Bacillus subtilis NrnB is expressed during sporulation and acts as a unique 3'-5' exonuclease.

All cells employ a combination of endo- and exoribonucleases to degrade long RNA polymers to fragments 2-5 nucleotides in length. These short RNA fragments are processed to monoribonucleotides by nanoRNases. Genetic depletion of nanoRNases has been shown to increase abundance of short RNAs. This deleteriously affects viability, virulence, and fitness, indicating that short RNAs are a metabolic burden. Previously, we provided evidence that NrnA is the housekeeping nanoRNase for Bacillus subtilis. Herein, we investigate the biological and biochemical functions of the evolutionarily related protein, B. subtilis NrnB (NrnBBs). These experiments show that NrnB is surprisingly different from NrnA. While NrnA acts at the 5' terminus of RNA substrates, NrnB acts at the 3' terminus. Additionally, NrnA is expressed constitutively under standard growth conditions, yet NrnB is selectively expressed during endospore formation. Furthermore, NrnA processes only short RNAs, while NrnB unexpectedly processes both short RNAs and longer RNAs. Indeed, inducible expression of NrnB can even complement the loss of the known global 3'-5' exoribonucleases, indicating that it acts as a general exonuclease. Together, these data demonstrate that NrnB proteins, which are widely found in Firmicutes, Epsilonproteobacteria and Archaea, are fundamentally different than NrnA proteins and may be used for specialized purposes.

In Vitro Analysis of Bacterial Microcompartments and Shell Protein Superstructures by Confocal Microscopy.

The shell proteins that comprise bacterial microcompartments (BMCs) can self-assemble into an array of superstructures such as nanotubes, flat sheets, and icosahedra. The physical characterization of BMCs and these superstructures typically relies on electron microscopy, which decouples samples from their solution context. We hypothesize that an investigation of fluorescently tagged BMCs and shell protein superstructures in vitro using high-resolution confocal microscopy will lead to new insights into the solution behavior of these entities. We find that confocal imaging is able to capture nanotubes and sheets previously reported by transmission electron microscopy (TEM). Using a combination of fluorescent tags, we present qualitative evidence that these structures intermix with one another in a hetero- and homotypic fashion. Complete BMCs are also able to accomplish intermixing as evidenced by colocalization data. Finally, a simple colocalization experiment suggests that fluorescently modified encapsulation peptides (EPs) may prefer certain shell protein binding partners. Together, these data demonstrate that high-resolution confocal microscopy is a powerful tool for investigating microcompartment-related structures in vitro, particularly for colocalization analyses. These results also support the notion that BMCs may intermix protein components, presumably from the outer shell. IMPORTANCE Microcompartments are large, organelle-like structures that help bacteria catabolize targeted metabolites while also protecting the cytosol against highly reactive metabolic intermediates. Their protein shell self-assembles into a polyhedral structure of approximately 100 to 200 nm in diameter. Inside the shell are thousands of copies of cargo enzymes, which are responsible for a specific metabolic pathway. While different approaches have revealed high-resolution structures of individual microcompartment proteins, it is less clear how these factors self-assemble to form the full native structure. In this study, we show that laser scanning confocal microscopy can be used to study microcompartment proteins. We find that this approach allows researchers to investigate the interactions and potential exchange of shell protein subunits in solution. From this, we conclude that confocal microscopy offers advantages for studying the in vitro structures of other microcompartments as well as carboxysomes and other bacterial organelles.

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.

Nano-RNases: oligo- or dinucleases?

Diribonucleotides arise from two sources: turnover of RNA transcripts (rRNA, tRNA, mRNA, and others) and linearization of cyclic-di-nucleotide signaling molecules. In both cases, there appears to be a requirement for a dedicated set of enzymes that will cleave these diribonucleotides into mononucleotides. The first enzyme discovered to mediate this activity is oligoribonuclease (Orn) from Escherichia coli. In addition to being the enzyme that cleaves dinucleotides and potentially other short oligoribonucleotides, Orn is also the only known exoribonuclease enzyme that is essential for E. coli, suggesting that removal of the shortest RNAs is an essential cellular function. Organisms naturally lacking the orn gene encode other nanoRNases (nrn) that can complement the conditional E. coli orn mutant. This review covers the history and recent advances in our understanding of these enzymes and their substrates. In particular, we focus on (i) the sources of diribonucleotides; (ii) the discovery of exoribonucleases; (iii) the structural features of Orn, NrnA/NrnB, and NrnC; (iv) the enzymatic activity of these enzymes against diribonucleotides versus other substrates; (v) the known physiological consequences of accumulation of linear dinucleotides; and (vi) outstanding biological questions for diribonucleotides and diribonucleases.

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.

Structural characterization of NrnC identifies unifying features of dinucleotidases.

RNA degradation is fundamental for cellular homeostasis. The process is carried out by various classes of endolytic and exolytic enzymes that together degrade an RNA polymer to mono-ribonucleotides. Within the exoribonucleases, nano-RNases play a unique role as they act on the smallest breakdown products and hence catalyze the final steps in the process. We recently showed that oligoribonuclease (Orn) acts as a dedicated diribonucleotidase, defining the ultimate step in RNA degradation that is crucial for cellular fitness (Kim et al., 2019). Whether such a specific activity exists in organisms that lack Orn-type exoribonucleases remained unclear. Through quantitative structure-function analyses, we show here that NrnC-type RNases share this narrow substrate length preference with Orn. Although NrnC and Orn employ similar structural features that distinguish these two classes of dinucleotidases from other exonucleases, the key determinants for dinucleotidase activity are realized through distinct structural scaffolds. The structures, together with comparative genomic analyses of the phylogeny of DEDD-type exoribonucleases, indicate convergent evolution as the mechanism of how dinucleotidase activity emerged repeatedly in various organisms. The evolutionary pressure to maintain dinucleotidase activity further underlines the important role these analogous proteins play for cell growth.

A NusG Specialized Paralog That Exhibits Specific, High-Affinity RNA-Binding Activity.

Bacterial NusG associates with RNA polymerase (RNAP) through its N-terminal domain, while the C-terminal domain (CTD) forms dynamic interactions with Rho, S10, NusB and NusA to affect transcription elongation. While virtually all bacteria encode for a core NusG, many also synthesize paralogs that transiently bind RNAP to alter expression of targeted genes. Yet, despite the importance of the genes they regulate, most of the subfamilies of NusG paralogs (e.g., UpxY, TaA, ActX and LoaP) have not been investigated in depth. Herein, we discover that LoaP requires a small RNA hairpin located within the 5' leader region of its targeted operons. LoaP binds the RNA element with nanomolar affinity and high specificity, in contrast to other NusG proteins, which have not been shown to exhibit RNA-binding activity. These data reveal a sequence feature that can be used to identify LoaP-regulated operons. This discovery also expands the repertoire of macromolecular interactions exhibited by the NusG CTD during transcription elongation to include an RNA ligand.

Riboswitch-Mediated Detection of Metabolite Fluctuations During Live Cell Imaging of Bacteria.

Riboswitches are a class of noncoding RNAs that regulate gene expression in response to changes in intracellular metabolite concentrations. When riboswitches are placed upstream of genetic reporters, the degree of reporter activity reflects the relative abundance of the metabolite that is sensed by the riboswitch. This method describes how reporters for live cell imaging, such as yellow fluorescent protein (YFP), can be placed under genetic control by metabolite-sensing riboswitches in the bacterium Bacillus subtilis. Specifically, a protocol for generating a fluorescent YFP reporter, based on a c-di-GMP responsive riboswitch, is outlined below.

An autoinhibitory mechanism controls RNA-binding activity of the nitrate-sensing protein NasR.

The ANTAR domain harnesses RNA-binding activity to promote transcription attenuation. Although several ANTAR proteins have been analyzed by high-resolution structural analyses, the residues involved in RNA-recognition and transcription attenuation have not been identified. Nor is it clear how signal-responsive domains are allosterically coupled with ANTAR domains for control of gene expression. Herein, we examined the sequence conservation of ANTAR domains to find residues that may associate with RNA. We subjected the corresponding positions of Klebsiella oxytoca NasR to site-directed alanine substitutions and measured RNA-binding activity. This revealed a functionally important patch of residues that forms amino acid pairing interactions with residues from NasR's nitrate-sensing NIT domain. We hypothesize these amino acid pairing interactions are part of an autoinhibitory mechanism that holds the structure in an "off" state in the absence of nitrate signal. Indeed, mutational disruption of these interactions resulted in constitutively active proteins, freed from autoinhibition and no longer influenced by nitrate. Moreover, sequence analyses suggested the autoinhibitory mechanism has been evolutionarily maintained by NasR proteins. These data reveal a molecular mechanism for how NasR couples its nitrate signal to RNA-binding activity, and generally show how signal-responsive domains of one-component regulatory proteins have evolved to exert control over RNA-binding ANTAR domains.

A dedicated diribonucleotidase resolves a key bottleneck for the terminal step of RNA degradation.

Degradation of RNA polymers, an ubiquitous process in all cells, is catalyzed by specific subsets of endo- and exoribonucleases that together recycle RNA fragments into nucleotide monophosphate. In γ-proteobacteria, 3-'5' exoribonucleases comprise up to eight distinct enzymes. Among them, Oligoribonuclease (Orn) is unique as its activity is required for clearing short RNA fragments, which is important for cellular fitness. However, the molecular basis of Orn's unique cellular function remained unclear. Here, we show that Orn exhibits exquisite substrate preference for diribonucleotides. Crystal structures of substrate-bound Orn reveal an active site optimized for diribonucleotides. While other cellular RNases process oligoribonucleotides down to diribonucleotide entities, Orn is the one and only diribonucleotidase that completes the terminal step of RNA degradation. Together, our studies indicate RNA degradation as a step-wise process with a dedicated enzyme for the clearance of a specific intermediate pool, diribonucleotides, that affects cellular physiology and viability.

Single-Cell Microscopy Reveals That Levels of Cyclic di-GMP Vary among Bacillus subtilis Subpopulations.

The synthesis of signaling molecules is one strategy bacteria employ to sense alterations in their environment and rapidly adjust to those changes. In Gram-negative bacteria, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates the transition from a unicellular motile state to a multicellular sessile state. However, c-di-GMP signaling has been less intensively studied in Gram-positive organisms. To that end, we constructed a fluorescent yfp reporter based on a c-di-GMP-responsive riboswitch to visualize the relative abundance of c-di-GMP for single cells of the Gram-positive model organism Bacillus subtilis Coupled with cell-type-specific fluorescent reporters, this riboswitch reporter revealed that c-di-GMP levels are markedly different among B. subtilis cellular subpopulations. For example, cells that have made the decision to become matrix producers maintain higher intracellular c-di-GMP concentrations than motile cells. Similarly, we find that c-di-GMP levels differ between sporulating and competent cell types. These results suggest that biochemical measurements of c-di-GMP abundance are likely to be inaccurate for a bulk ensemble of B. subtilis cells, as such measurements will average c-di-GMP levels across the population. Moreover, the significant variation in c-di-GMP levels between cell types hints that c-di-GMP might play an important role during B. subtilis biofilm formation. This study therefore emphasizes the importance of using single-cell approaches for analyzing metabolic trends within ensemble bacterial populations.IMPORTANCE Many bacteria have been shown to differentiate into genetically identical yet morphologically distinct cell types. Such population heterogeneity is especially prevalent among biofilms, where multicellular communities are primed for unexpected environmental conditions and can efficiently distribute metabolic responsibilities. Bacillus subtilis is a model system for studying population heterogeneity; however, a role for c-di-GMP in these processes has not been thoroughly investigated. Herein, we introduce a fluorescent reporter, based on a c-di-GMP-responsive riboswitch, to visualize the relative abundance of c-di-GMP for single B. subtilis cells. Our analysis shows that c-di-GMP levels are conspicuously different among B. subtilis cellular subtypes, suggesting a role for c-di-GMP during biofilm formation. These data highlight the utility of riboswitches as tools for imaging metabolic changes within individual bacterial cells. Analyses such as these offer new insight into c-di-GMP-regulated phenotypes, especially given that other biofilms also consist of multicellular communities.

Ethanolamine Utilization and Bacterial Microcompartment Formation Are Subject to Carbon Catabolite Repression.

Ethanolamine (EA) is a compound prevalent in the gastrointestinal (GI) tract that can be used as a carbon, nitrogen, and/or energy source. Enterococcus faecalis, a GI commensal and opportunistic pathogen, contains approximately 20 ethanolamine utilization (eut) genes encoding the necessary regulatory, enzymatic, and structural proteins for this process. Here, using a chemically defined medium, two regulatory factors that affect EA utilization were examined. First, the functional consequences of loss of the small RNA (sRNA) EutX on the efficacy of EA utilization were investigated. One effect observed, as loss of this negative regulator causes an increase in eut gene expression, was a concomitant increase in the number of catabolic bacterial microcompartments (BMCs) formed. However, despite this increase, the growth of the strain was repressed, suggesting that the overall efficacy of EA utilization was negatively affected. Second, utilizing a deletion mutant and a complement, carbon catabolite control protein A (CcpA) was shown to be responsible for the repression of EA utilization in the presence of glucose. A predicted cre site in one of the three EA-inducible promoters, PeutS, was identified as the target of CcpA. However, CcpA was shown to affect the activation of all the promoters indirectly through the two-component system EutV and EutW, whose genes are under the control of the PeutS promoter. Moreover, a bioinformatics analysis of bacteria predicted to contain CcpA and cre sites revealed that a preponderance of BMC-containing operons are likely regulated by carbon catabolite repression (CCR).IMPORTANCE Ethanolamine (EA) is a compound commonly found in the gastrointestinal (GI) tract that can affect the behavior of human pathogens that can sense and utilize it, such as Enterococcus faecalis and Salmonella Therefore, it is important to understand how the genes that govern EA utilization are regulated. In this work, we investigated two regulatory factors that control this process. One factor, a small RNA (sRNA), is shown to be important for generating the right levels of gene expression for maximum efficiency. The second factor, a transcriptional repressor, is important for preventing expression when other preferred sources of energy are available. Furthermore, a global bioinformatics analysis revealed that this second mechanism of transcriptional regulation likely operates on similar genes in related bacteria.

Processive Antitermination.

Transcription is a discontinuous process, where each nucleotide incorporation cycle offers a decision between elongation, pausing, halting, or termination. Many cis-acting regulatory RNAs, such as riboswitches, exert their influence over transcription elongation. Through such mechanisms, certain RNA elements can couple physiological or environmental signals to transcription attenuation, a process where cis-acting regulatory RNAs directly influence formation of transcription termination signals. However, through another regulatory mechanism called processive antitermination (PA), RNA polymerase can bypass termination sites over much greater distances than transcription attenuation. PA mechanisms are widespread in bacteria, although only a few classes have been discovered overall. Also, although traditional, signal-responsive riboswitches have not yet been discovered to promote PA, it is increasingly clear that small RNA elements are still oftentimes required. In some instances, small RNA elements serve as loading sites for cellular factors that promote PA. In other instances, larger, more complicated RNA elements participate in PA in unknown ways, perhaps even acting alone to trigger PA activity. These discoveries suggest that what is now needed is a systematic exploration of PA in bacteria, to determine how broadly these transcription elongation mechanisms are utilized, to reveal the diversity in their molecular mechanisms, and to understand the general logic behind their cellular applications. This review covers the known examples of PA regulatory mechanisms and speculates that they may be broadly important to bacteria.

A Subset of Exoribonucleases Serve as Degradative Enzymes for pGpG in c-di-GMP Signaling.

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that regulates processes, such as biofilm formation and virulence. During degradation, c-di-GMP is first linearized to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG) and subsequently hydrolyzed to two GMPs by a previously unknown enzyme, which was recently identified in Pseudomonas aeruginosa as the 3'-to-5' exoribonuclease oligoribonuclease (Orn). Mutants of orn accumulated pGpG, which inhibited the linearization of c-di-GMP. This product inhibition led to elevated c-di-GMP levels, resulting in increased aggregate and biofilm formation. Thus, the hydrolysis of pGpG is crucial to the maintenance of c-di-GMP homeostasis. How species that utilize c-di-GMP signaling but lack an orn ortholog hydrolyze pGpG remains unknown. Because Orn is an exoribonuclease, we asked whether pGpG hydrolysis can be carried out by genes that encode protein domains found in exoribonucleases. From a screen of these genes from Vibrio cholerae and Bacillus anthracis, we found that only enzymes known to cleave oligoribonucleotides (orn and nrnA) rescued the P. aeruginosa Δorn mutant phenotypes to the wild type. Thus, we tested additional RNases with demonstrated activity against short oligoribonucleotides. These experiments show that only exoribonucleases previously reported to degrade short RNAs (nrnA, nrnB, nrnC, and orn) can also hydrolyze pGpG. A B. subtilisnrnA nrnB mutant had elevated c-di-GMP, suggesting that these two genes serve as the primary enzymes to degrade pGpG. These results indicate that the requirement for pGpG hydrolysis to complete c-di-GMP signaling is conserved across species. The final steps of RNA turnover and c-di-GMP turnover appear to converge at a subset of RNases specific for short oligoribonucleotides.IMPORTANCE The bacterial bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) signaling molecule regulates complex processes, such as biofilm formation. c-di-GMP is degraded in two-steps, linearization into pGpG and subsequent cleavage to two GMPs. The 3'-to-5' exonuclease oligoribonuclease (Orn) serves as the enzyme that degrades pGpG in Pseudomonas aeruginosa Many phyla contain species that utilize c-di-GMP signaling but lack an Orn homolog, and the protein that functions to degrade pGpG remains uncharacterized. Here, systematic screening of genes encoding proteins containing domains found in exoribonucleases revealed a subset of genes encoded within the genomes of Bacillus anthracis and Vibrio cholerae that degrade pGpG to GMP and are functionally analogous to Orn. Feedback inhibition by pGpG is a conserved process, as strains lacking these genes accumulate c-di-GMP.

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.

LoaP is a broadly conserved antiterminator protein that regulates antibiotic gene clusters in Bacillus amyloliquefaciens.

A valuable resource available in the search for new natural products is the diverse microbial life that spans the planet. A large subset of these microorganisms synthesize complex specialized metabolites exhibiting biomedically important activities. A limiting step to the characterization of these compounds is an elucidation of the genetic regulatory mechanisms that oversee their production. Although proteins that control transcription initiation of specialized metabolite gene clusters have been identified, those affecting transcription elongation have not been broadly investigated. In this study, we analysed the phylogenetic distribution of the large, widespread NusG family of transcription elongation proteins and found that it includes a cohesive outgroup of paralogues (herein coined LoaP), which are often positioned adjacent or within gene clusters for specialized metabolites. We established Bacillus amyloliquefaciens LoaP as a paradigm for this protein subgroup and showed that it regulated the transcriptional readthrough of termination sites located within two different antibiotic biosynthesis operons. Both of these antibiotics have been implicated in plant-protective activities, demonstrating that LoaP controls an important regulon of specialized metabolite genes for this microorganism. These data therefore reveal transcription elongation as a point of regulatory control for specialized metabolite pathways and introduce a subgroup of NusG proteins for this purpose.

Bacterial riboswitches cooperatively bind Ni(2+) or Co(2+) ions and control expression of heavy metal transporters.

Bacteria regularly encounter widely varying metal concentrations in their surrounding environment. As metals become depleted or, conversely, accrue to toxicity, microbes will activate cellular responses that act to maintain metal homeostasis. A suite of metal-sensing regulatory ("metalloregulatory") proteins orchestrate these responses by allosterically coupling the selective binding of target metals to the activity of DNA-binding domains. However, we report here the discovery, validation, and structural details of a widespread class of riboswitch RNAs, whose members selectively and tightly bind the low-abundance transition metals, Ni(2+) and Co(2+). These riboswitches bind metal cooperatively, and with affinities in the low micromolar range. The structure of a Co(2+)-bound RNA reveals a network of molecular contacts that explains how it achieves cooperative binding between adjacent sites. These findings reveal that bacteria have evolved to utilize highly selective metalloregulatory riboswitches, in addition to metalloregulatory proteins, for detecting and responding to toxic levels of heavy metals.