A novel bioinformatic method for the identification of antimicrobial peptides in metagenomes.

AIMS: This study aimed to develop a new bioinformatic approach for the identification of novel antimicrobial peptides (AMPs), which did not depend on sequence similarity to known AMPs held within databases, but on structural mimicry of another antimicrobial compound, in this case an ultrashort, synthetic, cationic lipopeptide (C12-OOWW-NH2). METHODS AND RESULTS: When applied to a collection of metagenomic datasets, our outlined bioinformatic method successfully identified several short (8-10aa) functional AMPs, the activity of which was verified via disk diffusion and minimum inhibitory concentration assays against a panel of 12 bacterial strains. Some peptides had activity comparable to, or in some cases, greater than, those from published studies that identified AMPs using more conventional methods. We also explored the effects of modifications, including extension of the peptides, observing an activity peak at 9-12aa. Additionally, the inclusion of a C-terminal amide enhanced activity in most cases. Our most promising candidate (named PB2-10aa-NH2) was thermally stable, lipid-soluble, and possessed synergistic activity with ethanol but not with a conventional antibiotic (streptomycin). CONCLUSIONS: While several bioinformatic methods exist to predict AMPs, the approach outlined here is much simpler and can be used to quickly scan huge datasets. Searching for peptide sequences bearing structural similarity to other antimicrobial compounds may present a further opportunity to identify novel AMPs with clinical relevance, and provide a meaningful contribution to the pressing global issue of AMR.

Geochemical properties of blue carbon sediments through an elevation gradient: study of an anthropogenically impacted coastal lagoon.

Global research is showing that coastal blue carbon ecosystems are vulnerable to climate change driven threats including accelerated sea-level rise and prolonged periods of drought. Furthermore, direct anthropogenic impacts present immediate threats through deterioration of coastal water quality, land reclamation, long-term impact to sediment biogeochemical cycling. These threats will invariably alter the future efficacy of carbon (C) sequestration processes and it is imperative that currently existing blue carbon habitats be protected. Knowledge of underlying biogeochemical, physical and hydrological interactions occurring in functioning blue carbon habitats is essential for developing strategies to mitigate threats, and promote conditions to optimise C sequestration/storage. In this current work, we investigated how sediment geochemistry (0-10 cm depth) responds to elevation, an edaphic factor driven by long-term hydrological regimes consequently exerting control over particle sedimentation rates and vegetation succession. This study was performed in an anthropogenically impacted blue carbon habitat along a coastal ecotone encompassing an elevation gradient transect from intertidal sediments (un-vegetated and covered daily by tidal water), through vegetated salt marsh sediments (periodically covered by spring tides and flooding events), on Bull Island, Dublin Bay. We determined the quantity and distributions of bulk geochemical characteristics in sediments through the elevation gradient, including total organic carbon (TOC), total nitrogen (TN), total metals, silt, clay, and also, 16 individual polyaromatic hydrocarbon's (PAH's) as an indication of anthropogenic input. Elevation measurements for sample sites were determined on this gradient using a LiDAR scanner accompanied by an IGI inertial measurement unit (IMU) on board a light aircraft. Considering the gradient from the Tidal mud zone (T), through the low-mid marsh (M) to the most elevated upper marsh (H), there were significant differences between all zones for many measured environmental variables. The results of significance testing using Kruskal-Wallis analysis revealed, that %C, %N, PAH (µg/g), Mn (mg/kg), TOC:NH4 + and pH are significantly different between all zones on the elevation gradient. The highest values for all these variables exists (excluding pH which followed a reverse trend) in zone H, decreasing in zone M and lowest in the un-vegetated zone T. TC content is 16 fold higher overall in vegetated (3.43 -21.84%) than uninhabited (0.21-0.56%) sediments. TN was over 50 times higher (0.24-1.76%), more specifically increasing in % mass on approach to the upper salt marsh with distance from the tidal flats sediments zone T (0.002-0.05%). Clay and silt distributions were greatest in vegetated sediments, increasing in % content towards upper marsh zones The retention of water, metals, PAHs, mud, chloride ions, NH4 +, PO4 3- and SO4 2- increased with elevated C concentrations, concurrently where pH significantly decreased. Sediments were categorized with respect to PAH contamination where all SM samples were placed in the high polluted category. The results highlight the ability of Blue C sediments to immobilise increasing levels of C, N, and metals, and PAH with over time and with both lateral and vertical expansion. This study provides a valuable data set for an anthropogenically impacted blue carbon habitat predicted to suffer from sea-level rise and exponential urban development. Graphical abstract: Summarized results from this study demonstrating the geochemical changes through an elevation gradient, with a transect encompassing intertidal sediments through supratidal salt marsh sediments within Bull Island's blue carbon lagoon zones. Supplementary Information: The online version contains supplementary material available at 10.1007/s10533-022-00974-0.

Biogeochemical properties of blue carbon sediments influence the distribution and monomer composition of bacterial polyhydroxyalkanoates (PHA).

Coastal wetlands are highly efficient 'blue carbon' sinks which contribute to mitigating climate change through the long-term removal of atmospheric CO2 and capture of carbon (C). Microorganisms are integral to C sequestration in blue carbon sediments and face a myriad of natural and anthropogenic pressures yet their adaptive responses are poorly understood. One such response in bacteria is the alteration of biomass lipids, specifically through the accumulation of polyhydroxyalkanoates (PHAs) and alteration of membrane phospholipid fatty acids (PLFA). PHAs are highly reduced bacterial storage polymers that increase bacterial fitness in changing environments. In this study, we investigated the distribution of microbial PHA, PLFA profiles, community structure and response to changes in sediment geochemistry along an elevation gradient from intertidal to vegetated supratidal sediments. We found highest PHA accumulation, monomer diversity and expression of lipid stress indices in elevated and vegetated sediments where C, nitrogen (N), PAH and heavy metals increased, and pH was significantly lower. This was accompanied by a reduction in bacterial diversity and a shift to higher abundances of microbial community members favouring complex C degradation. Results presented here describe a connection between bacterial PHA accumulation, membrane lipid adaptation, microbial community composition and polluted C rich sediments. Graphical Abstract: Geochemical, microbiological and polyhydroxyalkanoate (PHA) gradient in a blue carbon zone. Supplementary Information: The online version contains supplementary material available at 10.1007/s10533-022-01008-5.

Expression, purification and crystallization of a novel metagenome-derived salicylaldehyde dehydrogenase from Alpine soil.

Salicylaldehyde dehydrogenase (SALD) catalyses the last reaction in the upper pathway of naphthalene degradation: the oxidation of salicylaldehyde to salicylate. This enzyme has been isolated and studied from a few organisms that belong to the betaproteobacteria and gammaproteobacteria, predominantly Pseudomonas putida. Furthermore, there is only one crystal structure of this enzyme, which was obtained from P. putida G7. Here, crystallographic studies and analysis of the crystal structure of an Alpine soil metagenome-derived SALD (SALDAP) from an alphaproteobacterium are presented. The SALDAP gene was discovered using gene-targeted sequence assembly and it was cloned into a pLATE51 vector. The recombinant protein was overexpressed in Escherichia coli BL21 (DE3) cells and the soluble protein was purified to homogeneity. The protein crystallized at 20°C and diffraction data from the crystals were collected at a resolution of 1.9 Å. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 116.8, b = 121.7, c = 318.0 Å. Analysis of the crystal structure revealed its conformation to be similar to the organization of the aldehyde dehydrogenase superfamily with three domains: the catalytic, NAD+-binding and bridging domains. The crystal structure of NahF from P. putida G7 was found to be the best structural homologue of SALDAP, even though the enzymes share only 48% amino-acid identity. Interestingly, a carboxylic acid (protocatechuic acid) was found to be a putative ligand of the enzyme and differential scanning fluorimetry was employed to confirm ligand binding. These findings open up the possibility of studying the mechanism(s) of product inhibition and biocatalysis of carboxylic acids using this enzyme and other related aldehyde dehydrogenases.

Monooxygenase- and Dioxygenase-Catalyzed Oxidative Dearomatization of Thiophenes by Sulfoxidation, cis-Dihydroxylation and Epoxidation.

Enzymatic oxidations of thiophenes, including thiophene-containing drugs, are important for biodesulfurization of crude oil and drug metabolism of mono- and poly-cyclic thiophenes. Thiophene oxidative dearomatization pathways involve reactive metabolites, whose detection is important in the pharmaceutical industry, and are catalyzed by monooxygenase (sulfoxidation, epoxidation) and dioxygenase (sulfoxidation, dihydroxylation) enzymes. Sulfoxide and epoxide metabolites of thiophene substrates are often unstable, and, while cis-dihydrodiol metabolites are more stable, significant challenges are presented by both types of metabolite. Prediction of the structure, relative and absolute configuration, and enantiopurity of chiral metabolites obtained from thiophene enzymatic oxidation depends on the substrate, type of oxygenase selected, and molecular docking results. The racemization and dimerization of sulfoxides, cis/trans epimerization of dihydrodiol metabolites, and aromatization of epoxides are all factors associated with the mono- and di-oxygenase-catalyzed metabolism of thiophenes and thiophene-containing drugs and their applications in chemoenzymatic synthesis and medicine.

Percutaneous coronary intervention versus coronary artery bypass grafting in patients with reduced ejection fraction.

OBJECTIVE: The aim of this study was to evaluate comparative outcomes for percutaneous coronary intervention (PCI) versus coronary artery bypass grafting (CABG) in patients with reduced ejection fraction. METHODS: All patients from the University of Pittsburgh Medical Center from 2011 to 2018 who had reduced preoperative ejection fraction (<50%) and underwent CABG or PCI for coronary revascularization were included in this study. Patients were risk-adjusted with propensity matching (1:1) and primary outcomes included long-term survival, readmission, and major adverse cardiac and cerebrovascular events (MACCE). RESULTS: A total of 2000 patients were included in the current study, consisting of CABG (n = 1553) and PCI (n = 447) cohorts with a mean ejection fraction of 35% ± 9.53%. Propensity matching yielded a 1:1 match with 324 patients in each cohort, controlling for all baseline characteristics. Thirty-day mortality was similar for PCI versus CABG (6.2% vs 4.9%; P = .49). Overall mortality over the study follow-up period (median, 3.23 years; range, 1.83-4.98 years) was significantly higher for the PCI cohort (37.4% vs 21.3%; P < .001). Total hospital readmissions (24.1% vs 12.9%; P = .001), cardiac readmissions (20.4% vs 11.1%; P = .001), myocardial infarction event (7.7% vs 1.8%; P = .001), MACCE (41.4% vs 23.8%; P < .001), and repeat revascularization (6.5% vs 2.6%; P = .02) occurred more frequently in the PCI cohort. Freedom from MACCE at 1 year (74.4% vs 87.0%; P < .001) and 5 years (54.5% vs 74.0%; P < .001) was significantly lower for the PCI cohort. On multivariable cox regression analysis, CABG (hazard ratio, 0.57; 95% confidence interval, 0.44-0.73; P < .001) was significantly associated with improved survival. Prior liver disease, dialysis, diabetes, and peripheral artery disease were the most significant predictors of mortality. The cumulative incidence of hospital readmission was lower for the CABG cohort (hazard ratio, 0.51; 95% confidence interval, 0.37-0.71; P < .001). Multivariable cox regression for MACCE (hazard ratio, 0.48; 95% confidence interval, 0.39-0.58; P < .001) showed significantly fewer events for the CABG cohort. CONCLUSIONS: Patients with reduced ejection fraction who underwent CABG had significantly improved survival, lower MACCE, and fewer repeat revascularization procedures compared with patients who underwent PCI.

Coronary Artery Bypass Grafting vs Percutaneous Coronary Intervention in Patients With Diabetes.

As percutaneous coronary intervention (PCI) continues to evolve, comparative outcomes for PCI vs coronary artery bypass grafting (CABG) remain relevant in diabetic patients. All revascularization procedures in patients with coronary artery disease and diabetes mellitus from 2010 to 2018 were included. Propensity matching was used to identify equivalent cohorts to compare revascularization strategies. Primary outcomes included 30-day, 1-year, and 5-year mortality. Multivariable analysis was used to define factors associated with major adverse cardiovascular and cerebrovascular events (MACCE). A total of 2869 patients with diabetes were divided into PCI (n = 653) and CABG (n = 2216) cohorts. Propensity matching yielded a 1:1 match consisting of 552 patients in each cohort (CABG vs PCI). Total median follow-up was 3.28 years (range: 1.83-5.00). Following propensity matching in patients with no prior PCI (1:1; n = 279), mortality remained significantly higher in the PCI cohort at 1 year (13.98% vs 7.53%; P = 0.014) and 5 years (26.88% vs 16.85%; P < 0.004). Hospital readmissions were higher for PCI patients at 1 year (16.49% vs 9.32%; P < 0.0122) and 5 years (19.71% vs 11.83%; P = 0.011). MACCE occurred more frequently in the PCI cohort (32.97% vs 21.51%; P = 0.002). Need for subsequent revascularization (6.45% vs 2.51%; P = 0.024) were significantly higher in the PCI cohort, and time interval to revascularization was significantly longer in the CABG cohort (3.48 [2.11-5.17] vs 2.62 [1.33-4.25] years; P < 0.001). The current study reports improved survival, fewer long-term hospital readmissions, and reduced MACCE and need for repeat revascularization in the CABG cohort. Given these data, patients with diabetes mellitus and coronary artery disease may fare better with surgical revascularization, compared to PCI.

Magnetic-Field Manipulation of Naturally Occurring Microbial Chiral Peptides to Regulate Gas-Hydrate Formation.

Clathrate hydrates are nonstoichiometric crystalline inclusion compounds, wherein a water host lattice entraps small guest molecules in cavities, with methane hydrates being the most widespread in nature. Recent studies have shown that proteins and polypeptides produced by micro-organisms can accelerate methane-hydrate formation. However, the role of magnetic fields and chirality in such phenomena is heretofore unclear. Here, we find prima facie evidence of differently oriented magnetic fields of varying strength showing intricate control on the hydrate-formation kinetics by R and S versions of a prototypical aromatic peptide derived from a naturally occurring, hydrate-promoting source. We also discuss the wider implications of these results on chirality in the biosphere and hydrates in the environment.

Engineering Peptides to Catalyze and Control Stabilization of Gas Hydrates: Learning From Nature.

Clathrate hydrates are nonstoichiometric crystalline inclusion compounds. Water acts as a "host lattice" and traps small guest molecules in stable cavities. One example, methane hydrates, are especially prevalent in situ at the seafloor. Although microorganism-produced proteins and polypeptides, including marine methylotroph porin proteins, can accelerate methane hydrate formation under conditions simulating their natural occurrence at the seafloor, the role that particular peptide sequences play in biocatalytic hydrate kinetics enhancement is unclear, especially the underlying molecular-level mechanisms. Here, we reveal the peptide-focused regulation of microorganisms' role in managing marine hydrates via an approximation mechanism of enzymatic catalysis accelerating hydrate formation. Aside from control of hydrate kinetics per se, we speculate that this peptide-centric mechanistic understanding could lead to a re-evaluation of the extent and geological importance of bioregulation of methane turnover in the biosphere.

Toluene Dioxygenase-Catalyzed cis-Dihydroxylation of Quinolines: A Molecular Docking Study and Chemoenzymatic Synthesis of Quinoline Arene Oxides.

Molecular docking studies of quinoline and 2-chloroquinoline substrates at the active site of toluene dioxygenase (TDO), were conducted using Autodock Vina, to identify novel edge-to-face interactions and to rationalize the observed stereoselective cis-dihydroxylation of carbocyclic rings and formation of isolable cis-dihydrodiol metabolites. These in silico docking results of quinoline and pyridine substrates, with TDO, also provided support for the postulated cis-dihydroxylation of electron-deficient pyridyl rings, to give transient cis-dihydrodiol intermediates and the derived hydroxyquinolines. 2-Chloroquinoline cis-dihydrodiol metabolites were used as precursors in the chemoenzymatic synthesis of enantiopure arene oxide and arene dioxide derivatives of quinoline, in the context of its possible mammalian metabolism and carcinogenicity.

Chemoenzymatic Synthesis of (-)-Ribisins A and B from Dibenzo[b,d]furan.

cis-Dihydrodiols, derived from monocyclic aromatic compounds, are valuable chiral pool intermediates for the synthesis of cyclic natural products. A drawback of this approach, to the synthesis of polycyclic secondary metabolites, is that additional rings must be annulated. To date, relatively few chiral natural products have been synthesized from polycyclic arene cis-dihydrodiols. Fungal metabolites, (-)-ribisins A and B, have now been obtained by functional group manipulation of a tricyclic arene metabolite, obtained from toluene dioxygenase-catalyzed regioselective and stereoselective cis-dihydroxylations of dibenzo[b,d]furan. The synthetic sequences were marginally shorter than the alternative routes, using monocyclic arene cis-dihydrodiols, and required no carbon-carbon bond-forming reactions.

Spectroscopic Characterisation of the Naphthalene Dioxygenase from Rhodococcus sp. Strain NCIMB12038.

Polycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are potential health risks due to their carcinogenic and mutagenic effects. Bacteria from the genus Rhodococcus are able to metabolise a wide variety of pollutants such as alkanes, aromatic compounds and halogenated hydrocarbons. A naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038 has been characterised for the first time, using electron paramagnetic resonance (EPR) spectroscopy and UV-Vis spectrophotometry. In the native state, the EPR spectrum of naphthalene 1,2-dioxygenase (NDO) is formed of the mononuclear high spin Fe(III) state contribution and the oxidised Rieske cluster is not visible as EPR-silent. In the presence of the reducing agent dithionite a signal derived from the reduction of the [2Fe-2S] unit is visible. The oxidation of the reduced NDO in the presence of O2-saturated naphthalene increased the intensity of the mononuclear contribution. A study of the "peroxide shunt", an alternative mechanism for the oxidation of substrate in the presence of H2O2, showed catalysis via the oxidation of mononuclear centre while the Rieske-type cluster is not involved in the process. Therefore, the ability of these enzymes to degrade recalcitrant aromatic compounds makes them suitable for bioremediative applications and synthetic purposes.

Characterisation of a solvent-tolerant haloarchaeal (R)-selective transaminase isolated from a Triassic period salt mine.

Transaminase enzymes (TAms) are becoming increasingly valuable in the chemist's toolbox as a biocatalytic route to chiral amines. Despite high profile successes, the lack of (R)-selective TAms and robustness under harsh industrial conditions continue to prove problematic. Herein, we report the isolation of the first haloarchaeal TAm (BC61-TAm) to be characterised for the purposes of pharmaceutical biocatalysis. BC61-TAm is an (R)-selective enzyme, cloned from an extremely halophilic archaeon, isolated from a Triassic period salt mine. Produced using a Haloferax volcanii-based expression model, the resulting protein displays a classic halophilic activity profile, as well as thermotolerance (optimum 50 °C) and organic solvent tolerance. Molecular modelling predicts the putative active site residues of haloarchaeal TAms, with molecular dynamics simulations providing insights on the basis of BC61-TAm's organic solvent tolerance. These results represent an exciting advance in the study of transaminases from extremophiles, providing a possible scaffold for future discovery of biocatalytic enzymes with robust properties.

The distribution of novel bacterial laccases in alpine paleosols is directly related to soil stratigraphy.

Bacterial laccases are now known to be abundant in soil and to function outside of the cell facilitating the bacterial degradation of lignin. In this study we wanted to test the hypotheses that: i) Such enzymes can be identified readily in stratified paleosols using metagenomics approaches, ii) The distribution of these genes as potential 'public good' proteins in soil is a function of the soil environment, iii) Such laccase genes can be readily retrieved and expressed in E. coli cloning systems to demonstrate that de novo assembly processes can be used to obtain similar metagenome-derived enzyme activities. To test these hypotheses, in silico gene-targeted assembly was employed to identify genes encoding novel type B two-domain bacterial laccases from alpine soil metagenomes sequenced on an Illumina MiSeq sequencer. The genes obtained from different strata were heterologously cloned, expressed and the gene products were shown to be active against two classical laccase substrates. The use of a metagenome-driven pipeline to obtain such active biocatalysts has demonstrated the potential for gene mining to be applied systematically for the discovery of such enzymes. These data ultimately further demonstrate the application of soil pedology methods to environmental enzyme discovery. As an interdisciplinary effort, we can now establish that paleosols can serve as a useful source of novel biocatalytic enzymes for various applications. We also, for the first time, link soil stratigraphy to enzyme profiling for widespread functional gene activity in paleosols.

Analysis of viral and bacterial communities in groundwater associated with contaminated land.

This work aimed at the comprehensive analysis of total microbial communities inhabiting a typical hydrocarbon-polluted site, where chemical characteristics of the groundwater were readily available. To achieve this, a joint metagenomic characterization of bacteria and viruses surrounding a contaminant plume was performed over a one-year period. The results presented demonstrated that both potential hydrocarbon degraders and their bacteriophages were dominant around the plume, and that the viral and bacterial diversities found at the site were probably influenced by the pH of the groundwater. Niche-specific and dispersed associations between phages and bacteria were identified. The niche phage-host associations were found at the edge of the site and at the core of the plume where pH was the highest (9.52). The identified host populations included several classes of bacteria (e.g. Clostridia and Proteobacteria). Thirty-six viral generalists were also discovered, with BGW-G9 having the broadest host range across 23 taxa, including Pseudomonas, Polycyclovorans, Methylocaldum and Candidatus Magnetobacterium species. The phages with broad host ranges are presumed to have significant effects on prokaryotic production and horizontal gene transfer, and therefore impact the biodegradation processes conducted by various bacteria of the environment studied. This study for the first time characterized the phages and their bacterial hosts associated with a contaminant plume.

Effect of soil horizon stratigraphy on the microbial ecology of alpine paleosols.

There is remarkable potential for research at the interface between the earth sciences and environmental microbiology that may lead to advances in our understanding of the role of bacterial communities in the surface or subsurface environment of our planet. One mainstay of sedimentary classification is the concept of differential soil and/or paleosol horizons being the result of primarily physical and chemical weathering, with relatively little understanding of how microbial communities between these stratified horizons differ, if at all. In this study we evaluate the differences in microbial community taxonomy and biogeochemical functional potential between stratified soil horizons in an alpine paleosol environment using next-generation sequencing (NGS) shotgun sequencing. Paleosols represent a unique environment to study the effect of differences soil horizon environments on the microbial community due to their relative isolation, and the fact that three distinct stratified soil horizons can be identified within the top 30 cm of the soil. This enables us to assess variation in microbial community composition that will be relatively distinct from variation due to distance alone. We test the hypothesis that variation in soil community composition is linked to variation in the physical and chemical parameters that define stratigraphy. Multivariate statistical analysis of sequencing reads from soil horizons across five sampling sites revealed that 1223 microbial genera vary significantly and consistently in abundance across stratified soil horizons at class level. Specifically Ktedonobacter, Bacilli and Betaproteobacteria responded most strongly to soil depth. Alpha diversity showed a positive correlation with soil depth. Beta diversity, however, did not differ significantly between horizons. Genes involved in carbohydrate and nitrogen metabolism were found to be more abundant in Ah horizon samples. Closer inspection of carbohydrate metabolism genes revealed that genes involved in CO2 fixation, fermentation and saccharide metabolism decreased in abundance with depth while one­carbon metabolism increased down profile.

Magnetic-field effects on methane-hydrate kinetics and potential geophysical implications: Insights from non-equilibrium molecular dynamics.

We have conducted non-equilibrium molecular-dynamics (NEMD) simulation to show that externally-applied magnetic fields, including their reversals in direction, have important effects on gas-release dynamics from methane hydrates. In particular, we apply fluctuation-dissipation analysis in the guise of Onsager's hypothesis to study hydrate kinetics at lower applied-field intensities, including temporary hydrate destabilisation in the wake of field-polarity switch; we scale down to the lowest practicable field intensities, of the order of 1 T. We conjecture, that these NEMD-based findings, particularly those involving polarity switch, may have ramifications for superchron-related Earth's magnetic-field polarity swaps affecting methane release into the geosphere, although a good deal of further work would be needed to provide a more definitive causal link.

Characterization of a novel ω-transaminase from a Triassic salt mine metagenome.

Chiral amines are valuable building blocks for the pharmaceutical industry, and are increasingly synthesized by transaminase-mediated (TAm) synthesis. Currently available TAms, primarily isolated from the genomes of cultured mesophilic bacteria, often suffer from a number of drawbacks, including poor substrate range and an inability to tolerate the harsh conditions often demanded by industrial processes. These characteristics have, in part, driven the search for novel biocatalysts from both metagenomic sources and extreme environments. Herein, we report the isolation and characterization of an ω-TAm from a metagenome of a Triassic salt mine in Kilroot, N. Ireland, an extremely hypersaline environment formed circa 220-250 mya. The gene sequence was identified based on homology with existing bacterial TAms, synthesized within a pET28a(+) plasmid and expressed in E. coli BL21 DE3 cells. The resultant 49 kDa protein accepted (S)-methylbenzylamine (MBA) as amino donor and had a specific activity of 0.54 U/mg using α-ketoglutarate (ΑKG) as substrate. Molecular modeling and substrate docking indicated the presence of key residues, conserved in a number of other TAms. Despite the hypersaline environment from which it was isolated, the enzyme displayed low halotolerance, highlighting that not all biocatalysts will demonstrate the extreme characteristics associated with their source environment. This study does however reinforce the viability of mining metagenomic datasets as a means of discovering novel and functional biocatalysts, and adds to a currently scant list of such examples in the field of TAms.

Draft Genome Sequence of the Predatory Marine Bacterium Halobacteriovorax sp. Strain JY17.

A draft genome sequence of Halobacteriovorax sp. strain JY17 was assembled from a metagenomic data set. The 3.47-Mbp genome of this unusual predatory bacterium contains 3,263 protein-coding sequences, 33 tRNAs, and 2 copies each of the 16S, 23S, and 5S rRNA genes. This is only the third sequenced representative of this genus.

Draft Genome Sequences of Pseudomonas putida UV4 and UV4/95, Toluene Dioxygenase-Expressing Producers of cis-1,2-Dihydrodiols.

Here, we present draft genome sequences of Pseudomonas putida strains UV4 and UV4/95, which demonstrate an ability to conduct a wide range of industrially important biotransformations of arenes, alkenes, and phenols.