EPS II is essential for biofilm formation in Sinorhizobium meliloti.

RINAUDI, L., ALMENGOR, A., GIORDANO, W., GONZÁLEZ J.E.

Austin, Texas, USA.

Conferencia; Third American Society for Microbiology Conference on Cell-Cell Communication in Bacteria. Austin, Texas, USA. October 7-10, 2007.; 2007

American Society for Microbiology

S. meliloti has the ability to synthesize two different exopolysaccharides, succinoglycan and EPS II, production of which is quorum sensing dependent. Preliminary evidence suggests that in the rhizobia-plant symbiosis EPS and quorum sensing are involved on biofilm formation. In order to establish the role of S. meliloti exopolysaccharides on biofilm formation, we performed a microplate assay to determine the biofilm proficiency of wild type strains as well as succinoglycan and/or EPS II biosynthesis mutants. Two different strain backgrounds were used; Rm1021 carries an insertional element within expR gene and does not produce EPS II under normal conditions; Rm8530, that contains a functional copy of this gene, is able to make the EPS II polymer. ExpR is a LuxR homologue whose function includes the activation of EPS II production in the presence of N-acyl homoserine lactones produced by the sinR/sinI quorum system in S. meliloti. In addition we used Confocal Laser Scaning Microscopy to analyze the characteristics of a S. meliloti biofilm formed in vitro over a 15-day time course experiment in chambered cover slides. When biofilm formation was tested on S. meliloti we found that Rm8530 forms a significantly better biofilm than Rm1021, which suggests that expR plays a role on biofilm formation. In the Rm8530 background, where large amounts of biofilm could be detected, introduction of an exoY mutation (which results in the inability to produce succinoglycan) results in no reduction in biofilm formation but an expA mutant (which blocks synthesis of EPS II) shows a marked decrease in biofilm formation, suggesting that EPS II is responsible for most of the biofilm formation in Rm8530. During the first 5 days post inoculation biofilms permeated by large channels but without any organization were observed in Rm8530 and Rm8530 exoY, followed by eventual dispersal of the cells. Starting at day 7 post inoculation these two strains developed a different kind of biofilm, where rhizobia formed very organized honeycombs-like structures. A sinI derivative was unable to form biofilm under our conditions. We also examined S. meliloti attachment to alfalfa roots. Rm8530 as well as Rm8530 exoY develop biofilms that cover the entire surface of the root, including the root hairs, while Rm1021 and Rm8530 expA form clusters of cells attached predominantly to the principal root. Our results suggest that succinoglycan does not play a major role on biofilm formation. On the other hand, EPS II seems to play a crucial role on biofilm development and perhaps proper attachment to the root hairs.