Fig 3. Mutation and complementation of rel _Bbu in B. burgdorferi.

(A) The rel _Bbu mutant (rel _Bbu ^-) was constructed by replacing the rel _Bbu gene in strain B31-5A4 with the streptomycin resistance gene aadA fused to the B. burgdorferi flgB promoter and the B. subtilis trpL terminator. The rel _Bbu mutant was complemented in trans by transformation with the Borrelia shuttle vector pBSV2 containing the rel _Bbu gene fused to either the flacp inducible promoter (pBS-flacp-rel _Bbu) or its native promoter (pBS-rel _Bbu). (B) RT-PCR analysis of RNA isolated from wild-type (lane 1), rel _Bbu ^- (lane 2), rel _Bbu ^- pBS-flacp-rel _Bbu (lane 3), or rel _Bbu ^- pBS-rel _Bbu (lane 4) strains. Samples were incubated with (+RT) or without (-RT) reverse transcriptase, and rel _Bbu and flaB transcripts were detected by PCR using primer pairs rsh 981F/rsh 1984R and flaB 423F/flaB 542R, respectively. Products were separated on 1% (rel _Bbu) or 2% (flaB) agarose gels and stained with ethidium bromide. ntc = no template control. (C) Production of (p)ppGpp in the wild-type (WT), rel _Bbu ^- and rel _Bbu ^- pBS-rel _Bbu (rel _Bbu ^- comp) strains. ³²P-labeled cultures were grown to log phase, shifted to RPMI for 6 h and nucleotides were extracted and analyzed by TLC.