Mutualism reduces the severity of gene disruptions in predictable ways across microbial communities.

Predicting evolution in microbial communities is critical for problems from human health to global nutrient cycling. Understanding how species interactions impact the distribution of fitness effects for a focal population would enhance our ability to predict evolution. Specifically, does the type of ecological interaction, such as mutualism or competition, change the average effect of a mutation (i.e., the mean of the distribution of fitness effects)? Furthermore, how often does increasing community complexity alter the impact of species interactions on mutant fitness? To address these questions, we created a transposon mutant library in Salmonella enterica and measured the fitness of loss of function mutations in 3,550 genes when grown alone versus competitive co-culture or mutualistic co-culture with Escherichia coli and Methylorubrum extorquens. We found that mutualism reduces the average impact of mutations, while competition had no effect. Additionally, mutant fitness in the 3-species communities can be predicted by averaging the fitness in each 2-species community. Finally, we discovered that in the mutualism S. enterica obtained vitamins and more amino acids than previously known. Our results suggest that species interactions can predictably impact fitness effect distributions, in turn suggesting that evolution may ultimately be predictable in multi-species communities.

Mutualism reduces the severity of gene disruptions in predictable ways across microbial communities.

Predicting evolution in microbial communities is critical for problems from human health to global nutrient cycling. Understanding how species interactions impact the distribution of fitness effects for a focal population would enhance our ability to predict evolution. Specifically, it would be useful to know if the type of ecological interaction, such as mutualism or competition, changes the average effect of a mutation (i.e., the mean of the distribution of fitness effects). Furthermore, how often does increasing community complexity alter the impact of species interactions on mutant fitness? To address these questions, we created a transposon mutant library in Salmonella enterica and measured the fitness of loss of function mutations in 3,550 genes when grown alone versus competitive co-culture or mutualistic co-culture with Escherichia coli and Methylorubrum extorquens. We found that mutualism reduces the average impact of mutations, while competition had no effect. Additionally, mutant fitness in the 3-species communities can be predicted by averaging the fitness in each 2-species community. Finally, the fitness effects of several knockouts in the mutualistic communities were surprising. We discovered that S. enterica is obtaining a different source of carbon and more vitamins and amino acids than we had expected. Our results suggest that species interactions can predictably impact fitness effect distributions, in turn suggesting that evolution may ultimately be predictable in multi-species communities.

Transcriptional Profiles of a Foliar Fungal Endophyte (Pestalotiopsis, Ascomycota) and Its Bacterial Symbiont (Luteibacter, Gammaproteobacteria) Reveal Sulfur Exchange and Growth Regulation during Early Phases of Symbiotic Interaction.

Symbiosis with bacteria is widespread among eukaryotes, including fungi. Bacteria that live within fungal mycelia (endohyphal bacteria) occur in many plant-associated fungi, including diverse Mucoromycota and Dikarya. Pestalotiopsis sp. strain 9143 is a filamentous ascomycete isolated originally as a foliar endophyte of Platycladus orientalis (Cupressaceae). It is infected naturally with the endohyphal bacterium Luteibacter sp. strain 9143, which influences auxin and enzyme production by its fungal host. Previous studies have used transcriptomics to examine similar symbioses between endohyphal bacteria and root-associated fungi such as arbuscular mycorrhizal fungi and plant pathogens. However, currently there are no gene expression studies of endohyphal bacteria of Ascomycota, the most species-rich fungal phylum. To begin to understand such symbioses, we developed methods for assessing gene expression by Pestalotiopsis sp. and Luteibacter sp. when grown in coculture and when each was grown axenically. Our assays showed that the density of Luteibacter sp. in coculture was greater than in axenic culture, but the opposite was true for Pestalotiopsis sp. Dual-transcriptome sequencing (RNA-seq) data demonstrate that growing in coculture modulates developmental and metabolic processes in both the fungus and bacterium, potentially through changes in the balance of organic sulfur via methionine acquisition. Our analyses also suggest an unexpected, potential role of the bacterial type VI secretion system in symbiosis establishment, expanding current understanding of the scope and dynamics of fungal-bacterial symbioses. IMPORTANCE Interactions between microbes and their hosts have important outcomes for host and environmental health. Foliar fungal endophytes that infect healthy plants can harbor facultative endosymbionts called endohyphal bacteria, which can influence the outcome of plant-fungus interactions. These bacterial-fungal interactions can be influential but are poorly understood, particularly from a transcriptome perspective. Here, we report on a comparative, dual-RNA-seq study examining the gene expression patterns of a foliar fungal endophyte and a facultative endohyphal bacterium when cultured together versus separately. Our findings support a role for the fungus in providing organic sulfur to the bacterium, potentially through methionine acquisition, and the potential involvement of a bacterial type VI secretion system in symbiosis establishment. This work adds to the growing body of literature characterizing endohyphal bacterial-fungal interactions, with a focus on a model facultative bacterial-fungal symbiosis in two species-rich lineages, the Ascomycota and Proteobacteria.

Experimental evolution of the megaplasmid pMPPla107 in Pseudomonas stutzeri enables identification of genes contributing to sensitivity to an inhibitory agent.

Horizontally transferred elements, such as plasmids, can burden host cells with various metabolic and fitness costs and may lead to other potentially detrimental phenotypic effects. Acquisition of the Pseudomonas syringae megaplasmid pMPPla107 by various Pseudomonads causes sensitivity to a growth-inhibiting substance that is produced in cultures by Pseudomonads during growth under standard laboratory conditions. After approximately 500 generations of laboratory passage of Pseudomonas stutzeri populations containing pMPPla107, strains from two out of six independent passage lines displayed resistance to this inhibitory agent. Resistance was transferable and is, therefore, associated with mutations occurring on pMPPla107. Resequencing experiments demonstrated that resistance is likely due to a large deletion on the megaplasmid in one line, and to a nonsynonymous change in an uncharacterized megaplasmid locus in the other strain. We further used allele exchange experiments to confirm that resistance is due to this single amino acid change in a previously uncharacterized megaplasmid protein, which we name SkaA. These results provide further evidence that costs and phenotypic changes associated with horizontal gene transfer can be compensated through single mutational events and emphasize the power of experimental evolution and resequencing to better understand the genetic basis of evolved phenotypes. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Chemical footprinting and kinetic assays reveal dual functions for highly conserved eukaryotic tRNAHis guanylyltransferase residues.

tRNAHis guanylyltransferase (Thg1) adds a single guanine to the -1 position of tRNAHis as part of its maturation. This seemingly modest addition of one nucleotide to tRNAHis ensures translational fidelity by providing a critical identity element for the histidyl aminoacyl tRNA synthetase (HisRS). Like HisRS, Thg1 utilizes the GUG anticodon for selective tRNAHis recognition, and Thg1-tRNA complex structures have revealed conserved residues that interact with anticodon nucleotides. Separately, kinetic analysis of alanine variants has demonstrated that many of these same residues are required for catalytic activity. A model in which loss of activity with the variants was attributed directly to loss of the critical anticodon interaction has been proposed to explain the combined biochemical and structural results. Here we used RNA chemical footprinting and binding assays to test this model and further probe the molecular basis for the requirement for two critical tRNA-interacting residues, His-152 and Lys-187, in the context of human Thg1 (hThg1). Surprisingly, we found that His-152 and Lys-187 alanine-substituted variants maintain a similar overall interaction with the anticodon region, arguing against the sufficiency of this interaction for driving catalysis. Instead, conservative mutagenesis revealed a new direct function for these residues in recognition of a non-Watson-Crick G-1:A73 bp, which had not been described previously. These results have important implications for the evolution of eukaryotic Thg1 from a family of ancestral promiscuous RNA repair enzymes to the highly selective enzymes needed for their essential function in tRNAHis maturation.

Just the Two of Us? A Family of Pseudomonas Megaplasmids Offers a Rare Glimpse into the Evolution of Large Mobile Elements.

Pseudomonads are ubiquitous group of environmental proteobacteria, well known for their roles in biogeochemical cycling, in the breakdown of xenobiotic materials, as plant growth promoters, and as pathogens of a variety of host organisms. We have previously identified a large megaplasmid present within one isolate of the plant pathogen Pseudomonas syringae, and here we report that a second member of this megaplasmid family is found within an environmental Pseudomonad isolate most closely related to Pseudomonas putida. Many of the shared genes are involved in critical cellular processes like replication, transcription, translation, and DNA repair. We argue that presence of these shared pathways sheds new light on discussions about the types of genes that undergo horizontal gene transfer (i.e., the complexity hypothesis) as well as the evolution of pangenomes. Furthermore, although both megaplasmids display a high level of synteny, genes that are shared differ by over 50% on average at the amino acid level. This combination of conservation in gene order despite divergence in gene sequence suggests that this Pseudomonad megaplasmid family is relatively old, that gene order is under strong selection within this family, and that there are likely many more members of this megaplasmid family waiting to be found in nature.

Outpatient Antibiotic Use and the Need for Increased Antibiotic Stewardship Efforts.

Antibiotic-resistant infections pose a growing threat to public health. Antibiotic use, regardless of whether it is warranted, is a primary factor in the development of resistance. In the United States, the majority of antibiotic health care expenditures are due to prescribing in outpatient settings. Much of this prescribing is inappropriate, with research showing that at least 30% of antibiotic use in outpatient settings is unnecessary. In this State of the Art Review article, we provide an overview of the latest research on outpatient antibiotic prescribing practices in the United States. Although many of the researchers in these studies describe antibiotic prescribing across all patient age groups, we highlight prescribing in pediatric populations when data are available. We then describe the various factors that can influence a physician's prescribing decisions and drive inappropriate antibiotic use and the potential role of behavioral science in enhancing stewardship interventions to address these drivers. Finally, we highlight the role that a wide range of health care stakeholders can play in aiding the expansion of outpatient stewardship efforts that are needed to fully address the threat of antibiotic resistance.

Characterization of 16S rRNA Processing with Pre-30S Subunit Assembly Intermediates from E. coli.

Ribosomal RNA (rRNA) is a major component of ribosomes and is fundamental to the process of translation. In bacteria, 16S rRNA is a component of the small ribosomal subunit and plays a critical role in mRNA decoding. rRNA maturation entails the removal of intervening spacer sequences contained within the pre-rRNA transcript by nucleolytic enzymes. Enzymatic activities involved in maturation of the 5'-end of 16S rRNA have been identified, but those involved in 3'-end maturation of 16S rRNA are more enigmatic. Here, we investigate molecular details of 16S rRNA maturation using purified in vivo-formed small subunit (SSU) assembly intermediates (pre-SSUs) from wild-type Escherichia coli that contain precursor 16S rRNA (17S rRNA). Upon incubation of pre-SSUs with E. coli S100 cell extracts or purified enzymes implicated in 16S rRNA processing, the 17S rRNA is processed into additional intermediates and mature 16S rRNA. These results illustrate that exonucleases RNase R, RNase II, PNPase, and RNase PH can process the 3'-end of pre-SSUs in vitro. However, the endonuclease YbeY did not exhibit nucleolytic activity with pre-SSUs under these conditions. Furthermore, these data demonstrate that multiple pathways facilitate 16S rRNA maturation with pre-SSUs in vitro, with the dominant pathways entailing complete processing of the 5'-end of 17S rRNA prior to 3'-end maturation or partial processing of the 5'-end with concomitant processing of the 3'-end. These results reveal the multifaceted nature of SSU biogenesis and suggest that E. coli may be able to escape inactivation of any one enzyme by using an existing complementary pathway.

Community-led comparative genomic and phenotypic analysis of the aquaculture pathogen Pseudomonas baetica a390T sequenced by Ion semiconductor and Nanopore technologies.

Pseudomonas baetica strain a390T is the type strain of this recently described species and here we present its high-contiguity draft genome. To celebrate the 16th International Conference on Pseudomonas, the genome of P. baetica strain a390T was sequenced using a unique combination of Ion Torrent semiconductor and Oxford Nanopore methods as part of a collaborative community-led project. The use of high-quality Ion Torrent sequences with long Nanopore reads gave rapid, high-contiguity and -quality, 16-contig genome sequence. Whole genome phylogenetic analysis places P. baetica within the P. koreensis clade of the P. fluorescens group. Comparison of the main genomic features of P. baetica with a variety of other Pseudomonas spp. suggests that it is a highly adaptable organism, typical of the genus. This strain was originally isolated from the liver of a diseased wedge sole fish, and genotypic and phenotypic analyses show that it is tolerant to osmotic stress and to oxytetracycline.

Patient specific computational models to optimize surgical correction for flatfoot deformity.

Several surgically corrective procedures are considered to treat Adult Acquired Flatfoot Deformity (AAFD) patients, relieve pain, and restore function. Procedure selection is based on best practices and surgeon preference. Recent research created patient specific models of AAFD to explore their predictive capabilities and examine effectiveness of the surgical procedure used to treat the deformity. The models' behavior was governed solely by patient bodyweight, soft tissue constraints, muscle loading, and joint contact without the assumption of idealized joints. The current work expanded those models to determine if an alternate procedure would be more effective for the individual. All procedures incorporated first a tendon transfer and then included one hindfoot procedure, the Medializing Calcaneal Osteotomy (MCO), and one of three lateral column procedures: Evans osteotomy, Calcaneocuboid Distraction Arthrodesis (CCDA), Z osteotomy, and the combination procedures MCO & Evans osteotomy, MCO & CCDA, and MCO & Z osteotomy. The combination MCO & Evans and MCO & Z procedures were shown to provide the greatest amount of correction for both forefoot abduction and hindfoot valgus. However, these two procedures significantly increased joint contact force, specifically at the calcaneocuboid joint, and ground reaction force along the lateral column. With exception to the lateral bands of the plantar fascia and middle spring ligament, the strain present in the plantar fascia, spring, and deltoid ligaments decreased after all procedures. The use of patient specific computational models provided the ability to investigate effects of alternate surgical corrections on restoring biomechanical function in these flatfoot patients. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1523-1531, 2017.

Sex Differences in Knee Flexion Angle During a Rapid Change of Direction While Running.

BACKGROUND: Females experience greater overall rates of athletic anterior cruciate ligament (ACL) injury than males. The specific mechanisms of the predisposition remain unclear. HYPOTHESIS: Modeling of knee kinematics has shown that the more extended the knee joint, the greater the strain on the ACL. The authors hypothesized that female athletes would have a lesser degree of knee flexion than male athletes at initial ground contact while performing change-of-direction cutting maneuvers. STUDY DESIGN: Controlled laboratory study. METHODS: Twenty female and 20 male high school soccer athletes with at least 1 year of experience were recruited for the study. Athletes were excluded if they had a history of any major lower limb injury or current knee pain causing a reduction in training and/or competition. Reflective markers were attached at the greater trochanter of the femur, the lateral epicondyle of the knee, and the lateral malleolus of the ankle to enable motion capture. Each athlete performed 6 change-of-direction maneuvers in random order in front of 2 cameras. Multiple regression analysis was used to determine differences between the sexes from the motion data captured; P < .05 defined significance. RESULTS: Statistically significant differences existed in knee flexion angles between male and female participants at the 90° and 135° cutting angles. At 90°, males and females showed initial contact knee flexion angles (mean ± SD) of 39.0° ± 6.8° and 29.3° ± 6.2°, respectively (P < .0001), and mean maximum flexion angles of 56.4° ± 6.9° and 49.7° ± 7.0°, respectively (P = .0036). At 135°, males and females showed mean initial contact knee flexion angles of 36.8° ± 7.9° and 29.7° ± 7.8°, respectively (P = .0053), and mean maximum flexion angles of 60.7° ± 8.1° and 51.6° ± 9.4°, respectively (P = .0017). CONCLUSION: The research conducted is intended to foster an awareness of injury disposition in female athletes and guide future endeavors to develop, test, and implement a proactive approach in lowering female noncontact athletic ACL injury rates. This project adds to the literature as wider side-cut maneuvers (≥90°) were studied, as compared with previous studies using small side-cut angles (<90°), offering a model for alternative sports actions.

Multiple phenotypic changes associated with large-scale horizontal gene transfer.

Horizontal gene transfer often leads to phenotypic changes within recipient organisms independent of any immediate evolutionary benefits. While secondary phenotypic effects of horizontal transfer (i.e., changes in growth rates) have been demonstrated and studied across a variety of systems using relatively small plasmids and phage, little is known about the magnitude or number of such costs after the transfer of larger regions. Here we describe numerous phenotypic changes that occur after a large-scale horizontal transfer event (∼1 Mb megaplasmid) within Pseudomonas stutzeri including sensitization to various stresses as well as changes in bacterial behavior. These results highlight the power of horizontal transfer to shift pleiotropic relationships and cellular networks within bacterial genomes. They also provide an important context for how secondary effects of transfer can bias evolutionary trajectories and interactions between species. Lastly, these results and system provide a foundation to investigate evolutionary consequences in real time as newly acquired regions are ameliorated and integrated into new genomic contexts.

Complete Genome Sequence of the Highly Transformable Pseudomonas stutzeri Strain 28a24.

Here, we report the complete genome sequence for an isolate of Pseudomonas stutzeri that is highly competent for natural transformation. This sequence enables insights into the genetic basis of natural transformation rate variations and provides an additional data point for genomic comparisons across a ubiquitous and highly diverse bacterial species.

Bigger is not always better: transmission and fitness burden of ∼1MB Pseudomonas syringae megaplasmid pMPPla107.

BACKGROUND: Horizontal gene transfer (HGT) is a widespread process that enables the acquisition of genes and metabolic pathways in single evolutionary steps. Previous reports have described fitness costs of HGT, but have largely focused on the acquisition of relatively small plasmids. We have previously shown that a Pseudomonas syringae pv. lachrymans strain recently acquired a cryptic megaplasmid, pMPPla107. This extrachromosomal element contributes hundreds of new genes to P. syringae and increases total genomic content by approximately 18%. However, this early work did not directly explore transmissibility, stability, or fitness costs associated with acquisition of pMPPla107. RESULTS: Here, we show that pMPPla107 is self-transmissible across a variety of diverse pseudomonad strains, on both solid agar and within shaking liquid cultures, with conjugation dependent on a type IV secretion system. To the best of our knowledge, this is the largest self-transmissible megaplasmid known outside of Sinorhizobium. This megaplasmid can be lost from all novel hosts although the rate of loss depends on medium type and genomic background. However, in contrast, pMPPla107 is faithfully maintained within the original parent strain (Pla107) even under direct negative selection during laboratory assays. These results suggest that Pla107 specific stabilizing mutations have occurred either on this strain's chromosome or within the megaplasmid. Lastly, we demonstrate that acquisition of pMPPla107 by strains other than Pla107 imparts severe (20%) fitness costs under competitive conditions in vitro. CONCLUSIONS: We show that pMPPla107 is capable of transmitting and maintaining itself across multiple Pseudomonas species, rendering it one of the largest conjugative elements discovered to date. The relative stability of pMPPla107, coupled with extensive fitness costs, makes it a tractable model system for investigating evolutionary and genetic mechanisms of megaplasmid maintenance and a unique testing ground to explore evolutionary dynamics after HGT of large secondary elements.

Saccharomyces cerevisiae Thg1 uses 5'-pyrophosphate removal to control addition of nucleotides to tRNA(His.).

In eukaryotes, the tRNA(His) guanylyltransferase (Thg1) catalyzes 3'-5' addition of a single guanosine residue to the -1 position (G-1) of tRNA(His), across from a highly conserved adenosine at position 73 (A73). After addition of G-1, Thg1 removes pyrophosphate from the tRNA 5'-end, generating 5'-monophosphorylated G-1-containing tRNA. The presence of the 5'-monophosphorylated G-1 residue is important for recognition of tRNA(His) by its cognate histidyl-tRNA synthetase. In addition to the single-G-1 addition reaction, Thg1 polymerizes multiple G residues to the 5'-end of tRNA(His) variants. For 3'-5' polymerization, Thg1 uses the 3'-end of the tRNA(His) acceptor stem as a template. The mechanism of reverse polymerization is presumed to involve nucleophilic attack of the 3'-OH from each incoming NTP on the intact 5'-triphosphate created by the preceding nucleotide addition. The potential exists for competition between 5'-pyrophosphate removal and 3'-5' polymerase reactions that could define the outcome of Thg1-catalyzed addition, yet the interplay between these competing reactions has not been investigated for any Thg1 enzyme. Here we establish transient kinetic assays to characterize the pyrophosphate removal versus nucleotide addition activities of yeast Thg1 with a set of tRNA(His) substrates in which the identity of the N-1:N73 base pair was varied to mimic various products of the N-1 addition reaction catalyzed by Thg1. We demonstrate that retention of the 5'-triphosphate is correlated with efficient 3'-5' reverse polymerization. A kinetic partitioning mechanism that acts to prevent addition of nucleotides beyond the -1 position with wild-type tRNA(His) is proposed.

Kinetic analysis of 3'-5' nucleotide addition catalyzed by eukaryotic tRNA(His) guanylyltransferase.

The tRNA(His) guanylyltransferase (Thg1) catalyzes the incorporation of a single guanosine residue at the -1 position (G(-1)) of tRNA(His), using an unusual 3'-5' nucleotidyl transfer reaction. Thg1 and Thg1 orthologs known as Thg1-like proteins (TLPs), which catalyze tRNA repair and editing, are the only known enzymes that add nucleotides in the 3'-5' direction. Thg1 enzymes share no identifiable sequence similarity with any other known enzyme family that could be used to suggest the mechanism for catalysis of the unusual 3'-5' addition reaction. The high-resolution crystal structure of human Thg1 revealed remarkable structural similarity between canonical DNA/RNA polymerases and eukaryotic Thg1; nevertheless, questions regarding the molecular mechanism of 3'-5' nucleotide addition remain. Here, we use transient kinetics to measure the pseudo-first-order forward rate constants for the three steps of the G(-1) addition reaction catalyzed by yeast Thg1: adenylylation of the 5' end of the tRNA (k(aden)), nucleotidyl transfer (k(ntrans)), and removal of pyrophosphate from the G(-1)-containing tRNA (k(ppase)). This kinetic framework, in conjunction with the crystal structure of nucleotide-bound Thg1, suggests a likely role for two-metal ion chemistry in all three chemical steps of the G(-1) addition reaction. Furthermore, we have identified additional residues (K44 and N161) involved in adenylylation and three positively charged residues (R27, K96, and R133) that participate primarily in the nucleotidyl transfer step of the reaction. These data provide a foundation for understanding the mechanism of 3'-5' nucleotide addition in tRNA(His) maturation.

Specimen specific, 3D modeling of the elbow--prediction of strain in the medial collateral ligament.

In this project 3D interactive models of twelve cadaver elbows are developed using the author's kinematic simulation software. The effective flexion-extension axes for each specimen's model are iteratively defined based upon congruent joint motion and individual limits in range-of-motion. Origins and insertions of both parts of the medial collateral ligament are digitized following careful dissection of each specimen. Ligament paths are then defined using cubic B-spline models of the principal fibers of each part, flexion extension motion of each elbow is carried out in real-time and the strain of each fiber model is calculated. Results indicate the existence of two distinct populations of medial collateral ligament - one whose anterior part stretches during flexion of the elbow and the other whose anterior part stretches during extension.

Experimental arrest of cerebral blood flow in human subjects: the red wing studies revisited.

Loss of consciousness in pilots during rapid ascent after bombing missions was a major problem in World War II, and experiments were undertaken to study the cause of this phenomenon. Postulating impaired cerebral blood flow as a likely mechanism, the investigators developed a neck device, the KRA Cuff, which when inflated could shut off blood supply to the brain. With cessation of blood flow for up to 100 seconds, the investigators observed a sequence of responses, including unconsciousness, followed by dilated pupils, tonic/clonic movements, loss of bladder and eventually bowel control, and appearance of pathological reflexes. This study, carried out in prisoners and patients with schizophrenia in 1941-42, largely disappeared from public discourse for a number of years. It has received occasional attention subsequently and been considered controversial. Recently discovered records, including extensive written and photographic data from the studies, shed new light on the methods and motives of the research team. We describe here this new information and its implications for the scientific and ethical assessment of the study.

tRNA(His) guanylyltransferase (THG1), a unique 3'-5' nucleotidyl transferase, shares unexpected structural homology with canonical 5'-3' DNA polymerases.

All known DNA and RNA polymerases catalyze the formation of phosphodiester bonds in a 5' to 3' direction, suggesting this property is a fundamental feature of maintaining and dispersing genetic information. The tRNA(His) guanylyltransferase (Thg1) is a member of a unique enzyme family whose members catalyze an unprecedented reaction in biology: 3'-5' addition of nucleotides to nucleic acid substrates. The 2.3-Å crystal structure of human THG1 (hTHG1) reported here shows that, despite the lack of sequence similarity, hTHG1 shares unexpected structural homology with canonical 5'-3' DNA polymerases and adenylyl/guanylyl cyclases, two enzyme families known to use a two-metal-ion mechanism for catalysis. The ability of the same structural architecture to catalyze both 5'-3' and 3'-5' reactions raises important questions concerning selection of the 5'-3' mechanism during the evolution of nucleotide polymerases.