Abstract
Controlled RNA degradation is a crucial process in bacterial cell biology for maintaining proper transcriptome homeostasis and adaptation to changing environments. mRNA turnover in many Gram-positive bacteria involves a specialized ribonuclease called RNase J (RnJ). To date, however, nothing is known about this process in the diphtheria-causative pathogen Corynebacterium diphtheriae, nor is known the identity of this ribonuclease in this organism. Here, we report that C. diphtheriae DIP1463 encodes a predicted RnJ homolog, comprised of a conserved N-terminal β-lactamase domain, followed by β-CASP and C-terminal domains. A recombinant protein encompassing the β-lactamase domain alone displays 5′-exoribonuclease activity, which is abolished by alanine-substitution of the conserved catalytic residues His186 and His188. Intriguingly, deletion of DIP1463/rnj in C. diphtheriae reduces bacterial growth and generates cell shape abnormality with markedly augmented cell width. Comparative RNA-seq analysis revealed that RnJ controls a large regulon encoding many factors predicted to be involved in biosynthesis, regulation, transport, and iron acquisition. One upregulated gene in the ∆rnj mutant is ftsH, coding for a membrane protease (FtsH) involved in cell division, whose overexpression in the wild-type strain also caused cell-width augmentation. Critically, the ∆rnj mutant is severely attenuated in virulence in a Caenorhabditis elegans model of infection, while the FtsH-overexpressing and toxin-less strains exhibit full virulence as the wild-type strain. Evidently, RNase J is a key ribonuclease in C. diphtheriae that post-transcriptionally influences the expression of numerous factors vital to corynebacterial cell physiology and virulence. Our findings have significant implications for basic biological processes and mechanisms of corynebacterial pathogenesis.
Highlights
Messenger RNA turnover is crucial in recycling nucleotides and controlling gene expression that permit bacteria to adapt with environmental changes [1,2,3,4]
While a wide variety of RNases, which partake in Messenger RNA (mRNA) maturation and degradation and in turn regulation of gene expression, have been identified and characterized in many bacterial systems [5], to date there is a scarcity of information regarding these processes in the actinobacterium C. diphtheriae—a human pathogen of major significance that was contained in the past century by aggressive vaccination around the world
Bioinformatics and the biochemical and genetic studies presented here demonstrate that the C. diphtheriae DIP1463 encodes a member of the class D ribonuclease, RNase J, and that this ribonuclease acts to modulate the expression of a large number of genes involved in many cellular processes, including cell division, metabolism, and pathogenicity
Summary
Messenger RNA (mRNA) turnover is crucial in recycling nucleotides and controlling gene expression that permit bacteria to adapt with environmental changes [1,2,3,4]. As an essential enzyme in Escherichia coli [6], the endonuclease RNase E is part of the multiprotein RNA degradosome complex that contains PNPase (a 30 -50 exonuclease), RhlB (DEAD-box family helicase), and enolase [7]. RNase E cleaves polyribosomal mRNAs into small fragments, which are further degraded into nucleotides by exoribonucleases, such as PNPase, RNase. In bacterial species that lack RNase E, including the Gram-positive Firmicutes Staphylococcus aureus and Bacillus subtilis [9], RNase Y is the functional equivalent of RNase E that was shown to interact with the degradosome partners in vivo [10]
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