RNase I Modulates Escherichia coli Motility, Metabolism, and Resistance

Abstract

Bacteria are constantly adapting to their environment by sensing extracellular factors that trigger production of intracellular signaling molecules, known as second messengers. Recently, 2′,3′-cyclic nucleotide monophosphates (2′,3′-cNMPs) were identified in Escherichia coli and have emerged as possible novel signaling molecules. 2′,3′-cNMPs are produced through endonucleolytic cleavage of short RNAs by the T2 endoribonuclease, RNase I; however, the physiological roles of RNase I remain unclear. Our transcriptomic analysis suggests that RNase I is involved in modulating numerous cellular processes, including nucleotide metabolism, motility, acid sensitivity, metal homeostasis, and outer membrane morphology. Through a combination of deletion strain and inhibitor studies, we demonstrate that RNase I plays a previously unknown role in E. coli stress resistance by affecting pathways that are part of the defense mechanisms employed by bacteria when introduced to external threats, including antibiotics. Thus, this work provides insight into the emerging roles of RNase I in bacterial signaling and physiology and highlights the potential of RNase I as a target for antibacterial adjuvants.

Cellular Effects of 2′,3′-Cyclic Nucleotide Monophosphates in Gram-Negative Bacteria

“…CNPase was chosen for its substrate specificity; unlike other cyclic nucleotide phosphodiesterases, it does not hydrolyze 3′,5′-cNMPs and acts primarily on 2′,3′ cyclic phosphates of 2′,3′-cyclic mononucleotides, as well as 2′,3′-cyclic phosphates on the 3′ terminus of oligoribonucleotides (reviewed in reference 28 ). Both RNase I deletion and expression of CNPase reduce 2′,3′-cNMP levels; however, relative to our recently reported data comparing gene expression in an E. coli Δ rna mutant to that in the WT strain ( 26 ), a comparison of a WT strain expressing CNPase versus one expressing CNP-inact allows us to uncover processes that are modulated directly by 2′,3′-cyclic phosphate-containing molecules, while excluding any confounding effects of deleting RNase I. This is particularly important, as our previous work identified roles for RNase I that are not linked to cytoplasmic 2′,3′-cNMP levels.…”

“…Our group previously found that RNase I (encoded by the rna gene) generates all detectable 2′,3′-cNMPs in E. coli ( 26 ); however, since RNase I affects nucleotide metabolism in both the cytoplasm and the periplasm ( 24 , 26 ), we required methods to modulate 2′,3′-cNMP levels independently of RNase I expression to determine which cellular effects are directly attributable to fluctuations in 2′,3′-cNMP pools. To this end, we leveraged the catalytic domain of Rattus norvegicus cyclic nucleotide phosphodiesterase ( 29 ) (CNPase; UniProtKB P13233 for full-length protein) to hydrolyze 2′,3′-cNMPs in RNase I + (wild-type [WT]) cells.…”

“…Previous work validated that E. coli expressing CNPase from plasmid pKT-CNP exhibits subquantifiable levels of 2′,3′-cAMP and -cGMP, and ∼25-fold and ∼15-fold lower levels of 2′,3′-cCMP and -cUMP, respectively ( 24 ). Therefore, expression of CNPase enabled identification of 2′,3′-cNMP-linked processes through transcriptomic profiling of WT E. coli with reduced 2′,3′-cNMP levels, eliminating the confounding effects inherent to using the E. coli Δ rna strain as in our previous transcriptomic analysis ( 26 ). RNA-seq revealed 519 genes that were significantly differentially expressed (adjusted P [ P adj ] < 0.05; >2-fold change in expression) in WT E. coli expressing CNPase compared to expression in equivalent cultures expressing a catalytically inactive CNPase variant (CNP-inact) (see Data Set S1 in the supplemental material).…”

“…Within E. coli , RNase I, an RNase T2 family member ( 25 ), generates all detectable 2′,3′-cNMPs through hydrolysis of RNA, providing the first insight into the biosynthetic origin of these atypical nucleotides in any organism ( 24 ). Additional experiments identified physiological functions for 2′,3′-cNMPs and RNase I in biofilm production ( 24 ); separately, RNase I was also found to modulate E. coli motility, resistance to β-lactams and acid stress, and outer membrane structure ( 26 ). These preliminary findings suggest that there are additional 2′,3′-cNMP- and RNase I-dependent processes yet to be discovered.…”

E. coli RNase I exhibits a strong Ca2+-dependent inherent double-stranded RNase activity

“…High concentrations of free Ca 2+ have been reported in the E. coli periplasm ( 31 ), indicating that availability of Ca 2+ may not be a limiting factor. While there still is some uncertainty on the cellular role of RNase I (reviewed by Deutscher ( 32 )), new studies showed that bacterial RNase I is involved in the regulation of a multitude of processes from motility, metabolism, and resistance ( 33 , 34 ). These regulatory functions have been linked to the generation of 2′,3′-cyclic nucleotide monophosphates (2′,3′-cNMPs) by RNase I through RNA scavenging ( 35 , 36 ), where the ability to efficiently degrade single-stranded as well as double-stranded or highly structured RNA is highly desirable.…”