Effects caused by iron in surface motility and biofilm formation in different
strains. A) Surface motility and development of structured biofilms are inversely regulated by iron levels in Rm1021. Under low-iron conditions, the iron regulator RirA is free and its repressor activity is low allowing high expression of rhb genes and the corresponding production of siderophore Rhb1021 (+Rb) in the reference strain Rm1021. Amphiphilic properties associated with Rhb1021 facilitate surface spreading and the formation of flat, unstructured biofilms. Under iron-replete conditions, the number of RirA molecules bound to Fe-S clusters rises leading to the repression of rhb genes and as a result, Rhb1021 production is abolished (−Rb), thereby preventing surface motility. This together with putative additional functions regulated by iron favors biofilm formation. Most likely, even under iron-replete conditions, some areas within a wild type biofilm might suffer low iron availability, which could trigger Rhb1021 production and impact biofilm architecture. B) The inability to produce Rhb1021 caused by rhb loss-of-function abolishes surface motility and reduces biofilm thickness regardless of iron levels in the medium. C) Uncontrolled Rhb1021 synthesis in the rirA mutant allows surface translocation under iron-replete conditions but prevents the formation of structured mature biofilms. D) Unlike Rm1021, the wild type strain GR4 does not produce Rhb1021 and does not show surface translocation under low-iron conditions. However, like in Rm1021, iron-replete conditions promote the formation of structured and thick biofilms.