brasilense Sp7 was greater in media containing NaNO3 compared to NH4Cl or N-lacking media. These results could be explained given that NO is produced in huge amounts in containing medium compared to supplemented ones. Moreover, the fact that exogenous NO donor GSK J4 not only increased biofilm formation in the wt strain but also reversed the phenotype of biofilm formation in the napA::Tn5

mutant further supports the hypothesis that NO is a signal for biofilm formation in A. brasilense (Fig. 4). Interestingly, the response to exogenous NO supply was not only limited to NO-producing conditions (e.g. KNO3-containing media; Fig. 3a). In NH4Cl-containing media, both strains also showed an increase in biofilm formation but in much less size this website than the biofilms produced in KNO3-supplemented medium (note the log y-axis scale, Fig 4b). This result indicates that the mechanism involved in NO responses in A. brasilense could be functional in both N sources. Rhizobacteria can encounter both forms of N in the soil, and . In fact, the spatial and temporal availability of and in soils is highly heterogeneous, within centimeters from the roots and changing over the course of a day (Bloom et al., 2003). In this context, biofilm formation by Azospirillum could be strongly influenced by the availability of N forms in the microsites of the soil. Our results are

in agreement with this hypothesis and point to strengthen the critical role played by NO. As plant roots are common sites for biofilm formation (Danhorn & Fuqua, 2007), the importance of NO as a regulator of the process in PGPR and the mechanisms involved are worthy areas of research. It was described that in N. europaea, Nitrosolobus multiformis, and Nitrospira briensis, NO activate gene transcription required for attachment and initial formation of biofilm (Schmidt et al., 2004). The switch into biofilm growing mode was dependent on NO concentration in the medium. At high NO concentrations, cells produced biofilm for long periods, while the gradual depletion of NO correlated with an increase of motility. Nitrite in supernatants 3-mercaptopyruvate sulfurtransferase of static cultures of Sp245

wt strain was detected in higher quantities from d1 to d5 (Fig. 3a) while biofilm formation was only observed until d3 and it was notably higher on d5 (Fig. 2). Taking into account that static growth of this strain was constant along the full assay (ca. 0.4 OD540nm, Fig. 1), this could indicate that the presence of NO signal on d1 is not sufficient to trigger biofilm formation until d3 (Figs 2 and 3a). A possible shift between NO synthesis (d1) and well-developed biofilm (d3) could be happening. The change from planktonic mode of life to biofilm form includes several physiological switches and the novo synthesis of bacterial cell wall components as well as extracellular matrix compounds (Hengge, 2009; Karatan & Watnick, 2009). Our results indicate that NO acts positively and is an early signal in biofilm formation in A.