than by the wild-type strain, in correlation with a significant increase in their flocculation in batch culture. In contrast, neither calcofluor-stained extracellular micro fibrils nor flocculation was detectable in ANU843C2+ cells. To clarify the role of cellulose synthesis in Rhizobium cell aggregation and attachment, we analyzed the ability of these mutants to produce biofilms on different surfaces. Alteration of wild-type CelC2 levels resulted in a reduced ability of bacteria to form biofilms both in abiotic surfaces and in planta.ConclusionsOur results support a key role of the CelC2 cellulase in cellulose biosynthesis by modulating the length of the cellulose fibrils that mediate firm adhesion among Rhizobium bacteria leading to biofilm formation. Rhizobium cellulose is an essential component of the biofilm polysaccharidic matrix architecture and either an excess or a defect of this “building material” seem to collapse the biofilm structure. These results position cellulose hydrolytic enzymes as excellent anti-biofilm candidates.
Highlights
The synthesis of cellulose is among the most important but poorly understood biochemical processes, especially in bacteria, due to its complexity and high degree of regulation
Cellulose production is widespread among rhizobia Currently, the known diversity of bacteria that can establish symbiotic nitrogen fixing root nodules with legumes, mostly resides within the order Hyphomicrobiales, which includes about 83 species distributed in different families and genera
We examined most of the official type strains of each of these taxa for cellulose production by Congo red staining, and all were found to be positive to varying degrees (Figure 1B-C, Additional file 1)
Summary
The synthesis of cellulose is among the most important but poorly understood biochemical processes, especially in bacteria, due to its complexity and high degree of regulation. Root attachment and colonization by rhizobia follow the twophase sequence of events previously described for bacteria in general [1] Several bacterial proteins, such as adhesins, and flagellar proteins [2,3], have been proposed to be important factors for the early reversible, specific binding events, whereas bacterial cell-surface polysaccharides are the main components involved in the later, irreversible attachment stages. Some of these cell surface components include exopolysaccharide, lipopolysaccharides, and cyclic β-1,2-glucans [4,5,6], but mainly cellulose fibrils, which mediate firm adhesion of the bacteria to root hairs and anchor bacteria to the root surface [1,7,8]. Hapten-inhibitable root hair lectins are involved in this dynamic attachment process [9,10,11,12]
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