Fluorescent Proteins, Promoters, and Selectable Markers for Applications in the Lyme Disease Spirochete Borrelia burgdorferi

Abstract

Genetic manipulation of the Lyme disease spirochete B. burgdorferi remains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest, B. burgdorferi displays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacterium’s transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use in B. burgdorferi by generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and promoters of varied strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents.

Recent discoveries and advancements in research on the Lyme disease spirochete Borrelia burgdorferi

“…An additional advance in B. burgdorferi research is in the introduction of constitutive promoters of different strengths. Although strong promoters from the flaB and flgB genes have been used historically, transcriptome data have now been used to identify constitutively weak (P 0526 ) and moderate (P 0826 , P resT , P 0031 , and P 0026 ) promoters which will be useful for expressing more biologically relevant levels of a gene of interest 57 . Furthermore, monomeric fluorescent markers have been adapted for B. burgdorferi , including cyan, green, yellow, red, and infrared fluorescent proteins.…”

“…The infrared marker requires the addition of exogenous biliverdin, but the excitation wavelength is less toxic to cells than other markers and results in less autofluorescence. These markers will inevitably prove useful in co-localization studies and other research and allow simultaneous imaging of up to four distinct proteins in a cell 57 . In the same publication, the options for antibiotic markers were expanded.…”

“…In the same publication, the options for antibiotic markers were expanded. Historically, kanamycin ( aphI ), gentamicin ( aacC1 ), and streptomycin ( aadA ) resistance genes have been the available markers of choice 57 . Now resistance markers that enzymatically inactivate the translation inhibitors hygromycin B and blasticidin S have been adapted for use in B. burgdorferi 57 .…”

“…Historically, kanamycin ( aphI ), gentamicin ( aacC1 ), and streptomycin ( aadA ) resistance genes have been the available markers of choice 57 . Now resistance markers that enzymatically inactivate the translation inhibitors hygromycin B and blasticidin S have been adapted for use in B. burgdorferi 57 . Since these drugs are not used clinically and exhibit minimal cross-resistance with the other markers, they can be exploited for the genetic manipulation of B. burgdorferi 57 .…”

“…Now resistance markers that enzymatically inactivate the translation inhibitors hygromycin B and blasticidin S have been adapted for use in B. burgdorferi 57 . Since these drugs are not used clinically and exhibit minimal cross-resistance with the other markers, they can be exploited for the genetic manipulation of B. burgdorferi 57 . These laboratory tools will facilitate further research into B. burgdorferi infection and physiology, which will likely contribute to novel strategies to prevent and treat Lyme disease.…”