Abstract Borrelia burgdorferi (Bb), the etiological agent of Lyme disease, maintains itself in nature via a complex life cycle involving an arthropod (tick) vector and small mammals. During its cycle between ticks and mammals, Bb undergoes dramatic adaptive changes in order to interact with and adapt to these two disparate niches. A substantial number of studies have indicated that the RpoN-RpoS regulatory pathway (aka the σ54-σS cascade) plays a central role in host adaptation and virulence gene expression in Bb. Specifically, this pathway controls expression of major virulence-associated outer membrane lipoproteins including OspC, DbpA, and DbpB. Recently, we found that, in addition to the bacterial enhancer binding protein Rrp2, a Fur/PerR homologue called BosR is also required for the activation of the RpoN-RpoS pathway. More importantly, an overwhelming body of evidence supports that the BosR/RpoN/RpoS network governs expression of additional effector proteins associated with Bb fitness, survival, and pathogenesis. We performed global transcriptome profiling using microarray and RNA-seq and have identified potential BosR/RpoN/RpoS-regulated genes (other than ospC, dbpA and dbpB). However, virtually nothing is known regarding the functions and contributions of these genes to the tick-mammal infectious life cycle of Bb. In this proposal, we will directly examine the impact of two BosR/RpoN/RpoS-regulated genes on Bb's parasitic strategies. In Specific Aim 1, we shall systematically examine gene expression in Bb's native tick and mammal environments, which will pinpoint the potential life- cycle-specific roles of the gene targets. In Specific Aim 2, we shall define the roles of these genes in bacterial pathogenesis via genetic mutagenesis approaches and a Lyme borreliosis tick-mouse model system. Accomplishing the two Specific Aims of this proposal will advance our understanding of virulence expression control in Bb, and lead to the identification of new virulence factors. Defining the contributions of BosR/RpoN/RpoS-regulated genes to borrelial physiology and pathogenesis, and the mechanistic details underlying the respective gene functions, will be the focus of our future research, which may ultimately lead to the development of new strategies to prevent and/or treat Lyme disease.