Downloaded from www.microbiologyresearch.org by IP: 54.162.190.106 On: Fri, 26 Feb 2016 07:26:05 Analysis of the determinants of bba64 (P35) gene expression in Borrelia burgdorferi using a gfp reporter Aarti Gautam, Marianne Hathaway, Natalie McClain, Geeta Ramesh and Ramesh Ramamoorthy Correspondence Ramesh Ramamoorthy rramesh@tulane.edu Division of Bacteriology and Parasitology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, LA 70433, USA Received 13 July 2007 Revised 21 September 2007 Accepted 4 October 2007 The bba64 (P35) gene of Borrelia burgdorferi, the agent of Lyme disease, encodes a surface-exposed lipoprotein. The expression of bba64 in vitro is tightly regulated and dependent on several environmental factors. In nature, its expression is induced in the tick vector during feeding and maintained during infection of the vertebrate host. The pattern of expression of bba64 suggests that it imparts a critical function to the pathogen. A previous study has shown that the expression of bba64 is down-regulated in the absence of RpoS, suggesting that the alternative sigma factor may be involved in its expression. A DNA-binding protein has also been shown to specifically recognize a sequence in the 59 regulatory region of the gene. Therefore, the contribution of these putative determinants to the differential expression of bba64 was investigated. The role of RpoS was critically evaluated by genetic complementation of the rpoS mutant using a chromosomally targeted copy of the wild-type gene. The results confirm that RpoS is indeed required for the expression of bba64. The role of the upstream DNA-binding site was examined using bba64 promoter–gfp transcriptional fusions in a shuttle vector. The DNA-binding site was studied by targeting mutations to an inverted repeat sequence (IRS), the most prominent feature within the binding site, as well as by deletion of the entire sequence upstream of the basal promoter. Quantitative assessment of gene expression demonstrated that neither the IRS nor the sequence upstream of the promoter was essential for expression. Moreover, the expression of the reporter (GFP) appeared to remain RpoS-dependent in all cases, based on the co-expression of GFP and OspC in a subpopulation of spirochaetes and the selective expression of GFP in the stationary phase. Collectively, the data indicate that RpoS is the sole determinant of differential bba64 expression in cultured spirochaetes. INTRODUCTION Borrelia burgdorferi, the spirochaetal agent of Lyme disease, is maintained in nature via a complex enzootic life cycle involving Ixodes ticks and small rodents. To survive in this enzootic cycle, B. burgdorferi must adapt physiologically to diverse environments. Central to its adaptation process is the differential expression of proteins in response to changes in the environment, especially as this organism traverses from its tick vector to the mammalian host and vice versa. The genome of B. burgdorferi strain B31 is composed of a linear chromosome, nine circular plasmids and 12 linear plasmids (Casjens et al., 2000; Fraser et al., 1997). One of the genetic elements that display prolific differential expression in response to environmental signals is linear plasmid 54 (lp54) (Brooks et al., 2003; Carroll et al., 2000; Clifton et al., 2006; Ojaimi et al., 2003; Revel et al., 2002; Tokarz et al., 2004). lp54 of B. burgdorferi B31 consists of 76 ORFs that include lipoproteins such as OspA and OspB (Barbour & Garon, 1987) and decorin-binding proteins A (DbpA) and B (DbpB) (Hagman et al., 1998). In addition to these immunogenic proteins, lp54 also carries eight out of the 14 members of gene family 54. Paralogues of this gene family exhibit significant intrafamily sequence divergence, with amino acid similarity and identity values as low as 7.35 and 5.4 %, respectively (McDowell et al., 2005). Two members of this family, BBA64 (Gilmore et al., 1997) and BBA66, have been localized to the surface of the spirochaete (Brooks et al., 2006). Members of gene family 54 display distinct expression patterns. Some members (bba64 and bba66) of the family are silent during the unfed-tick phase (Gilmore et al., 2001; Tokarz et al., 2004) but are turned on during tick feeding Abbreviations: Ab, antibody; IRS, inverted repeat sequence. A supplementary table listing the reagents and settings used for confocal microscopy is available with the online version of this paper. Microbiology (2008), 154, 275–285 DOI 10.1099/mic.0.2007/011676-0 2007/011676 G 2008 SGM Printed in Great Britain 275