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.

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.

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.

Host suppression of quorum sensing during catheter-associated urinary tract infections.

Chronic bacterial infections on medical devices, including catheter-associated urinary tract infections (CAUTI), are associated with bacterial biofilm communities that are refractory to antibiotic therapy and resistant to host immunity. Previously, we have shown that Pseudomonas aeruginosa can cause CAUTI by forming a device-associated biofilm that is independent of known biofilm exopolysaccharides. Here, we show by RNA-seq that host urine alters the transcriptome of P. aeruginosa by suppressing quorum sensing regulated genes. P. aeruginosa produces acyl homoserine lactones (AHLs) in the presence of urea, but cannot perceive AHLs. Repression of quorum sensing by urine implies that quorum sensing should be dispensable during infection of the urinary tract. Indeed, mutants defective in quorum sensing are able to colonize similarly to wild-type in a murine model of CAUTI. Quorum sensing-regulated processes in clinical isolates are also inhibited by urea. These data show that urea in urine is a natural anti-quorum sensing mechanism in mammals.

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.

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.

Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems.

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele, in contrast to the wild type MshE, was unable to complement the ΔmshE mutant for the production of extracellular MshA to the cell surface, reduction in flagella swimming motility, attachment to surfaces and formation of biofilms. Testing homologs of MshE for binding to c-di-GMP identified the type II secretion ATPase of Pseudomonas aeruginosa (PA14_29490) as a c-di-GMP receptor, indicating that type II secretion and type IV pili are both regulated by c-di-GMP.

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.

Assessment of the requirements for magnesium transporters in Bacillus subtilis.

Magnesium is the most abundant divalent metal in cells and is required for many structural and enzymatic functions. For bacteria, at least three families of proteins function as magnesium transporters. In recent years, it has been shown that a subset of these transport proteins is regulated by magnesium-responsive genetic control elements. In this study, we investigated the cellular requirements for magnesium homeostasis in the model microorganism Bacillus subtilis. Putative magnesium transporter genes were mutationally disrupted, singly and in combination, in order to assess their general importance. Mutation of only one of these genes resulted in strong dependency on supplemental extracellular magnesium. Notably, this transporter gene, mgtE, is known to be under magnesium-responsive genetic regulatory control. This suggests that the identification of magnesium-responsive genetic mechanisms may generally denote primary transport proteins for bacteria. To investigate whether B. subtilis encodes yet additional classes of transport mechanisms, suppressor strains that permitted the growth of a transporter-defective mutant were identified. Several of these strains were sequenced to determine the genetic basis of the suppressor phenotypes. None of these mutations occurred in transport protein homologues; instead, they affected housekeeping functions, such as signal recognition particle components and ATP synthase machinery. From these aggregate data, we speculate that the mgtE protein provides the primary route of magnesium import in B. subtilis and that the other putative transport proteins are likely to be utilized for more-specialized growth conditions.

The mechanism for RNA recognition by ANTAR regulators of gene expression.

ANTAR proteins are widespread bacterial regulatory proteins that have RNA-binding output domains and utilize antitermination to control gene expression at the post-initiation level. An ANTAR protein, EutV, regulates the ethanolamine-utilization genes (eut) in Enterococcus faecalis. Using this system, we present genetic and biochemical evidence of a general mechanism of antitermination used by ANTARs, including details of the antiterminator structure. The novel antiterminator structure consists of two small hairpins with highly conserved terminal loop residues, both features being essential for successful antitermination. The ANTAR protein dimerizes and associates with its substrate RNA in response to signal-induced phosphorylation. Furthermore, bioinformatic searches using this conserved antiterminator motif identified many new ANTAR target RNAs in phylogenetically diverse bacterial species, some comprising complex regulons. Despite the unrelatedness of the species in which they are found, the majority of the ANTAR-associated genes are thematically related to nitrogen management. These data suggest that the central tenets for gene regulation by ANTAR antitermination occur widely in nature to specifically control nitrogen metabolism.