lacZ reporter system for use in Borrelia burgdorferi

Abstract

Regulation of gene expression is critical for the ability of Borrelia burgdorferi to adapt to different environments during its natural infectious cycle. Reporter genes have been used successfully to study gene regulation in multiple organisms. We have introduced a lacZ gene into B. burgdorferi, and we show that B. burgdorferi produces a protein with detectable β-galactosidase activity in both liquid and solid media when lacZ is expressed from a constitutive promoter. Furthermore, when lacZ is expressed from the ospC promoter, β-galactosidase activity is detected only in B. burgdorferi clones that express ospC, and it accurately monitors endogenous gene expression. The addition of lacZ to the repertoire of genetic tools available for use in B. burgdorferi should contribute to a better understanding of how B. burgdorferi gene expression is regulated during the infectious cycle.

FIG. 1.

Schematic diagram of the lacZ shuttle vectors. The E. coli lacZ gene was cloned into pBSV2G_dvB2, creating pBH-lacZEc (A, top). A B. burgdorferi codon-optimized lacZ gene, lacZ_Bb, was cloned directly into pBSV2G, creating pBH-lacZBb (B, top). The flaB promoter from B. burgdorferi (flaBp) was added to each construct, yielding pBHflaBp-lacZEc (A, bottom) and pBHflaBp-lacZBb (B, bottom), respectively.

FIG. 2.

β-Galactosidase activity in E. coli and B. burgdorferi lysates. Modified Miller ONPG assays (22) were performed on Δlac E. coli and B. burgdorferi harboring pBH-lacZEc, pBHflaBp-lacZEc, pBH-lacZBb, and pBHflaBp-lacZBb. β-Galactosidase activity units (Miller units) are reported per mg of protein, and the levels represent means ± standard errors of the means. Background activity of bacteria without the shuttle vector was subtracted from reported values. ***, P < 0.001; **, P < 0.01 (compared to respective promoterless constructs or as indicated based on a Student two-tailed t test).

FIG. 3.

lacZ for blue-white screening of B. burgdorferi colonies. BSK solid medium without phenol red was used to grow B31-A34 clones harboring pBH-lacZEc (top left), pBHflaBp-lacZEc (top right), pBH-lacZBb (bottom left), and pBHflaBp-lacZBb (bottom right). Approximately 0.5 ml of X-Gal in DMSO (20 mg/ml) was added to plates after colony formation, and plates were incubated overnight before photographs were taken.

FIG. 4.

lacZ expression and β-galactosidase activity as a reporter of ospC expression. (A) β-Galactosidase activity in B. burgdorferi B31-A34 and B312 harboring pBH-lacZBb, pBHospCp-lacZBb, and pBHflaBp-lacZBb, as indicated. Units are reported per mg of protein, and levels represent means ± standard errors of the means. The background activity of bacteria lacking a shuttle vector was subtracted from reported values. ND, no detectable activity above background (i.e., without plasmid) levels. ***, P < 0.001; **, P < 0.01 (compared to respective promoterless constructs or as indicated based on a Student two-tailed t test). (B) Transcript levels of ospC and lacZ_Bb in B31-A34 and B312 clones harboring lacZ_Bb, as indicated, were measured by quantitative RT-PCR with primer/probe sets specific for ospC and lacZ_Bb, respectively, and normalized to the chromosomal flaB gene transcript. Values represent means ± standard errors of the means. cDNA from a RT-PCR mixture without reverse transcriptase was used to control for DNA contamination and was subtracted from reported values. (C to E) BSK solid medium without phenol red was used to grow B31-A34 harboring pBHospCp-lacZBb (C), B312 harboring pBHospCp-lacZ_Bb (D), or B312 harboring pBHflaBp-lacZBb (E). X-Gal was added after colony formation, and plates were incubated overnight before photographs were taken. Magnified insets have been added to panels D and E to illustrate differences between B312 colonies harboring pBHospCp-lacZBb versus pBHflaBp-lacZBb.