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

Organismal adaptations to environmental stimuli are governed by intracellular signaling molecules such as nucleotide second messengers. Recent studies have identified functional roles for the noncanonical 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 enterica serovar 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 that 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 as 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.

RNase I regulates Escherichia coli 2',3'-cyclic nucleotide monophosphate levels and biofilm formation.

Regulation of nucleotide and nucleoside concentrations is critical for faithful DNA replication, transcription, and translation in all organisms, and has been linked to bacterial biofilm formation. Unusual 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) recently were quantified in mammalian systems, and previous reports have linked these nucleotides to cellular stress and damage in eukaryotes, suggesting an intriguing connection with nucleotide/nucleoside pools and/or cyclic nucleotide signaling. This work reports the first quantification of 2',3'-cNMPs in Escherichia coli and demonstrates that 2',3'-cNMP levels in E. coli are generated specifically from RNase I-catalyzed RNA degradation, presumably as part of a previously unidentified nucleotide salvage pathway. Furthermore, RNase I and 2',3'-cNMP levels are demonstrated to play an important role in controlling biofilm formation. This work identifies a physiological role for cytoplasmic RNase I and constitutes the first progress toward elucidating the biological functions of bacterial 2',3'-cNMPs.

RNase I Modulates Escherichia coli Motility, Metabolism, and Resistance.

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.

Exploring the Links between Nucleotide Signaling and Quorum Sensing Pathways in Regulating Bacterial Virulence.

The survival of all organisms depends on implementation of appropriate phenotypic responses upon perception of relevant environmental stimuli. Sensory inputs are propagated via interconnected biochemical and/or electrical cascades mediated by diverse signaling molecules, including gases, metal cations, lipids, peptides, and nucleotides. These networks often comprise second messenger signaling systems in which a ligand (the primary messenger) binds to an extracellular receptor, thereby altering the intracellular concentration of a second messenger molecule which ultimately modulates gene expression through interaction with various effectors. The identification of intersections of these signaling pathways, such as nucleotide second messengers and quorum sensing, provides new insights into the mechanisms by which bacteria use multiple inputs to regulate cellular metabolism and phenotypes. Further investigations of the overlap between bacterial signaling pathways may yield new targets and methods to control bacterial behavior, such as biofilm formation and virulence.

Oxygen-Dependent Globin Coupled Sensor Signaling Modulates Motility and Virulence of the Plant Pathogen Pectobacterium carotovorum.

Bacterial pathogens utilize numerous signals to identify the presence of their host and coordinate changes in gene expression that allow for infection. Within plant pathogens, these signals typically include small molecules and/or proteins from their plant hosts and bacterial quorum sensing molecules to ensure sufficient bacterial cell density for successful infection. In addition, bacteria use environmental signals to identify conditions when the host defenses are weakened and potentially to signal entry into an appropriate host/niche for infection. A globin coupled sensor protein (GCS), termed PccGCS, within the soft rot bacterium Pectobacterium carotovorum ssp. carotovorum WPP14 has been identified as an O2 sensor and demonstrated to alter virulence factor excretion and control motility, with deletion of PccGCS resulting in decreased rotting of a potato host. Using small molecules that modulate bacterial growth and quorum sensing, PccGCS signaling also has been shown to modulate quorum sensing pathways, resulting in the PccGCS deletion strain being more sensitive to plant-derived phenolic acids, which can function as quorum sensing inhibitors, and exhibiting increased N-acylhomoserine lactone (AHL) production. These findings highlight a role for GCS proteins in controlling key O2-dependent phenotypes of pathogenic bacteria and suggest that modulating GCS signaling to limit P. carotovorum motility may provide a means to decrease rotting of plant hosts.

Identification of Ellagic Acid Rhamnoside as a Bioactive Component of a Complex Botanical Extract with Anti-biofilm Activity.

Staphylococcus aureus is a leading cause of hospital-acquired infections. It is listed among the top "serious threats" to human health in the USA, due in large part to rising rates of resistance. Many S. aureus infections are recalcitrant to antibiotic therapy due to their ability to form a biofilm, which acts not only as a physical barrier to antibiotics and the immune system, but results in differences in metabolism that further restricts antibiotic efficacy. Development of a modular strategy to synthesize a library of phenolic glycosides allowed for bioactivity testing and identification of anti-biofilm compounds within an extract of the elmleaf blackberry (Rubus ulmifolius). Two ellagic acid (EA) derivatives, EA xyloside and EA rhamnoside, have been identified as components of the Rubus extract. In addition, EA rhamnoside has been identified as an inhibitor of biofilm formation, with activity comparable to the complex extract 220D-F2 (composed of a mixture of EA glycosides), and confirmed by confocal laser scanning microscopy analyses.

A facile and sensitive method for quantification of cyclic nucleotide monophosphates in mammalian organs: basal levels of eight cNMPs and identification of 2',3'-cIMP.

A sensitive, versatile and economical method to extract and quantify cyclic nucleotide monophosphates (cNMPs) using LC-MS/MS, including both 3',5'-cNMPs and 2',3'-cNMPs, in mammalian tissues and cellular systems has been developed. Problems, such as matrix effects from complex biological samples, are addressed and have been optimized. This protocol allows for comparison of multiple cNMPs in the same system and was used to examine the relationship between tissue levels of cNMPs in a panel of rat organs. In addition, the study reports the first identification and quantification of 2',3'-cIMP. The developed method will allow for quantification of cNMPs levels in cells and tissues with varying disease states, which will provide insight into the role(s) and interplay of cNMP signalling pathways.