1 A Novel Gene Amplification Causes Up-regulation of the PatAB ABC Transporter and 1 Fluoroquinolone Resistance in Streptococcus pneumoniae. 2

Abstract 18 Over-expression of the ABC transporter genes patA and patB confers efflux-mediated 19 fluoroquinolone resistance in Streptococcus pneumoniae, and is also linked to 20 pneumococcal stress responses. Although up-regulation of patAB has been observed in 21 many laboratory mutants and clinical isolates, regulatory mechanisms controlling expression 22 of these genes are unknown. In this study, we aimed to identify the cause of high-level 23 constitutive over-expression of patAB in M184, a multidrug resistant mutant of S. 24 pneumoniae R6. 25 Using a whole genome transformation and sequencing approach, we identified a novel 26 duplication of a 9.2 kb region of the M184 genome, which included the patAB genes. This 27 duplication did not affect growth and was semi-stable with a low segregation rate. 28 Expression levels of patAB in M184 were much higher than could be fully explained by 29 doubling of gene dosage alone, and inactivation of the first copy of patA had no effect on 30 multidrug resistance. Using a GFP reporter system, increased patAB expression was ascribed 31 to transcriptional read-through from a tRNA gene upstream of the second copy of patAB. 3