Functional Prokaryotic-Like Deoxycytidine Triphosphate Deaminases and Thymidylate Synthase in Eukaryotic Social Amoebae: Vertical, Endosymbiotic, or Horizontal Gene Transfer?

The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine triphosphate into deoxyuridine triphosphate, whereas eukaryotes and gram-positive bacteria instead use deoxycytidine monophosphate deaminase to transform deoxycytidine monophosphate to deoxyuridine monophosphate. It is then unusual that in addition to deoxycytidine monophosphate deaminases, the eukaryote Dictyostelium discoideum has 2 deoxycytidine triphosphate deaminases (Dcd1Dicty and Dcd2Dicty). Expression of either DcdDicty can fully rescue the slow growth of an Escherichia coli dcd knockout. Both DcdDicty mitigate the hydroxyurea sensitivity of a Schizosaccharomyces pombe deoxycytidine monophosphate deaminase knockout. Phylogenies show that Dcd1Dicty homologs may have entered the common ancestor of the eukaryotic groups of Amoebozoa, Obazoa, Metamonada, and Discoba through an ancient horizontal gene transfer from a prokaryote or an ancient endosymbiotic gene transfer from a mitochondrion, followed by horizontal gene transfer from Amoebozoa to several other unrelated groups of eukaryotes. In contrast, the Dcd2Dicty homologs were a separate horizontal gene transfer from a prokaryote or a virus into either Amoebozoa or Rhizaria, followed by a horizontal gene transfer between them. ThyXDicty, the D. discoideum thymidylate synthase, another enzyme of the deoxythymidine triphosphate biosynthesis pathway, was suggested previously to be acquired from the ancestral mitochondria or by horizontal gene transfer from alpha-proteobacteria. ThyXDicty can fully rescue the E. coli thymidylate synthase knockout, and we establish that it was obtained by the common ancestor of social amoebae not from mitochondria but from a bacterium. We propose horizontal gene transfer and endosymbiotic gene transfer contributed to the enzyme diversity of the deoxythymidine triphosphate synthesis pathway in most social amoebae, many Amoebozoa, and other eukaryotes.

Genome sequences of six cluster CT and two cluster DJ bacteriophages that infect Gordonia rubripertincta.

We report the genome sequences of eight bacteriophages isolated using Gordonia rubripertincta NRRL B-16540-SEA. Based on gene content similarity to phages in the Actinobacteriophage database, six of the phages are assigned to phage cluster CT while two are assigned to cluster DJ.

Instructional Models for Course-Based Research Experience (CRE) Teaching.

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching.

Glycopeptidome of a heavily N-glycosylated cell surface glycoprotein of Dictyostelium implicated in cell adhesion.

Genetic analysis has implicated the cell surface glycoprotein gp130 in cell interactions of the social amoeba Dictyostelium, and information about the utilization of the 18 N-glycosylation sequons present in gp130 is needed to identify critical molecular determinants of its activity. Various glycomics strategies, including mass spectrometry of native and derivatized glycans, monosaccharide analysis, exoglycosidase digestion, and antibody binding, were applied to characterize a nonanchored version secreted from Dictyostelium. s-gp130 is modified by a predominant Man(8)GlcNAc(4) species containing bisecting and intersecting GlcNAc residues and additional high-mannose N-glycans substituted with sulfate, methyl-phosphate, and/or core alpha 3-fucose. Site mapping confirmed the occupancy of 15 sequons, some variably, and glycopeptide analysis confirmed 14 sites and revealed extensive heterogeneity at most sites. Glycopeptide glycoforms ranged from Man(6) to Man(9), GlcNAc(0-2) (peripheral), Fuc(0-2) (including core alpha 3 and peripheral), (SO(4))(0-1), and (MePO(4))(0-1), which represented elements of virtually the entire known cellular N-glycome as inferred from prior metabolic labeling and mass spectrometry studies. gp130, and a family of 14 related predicted glycoproteins whose polypeptide sequences are rapidly diverging in the Dictyostelium lineage, may contribute a functionally important shroud of high-mannose N-glycans at the interface of the amoebae with each other, their predators and prey, and the soil environment.

Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus.

OBJECTIVE: The nuclear and mitochondrial genomes of Dictyostelium discoideum, a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectively. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. RESULTS: The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the shortened N-termini that arose from the loss of residues 1-37. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

Glycoprotein gp130 of dictyostelium discoideum influences macropinocytosis and adhesion.

Glycoprotein gp130, found on the plasma membrane of Dictyostelium discoideum amoebae, was postulated previously to play a role in phagocytosis. The gene for gp130 was cloned and when translated, yielded a 768 amino acid preproprotein of 85.3 kDa. It had nearly 40% similarity to the 138 kDa family of glycoproteins implicated in sexual cell fusion during macrocyst formation in D. discoideum. The difference between the calculated size and observed M(r) of 130 kDa on protein gels likely was due to N-glycosylation that was confirmed by lectin blots. Consistent with its surface-exposure, an antibody raised against recombinant protein stained the plasma membrane of D. discoideum amoebae. Gp130 and its transcripts were high during axenic growth of cells, but relatively low during growth on bacteria. The gene for gp130 was disrupted and cell lines lacking the glycoprotein were efficient phagocytes, indicating that gp130 was dispensable for phagocytosis. Gp130-null cells were similar in size to parent DH1 cells, had enhanced macropinocytosis and grew faster to higher densities. They also exhibited weaker cell-substrate adhesion but displayed greater cell-cell cohesion. Collectively, the data indicated that gp130 influenced macropinocytosis and played a role in adhesion during vegetative growth.

Influenza D virus M2 protein exhibits ion channel activity in Xenopus laevis oocytes.

BACKGROUND: A new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a "mixing vessel" for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target. RESULTS: To test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2-cMyc gene was synthesized and injected into Xenopus oocytes. The translation products of DM2-cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analyses. The DM2-cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (Vmid) values in Boltzmann modeling for oocytes injected with DM2-cMyc RNA or a buffer were -152 and -200 mV, respectively. Assuming the same expression level in the Xenopus oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by Vmid for CM2 and DM2 were -141 and -146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were -21 and -22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower Vmid values while showing no change in reversal potential. CONCLUSION: The M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule.

Structural insights into the mechanism defining substrate affinity in Arabidopsis thaliana dUTPase: the role of tryptophan 93 in ligand orientation.

BACKGROUND: Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) hydrolyzes dUTP to dUMP and pyrophosphate to maintain the cellular thymine-uracil ratio. dUTPase is also a target for cancer chemotherapy. However, the mechanism defining its substrate affinity remains unclear. Sequence comparisons of various dUTPases revealed that Arabidopsis thaliana dUTPase has a unique tryptophan at position 93, which potentially contributes to its degree of substrate affinity. To better understand the roles of tryptophan 93, A. thaliana dUTPase was studied. RESULTS: Enzyme assays showed that A. thaliana dUTPase belongs to a high-affinity group of isozymes, which also includes the enzymes from Escherichia coli and Mycobacterium tuberculosis. Enzymes from Homo sapiens and Saccharomyces cerevisiae are grouped as low-affinity dUTPases. The structure of the homo-trimeric A. thaliana dUTPase showed three active sites, each with a different set of ligand interactions between the amino acids and water molecules. On an α-helix, tryptophan 93 appears to keep serine 89 in place via a water molecule and to specifically direct the ligand. Upon being oriented in the active site, the C-terminal residues close the active site to promote the reaction. CONCLUSIONS: In the high-affinity group, the prefixed direction of the serine residues was oriented by a positively charged residue located four amino acids away, while low-affinity enzymes possess small hydrophobic residues at the corresponding sites.