Polyamine uptake and utilization and characterization of potA (bb0642) and potdD ( bb0639) in Borrelia burgdorferi

Date
2012
Authors
Romo, Jesus Antonio
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Abstract

Lyme disease is a multi-phasic, systemic infection caused by the spirochetal bacterium Borrelia burgdorferi. It is the major arthropod-borne disease in the US and is transmitted to mammals, including humans, by Ixodid ticks. This spirochetal pathogen has a limited metabolic capability, and therefore is intimately dependent on its hosts for survival. In silico analysis revealed that B. burgdorferi encodes for a series of co-transcribed genes (potA-D) for the uptake of polyamines, ubiquitous molecules necessary for many normal cell functions. The terminal open reading frame (ORF) of the operon encodes for potD, a substrate binding protein. potA and potD were cloned into E. coli expression vector pET23a, and the two recombinant proteins with a C-terminal 6-Histidine tag were expressed, purified, and used to generate mono-specific anti-serum in mice. These critical reagents have been used to determine that PotA is expressed at higher levels in the tick vector than vertebrate host, while PotD is expressed at the same levels in B. burgdorferi in both tick and mammalian host conditions highlighting a complex and potentially important function. Moreover, increasing concentrations of select polyamines causes a decreased levels of both PotA and PotD. Protease accessibility assays performed on two strains of B. burgdorferi, A3 and MSK5, show that PotD is not surface exposed, indicating a similar organization to the Pot system of E. coli. A plasmid construct allowing for deletion of potD from the B. burgdorferi chromosome has been generated and used to transform B. burgdorferi. Analysis of this deletion strain will be an extremely useful tool in delineating the role of polyamine transport in B. burgdorferi.

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Keywords
Borrelia, Polyamines, Transport
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Department
Integrative Biology