Insights into the complex regulation of rpoS in Borrelia burgdorferi

Abstract

SummaryCo-ordinated regulation of gene expression is required for the transmission and survival of Borrelia burgdorferi in different hosts. The sigma factor RpoS (s S ), as regulated by RpoN (s 54 ), has been shown to regulate key virulence factors (e.g. OspC) required for these processes. As important, multiple signals (e.g. temperature, pH, cell density, oxygen) have been shown to increase the expression of s S -dependent genes; however, little is known about the signal transduction mechanisms that modulate the expression of rpoS. In this report we show that: (i) rpoS has a s 54 -dependent promoter that requires Rrp2 to activate transcription; (ii) Rrp2D123, a constitutively active form of Rrp2, activated s 54 -dependent transcription of rpoS/P-lacZ reporter constructs in Escherichia coli; (iii) quantitative reverse transcription polymerase chain reaction (QRT-PCR) experiments with reporter cat constructs in B. burgdorferi indicated that Rrp2 activated transcription of rpoS in an enhancerindependent fashion; and finally, (iv) rpoN is required for cell density-and temperature-dependent expression of rpoS in B. burgdorferi, but histidine kinase Hk2, encoded by the gene immediately upstream of rrp2, is not essential. Based on these findings, a model for regulation of rpoS has been proposed which provides mechanisms for multiple signalling pathways to modulate the expression of the s S regulon in B. burgdorferi.

Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles ofBorrelia burgdorferi

“…In addition, the genome of B. burgdorferi codes for a limited number of regulators to control myriad metabolic processes, leading to overlapping or discrete networks responding to different environmental signals (32). Currently, much of the information on gene regulation in B. burgdorferi has been obtained from the analysis of a central regulatory pathway mediated by Rrp2-RpoN-RpoS (17,20,31,41,86). The activation of this pathway facilitates a concerted expression of a variety of borrelial determinants, such as OspC, DbpA/B, and BBK32, known to be involved in the pathogenic mechanisms of B. burgdorferi (41,86).…”

“…A central regulatory pathway comprising Rrp2-RpoN-RpoS has been characterized as regulating outer surface protein C (OspC), decorin binding protein A (DbpA), fibronectin binding protein BBK32, and several other borrelial determinants involved in various pathogenic processes (11, 17-20, 41, 88). Mutations in one or more of these regulators have also been characterized as having significant global effects on the transcriptional levels of a large number of ORFs in B. burgdorferi (12,17,20,31,61). Recently, it has also been shown that inactivation of ospAB constitutively activates the Rrp2-RpoNRpoS pathway, indicating mechanisms that are unique to B. burgdorferi and to its survival strategies in the tick vector and mammalian hosts (40).…”

lacZ Reporter System for Use in Borrelia burgdorferi

“…1A). BamHI and XhoI were the enzymes used for creating promoter-lacZ fusions in pPBMB101, which has a ribosome binding site (RBS) between the XhoI site and the lacZ start codon (8).…”

“…The reporter gene transcript and encoded protein are the same irrespective of which promoter is used to express it. The lacZ gene is one of the most widely used reporter genes, and B. burgdorferi promoters have previously been fused to lacZ, but ␤-galactosidase activity was assessed in E. coli, not B. burgdorferi (8,32). However, since many studies have detected significant differences in the expression profiles of B. burgdorferi and E. coli (1,12,33,34), using lacZ as a reporter gene directly in B. burgdorferi will allow a more accurate analysis of native promoter activity.…”

The Fur homologue BosR requires Arg39 to activate rpoS transcription in Borrelia burgdorferi and thereby direct spirochaete infection in mice

“…The binding site for RpoN within the rpoS promoter is known (Smith et al, 2007;Studholme & Buck, 2000). However, even though Rrp2 has a predicted DNA-binding domain, it appears that Rrp2 acts on RpoN without the benefit of binding DNA (Blevins et al, 2009;Burtnick et al, 2007;Yang et al, 2003). In vitro, BosR binds multiple sites within the rpoS promoter (Ouyang et al, 2011(Ouyang et al, , 2014(Ouyang et al, , 2015.…”