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

Abstract

Organismal adaptations to environmental stimuli are governed by intracellular signaling molecules such as nucleotide second messengers. Recent studies have identified functional roles for the non-canonical 2´,3´-cyclic nucleotide monophosphates (2´,3´-cNMPs) in both eukaryotes and prokaryotes. In Escherichia coli , 2´,3´-cNMPs are produced by RNase I-catalyzed RNA degradation, and these cyclic nucleotides modulate biofilm formation through unknown mechanisms. The present work dissects cellular processes in E. coli and Salmonella Typhimurium that are modulated by 2´,3´-cNMPs through the development of cell-permeable 2´,3´-cNMP analogs and a 2´,3´-cyclic nucleotide phosphodiesterase. Utilization of these chemical and enzymatic tools, in conjunction with phenotypic and transcriptomic investigations, identified pathways regulated by 2´,3´-cNMPs, including flagellar motility and biofilm formation, and by oligoribonucleotides with 3’-terminal 2´,3´-cyclic phosphates, including responses to cellular stress. Furthermore, interrogation of metabolomic and organismal databases has identified 2´,3´-cNMPs in numerous organisms and homologs of the E. coli metabolic proteins that are involved in key eukaryotic pathways. Thus, the present work provides key insights into the roles of these understudied facets of nucleotide metabolism and signaling in prokaryotic physiology and suggest broad roles for 2´,3´-cNMPs among bacteria and eukaryotes. IMPORTANCE Bacteria adapt to environmental challenges by producing intracellular signaling molecules which control downstream pathways and alter cellular processes for survival. Nucleotide second messengers serve to transduce extracellular signals and regulate a wide array of intracellular pathways. Recently, 2´,3´-cyclic nucleotide monophosphates (2´,3´-cNMPs) were identified for contributing to the regulation of cellular pathways in eukaryotes and prokaryotes. In this study we define previously unknown cell processes that are affected by fluctuating 2´,3´-cNMP levels or RNA oligomers with 2´,3´-cyclic phosphate termini in E. coli and Salmonella Typhimurium, providing a framework for studying novel signaling networks in prokaryotes. Furthermore, we utilize metabolomics databases to identify additional prokaryotic and eukaryotic species that generate 2´,3´-cNMPs as a resource for future studies.

Binding of 2′,3′-Cyclic Nucleotide Monophosphates to Bacterial Ribosomes Inhibits Translation

“… 16 These results suggest that cells can modulate protein synthesis by altering cytoplasmic 2′,3′-cNMP levels. The fluctuations in 2′,3-cNMP concentration (ranging from <500 fM from >100 μM) over the growth phases 16 , 29 may help to modulate translation as the cells alter behavior and physiological state. Similarly, the increase in 2′,3′-cNMP levels upon amino acid starvation 16 may allow the cells to rapidly inhibit translation and preserving amino acid stores.…”

“…Bacterial growth and 2′,3′-cNMP isolation was performed as previously described, with minor modifications. , Isolated colonies were grown overnight in 2 mL of M9 minimal media (supplemented with 0.2% casamino acids, 0.4% glucose) and inoculated 1:100 into fresh media containing 100 μM IPTG and, if necessary, 100 μg/mL ampicillin (DOT Scientific). Growths occurred in 50 mL VWR centrifuge tubes (sterile, polypropylene, with caps loose for aeration; Cat: 21008-242), under 37 °C incubation and 225 rpm shaking.…”

“…To investigate the role(s) of 2′,3′-cNMPs, our groups have developed tools to modulate 2′,3′-cNMP levels and have demonstrated that decreased 2′,3′-cNMP levels lead to widespread changes in the transcriptomes of E. coli and Salmonella Typhimurium. 29 …”

“…To investigate the role(s) of 2′,3′-cNMPs, our groups have developed tools to modulate 2′,3′-cNMP levels and have demonstrated that decreased 2′,3′-cNMP levels lead to widespread changes in the transcriptomes of E. coli and Salmonella Typhimurium. 29 The broad changes in transcription observed when 2′,3′-cNMP levels are decreased suggest that bacteria sense 2′,3′-cNMP levels, likely through binding to cellular proteins. Indeed, recent work in Arabidopsis demonstrated that 2′,3′-cAMP is bound by the stress granule and that treatment with 2′,3′-cAMP resulted in increased stress granule production.…”

“…Another class of cellular nucleotides, termed 2′,3′-cyclic nucleotide monophosphates (2′,3′-cNMPs), has been recently (re)­discovered in bacteria and eukaryotes. − 2′,3′-cNMPs are products of cellular RNA degradation, , and these molecules have been linked to cellular stress in mammals − and Arabidopsis . In E. coli and Salmonella enterica Typhimurium ( Salmonella Typhimurium), 2′,3′-cNMPs are upregulated under conditions of amino acid starvation, mRNA decay, ribosome recycling, and alkylating agents. , In addition, 2′,3′-cNMP levels have been demonstrated to fluctuate with the growth phase in both E. coli and Salmonella , suggesting that 2′,3′-cNMPs may be involved in the transition from the exponential to stationary phase. To investigate the role(s) of 2′,3′-cNMPs, our groups have developed tools to modulate 2′,3′-cNMP levels and have demonstrated that decreased 2′,3′-cNMP levels lead to widespread changes in the transcriptomes of E. coli and Salmonella Typhimurium …”