Putative Transfer Research Articles

NanoSIMS and tissue autoradiography reveal symbiont carbon fixation and organic carbon transfer to giant ciliate host

The giant colonial ciliate Zoothamnium niveum harbors a monolayer of the gammaproteobacteria Cand. Thiobios zoothamnicoli on its outer surface. Cultivation experiments revealed maximal growth and survival under steady flow of high oxygen and low sulfide concentrations. We aimed at directly demonstrating the sulfur-oxidizing, chemoautotrophic nature of the symbionts and at investigating putative carbon transfer from the symbiont to the ciliate host. We performed pulse-chase incubations with 14C- and 13C-labeled bicarbonate under varying environmental conditions. A combination of tissue autoradiography and nanoscale secondary ion mass spectrometry coupled with transmission electron microscopy was used to follow the fate of the radioactive and stable isotopes of carbon, respectively. We show that symbiont cells fix substantial amounts of inorganic carbon in the presence of sulfide, but also (to a lesser degree) in the absence of sulfide by utilizing internally stored sulfur. Isotope labeling patterns point to translocation of organic carbon to the host through both release of these compounds and digestion of symbiont cells. The latter mechanism is also supported by ultracytochemical detection of acid phosphatase in lysosomes and in food vacuoles of ciliate cells. Fluorescence in situ hybridization of freshly collected ciliates revealed that the vast majority of ingested microbial cells were ectosymbionts.

Comparative analysis of inverted repeats of polypod fern (Polypodiales) plastomes reveals two hypervariable regions

BackgroundFerns are large and underexplored group of vascular plants (~ 11 thousands species). The genomic data available by now include low coverage nuclear genomes sequences and partial sequences of mitochondrial genomes for six species and several plastid genomes.ResultsWe characterized plastid genomes of three species of Dryopteris, which is one of the largest fern genera, using sequencing of chloroplast DNA enriched samples and performed comparative analysis with available plastomes of Polypodiales, the most species-rich group of ferns. We also sequenced the plastome of Adianthum hispidulum (Pteridaceae). Unexpectedly, we found high variability in the IR region, including duplication of rrn16 in D. blanfordii, complete loss of trnI-GAU in D. filix-mas, its pseudogenization due to the loss of an exon in D. blanfordii. Analysis of previously reported plastomes of Polypodiales demonstrated that Woodwardia unigemmata and Lepisorus clathratus have unusual insertions in the IR region. The sequence of these inserted regions has high similarity to several LSC fragments of ferns outside of Polypodiales and to spacer between tRNA-CGA and tRNA-TTT genes of mitochondrial genome of Asplenium nidus. We suggest that this reflects the ancient DNA transfer from mitochondrial to plastid genome occurred in a common ancestor of ferns. We determined the marked conservation of gene content and relative evolution rate of genes and intergenic spacers in the IRs of Polypodiales. Faster evolution of the four intergenic regions had been demonstrated (trnA- orf42, rrn16-rps12, rps7-psbA and ycf2-trnN).ConclusionsIRs of Polypodiales plastomes are dynamic, driven by such events as gene loss, duplication and putative lateral transfer from mitochondria.

Ecophysiology of Freshwater Verrucomicrobia Inferred from Metagenome-Assembled Genomes.

Microbes are critical in carbon and nutrient cycling in freshwater ecosystems. Members of the Verrucomicrobia are ubiquitous in such systems, and yet their roles and ecophysiology are not well understood. In this study, we recovered 19 Verrucomicrobia draft genomes by sequencing 184 time-series metagenomes from a eutrophic lake and a humic bog that differ in carbon source and nutrient availabilities. These genomes span four of the seven previously defined Verrucomicrobia subdivisions and greatly expand knowledge of the genomic diversity of freshwater Verrucomicrobia. Genome analysis revealed their potential role as (poly)saccharide degraders in freshwater, uncovered interesting genomic features for this lifestyle, and suggested their adaptation to nutrient availabilities in their environments. Verrucomicrobia populations differ significantly between the two lakes in glycoside hydrolase gene abundance and functional profiles, reflecting the autochthonous and terrestrially derived allochthonous carbon sources of the two ecosystems, respectively. Interestingly, a number of genomes recovered from the bog contained gene clusters that potentially encode a novel porin-multiheme cytochrome c complex and might be involved in extracellular electron transfer in the anoxic humus-rich environment. Notably, most epilimnion genomes have large numbers of so-called "Planctomycete-specific" cytochrome c-encoding genes, which exhibited distribution patterns nearly opposite to those seen with glycoside hydrolase genes, probably associated with the different levels of environmental oxygen availability and carbohydrate complexity between lakes/layers. Overall, the recovered genomes represent a major step toward understanding the role, ecophysiology, and distribution of Verrucomicrobia in freshwater. IMPORTANCE Freshwater Verrucomicrobia spp. are cosmopolitan in lakes and rivers, and yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia spp. are restricted to one subdivision of this phylum. Here, we greatly expanded the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent the first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia to be potential (poly)saccharide degraders and suggested their adaptation to carbon sources of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called "Planctomycete-specific" cytochrome c-encoding genes and identified their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology, and distribution of freshwater Verrucomicrobia, while highlighting their potential role in freshwater carbon cycling.

Modulating proposed electron transfer pathways in P450BM3 led to improved activity and coupling efficiency

Electrochemical in vitro reduction of P450 enzymes is a promising alternative to in vivo applications. Previously we presented three engineered P450BM3 variants for aniline hydroxylation, equipped with a carbon nanotube binding-peptide (CNT-tag) for self-assembly on CNT electrodes. Compared to wildtype P450BM3 the NADPH-dependent activity was enhanced, but the coupling efficiency remained low. For P450BM3 Verma, Schwaneberg and Roccatano (2014, Biopolymers 101, 197–209) calculated putative electron transfer pathways (eTPs) by MD simulations. We hypothesised that knockouts of these transfer pathways would alter the coupling efficiency of the system. The results revealed no improved system for the electrically-driven P450s. For the NADPH-driven P450s, however, the most active eTP-mutant showed a 13-fold increased activity and a 32-fold elevated coupling efficiency using NADPH as reducing equivalent. This suggests an alternative principle of electron transport for the reduction by NADPH and an electrode, respectively. The work presents moreover a tool to improve the coupling and activity of P450s with non-natural substrates.

Mitochondrial genome of Abraxas suspecta (Lepidoptera: Geometridae)and comparative analysis with other Lepidopterans.

In this study, a complete mitochondrial genome (mitogenome) sequence of Abraxas suspecta (Lepidoptera: Geometridae) is isolated and characterized. The complete DNA is 15,547 bp length and contains 2 ribosomal RNA genes, 23 putative transfer RNA (tRNA) genes including an extra tRNAAsn (AUU), 13 protein-coding genes and an adenine (A) + thymine (T)-rich region. The nucleotide composition and gene organization are identical to those of other lepidopteran, except for the presence of an extra copy of trnN (AUU). Of the 38 genes, twenty-five genes (9 PCGs and 16 tRNAs) are encoded by heavy strand (H-strand), while thirteen are encoded by light strand (L-strand). Among the 13 PCGs, 12 PCGs employ ATN as initiation codon, while cytochrome c oxidase subunit 1 (cox1) utilizes CGA as initiation codon. Four of the 13 PCGs have the incomplete termination codon T, while the remainder terminated with the canonical stop codon. All tRNA genes are folded into the typical clover-leaf structure of mitochondrial tRNAs, except for the tRNASer (AGN) gene, in which the DHU arm fails to form a stable stem-loop structure. The A+T-rich region is 532 bp long, and contains some conserved regions, including 'ATAGA' motif followed by a 17bp poly-T stretch, a microsatellite-like element (AT)8(AAT)3 and also a poly-A element. A short Phylogenetic analysis based on 13 PCGs using maximum likelihood (ML) and Bayesian inference (BI) revealed that A. suspecta resides in the Geometridae family. We present the method and approach to use moths as model organisms for further genetic and evolutionary biology studies.

Creatine kinase in human erythrocytes: A genetic anomaly reveals presence of soluble brain-type isoform

For maintaining energy homeostasis, creatine kinase (CK) is present at elevated levels in tissues with high and/or fluctuating energy requirements such as muscle, brain, and epithelia, while there is very few CK, if any, in peripheral blood cells. However, an ectopic expression of brain-type creatine kinase (BCK) has been reported for platelets and leukocytes in an autosomal dominant inherited anomaly named CKBE. Here we investigated CK in erythrocytes of CKBE individuals from eight unrelated families. The data revealed a varying but significant increase of CK activity in CKBE individuals as compared to controls, reaching an almost 800-fold increase in two CKBE individuals which also had increased erythrocyte creatine. Immunoblotting with highly specific antibodies confirmed that the expressed CK isoform is BCK. Cell fractionation evidenced soluble BCK, suggesting cytosolic and not membrane localization of erythrocyte CK as reported earlier. These results are discussed in the context of putative CK energy buffering and transfer functions in red blood cells.

Mass Transport Phenomena in Lipid Oxidation and Antioxidation.

In lipid dispersions, the ability of reactants to move from one lipid particle to another is an important, yet often ignored, determinant of lipid oxidation and its inhibition by antioxidants. This review describes three putative interparticle transfer mechanisms for oxidants and antioxidants: (a) diffusion, (b) collision-exchange-separation, and (c) micelle-assisted transfer. Mechanism a involves the diffusion of molecules from one particle to another through the intervening aqueous phase. Mechanism b involves the transfer of molecules from one particle to another when the particles collide with each other. Mechanism c involves the solubilization of molecules in micelles within the aqueous phase and then their transfer between particles. During lipid oxidation, the accumulation of surface-active lipid hydroperoxides (LOOHs) beyond their critical micelle concentration may shift their mass transport from the collision-exchange-separation pathway (slow transfer) to the micelle-assisted mechanism (fast transfer), which may account for the transition from the initiation to the propagation phase. Similarly, the cut-off effect governing antioxidant activity in lipid dispersions may be due to the fact that above a certain hydrophobicity, the transfer mechanism for antioxidants changes from diffusion to collision-exchange-separation. This hypothesis provides a simple model to rationalize the design and formulation of antioxidants and dispersed lipids.

Uptake and impact of natural diet-derived small RNA in invertebrates: Implications for ecology and agriculture

ABSTRACTThe putative transfer and gene regulatory activities of diet-derived small RNAs (sRNAs) in ingesting animals are still debated. The existence of natural uptake of diet-derived sRNA by invertebrate species could have significant implication for our understanding of ecological relationships and could synergize with efforts to use RNA interference (RNAi) technology in agriculture. Here, we synthesize information gathered from studies in invertebrates using natural or artificial dietary delivery of sRNA and from studies of sRNA in vertebrate animals and plants to review our current understanding of uptake and impact of natural diet-derived sRNA on invertebrates. Our understanding has been influenced and sometimes confounded by the diversity of invertebrates and ingested plants studied, our limited insights into how gene expression may be modulated by dietary sRNAs at the mechanistic level, and the paucity of studies focusing directly on natural uptake of sRNA. As such, we suggest 2 strategies to investigate this phenomenon more comprehensively and thus facilitate the realization of its potentially broad impact on ecology and agriculture in the future.

Classification of a Hypervirulent Aeromonas hydrophila Pathotype Responsible for Epidemic Outbreaks in Warm-Water Fishes.

Lineages of hypervirulent Aeromonas hydrophila (vAh) are the cause of persistent outbreaks of motile Aeromonas septicemia in warm-water fishes worldwide. Over the last decade, this virulent lineage of A. hydrophila has resulted in annual losses of millions of tons of farmed carp and catfish in the People's Republic of China and the United States (US). Multiple lines of evidence indicate US catfish and Asian carp isolates of A. hydrophila affiliated with sequence type 251 (ST251) share a recent common ancestor. To address the genomic context for the putative intercontinental transfer and subsequent geographic spread of this pathogen, we conducted a core genome phylogenetic analysis on 61 Aeromonas spp. genomes, of which 40 were affiliated with A. hydrophila, with 26 identified as epidemic strains. Phylogenetic analyses indicate all ST251 strains form a coherent lineage affiliated with A. hydrophila. Within this lineage, conserved genetic loci unique to A. hydrophila were identified, with some genes present in consistently higher copy numbers than in non-epidemic A. hydrophila isolates. In addition, results from analyses of representative ST251 isolates support the conclusion that multiple lineages are present within US vAh isolated from Mississippi, whereas vAh isolated from Alabama appear clonal. This is the first report of genomic heterogeneity within US vAh isolates, with some Mississippi isolates showing closer affiliation with the Asian grass carp isolate ZC1 than other vAh isolated in the US. To evaluate the biological significance of the identified heterogeneity, comparative disease challenges were conducted with representatives of different vAh genotypes. These studies revealed that isolate ZC1 yielded significantly lower mortality in channel catfish, relative to Alabama and Mississippi vAh isolates. Like other Asian vAh isolates, the ZC1 lineage contains all core genes for a complete type VI secretion system (T6SS). In contrast, more virulent US isolates retain only remnants of the T6SS (clpB, hcp, vgrG, and vasH) which may have functional implications. Collectively, these results characterize a hypervirulent A. hydrophila pathotype that affects farmed fish on multiple continents.

Melanin 'dust' or 'ghost'?

Melanin 'dust' or 'ghost'?

Spectral Characteristics of Noble Metal Nanoparticle–Molybdenum Disulfide Heterostructures

Energy and damping of resonances which occur in spectra of monolayer molybdenum disulfide (MoS2) decorated by noble metal nanospheres and prisms are examined by comparing discrete dipole approximation (DDA) simulations and transmission UV–vis measurements. Localized surface plasmon resonance (LSPR) as well as A and B exciton transitions of the semiconductor are characterized individually and in tandem on a silica substrate. Extinction energies for LSPR and direct bandgap transition of MoS2 estimated by DDA are within 2% of measured values for gold nanospheres and silver nanoprisms. Resonant near‐fields mapped at nanoparticle–MoS2 interfaces show plasmon damping via energy dissipation to the MoS2 and direct bandgap excitation. Linewidth analysis of resonant far‐field damping estimates energy transfer efficiencies for putative hot electron transfer, corroborated by experiment. Use of DDA simulations to characterize and inform induction and damping of electric resonance modes in nanoparticle–MoS2 heterostructures is illustrated.

Klebsiella pneumoniae Asparagine tDNAs Are Integration Hotspots for Different Genomic Islands Encoding Microcin E492 Production Determinants and Other Putative Virulence Factors Present in Hypervirulent Strains.

Due to the developing of multi-resistant and invasive hypervirulent strains, Klebsiella pneumoniae has become one of the most urgent bacterial pathogen threats in the last years. Genomic comparison of a growing number of sequenced isolates has allowed the identification of putative virulence factors, proposed to be acquirable mainly through horizontal gene transfer. In particular, those related with synthesizing the antibacterial peptide microcin E492 (MccE492) and salmochelin siderophores were found to be highly prevalent among hypervirulent strains. The determinants for the production of both molecules were first reported as part of a 13-kbp segment of K. pneumoniae RYC492 chromosome, and were cloned and characterized in E. coli. However, the genomic context of this segment in K. pneumoniae remained uncharacterized. In this work, we provided experimental and bioinformatics evidence indicating that the MccE492 cluster is part of a highly conserved 23-kbp genomic island (GI) named GIE492, that was integrated in a specific asparagine-tRNA gene (asn-tDNA) and was found in a high proportion of isolates from liver abscesses sampled around the world. This element resulted to be unstable and its excision frequency increased after treating bacteria with mitomycin C and upon the overexpression of the island-encoded integrase. Besides the MccE492 genetic cluster, it invariably included an integrase-coding gene, at least seven protein-coding genes of unknown function, and a putative transfer origin that possibly allows this GI to be mobilized through conjugation. In addition, we analyzed the asn-tDNA loci of all the available K. pneumoniae assembled chromosomes to evaluate them as GI-integration sites. Remarkably, 73% of the strains harbored at least one GI integrated in one of the four asn-tDNA present in this species, confirming them as integration hotspots. Each of these tDNAs was occupied with different frequencies, although they were 100% identical. Also, we identified a total of 47 asn-tDNA-associated GIs that were classified into 12 groups of homology differing in theencoded functionalities but sharing with GIE492 a conserved recombination module and potentially its mobility features. Most of these GIs encoded factors with proven or potential role in pathogenesis, constituting a major reservoir of virulence factors in this species.

Regulatory function of Arabidopsis lipid transfer protein 1 (LTP1) in ethylene response and signaling.

Ethylene as a gaseous plant hormone is directly involved in various processes during plant growth and development. Much is known regarding the ethylene receptors and regulatory factors in the ethylene signal transduction pathway. In Arabidopsis thaliana, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) can interact with and positively regulates the ethylene receptor ETHYLENE RESPONSE1 (ETR1). In this study we report the identification and characterization of an RTE1-interacting protein, a putative Arabidopsis lipid transfer protein 1 (LTP1) of unknown function. Through bimolecular fluorescence complementation, a direct molecular interaction between LTP1 and RTE1 was verified in planta. Analysis of an LTP1-GFP fusion in transgenic plants and plasmolysis experiments revealed that LTP1 is localized to the cytoplasm. Analysis of ethylene responses showed that the ltp1 knockout is hypersensitive to 1-aminocyclopropanecarboxylic acid (ACC), while LTP1 overexpression confers insensitivity. Analysis of double mutants etr1-2 ltp1 and rte1-3 ltp1 demonstrates a regulatory function of LTP1 in ethylene receptor signaling through the molecular association with RTE1. This study uncovers a novel function of Arabidopsis LTP1 in the regulation of ethylene response and signaling.

Metagenomic Analyses of the Autotrophic Fe(II)-Oxidizing, Nitrate-Reducing Enrichment Culture KS.

Nitrate-dependent ferrous iron [Fe(II)] oxidation (NDFO) is a well-recognized chemolithotrophic pathway in anoxic sediments. The neutrophilic chemolithoautotrophic enrichment culture KS originally obtained from a freshwater sediment (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl Environ Microbiol 62:1458-1460, 1996) has been used as a model system to study NDFO. However, the primary Fe(II) oxidizer in this culture has not been isolated, despite extensive efforts to do so. Here, we present a metagenomic analysis of this enrichment culture in order to gain insight into electron transfer pathways and the roles of different bacteria in the culture. We obtained a near-complete genome of the primary Fe(II) oxidizer, a species in the family Gallionellaceae, and draft genomes from its flanking community members. A search of the putative extracellular electron transfer pathways in these genomes led to the identification of a homolog of the MtoAB complex [a porin-multiheme cytochromec system identified in neutrophilic microaerobic Fe(II)-oxidizing Sideroxydans lithotrophicus ES-1] in a Gallionellaceae sp., and findings of other putative genes involving cytochromecand multicopper oxidases, such as Cyc2 and OmpB. Genome-enabled metabolic reconstruction revealed that this Gallionellaceae sp. lacks nitric oxide and nitrous oxide reductase genes and may partner with flanking populations capable of complete denitrification to avoid toxic metabolite accumulation, which may explain its resistance to growth in pure culture. This and other revealed interspecies interactions and metabolic interdependencies in nitrogen and carbon metabolisms may allow these organisms to cooperate effectively to achieve robust chemolithoautotrophic NDFO. Overall, the results significantly expand our knowledge of NDFO and suggest a range of genetic targets for further exploration.

Unique features of a global human ectoparasite identified through sequencing of the bed bug genome.

The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host–symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human–bed bug and symbiont–bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite.

Comparison of Dicyclohexylcarbodiimide (DCCD)-Induced Effects on Structure and Activity in Cytochrome c Oxidase (COX) from Bovine Heart and Rhodobacter Sphaeroides

Previous work has shown that DCCD inhibits bovine heart cytochrome c oxidase (COX) function by binding to Glu-90 in subunit III (SIII). The goal of this study was to compare the effects of DCCD modification on structure and electron transfer in COX from bovine heart and Rhodobacter sphaeroides. DCCD modification induced similar shifts in migration of SIII on SDS-PAGE in both enzymes, suggesting similar SIII modification. The concentration dependence for the inhibition of electron transfer by DCCD was also similar in both enzymes. The pH dependence of electron transfer activity in the DCCD-modified enzymes was acid shifted 0.7-0.9 pH unit in both enzymes. However, bovine heart COX treated with DCCD exhibited less inhibition of electron transfer activity at pH 9.5-10.0 than at lower pH, while DCCD treated R. sphaeroides COX had less inhibition at pH 6.5-7.0 than at higher pH. The heme circular dichroism spectrum showed that DCCD induced a red shift of 0.5-0.8 nm in both enzymes when monitored at both pH 7.0 and 10.0. Steady-state heme a reduction during electron transfer increased from 18±1% to 33±2% in DCCD-modified beef COX at pH 7.0, while in R. sphaeroides, it remained unchanged from 41±2% to 36±2%. In summary, our results indicate that DCCD, while binding at a similar site in SIII, leads to differential effects in the two enzymes. DCCD modification of SIII of bovine heart may cause blockage of the putative O2 transfer pathway, while in R. sphaeroides it may induce a slowed proton uptake. The different characteristics of inhibition may be due to variation in both the number of subunits and their structural homology between the two forms of COX forms.

Negligible uptake and transfer of diet-derived pollen microRNAs in adult honey bees

ABSTRACTThe putative transfer and gene regulatory activities of diet-derived miRNAs in ingesting animals are still debated. Importantly, no study to date has fully examined the role of dietary uptake of miRNA in the honey bee, a critical pollinator in both agricultural and natural ecosystems. After controlled pollen feeding experiments in adult honey bees, we observed that midguts demonstrated robust increases in plant miRNAs after pollen ingestion. However, we found no evidence of biologically relevant delivery of these molecules to proximal or distal tissues of recipient honey bees. Our results, therefore, support the premise that pollen miRNAs ingested as part of a typical diet are not robustly transferred across barrier epithelia of adult honey bees under normal conditions. Key future questions include whether other small RNA species in honey bee diets behave similarly and whether more specialized and specific delivery mechanisms exist for more efficient transport, particularly in the context of stressed barrier epithelia.

1H, 15N and 13C chemical shift assignment of the Gram-positive conjugative transfer protein TraHpIP501.

Conjugative transfer of DNA represents the most important transmission pathway in terms of antibiotic resistance and virulence gene dissemination among bacteria. TraH is a putative transfer protein of the type IV secretion system (T4SS) encoded by the Gram-positive (G+) conjugative plasmid pIP501. This molecular machine involves a multi-protein core complex spanning the bacterial envelope thereby serving as a macromolecular secretion channel. Here, we report the near complete (1)H, (13)C and (15)N resonance assignment of a soluble TraH variant comprising the C-terminal domain.

Transfer of energy pathway genes in microbial enhanced biological phosphorus removal communities.

BackgroundLateral gene transfer (LGT) is an important evolutionary process in microbial evolution. In sewage treatment plants, LGT of antibiotic resistance and xenobiotic degradation-related proteins has been suggested, but the role of LGT outside these processes is unknown. Microbial communities involved in Enhanced Biological Phosphorus Removal (EBPR) have been used to treat wastewater in the last 50 years and may provide insights into adaptation to an engineered environment. We introduce two different types of analysis to identify LGT in EBPR sewage communities, based on identifying assembled sequences with more than one strong taxonomic match, and on unusual phylogenetic patterns. We applied these methods to investigate the role of LGT in six energy-related metabolic pathways.ResultsThe analyses identified overlapping but non-identical sets of transferred enzymes. All of these were homologous with sequences from known mobile genetic elements, and many were also in close proximity to transposases and integrases in the EBPR data set. The taxonomic method had higher sensitivity than the phylogenetic method, identifying more potential LGTs. Both analyses identified the putative transfer of five enzymes within an Australian community, two in a Danish community, and none in a US-derived culture.ConclusionsOur methods were able to identify sequences with unusual phylogenetic or compositional properties as candidate LGT events. The association of these candidates with known mobile elements supports the hypothesis of transfer. The results of our analysis strongly suggest that LGT has influenced the development of functionally important energy-related pathways in EBPR systems, but transfers may be unique to each community due to different operating conditions or taxonomic composition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1752-5) contains supplementary material, which is available to authorized users.

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production.