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 timedependent 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.

Characterization of 6S RNA in the Lyme disease spirochete

“…(a) Northern blot analyses of total RNA isolated from wild-type (WT), ssrS mutant (ssrS null) and ssrS complemented (ssrS comp) strains temperature-shifted from 23°C and grown at 35°C until mid-log phase. RNA was separated on an 0.8% agarose gel, transferred to membranes and hybridized with 32 P-labeled probes to ospC, ospA, dbpA and flaB mRNA ( Archambault et al, 2013;Fraser et al, 1997). We found that processing of both the 5′ and 3′ ends of Bb6S RNA requires the endoribonuclease RNase Y (Figure 3e,f).…”

“…6S RNA is processed from a larger transcript in E. coli by mechanisms involving RNase BN or the endoribonucleases RNase G and RNase E (Chen, Dutta, & Deutscher, ; Kim & Lee, ) and further trimmed by exoribonucleases RNase T and RNase PH (Li, Pandit, & Deutscher, ). B. burgdorferi has a limited repertoire of ribonucleases, compared to E. coli and B. subtilis , and lacks genes encoding RNase G and RNase E; the only predicted endoribonuclease homologs are RNase III, RNase M5, RNase P, RNase Y, RNase Z, YbeY and RNase HII (Anacker, Drecktrah, LeCoultre, Lybecker, & Samuels, ; Archambault, Borchert, Bergeron, Snow, & Schlax, ; Fraser et al, ). The size of Bb6S RNA was assayed by Northern blot in individual RNase mutant strains to investigate Bb6S RNA processing.…”

“…RNase E and RNase G, along with exoribonucleases, process the pre‐6S RNA to the mature form (Chae et al, ; Kim & Lee, ). In B. burgdorferi , ygfA is not adjacent to ssrS and the genome lacks homologs of RNase E and RNase G (Anacker et al, ; Archambault et al, ; Fraser et al, ). We found that processing of both the 5′ and 3′ ends of Bb6S RNA requires the endoribonuclease RNase Y (Figure e,f).…”

“…This is only the second identification of an sRNA-protein interaction in B. burgdorferi (Lybecker, Abel, Feig, & Samuels, 2010;Lybecker & Samuels, 2017). 6S RNA is processed from a larger transcript in E. coli by mechanisms involving RNase BN or the endoribonucleases RNase G and RNase E (Chen, Dutta, & Deutscher, 2016;Kim & Lee, 2004) and further trimmed by exoribonucleases RNase T and RNase PH (Li, Pandit, & Deutscher, 1998 (Anacker, Drecktrah, LeCoultre, Lybecker, & Samuels, 2018;Archambault, Borchert, Bergeron, Snow, & Schlax, 2013;Fraser et al, 1997). The size of Bb6S RNA was assayed by Northern blot in individual RNase mutant strains to investigate Bb6S RNA processing.…”

The Rho-Independent Transcription Terminator for the porA Gene Enhances Expression of the Major Outer Membrane Protein and Campylobacter jejuni Virulence in Abortion Induction

“…It also indicates that T porA affects MOMP expression in C. jejuni. (50,51). The intrinsic resistance is primarily due to the function of the CmeABC efflux system (52,53).…”