Measurements of mRNA degradation in Borrelia burgdorferi

Abstract

The importance of gene regulation in the enzootic cycle of Borrelia burgdorferi, the spirochete that causes Lyme disease, is well established. B. burgdorferi regulates gene expression in response to changes in environmental stimuli associated with changing hosts. In this study, we monitored mRNA decay in B. burgdorferi following transcriptional arrest with actinomycin D. The time-dependent decay of transcripts encoding RNA polymerase subunits (rpoA and rpoS), ribosomal proteins (rpsD, rpsK, rpsM, rplQ, and rpsO), a nuclease (pnp), outer surface lipoproteins (ospA and ospC), and a flagellar protein (flaB) have different profiles and indicate half-lives ranging from approximately 1 min to more than 45 min in cells cultured at 35°C. Our results provide a first step in characterizing mRNA decay in B. burgdorferi and in investigating its role in gene expression and regulation.

Fig 1

Actinomycin D prevents induction of transcription of ospC controlled by an IPTG-inducible promoter. Quantitative RT-PCR amplification curves are shown for ospC (A) and flaB (B) transcripts. A3-LS-flacp-ospC cultures at mid-log growth phase were split, and half of the initial culture was treated with actinomycin D (final concentration, 150 μg ml⁻¹). Thirty minutes later, cultures were split again and treated with IPTG or used as controls lacking an inducer. Cells were cultured for an additional 3 h before RNA isolation and qRT-PCR amplification. ●, Lacking both actinomycin D and IPTG (C_{T ospC} = 22.22; C_{T flaB} = 12.42); ○, lacking actinomycin D but containing IPTG (C_{T ospC} = 17.51; C_{T flaB} = 12.30); ▼, containing actinomycin D but lacking IPTG (C_{T ospC} = 22.57; C_{T flaB} = 12.96); Δ, containing both actinomycin D and IPTG (C_{T ospC} = 22.70; C_{T flaB} = 12.87). Ten ng of total RNA was used in each reaction mixture. dRn, magnitude of the fluorescence signal over background fluorescence generated at each time point.

Fig 2

Transcriptional arrest initiated by addition of actinomycin D or by the removal of inducer results in similar patterns of rpoS mRNA degradation in strain 297-LK-flacp-rpoS. Transcription of rpoS was induced by adding 1 mM (final concentration) IPTG to growing 297-LK-flacp-rpoS cultures. Twenty-four h later, cells were washed, split, and resuspended in media without inducer or with inducer plus 150 μg ml⁻¹ actinomycin D. Aliquots from each culture were collected at time points 5, 10, 20, 45, 240, and 1,260 min after resuspension. The fraction of rpoS (A) and flaB (B) mRNA remaining at time points after transcriptional arrest brought about by removal of inducer (filled circles) or by addition of actinomycin D (empty circles) was determined. Means ± standard deviations are plotted against time (n = 2; with duplicate qRT-PCRs). A horizontal line representing 50% of the original mRNA level has been plotted as a reference in each graph. Raw data (C_T values) are included in Table S1 in the supplemental material.

Fig 3

mRNA decay curves for diverse genes in B. burgdorferi. The fraction of individual mRNA transcripts remaining at time points following the addition of actinomycin D. Three biological replicates (n = 3) were performed (represented by squares, circles, and triangles) with duplicate qRT-PCRs (open versus filled shapes). A horizontal line representing 50% of the original mRNA level has been plotted as a reference. (A) rpoS encoding alternative sigma factor RpoS (σ^s). (B) rpoA encoding the alpha subunit of RNA polymerase. (C) rplQ encoding ribosomal protein L17. (D) rpsD encoding ribosomal protein S4. (E) rpsK encoding ribosomal protein S11. (F) rpsM encoding ribosomal protein S13. (G) rpsO encoding ribosomal protein S15. (H) Region overlapping rpsO and pnp. (I) pnp encoding polynucleotide phosphorylase. (J) ospA encoding outer surface lipoprotein A. (K) ospC encoding outer surface lipoprotein C. (L) flaB encoding flagellar protein B.

Fig 4

Comparison of mRNA half-lives in polycistronic transcripts. The expected operon configuration for genes used in this study are shown with the calculated half-life of the amplified product labeled in parentheses. Genes encoding RNA polymerase subunits are blue, ribosomal proteins are red, nucleases are green, outer surface lipoproteins are black, and flagellar proteins are yellow. Gene loci that were not tested are colored gray.