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Ifill, Gyles Anderson

University of British Columbia

In many Gram-negative pathogens, gene transcription is often facilitated by post-transcriptional regulatory mechanisms. In Bordetella pertussis, several regulatory RNAs have been identified, but their targets and impacts on virulence factor production have not been assessed. RNase III and RNase E, endonucleases with dominant roles in regulatory RNA processing, were mutated in B. pertussis to determine gene loci regulated at the post-transcriptional level. RNA-Seq analysis of these strains identified that ~25% of the B. pertussis transcriptome was affected in each endonuclease mutant. Substantial impacts were observed on genes associated with amino acid uptake, bacterial secretion, and many virulence factors. Comparing these findings to the regulon of the RNA chaperone Hfq, 120 genes, and 19 operons were identified as potentially influenced at the post-transcriptional level. Amongst these, the lipid A glucosamine modification (lgm) locus was one of the most upregulated gene loci in the RNase E mutant strain. The lgm locus is a 5 gene operon consisting of four genes in one orientation (lgmA-D) and a fifth open reading frame (lgmE) overlapping in the opposite orientation. Given lipid A modifications in Gram-negative bacteria are typically controlled by complex regulatory networks, it was proposed that several overlapping systems are involved in lgm locus regulation, including the role of a cis-encoded antisense RNA. As shown by luciferase reporter assays, the lgm locus responds to Bvg phase, nutrient availability, low pH, and increased C02%. These assays also identified a reciprocal relationship in activation of the diametrically opposed lgmA and lgmE promoters, suggestive of asRNA regulation. It was then proposed that lgmE acts as dual-function RNA as the lgmE ORF has all the characteristics of a translated protein. It is shown that lgmE encodes a functional, small membrane-associated protein, and deletion and overexpression of lgmE negatively impact lgm locus activity. Furthermore, structural predictions and modelling of protein-protein interactions suggest LgmE may form a membrane anchor for localization with LgmB. Overall, the lgm locus forms a non-contiguous operon whose activity is modulated at the transcriptional, post-transcriptional, and post-translational levels, with these integrated mechanisms intersecting as a means to fine-tune lipid A modification in B. pertussis.

Text

2023-01-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/80589

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/