word count: 249 7 8 Main text word count: 4,284 9 10 11 12 1. Abstract 24Uropathogenic Escherichia coli (UPEC) are the most common etiological agent 25 of uncomplicated urinary tract infection (UTI). An important mechanism of gene 26 regulation in UPEC is phase variation that involves inversion of a promoter-containing 27 DNA element via enzymatic activity of tyrosine recombinases, resulting in biphasic, ON 28 or OFF expression of target genes. The UPEC reference strain CFT073 has five 29 tyrosine site-specific recombinases that function at two previously characterized 30 promoter inversion systems, fimS and hyxS. Three of the five recombinases are located 31 proximally to their cognate target elements, which is typical of promoter inversion 32 systems. The genes for the other two recombinases, IpuA and IpuB are located distal 33 from these sites. Here, we identified and characterized a third phase variable invertible 34 element in CFT073, ipuS located proximal to ipuA and ipuB. The inversion of ipuS is 35 catalyzed by four of the five CFT073 recombinases. Orientation of the element drives 36 transcription of a two-gene operon containing ipuR, a predicted LuxR-type regulator, 37 and upaE, a predicted autotransporter. We show that the predicted autotransporter 38UpaE is surface-located and facilitates biofilm formation as well as adhesion to 39 extracellular matrix proteins in a K-12 recombinant background. Consistent with this 40 phenotype, the ipuS ON condition in CFT073 results in defective swimming motility, 41 increased adherence to human kidney epithelial cells, and a positive competitive kidney 42 colonization advantage in experimental mouse UTI infections. Overall, the identification 43 of a third phase-switch in UPEC that is regulated by a shared set of recombinases 44 describes a complex phase-variable virulence network in UPEC. 45 46 Importance 47 Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary 48 tract infection (UTI). ON versus OFF phase-switching by inversion of small DNA 49 elements at two chromosome sites in UPEC regulates the expression of important 50 virulence factors, including the type 1 fimbriae adhesion organelle. In this report, we 51 describe a third invertible element, ipuS, in the UPEC reference strain CFT073. The 52 inversion of ipuS controls the phase variable expression of upaE, an autotransporter 53 gene that encodes a surface protein involved in adherence to extracellular matrix 54 proteins and colonization of the kidneys in a murine model of UTI. 55 56 Introduction 57 Urinary tract infections (UTIs) are one of the most common infections diagnosed 58 in clinics and hospitals. Nearly 50% of women will experience a UTI in their lifetime with 59 treatment costs exceeding $3.5 billion annually in the United States (1, 2). The most 60 common etiological agent of uncomplicated UTIs are uropathogenic Escherichia coli 61 (UPEC), which account for ~80% of reported infections (3). The predicted reservoir of 62 UPEC is the colon and infection follows an ascending rout...