Expression ofspoTinBorrelia burgdorferiduring Serum Starvation

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Abstract
Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the tickIxodes scapularis. A 2.9-kb fragment containing a putativespoTgene was isolated fromB. burgdorferigenomic DNA by PCR amplification and cloned into a pBAD24 vector. The cloned gene complementedEscherichia colimutant strain CF1693, which contains deletions of both therelAandspoTgenes. ThespoTgene inE. coliencodes a bifunctional enzyme capable of synthesizing and degrading (p)ppGpp, which mediates the stringent response during carbon source starvation.B. burgdorferihas been reported to have a stress response to serum starvation. Thin-layer chromatography was used to detect (p)ppGpp extracted from H332PO4-labeledB. burgdorfericells starved for serum in RPMI.B. burgdorferi spoTgene expression was characterized during fatty acid starvation. Northern analysis ofspoTrevealed detectable message at 2.5 min of starvation in RPMI. Expression ofspoTduring serum starvation increased ∼6-fold during the 30 min that starvation conditions were maintained. Further, expression ofspoTdecreased when serum was added to serum-starved cells. Reverse transcriptase PCR (RT-PCR) was used to detectspoTmRNA from ∼106cells starved for serum in RPMI for 2.5 to 30 min or incubated in tick saliva for 15 min. Northern blot analysis suggests thatspoTtranscript was ∼900 nucleotides in length. RT-PCR amplification of the transcript using several sets of primers confirmed this finding. Additionally, a truncated clone containing only the first 950 bp of the 2,001-bpspoTopen reading frame was able to complementE. coliCF1693. The data suggest thatB. burgdorferiexhibits a stringent response to serum starvation and during incubation in tick saliva.