NrnA is a 5'-3' exonuclease that processes short RNA substrates in vivo and in vitro.

Bacterial RNases process RNAs until only short oligomers (2-5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2-4 nucleotides in length, all of which were processed from their 5' terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.

Chemical probing provides insight into the native assembly state of a bacterial microcompartment.

Bacterial microcompartments (BMCs) are widespread in bacteria and are used for a variety of metabolic purposes, including catabolism of host metabolites. A suite of proteins self-assembles into the shell and cargo layers of BMCs. However, the native assembly state of these large complexes remains to be elucidated. Herein, chemical probes were used to observe structural features of a native BMC. While the exterior could be demarcated with fluorophores, the interior was unexpectedly permeable, suggesting that the shell layer may be more dynamic than previously thought. This allowed access to cross-linking chemical probes, which were analyzed to uncover the protein interactome. These cross-links revealed a complex multivalent network among cargo proteins that contained encapsulation peptides and demonstrated that the shell layer follows discrete rules in its assembly. These results are consistent overall with a model in which biomolecular condensation drives interactions of cargo proteins before envelopment by shell layer proteins.

Loss of Ethanolamine Utilization in Enterococcus faecalis Increases Gastrointestinal Tract Colonization.

Enterococcus faecalis is paradoxically a dangerous nosocomial pathogen and a normal constituent of the human gut microbiome, an environment rich in ethanolamine. E. faecalis carries the eut (ethanolamine utilization) genes, which enable the catabolism of ethanolamine (EA) as a valuable source of carbon and/or nitrogen. EA catabolism was previously shown to contribute to the colonization and growth of enteric pathogens, such as Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli (EHEC), in the gut environment. We tested the ability of eut mutants of E. faecalis to colonize the gut using a murine model of gastrointestinal (GI) tract competition and report the surprising observation that these mutants outcompete the wild-type strain.IMPORTANCE Some bacteria that are normal, harmless colonizers of the human body can cause disease in immunocompromised patients, particularly those that have been heavily treated with antibiotics. Therefore, it is important to understand the factors that promote or negate these organisms' ability to colonize. Previously, ethanolamine, found in high concentrations in the GI tract, was shown to promote the colonization and growth of bacteria associated with food poisoning. Here, we report the surprising, opposite effect of ethanolamine utilization on the commensal colonizer E. faecalis, namely, that loss of this metabolic capacity made it a better colonizer.

Riboswitches. A riboswitch-containing sRNA controls gene expression by sequestration of a response regulator.

The ethanolamine utilization (eut) locus of Enterococcus faecalis, containing at least 19 genes distributed over four polycistronic messenger RNAs, appears to be regulated by a single adenosyl cobalamine (AdoCbl)-responsive riboswitch. We report that the AdoCbl-binding riboswitch is part of a small, trans-acting RNA, EutX, which additionally contains a dual-hairpin substrate for the RNA binding-response regulator, EutV. In the absence of AdoCbl, EutX uses this structure to sequester EutV. EutV is known to regulate the eut messenger RNAs by binding dual-hairpin structures that overlap terminators and thus prevent transcription termination. In the presence of AdoCbl, EutV cannot bind to EutX and, instead, causes transcriptional read through of multiple eut genes. This work introduces riboswitch-mediated control of protein sequestration as a posttranscriptional mechanism to coordinately regulate gene expression.

Transport of magnesium by a bacterial Nramp-related gene.

Magnesium is an essential divalent metal that serves many cellular functions. While most divalent cations are maintained at relatively low intracellular concentrations, magnesium is maintained at a higher level (∼0.5-2.0 mM). Three families of transport proteins were previously identified for magnesium import: CorA, MgtE, and MgtA/MgtB P-type ATPases. In the current study, we find that expression of a bacterial protein unrelated to these transporters can fully restore growth to a bacterial mutant that lacks known magnesium transporters, suggesting it is a new importer for magnesium. We demonstrate that this transport activity is likely to be specific rather than resulting from substrate promiscuity because the proteins are incapable of manganese import. This magnesium transport protein is distantly related to the Nramp family of proteins, which have been shown to transport divalent cations but have never been shown to recognize magnesium. We also find gene expression of the new magnesium transporter to be controlled by a magnesium-sensing riboswitch. Importantly, we find additional examples of riboswitch-regulated homologues, suggesting that they are a frequent occurrence in bacteria. Therefore, our aggregate data discover a new and perhaps broadly important path for magnesium import and highlight how identification of riboswitch RNAs can help shed light on new, and sometimes unexpected, functions of their downstream genes.

Multiple posttranscriptional regulatory mechanisms partner to control ethanolamine utilization in Enterococcus faecalis.

Ethanolamine, a product of the breakdown of phosphatidylethanolamine from cell membranes, is abundant in the human intestinal tract and in processed foods. Effective utilization of ethanolamine as a carbon and nitrogen source may provide a survival advantage to bacteria that inhabit the gastrointestinal tract and may influence the virulence of pathogens. In this work, we describe a unique series of posttranscriptional regulatory strategies that influence expression of ethanolamine utilization genes (eut) in Enterococcus, Clostridium, and Listeria species. One of these mechanisms requires an unusual 2-component regulatory system. Regulation involves specific sensing of ethanolamine by a sensor histidine kinase (EutW), resulting in autophosphorylation and subsequent phosphoryl transfer to a response regulator (EutV) containing a RNA-binding domain. Our data suggests that EutV is likely to affect downstream gene expression by interacting with conserved transcription termination signals located within the eut locus. Breakdown of ethanolamine requires adenosylcobalamin (AdoCbl) as a cofactor, and, intriguingly, we also identify an intercistronic AdoCbl riboswitch that has a predicted structure different from previously established AdoCbl riboswitches. We demonstrate that association of AdoCbl to this riboswitch prevents formation of an intrinsic transcription terminator element located within the intercistronic region. Together, these results suggest an intricate and carefully coordinated interplay of multiple regulatory strategies for control of ethanolamine utilization genes. Gene expression appears to be directed by overlapping posttranscriptional regulatory mechanisms, each responding to a particular metabolic signal, conceptually akin to regulation by multiple DNA-binding transcription factors.

An mRNA structure that controls gene expression by binding S-adenosylmethionine.

Riboswitches are metabolite-binding RNA structures that serve as genetic control elements for certain messenger RNAs. These RNA switches have been identified in all three kingdoms of life and are typically responsible for the control of genes whose protein products are involved in the biosynthesis, transport or utilization of the target metabolite. Herein, we report that a highly conserved RNA domain found in bacteria serves as a riboswitch that responds to the coenzyme S-adenosylmethionine (SAM) with remarkably high affinity and specificity. SAM riboswitches undergo structural reorganization upon introduction of SAM, and these allosteric changes regulate the expression of 26 genes in Bacillus subtilis. This and related findings indicate that direct interaction between small metabolites and allosteric mRNAs is an important and widespread form of genetic regulation in bacteria.