Document Type

Article

Date of Original Version

2003

Department

Cell & Molecular Biology

Abstract

Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the tick Ixodes scapularis. A 2.9-kb fragment containing a putative spoT gene was isolated from B. burgdorferi genomic DNA by PCR amplification and cloned into a pBAD24 vector. The cloned gene complemented Escherichia coli mutant strain CF1693, which contains deletions of both the relA and spoT genes. The spoT gene in E. coli encodes a bifunctional enzyme capable of synthesizing and degrading (p)ppGpp, which mediates the stringent response during carbon source starvation. B. burgdorferi has been reported to have a stress response to serum starvation. Thin-layer chromatography was used to detect (p)ppGpp extracted from H3 32PO4-labeled B. burgdorferi cells starved for serum in RPMI. B. burgdorferi spoT gene expression was characterized during fatty acid starvation. Northern analysis of spoT revealed detectable message at 2.5 min of starvation in RPMI. Expression of spoT during serum starvation increased _6-fold during the 30 min that starvation conditions were maintained. Further, expression of spoT decreased when serum was added to serum-starved cells. Reverse transcriptase PCR (RT-PCR) was used to detect spoT mRNA from ~106 cells starved for serum in RPMI for 2.5 to 30 min or incubated in tick saliva for 15 min. Northern blot analysis suggests that spoT transcript 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-bp spoT open reading frame was able to complement E. coli CF1693. The data suggest that B. burgdorferi exhibits a stringent response to serum starvation and during incubation in tick saliva.

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