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.

Cold atmospheric pressure plasma-antibiotic synergy in Pseudomonas aeruginosa biofilms is mediated via oxidative stress response.

Cold atmospheric-pressure plasma (CAP) has emerged as a potential alternative or adjuvant to conventional antibiotics for the treatment of bacterial infections, including those caused by antibiotic-resistant pathogens. The potential of sub-lethal CAP exposures to synergise conventional antimicrobials for the eradication of Pseudomonas aeruginosa biofilms is investigated in this study. The efficacy of antimicrobials following or in the absence of sub-lethal CAP pre-treatment in P. aeruginosa biofilms was assessed. CAP pre-treatment resulted in an increase in both planktonic and biofilm antimicrobial sensitivity for all three strains tested (PAO1, PA14, and PA10548), with both minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs) of individual antimicrobials, being significantly reduced following CAP pre-treatment of the biofilm (512-fold reduction with ciprofloxacin/gentamicin; and a 256-fold reduction with tobramycin). At all concentrations of antimicrobial used, the combination of sub-lethal CAP exposure and antimicrobials was effective at increasing time-to-peak metabolism, as measured by isothermal microcalorimetry, again indicating enhanced susceptibility. CAP is known to damage bacterial cell membranes and DNA by causing oxidative stress through the in situ generation of reactive oxygen and nitrogen species (RONS). While the exact mechanism is not clear, oxidative stress on outer membrane proteins is thought to damage/perturb cell membranes, confirmed by ATP and LDH leakage, allowing antimicrobials to penetrate the bacterial cell more effectively, thus increasing bacterial susceptibility. Transcriptomic analysis, reveals that cold-plasma mediated oxidative stress caused upregulation of P. aeruginosa superoxide dismutase, cbb3 oxidases, catalases, and peroxidases, and upregulation in denitrification genes, suggesting that P. aeruginosa uses these enzymes to degrade RONS and mitigate the effects of cold plasma mediated oxidative stress. CAP treatment also led to an increased production of the signalling molecule ppGpp in P. aeruginosa, indicative of a stringent response being established. Although we did not directly measure persister cell formation, this stringent response may potentially be associated with the formation of persister cells in biofilm cultures. The production of ppGpp and polyphosphate may be associated with protein synthesis inhibition and increase efflux pump activity, factors which can result in antimicrobial tolerance. The transcriptomic analysis also showed that by 6 h post-treatment, there was downregulation in ribosome modulation factor, which is involved in the formation of persister cells, suggesting that the cells had begun to resuscitate/recover. In addition, CAP treatment at 4 h post-exposure caused downregulation of the virulence factors pyoverdine and pyocyanin; by 6 h post-exposure, virulence factor production was increasing. Transcriptomic analysis provides valuable insights into the mechanisms by which P. aeruginosa biofilms exhibits enhanced susceptibility to antimicrobials. Overall, these findings suggest, for the first time, that short CAP sub-lethal pre-treatment can be an effective strategy for enhancing the susceptibility of P. aeruginosa biofilms to antimicrobials and provides important mechanistic insights into cold plasma-antimicrobial synergy. Transcriptomic analysis of the response to, and recovery from, sub-lethal cold plasma exposures in P. aeruginosa biofilms improves our current understanding of cold plasma biofilm interactions.

DePolymerase Predictor (DePP): a machine learning tool for the targeted identification of phage depolymerases.

Biofilm production plays a clinically significant role in the pathogenicity of many bacteria, limiting our ability to apply antimicrobial agents and contributing in particular to the pathogenesis of chronic infections. Bacteriophage depolymerases, leveraged by these viruses to circumvent biofilm mediated resistance, represent a potentially powerful weapon in the fight against antibiotic resistant bacteria. Such enzymes are able to degrade the extracellular matrix that is integral to the formation of all biofilms and as such would allow complementary therapies or disinfection procedures to be successfully applied. In this manuscript, we describe the development and application of a machine learning based approach towards the identification of phage depolymerases. We demonstrate that on the basis of a relatively limited number of experimentally proven enzymes and using an amino acid derived feature vector that the development of a powerful model with an accuracy on the order of 90% is possible, showing the value of such approaches in protein functional annotation and the discovery of novel therapeutic agents.

Mini-XT, a miniaturized tagmentation-based protocol for efficient sequencing of SARS-CoV-2.

BACKGROUND: The COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost 1 million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. RESULTS: We present the 'Mini-XT' miniaturized tagmentation-based library preparation protocol available on protocols.io ( https://doi.org/10.17504/protocols.io.bvntn5en ). SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. The final version of Mini-XT has been used to sequence 4384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. CONCLUSIONS: The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes eightfold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.

Small RNA F6 Provides Mycobacterium smegmatis Entry into Dormancy.

Regulatory small non-coding RNAs play a significant role in bacterial adaptation to changing environmental conditions. Various stresses such as hypoxia and nutrient starvation cause a reduction in the metabolic activity of Mycobacterium smegmatis, leading to entry into dormancy. We investigated the functional role of F6, a small RNA of M. smegmatis, and constructed an F6 deletion strain of M. smegmatis. Using the RNA-seq approach, we demonstrated that gene expression changes that accompany F6 deletion contributed to bacterial resistance against oxidative stress. We also found that F6 directly interacted with 5'-UTR of MSMEG_4640 mRNA encoding RpfE2, a resuscitation-promoting factor, which led to the downregulation of RpfE2 expression. The F6 deletion strain was characterized by the reduced ability to enter into dormancy (non-culturability) in the potassium deficiency model compared to the wild-type strain, indicating that F6 significantly contributes to bacterial adaptation to non-optimal growth conditions.

Novel Phage-Derived Depolymerase with Activity against Proteus mirabilis Biofilms.

The adherence of Proteus mirabilis to the surface of urinary catheters leads to colonization and eventual blockage of the catheter lumen by unique crystalline biofilms produced by these opportunistic pathogens, making P. mirabilis one of the leading causes of catheter-associated urinary tract infections. The Proteus biofilms reduce efficiency of antibiotic-based treatment, which in turn increases the risk of antibiotic resistance development. Bacteriophages and their enzymes have recently become investigated as alternative treatment options. In this study, a novel Proteus bacteriophage (vB_PmiS_PM-CJR) was isolated from an environmental sample and fully characterized. The phage displayed depolymerase activity and the subsequent genome analysis revealed the presence of a pectate lyase domain in its tail spike protein. The protein was heterologously expressed and purified; the ability of the purified tail spike to degrade Proteus biofilms was tested. We showed that the application of the tail spike protein was able to reduce the adherence of bacterial biofilm to plastic pegs in a MBEC (minimum biofilm eradication concentration) assay and improve the survival of Galleria mellonella larvae infected with Proteus mirabilis. Our study is the first to successfully isolate and characterize a biofilm depolymerase from a Proteus phage, demonstrating the potential of this group of enzymes in treatment of Proteus infections.

Microbiology of a NaCl stalactite 'salticle' in Triassic halite.

Large regions of Earth's surface are underlain by salt deposits that evaporated from ancient oceans and are populated by extreme halophilic microbes. Some of these halophiles may have been preserved over geological timescales within hypersaline fluid inclusions, but ingresses of water and/or anthropogenic activities can lead to the formation of alternative habitats, including NaCl stalactites or other speleothems. While the microbiology of ancient evaporites has been well studied, the ecology of these recently formed structures is less-well understood. Here, the microbiology of a NaCl stalactite ('salticle') in a Triassic halite mine is characterized. The specific aims were to determine the presence of fluid inclusions, determine the microbial structure of the salticle compared with a nearby brine-pool and surficial soil, and characterize the ecophysiological capabilities of this unique ecosystem. The salticle contained fluid inclusions, and their microbiome was composed of Euryarchaetota, Proteobacteria, and Actinobacteria, with Haloarchaea in greater abundance than brine-pool or soil microbiomes. The salticle metagenome exhibited a greater abundance of genes involved in osmoregulation, anaerobic respiration, UV resistance, oxidative stress, and stress-protein synthesis relative to the soil microbiome. We discuss the potential astrobiological implications of salticles as enclosed salt-saturated habitats that are protected from ionizing radiation and have a stable water activity.

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.

Profiling the microbial community of a Triassic halite deposit in Northern Ireland: an environment with significant potential for biodiscovery.

Kilroot salt mine, a Triassic halite deposit located in County Antrim, Northern Ireland, is the only permanent hypersaline environment on the island of Ireland. In this study, the microbiome of this unstudied environment was profiled for the first time using conventional and enhanced culturing techniques, and culture independent metagenomic approaches. Using both conventional isolation plates and iChip devices, 89 halophilic archaeal isolates from six known genera, and 55 halophilic or halotolerant bacterial isolates from 18 genera were obtained, based on 16S rRNA gene sequencing. The archaeal isolates were similar to those previously isolated from other ancient halite deposits, and as expected, numerous genera were identified in the metagenome which were not represented among the culturable isolates. Preliminary screening of a selection of isolates from this environment identified antimicrobial activities against a panel of clinically important bacterial pathogens from 15 of the bacterial isolates and one of the archaea. This, alongside previous studies reporting the discovery of novel biocatalysts from the Kilroot mine microbiome, suggests that this environment may be a new, untapped source of of chemical diversity with high biodiscovery potential.

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.

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.

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.

Spatial requirement for PAMO for transformation of non-native linear substrates.

Phenylacetone monooxygenase is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenases family, and therefore it is an ideal candidate for the synthesis of industrially relevant ester or lactone compounds. However, its limited substrate scope has largely limited its industrial applications. Linear substrates are interesting from an industrial point of view, it is thus necessary to identify the essential spatial requirement for achieving high conversions for non-native linear substrates. Here using molecular dynamics simulations, we compared the conversion of a non-native linear substrate 2-octanone and the native substrate phenylacetone, catalyzed by the WT enzyme and a quadruple variant P253F/G254A/R258M/L443F that exhibits significantly improved activity towards 2-octanone. We uncovered that a remarkable movement of L289 is crucial for a reshaping of the active site of the quadruple variant so as to prevent the aliphatic substrate from moving away from the C4a-peroxyflavin, thus enabling it to keep a catalytically relevant pose during the oxygenation process. By performing steady-state kinetic analysis of two single-mutation variants at position 258, we further validated that the L289 reposition is attributed to the combined effect of quadruple mutations. In order to further explore the substrate scope of PAMO we also studied the binding of cyclopentanone and 2-phenylcyclohexanone, which are the typical substrates of CPMO in group I and CHMO in group III, respectively. Our study provides fundamental atomic-level insights in rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels.

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.

Draft Genome Sequence of Rhodococcus sp. Strain NCIMB 12038, a Naphthalene-Degrading Bacterium.

We report here the draft genome sequence of Rhodococcus sp. strain NCIMB 12038, an industrially important bacterium, possessing a large and diverse repertoire of genes involved in the biotransformation of various organic compounds, including naphthalene.

Temporal dynamics of uncultured viruses: a new dimension in viral diversity.

Recent work has vastly expanded the known viral genomic sequence space, but the seasonal dynamics of viral populations at the genome level remain unexplored. Here we followed the viral community in a freshwater lake for 1 year using genome-resolved viral metagenomics, combined with detailed analyses of the viral community structure, associated bacterial populations and environmental variables. We reconstructed 8950 complete and partial viral genomes, the majority of which were not persistent in the lake throughout the year, but instead continuously succeeded each other. Temporal analysis of 732 viral genus-level clusters demonstrated that one-fifth were undetectable at specific periods of the year. Based on host predictions for a subset of reconstructed viral genomes, we for the first time reveal three distinct patterns of host-pathogen dynamics, where the viruses may peak before, during or after the peak in their host's abundance, providing new possibilities for modelling of their interactions. Time series metagenomics opens up a new dimension in viral profiling, which is essential to understand the full scale of viral diversity and evolution, and the ecological roles of these important factors in the global ecosystem.

Catalytic mechanism of phenylacetone monooxygenases for non-native linear substrates.

Phenylacetone monooxygenase (PAMO) is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenase family, and therefore it is an ideal candidate for the synthesis of industrially relevant compounds. However, its limited substrate scope has largely limited its industrial applications. In the present work, we provide, for the first time, the catalytic mechanism of PAMO for the native substrate phenylacetone as well as for a linear non-native substrate 2-octanone, using molecular dynamics simulations, quantum mechanics and quantum mechanics/molecular mechanics calculations. We provide a theoretical basis for the preference of the enzyme for the native aromatic substrate over non-native linear substrates. Our study provides fundamental atomic-level insights that can be employed in the rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels.

Metagenomic Characterisation of the Viral Community of Lough Neagh, the Largest Freshwater Lake in Ireland.

Lough Neagh is the largest and the most economically important lake in Ireland. It is also one of the most nutrient rich amongst the world's major lakes. In this study, 16S rRNA analysis of total metagenomic DNA from the water column of Lough Neagh has revealed a high proportion of Cyanobacteria and low levels of Actinobacteria, Acidobacteria, Chloroflexi, and Firmicutes. The planktonic virome of Lough Neagh has been sequenced and 2,298,791 2×300 bp Illumina reads analysed. Comparison with previously characterised lakes demonstrates that the Lough Neagh viral community has the highest level of sequence diversity. Only about 15% of reads had homologs in the RefSeq database and tailed bacteriophages (Caudovirales) were identified as a major grouping. Within the Caudovirales, the Podoviridae and Siphoviridae were the two most dominant families (34.3% and 32.8% of the reads with sequence homology to the RefSeq database), while ssDNA bacteriophages constituted less than 1% of the virome. Putative cyanophages were found to be abundant. 66,450 viral contigs were assembled with the largest one being 58,805 bp; its existence, and that of another 34,467 bp contig, in the water column was confirmed. Analysis of the contigs confirmed the high abundance of cyanophages in the water column.