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The Outer Membrane Protein TolC from Sinorhizobium meliloti Affects Protein Secretion, Polysaccharide Biosynthesis, Antimicrobial Resistance, and Symbiosis

¹IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; ²Institute for Genome Research and Systems Biology, Center for Biotechnology, Bielefeld University, P.O. 33501, Bielefeld, Germany; ³Institute of Biology III, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany

Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation relationship with Medicago sativa. During this process, it must cope with diverse environments and has evolved different types of transport systems that help its propagation in the plant roots. TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In this work, we have characterized the protein TolC from S. meliloti 2011. An insertional mutation in the tolC gene strongly affected the resistance phenotype to antimicrobial agents and induced higher susceptibility to osmotic and oxidative stresses. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type versus tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of open reading frame SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by a TolC-dependent secretion system. In the absence of TolC, neither succinoglycan nor galactoglucan were detected in the culture supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of nonfixing nitrogen nodules in M. sativa roots. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.

Additional keywords:EPS I, EPS II, type I secretion.