The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli.

Small proteins (<50 amino acids) are emerging as ubiquitous and important regulators in organisms ranging from bacteria to humans, where they commonly bind to and regulate larger proteins during stress responses. However, fundamental aspects of small proteins, such as their molecular mechanism of action, downregulation after they are no longer needed, and their evolutionary provenance, are poorly understood. Here, we show that the MntS small protein involved in manganese (Mn) homeostasis binds and inhibits the MntP Mn transporter. Mn is crucial for bacterial survival in stressful environments but is toxic in excess. Thus, Mn transport is tightly controlled at multiple levels to maintain optimal Mn levels. The small protein MntS adds a new level of regulation for Mn transporters, beyond the known transcriptional and post-transcriptional control. We also found that MntS binds to itself in the presence of Mn, providing a possible mechanism of downregulating MntS activity to terminate its inhibition of MntP Mn export. MntS is homologous to the signal peptide of SitA, the periplasmic metal-binding subunit of a Mn importer. Remarkably, the homologous signal peptide regions can substitute for MntS, demonstrating a functional relationship between MntS and these signal peptides. Conserved gene neighborhoods support that MntS evolved from the signal peptide of an ancestral SitA protein, acquiring a life of its own with a distinct function in Mn homeostasis.

Regulatory RNAs in bacteria.

Bacteria possess numerous and diverse means of gene regulation using RNA molecules, including mRNA leaders that affect expression in cis, small RNAs that bind to proteins or base pair with target RNAs, and CRISPR RNAs that inhibit the uptake of foreign DNA. Although examples of RNA regulators have been known for decades in bacteria, we are only now coming to a full appreciation of their importance and prevalence. Here, we review the known mechanisms and roles of regulatory RNAs, highlight emerging themes, and discuss remaining questions.

The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element.

The highly structured, cis-encoded RNA elements known as riboswitches modify gene expression upon binding a wide range of molecules. The yybP-ykoY motif was one of the most broadly distributed and numerous bacterial riboswitches for which the cognate ligand was unknown. Using a combination of in vivo reporter and in vitro expression assays, equilibrium dialysis, and northern analysis, we show that the yybP-ykoY motif responds directly to manganese ions in both Escherichia coli and Bacillus subtilis. The identification of the yybP-ykoY motif as a manganese ion sensor suggests that the genes that are preceded by this motif and encode a diverse set of poorly characterized membrane proteins have roles in metal homeostasis.

Structure-function analysis of manganese exporter proteins across bacteria.

Manganese (Mn) is an essential trace nutrient for organisms because of its role in cofactoring enzymes and providing protection against reactive oxygen species (ROS). Many bacteria require manganese to form pathogenic or symbiotic interactions with eukaryotic host cells. However, excess manganese is toxic, requiring cells to have manganese export mechanisms. Bacteria are currently known to possess two widely distributed classes of manganese export proteins, MntP and MntE, but other types of transporters likely exist. Moreover, the structure and function of MntP is not well understood. Here, we characterized the role of three structurally related proteins known or predicted to be involved in manganese transport in bacteria from the MntP, UPF0016, and TerC families. These studies used computational analysis to analyze phylogeny and structure, physiological assays to test sensitivity to high levels of manganese and ROS, and inductively coupled plasma-mass spectrometry (ICP-MS) to measure metal levels. We found that MntP alters cellular resistance to ROS. Moreover, we used extensive computational analyses and phenotypic assays to identify amino acids required for MntP activity. These negatively charged residues likely serve to directly bind manganese and transport it from the cytoplasm through the membrane. We further characterized two other potential manganese transporters associated with a Mn-sensing riboswitch and found that the UPF0016 family of proteins has manganese export activity. We provide here the first phenotypic and biochemical evidence for the role of Alx, a member of the TerC family, in manganese homeostasis. It does not appear to export manganese, but rather it intriguingly facilitates an increase in intracellular manganese concentration. These findings expand the available knowledge about the identity and mechanisms of manganese homeostasis proteins across bacteria and show that proximity to a Mn-responsive riboswitch can be used to identify new components of the manganese homeostasis machinery.

The Escherichia coli small protein MntS and exporter MntP optimize the intracellular concentration of manganese.

Escherichia coli does not routinely import manganese, but it will do so when iron is unavailable, so that manganese can substitute for iron as an enzyme cofactor. When intracellular manganese levels are low, the cell induces the MntH manganese importer plus MntS, a small protein of unknown function; when manganese levels are high, the cell induces the MntP manganese exporter and reduces expression of MntH and MntS. The role of MntS has not been clear. Previous work showed that forced MntS synthesis under manganese-rich conditions caused bacteriostasis. Here we find that when manganese is scarce, MntS helps manganese to activate a variety of enzymes. Its overproduction under manganese-rich conditions caused manganese to accumulate to very high levels inside the cell; simultaneously, iron levels dropped precipitously, apparently because manganese-bound Fur blocked the production of iron importers. Under these conditions, heme synthesis stopped, ultimately depleting cytochrome oxidase activity and causing the failure of aerobic metabolism. Protoporphyrin IX accumulated, indicating that the combination of excess manganese and iron deficiency had stalled ferrochelatase. The same chain of events occurred when mutants lacking MntP, the manganese exporter, were exposed to manganese. Genetic analysis suggested the possibility that MntS exerts this effect by inhibiting MntP. We discuss a model wherein during transitions between low- and high-manganese environments E. coli uses MntP to compensate for MntH overactivity, and MntS to compensate for MntP overactivity.

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli.

Small proteins (< 50 amino acids) are emerging as ubiquitous and important regulators in organisms ranging from bacteria to humans, where they commonly bind to and regulate larger proteins during stress responses. However, fundamental aspects of small proteins, such as their molecular mechanism of action, downregulation after they are no longer needed, and their evolutionary provenance are poorly understood. Here we show that the MntS small protein involved in manganese (Mn) homeostasis binds and inhibits the MntP Mn transporter. Mn is crucial for bacterial survival in stressful environments, but is toxic in excess. Thus, Mn transport is tightly controlled at multiple levels to maintain optimal Mn levels. The small protein MntS adds a new level of regulation for Mn transporters, beyond the known transcriptional and post-transcriptional control. We also found that MntS binds to itself in the presence of Mn, providing a possible mechanism of downregulating MntS activity to terminate its inhibition of MntP Mn export. MntS is homologous to the signal peptide of SitA, the periplasmic metal-binding subunit of a Mn importer. Remarkably, the homologous signal peptide regions can substitute for MntS, demonstrating a functional relationship between MntS and these signal peptides. Conserved gene-neighborhoods support that MntS evolved from an ancestral SitA, acquiring a life of its own with a distinct function in Mn homeostasis. Significance: This study demonstrates that the MntS small protein binds and inhibits the MntP Mn exporter, adding another layer to the complex regulation of Mn homeostasis. MntS also interacts with itself in cells with Mn, which could prevent it from regulating MntP. We propose that MntS and other small proteins might sense environmental signals and shut off their own regulation via binding to ligands (e.g., metals) or other proteins. We also provide evidence that MntS evolved from the signal peptide region of the Mn importer, SitA. Homologous SitA signal peptides can recapitulate MntS activities, showing that they have a second function beyond protein secretion. Overall, we establish that small proteins can emerge and develop novel functionalities from gene remnants.

Regulation of Bacterial Manganese Homeostasis and Usage During Stress Responses and Pathogenesis.

Manganese (Mn) plays a multifaceted role in the survival of pathogenic and symbiotic bacteria in eukaryotic hosts, and it is also important for free-living bacteria to grow in stressful environments. Previous research has uncovered components of the bacterial Mn homeostasis systems that control intracellular Mn levels, many of which are important for virulence. Multiple studies have also identified proteins that use Mn once it is inside the cell, including Mn-specific enzymes and enzymes transiently loaded with Mn for protection during oxidative stress. Emerging evidence continues to reveal proteins involved in maintaining Mn homeostasis, as well as enzymes that can bind Mn. For some of these enzymes, Mn serves as an essential cofactor. For other enzymes, mismetallation with Mn can lead to inactivation or poor activity. Some enzymes may even potentially be regulated by differential metallation with Mn or zinc (Zn). This review focuses on new developments in regulatory mechanisms that affect Mn homeostasis and usage, additional players in Mn import that increase bacterial survival during pathogenesis, and the interplay between Mn and other metals during Mn-responsive physiological processes. Lastly, we highlight lessons learned from fundamental research that are now being applied to bacterial interactions within larger microbial communities or eukaryotic hosts.

The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis.

Manganese is a critical micronutrient for cells, serving as an enzyme cofactor and protecting against oxidative stress. Yet, manganese is toxic in excess and little is known about its distribution in cells. Bacteria control intracellular manganese levels by the transcription regulator MntR. When this work began, the only Escherichia coli K-12 gene known to respond to manganese via MntR repression was mntH, which encodes a manganese importer. We show that mntS (formerly the small RNA gene rybA) is repressed by manganese through MntR and encodes an unannotated 42-amino-acid protein. Overproduction of MntS causes manganese sensitivity, while a lack of MntS perturbs proper manganese-dependent repression of mntH. We also provide evidence that mntP (formerly yebN), which encodes a putative efflux pump, is positively regulated by MntR. Deletion of mntP leads to profound manganese sensitivity and to elevated intracellular manganese levels. This work thus defines two new proteins involved in manganese homeostasis and suggests mechanisms for their action.

Adapting Undergraduate Research to Remote Work to Increase Engagement.

Demand for undergraduate research experiences typically outstrips the available laboratory positions, which could have been exacerbated during the remote work conditions imposed by the SARS-CoV-2/COVID-19 pandemic. This report presents a collection of examples of how undergraduates have been engaged in research under pandemic work restrictions. Examples include a range of projects related to fluid dynamics, cancer biology, nanomedicine, circadian clocks, metabolic disease, catalysis, and environmental remediation. Adaptations were made that included partnerships between remote and in-person research students and students taking on more data analysis and literature surveys, as well as data mining, computational, and informatics projects. In many cases, these projects engaged students who otherwise would have worked in traditional bench research, as some previously had. Several examples of beneficial experiences are reported, such as the additional time spent studying the literature, which gave students a heightened sense of project ownership, and more opportunities to integrate feedback into writing and research. Additionally, the more intentional and regular communication necessitated by remote work proved beneficial for all team members. Finally, online seminars and conferences have made participation possible for many more students, especially those at predominantly undergraduate institutions. Participants aim to adopt these beneficial practices in our research groups even after pandemic restrictions end.

Bacterial manganese sensing and homeostasis.

Manganese (Mn) plays a complex role in the survival of pathogenic and symbiotic bacteria in eukaryotic hosts and is also important for free-living bacteria to thrive in stressful environments. This review summarizes new aspects of regulatory strategies to control intracellular Mn levels and gives an overview of several newly identified families of bacterial Mn transporters. Recent illustrative examples of advances in quantification of intracellular Mn pools and characterization of the effects of Mn perturbations are highlighted. These discoveries help define mechanisms of Mn selectivity and toxicity and could enable new strategies to combat pathogenic bacteria and promote growth of desirable bacteria.

Characterization of novel alleles of the Escherichia coli umuDC genes identifies additional interaction sites of UmuC with the beta clamp.

Translesion synthesis is a DNA damage tolerance mechanism by which damaged DNA in a cell can be replicated by specialized DNA polymerases without being repaired. The Escherichia coli umuDC gene products, UmuC and the cleaved form of UmuD, UmuD', comprise a specialized, potentially mutagenic translesion DNA polymerase, polymerase V (UmuD'(2)C). The full-length UmuD protein, together with UmuC, plays a role in a primitive DNA damage checkpoint by decreasing the rate of DNA synthesis. It has been proposed that the checkpoint is manifested as a cold-sensitive phenotype that is observed when the umuDC gene products are overexpressed. Elevated levels of the beta processivity clamp along with elevated levels of the umuDC gene products, UmuD'C, exacerbate the cold-sensitive phenotype. We used this observation as the basis for genetic selection to identify two alleles of umuD' and seven alleles of umuC that do not exacerbate the cold-sensitive phenotype when they are present in cells with elevated levels of the beta clamp. The variants were characterized to determine their abilities to confer the umuD'C-specific phenotype UV-induced mutagenesis. The umuD variants were assayed to determine their proficiencies in UmuD cleavage, and one variant (G129S) rendered UmuD noncleaveable. We found at least two UmuC residues, T243 and L389, that may further define the beta binding region on UmuC. We also identified UmuC S31, which is predicted to bind to the template nucleotide, as a residue that is important for UV-induced mutagenesis.

Novel conserved motifs in Rev1 C-terminus are required for mutagenic DNA damage tolerance.

The genes encoding Rev1 and DNA polymerase zeta (Rev3/Rev7) are together required for the vast majority of DNA damage-induced mutations in eukaryotes from yeast to humans. Here, we provide insight into the critical role that the Saccharomyces cerevisiae Rev1 C-terminus plays in the process of mutagenic DNA damage tolerance. The Rev1 C-terminus was previously thought to be poorly conserved and therefore not likely to be important for mediating protein-protein interactions. However, through comprehensive alignments of the Rev1 C-terminus, we have identified novel and hitherto unrecognized conserved motifs that we show play an essential role in REV1-dependent survival and mutagenesis in S. cerevisiae, likely in its post-replicative gap-filling mode. We further show that the minimal C-terminal fragment of Rev1 containing these highly conserved motifs is sufficient to interact with Rev7.

Eukaryotic translesion polymerases and their roles and regulation in DNA damage tolerance.

DNA repair and DNA damage tolerance machineries are crucial to overcome the vast array of DNA damage that a cell encounters during its lifetime. In this review, we summarize the current state of knowledge about the eukaryotic DNA damage tolerance pathway translesion synthesis (TLS), a process in which specialized DNA polymerases replicate across from DNA lesions. TLS aids in resistance to DNA damage, presumably by restarting stalled replication forks or filling in gaps that remain in the genome due to the presence of DNA lesions. One consequence of this process is the potential risk of introducing mutations. Given the role of these translesion polymerases in mutagenesis, we discuss the significant regulatory mechanisms that control the five known eukaryotic translesion polymerases: Rev1, Pol zeta, Pol kappa, Pol eta, and Pol iota.

The critical mutagenic translesion DNA polymerase Rev1 is highly expressed during G(2)/M phase rather than S phase.

The Rev1 protein lies at the root of mutagenesis in eukaryotes. Together with DNA polymerase zeta (Rev3/7), Rev1 function is required for the active introduction of the majority of mutations into the genomes of eukaryotes from yeast to humans. Rev1 and polymerase zeta are error-prone translesion DNA polymerases, but Rev1's DNA polymerase catalytic activity is not essential for mutagenesis. Rather, Rev1 is thought to contribute to mutagenesis principally by engaging in crucial protein-protein interactions that regulate the access of translesion DNA polymerases to the primer terminus. This inference is based on the requirement of the N-terminal BRCT (BRCA1 C-terminal) domain of Saccharomyces cerevisiae Rev1 for mutagenesis and the interaction of the C-terminal region of mammalian Rev1 with several other translesion DNA polymerases. Here, we report that S. cerevisiae Rev1 is subject to pronounced cell cycle control in which the levels of Rev1 protein are approximately 50-fold higher in G(2) and throughout mitosis than during G(1) and much of S phase. Differential survival of a rev1Delta strain after UV irradiation at various points in the cell cycle indicates that this unanticipated regulation is physiologically relevant. This unexpected finding has important implications for the regulation of mutagenesis and challenges current models of error-prone lesion bypass as a process involving polymerase switching that operates mainly during S phase to rescue stalled replication forks.