Under laboratory conditions, S. meliloti can form three distinct types of biofilms, termed ‘flat,’‘structured,’

Smad inhibitor and ‘organized.’ EPS II-producing strain Rm8530, which has a mucoid phenotype, displays a highly structured architectural biofilm, in contrast to the unstructured one formed by non-EPS II-producing strain 1021. In experiments with Medicago sativa (alfalfa), strain Rm8530 expR+ formed biofilms covering the entire surface of the root, including root hairs, whereas strain Rm1021 formed clusters of cells adhering mainly to the main root (Rinaudi & González, 2009). Exopolysaccharides determine living conditions for microorganisms in biofilms, because they affect the porosity, density, water content, charge, hydrophobicity, and mechanical stability of biofilms (Flemming & Wingender, 2002). In S. meliloti, MucR controls exopolysaccharide production. To clarify the relationship between exopolysaccharide synthesis and biofilm formation, mucR expression was studied using transcriptional fusion to lacZ. The results indicated that mucR does not respond to changes in environmental conditions, and

does not play an important role in biofilm formation (Rinaudi et al., 2009). Biofilm formation in the Rm1021 strain is limited, and Adriamycin order does not appear to be mediated by the presence of exopolysaccharides. In the Rm8530 expR+ strain, biofilm formation is controlled by the ExpR/Sin quorum-sensing system, through production of EPS II. Levels of biofilm formation and phenotype observed by confocal microscopy in strain Rm1021 mucR− are similar

to those of wild-type Rm1021, suggesting that the low-molecular-weight fraction of EPS II could control the formation of biofilms both in vivo and in vitro (Rinaudi & González, 2009). Microscopic examination of S. meliloti cells within curled root hairs revealed small biofilm-type aggregates that could provide inocula for Sclareol root invasion, and rhizobial cells migrated down infection threads toward the root interior as biofilm-like filaments (Ramey et al., 2004). These authors also showed that Agrobacterium and rhizobia can form dense, structurally complex biofilms on root surfaces. As explained in the Introduction, it is very difficult to differentiate between structures now known as biofilms to what were previously described as bacterial aggregation, microcolony, agglutination, and flocculation. In this context, the agglutination to glass and the flocculation of R. leguminosarum observed more than two decades ago could be classic biofilms (Smit et al., 1987). Likewise, fluorescence protein-expressing S. meliloti attached to roots and forming infection threads, as documented by Gage et al. (1996), and later by our group (Giordano et al.