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Journal of Bacteriology, January 2003, p. 444-452, Vol. 185, No. 2
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.2.444-452.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Expression of spoT in Borrelia burgdorferi during Serum Starvation

Marc B. Concepcion and David R. Nelson*

Department of Cell and Molecular Biology, University of Rhode Island, Kingston, Rhode Island 02881

Received 3 June 2002/ Accepted 17 October 2002

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 H332PO4-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.

* Corresponding author. Mailing address: Department of Cell and Molecular Biology, 117 Morrill Hall, University of Rhode Island, Kingston, RI 02881. Phone: (401) 874-5902. Fax: (401) 874-2202. E-mail: dnelson{at}uri.edu.

Journal of Bacteriology, January 2003, p. 444-452, Vol. 185, No. 2
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.2.444-452.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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