Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and biofilm formation on plant roots and abiotic surfaces

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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. 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. Results ANU843 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 . Conclusions Our 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.

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Publié le 01 janvier 2012
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Robledo et al. Microbial Cell Factories 2012, 11 :125 http://www.microbialcellfactories.com/content/11/1/125
R E S E A R C H Open Access Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and biofilm formation on plant roots and abiotic surfaces M Robledo 1,5 , L Rivera 1 , Jose I Jiménez-Zurdo 2 , R Rivas 1 , F Dazzo 3 , E Velázquez 1 , E Martínez-Molina 1 , Ann M Hirsch 4 and Pedro F Mateos 1*
Abstract Background: 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. 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. Results: ANU843 celC mutants lacking (ANU843 Δ C2) 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 ANU843 Δ C2 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 . Conclusions: Our 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. Keywords: Cellulose biosynthesis, Rhizobium , Cellulases, Biofilm, Symbiosis
Background when flavonoids secreted by the plant induce Rhizobium Symbioses between diazotrophic rhizobia and legume nod genes, which are involved in the synthesis and secre-plants are of critical agronomic and environmental tion of lipo-chitooligosaccharide signal molecules, known importance, making crop production possible in nitrogen- as Nod factors. In response, plant root hairs deform limited soils without fertilizer supply. Rhizobia grow as and exhibit a typical marked curling to facilitate bacteria free-living organisms, but can also induce and colonize penetration. The interaction continues with the initiation root nodules in legume plants thereby establishing a part- of the root nodule, where bacterial cells are released into nership that benefits both organisms. This process begins the host cells. Eventually, upon a morphological differen-tiation into bacteroids, bacteria fix atmospheric dinitro-gen into ammonia. Among the many factors involved in * Correspondence: pfmg@usal.es ad de development of an effective symbiosis between rhizobia 1 SDaleapmaratnacma,eSnatloamdeanMciac,rSopbiaionlogíayGenéticaandCIALE,Universid and their host plants, those associated with adherence Full list of author information is available at the end of the article and colonization of bacteria on the surface of roots and © 2012 Robledo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.