Bacterial exopolysaccharides: biosynthesis, biological role and applications
Team : Leonilde Moreira, Arsénio M. Fialho, Jorge H. Leitão, Sílvia A. Sousa, Inês Silva, Isabel Sá-Correia, Dalila Mil-Homens
Extracellular polysaccharides (EPS) of bacterial origin have diverse functions in nature, such as in bacterium-plant interactions, virulence factors or protective agents. Due to their unique properties as rheology modification agents, stabilizers, emulsifiers and gelling agents, they also represent an important class of polymeric materials with interest in the Biotechnology field. Research carried out by our group is focused on the proteins directing EPS biosynthesis, on EPS-mediated interaction with hosts, either in symbiosis or pathogenesis, and on EPS potential industrial applications. The biological systems under study are the gellan gum from Sphingomonas elodea, cepacian from Burkholderia genus and the exopolysaccharides succinoglycan and galactoglucan fromSinorhizobium meliloti.
1. Biosynthesis and applications of gellan gum
Gellan is an extracellular polysaccharide produced by the non-pathogenic bacteria S. elodea ATCC 31461. It has approval in the US and EU for food use as a gelling, stabilizing and suspending agent, either alone or in combination with other hydrocolloids. In recent years, significant progress in understanding the relationship between gellan structure and properties and in the elucidation of the biosynthesis and engineering of this Biotechnological product has been made. Our research group has significantly contributed to this progress by characterizing many proteins involved in sugar nucleotide biosynthesis, repeat-unit formation, polymerization and secretion of the polymer. The crystal structure of UDP-glucose dehydrogenase UgdG catalyzing a NAD-dependent two-fold oxidation of UDP-glucose to UDP-glucuronic acid, one of the key components for gellan biosynthesis,was determined.
A large collection of mutants producing gellan-like polymers with different properties was obtained over the years and is being tested envisaging Biotechnological applications. One interesting and emerging application of the exopolysaccharide gellan in the biomedical field is its use as a 3D-scaffold material for tissue engineering due to its unique rheological properties, stability, biocompatibility and biodegradability.
2. Biosynthesis of cepacian produced by strains of the Burkholderia genus
The production of the exopolysaccharide cepacian is a common feature of clinical and environmental strains of the Burkholderiagenus. The structure of the repeat-unit of this EPS was determined; the pathway leading to the nucleotide sugar precursors necessary to cepacian biosynthesis was proposed ; and the bce gene cluster involved in cepacian biosynthesis was identified. Following the identification of the cepacian biosynthetic cluster, the functional analysis of several biosynthetic genes was performed. Studies aiming the identification of the remaining genes involved in cepacian biosynthesis by Burkholderia were pursued and the outcome was the extension of the bce cluster by another 9 genes. Furthermore, the crystal structure of UDP-glucose dehydrogenase BceC catalyzing a NAD-dependent oxidation of UDP-glucose into UDP-glucuronic acid, one of the nucleotide sugar precursors of cepacian, was determined. To assess mucoid phenotype switch mechanisms, global expression analysis of two clonal isolates was performed using an Affymetrix dual species Burkholderia custom microarray based on the genomes of B. multivorans ATCC 17616 and B. cenocepacia J2315.
3. Sinorhizobium meliloti proteins with a role in exopolysaccharide biosynthesis and in nitrogen fixation symbiosis
The TolC protein from Sinorhizobium meliloti has been demonstrated as required for establishing the symbiosis with the plantMedicago sativa as well as for protein and exopolysaccharide secretion and protection against osmotic and oxidative stresses. As a step towards understanding the physiology of the S. meliloti tolC mutant in free-living condition, its transcriptional profile was determined. The results showed an induction in the tolC mutant of a cytoplasmic and an extracytoplasmic stress response, possibly mediated by the CpxRA two-component regulatory system, and a repression of genes mainly involved in nitrogen metabolism, transport and cell division. Furthermore, the role of TolC in adhesion to root hairs is being evaluated.
- Ferreira AS, Silva IN, Oliveira VH, Becker JD, Givskov M, Ryan RP, Fernandes F, Moreira LM., "Comparative transcriptomic analysis of the Burkholderia cepacia tyrosine kinase bceF mutant reveals a role in tolerance to stress, biofilm formation, and virulence", Appl Environ Microbiol. 79(9):3009-20, 2013.
- Ferreira AS, Silva IN, Oliveira VH, Cunha R, Moreira LM., "Insights into the role of extracellular polysaccharides in Burkholderia adaptation to different environments", Front Cell Infect Microbiol. 1:16, 2011.
- Ferreira, A.S., Leitão, J.H., Silva, I.N., Pinheiro, P.F., Sousa, S.A., Ramos, C.G., Moreira, L.M. , "Distribution of cepacian biosynthetic genes among environmental and clinical strains of the Burkholderia genus and role of this exopolysaccharide on resistance to stress conditions ", Applied and Environmental Microbiology , 76: 441-450, 2010 .
- Rocha, J., Granja, A.T., Sá-Correia, I., Fialho ,A.M., Frazão, C. , "Cloning, expression, crystallization and preliminary crystallographic studies of UgdG, and UDP-glucose dehydrogenase from Sphingomonas elodea ATCC 31461", Acta Crystallographica, Section F, Structural Biology and Crystallization Communications , 66(1): 69-72, 2010.
- Rocha, J., Popescu, A.O., Sá-Correia, I., Fialho, A.M., Frazão, C. " Cloning, expression, purification, crystallization and preliminary crystallographic studies of BceC, a UDP-glucose dehydrogenase from Burkholderia cepacia IST408". Acta Crystallographica, Section F, Structural Biology and Crystallization Communications , 66: 269-271, 2010 .
- Santos, M.R., Cosme, A.M., Becker, J.D., Medeiros, J.M.C., Mata, M.F., Moreira, L.M., "Absence of functional TolC protein causes increased stress response gene expression in Sinorhizobium meliloti", BMC Microbiology . 10(1): 180, 2010.
- Fialho, A.M., Moreira, L.M., Granja, A.T., Popescu, A.O., Hoffmann, K., Sá-Correia, I., "Occurrence, production and applications of gellan: current state and perspectives". Applied Microbiology and Biotechnology, 79, 889-900, 2008.
- Cosme, A.M., Becker, A., Santos, M.R., Sharypova, L.A., Santos, P.M., Moreira, L.M., "The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance and symbiosis".Molecular Plant-Microbe Interactions , 21, 947-57, 2008
- Sá-Correia, I., Fialho, A. M., Videira, P.A., Moreira, L. M., Marques, A. R., Albano, H., "Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes, enzymes and exopolysaccharide production engineering", Journal of Industrial Microbiology and Biotechnology , 29, 170-176, 2002.