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  1. Bacterial chemotaxis is the directed movement of motile bacteria in gradients of chemoeffectors. This behavior is mediated by dedicated signal transduction pathways that couple environment sensing with changes in the direction of rotation of flagellar motors to ultimately affect the motility pattern. Azospirillum brasilense uses two distinct chemotaxis pathways, named Che1 and Che4, and four different response regulators (CheY1, CheY4, CheY6, and CheY7) to control the swimming pattern during chemotaxis. Each of the CheY homologs was shown to differentially affect the rotational bias of the polar flagellum and chemotaxis. The role, if any, of these CheY homologs in swarming, whichmore »depends on a distinct lateral flagella system or in attachment is not known. Here, we characterize CheY homologs’ roles in swimming, swarming, and attachment to abiotic and biotic (wheat roots) surfaces and biofilm formation. We show that while strains lacking CheY1 and CheY6 are still able to navigate air gradients, strains lacking CheY4 and CheY7 are chemotaxis null. Expansion of swarming colonies in the presence of gradients requires chemotaxis. The induction of swarming depends on CheY4 and CheY7, but the cells’ organization as dense clusters in productive swarms appear to depend on functional CheYs but not chemotaxis per se . Similarly, functional CheY homologs but not chemotaxis, contribute to attachment to both abiotic and root surfaces as well as to biofilm formation, although these effects are likely dependent on additional cell surface properties such as adhesiveness. Collectively, our data highlight distinct roles for multiple CheY homologs and for chemotaxis on swarming and attachment to surfaces.« less