Tuwongella immobilis gen. nov., sp. nov., a novel non-motile bacterium within the phylum Planctomycetes.

A gram-negative, budding, catalase negative, oxidase positive and non-motile bacterium (MBLW1T) with a complex endomembrane system has been isolated from a freshwater lake in southeast Queensland, Australia. Phylogeny based on 16S rRNA gene sequence analysis places the strain within the family Planctomycetaceae, related to Zavarzinella formosa (93.3 %), Telmatocola sphagniphila (93.3 %) and Gemmata obscuriglobus (91.9 %). Phenotypic and chemotaxonomic analysis demonstrates considerable differences to the type strains of the related genera. MBLW1T displays modest salt tolerance and grows optimally at pH values of 7.5-8.0 and at temperatures of 32-36 °C. Transmission electron microscopy analysis demonstrates the presence of a complex endomembrane system, however, without the typically condensed nucleoid structure found in related genera. The major fatty acids are 16 : 1 ω5c, 16 : 0 and 18 : 0. Based on discriminatory results from 16S rRNA gene sequence analysis, phenotypic, biochemical and chemotaxonomic analysis, MBLW1T should be considered as a new genus and species, for which the name Tuwongella immobilis gen. nov., sp. nov. is proposed. The type strain is MBLW1T (=CCUG 69661T=DSM 105045T).

Nuclear Pore-Like Structures in a Compartmentalized Bacterium.

Planctomycetes are distinguished from other Bacteria by compartmentalization of cells via internal membranes, interpretation of which has been subject to recent debate regarding potential relations to Gram-negative cell structure. In our interpretation of the available data, the planctomycete Gemmata obscuriglobus contains a nuclear body compartment, and thus possesses a type of cell organization with parallels to the eukaryote nucleus. Here we show that pore-like structures occur in internal membranes of G.obscuriglobus and that they have elements structurally similar to eukaryote nuclear pores, including a basket, ring-spoke structure, and eight-fold rotational symmetry. Bioinformatic analysis of proteomic data reveals that some of the G. obscuriglobus proteins associated with pore-containing membranes possess structural domains found in eukaryote nuclear pore complexes. Moreover, immunogold labelling demonstrates localization of one such protein, containing a ß-propeller domain, specifically to the G. obscuriglobus pore-like structures. Finding bacterial pores within internal cell membranes and with structural similarities to eukaryote nuclear pore complexes raises the dual possibilities of either hitherto undetected homology or stunning evolutionary convergence.

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics.

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100-1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.

Shining a light on dark sequencing: characterising errors in Ion Torrent PGM data.

The Ion Torrent Personal Genome Machine (PGM) is a new sequencing platform that substantially differs from other sequencing technologies by measuring pH rather than light to detect polymerisation events. Using re-sequencing datasets, we comprehensively characterise the biases and errors introduced by the PGM at both the base and flow level, across a combination of factors, including chip density, sequencing kit, template species and machine. We found two distinct insertion/deletion (indel) error types that accounted for the majority of errors introduced by the PGM. The main error source was inaccurate flow-calls, which introduced indels at a raw rate of 2.84% (1.38% after quality clipping) using the OneTouch 200 bp kit. Inaccurate flow-calls typically resulted in over-called short-homopolymers and under-called long-homopolymers. Flow-call accuracy decreased with consecutive flow cycles, but we also found significant periodic fluctuations in the flow error-rate, corresponding to specific positions within the flow-cycle pattern. Another less common PGM error, high frequency indel (HFI) errors, are indels that occur at very high frequency in the reads relative to a given base position in the reference genome, but in the majority of instances were not replicated consistently across separate runs. HFI errors occur approximately once every thousand bases in the reference, and correspond to 0.06% of bases in reads. Currently, the PGM does not achieve the accuracy of competing light-based technologies. However, flow-call inaccuracy is systematic and the statistical models of flow-values developed here will enable PGM-specific bioinformatics approaches to be developed, which will account for these errors. HFI errors may prove more challenging to address, especially for polymorphism and amplicon applications, but may be overcome by sequencing the same DNA template across multiple chips.

High-pressure freezing/freeze substitution and transmission electron microscopy for characterization of metal oxide nanoparticles within sunscreens.

AIMS: To date, the description of a single, suitable method to observe in detail metal oxide nanoparticles in situ within sunscreens is currently lacking, despite growing concern as to how they interact with humans. This study explores the usefulness of transmission electron microscopy to characterize the nanoparticles in sunscreens. MATERIALS & METHODS: High-pressure freezing then freeze substitution was used to prepare resin-embedded commercial sunscreen samples, and ultrathin sections of these were observed with transmission electron microscopy. Conventional room temperature processing for resin embedding was also trialed. RESULTS: High-pressure frozen/freeze substituted samples provided clear visualization of the size and shape of the nanoparticles and agglomerates and allowed further characterization of the composition and crystal form of the metal oxides, while conventionally processed chemically fixed samples were subject to distribution/agglomeration artifacts. CONCLUSION: Transmission electron microscopy of high-pressure frozen/freeze substituted samples is an ideal method to completely observe metal oxide nanoparticles in situ in sunscreens.

Gold nanoparticle penetration and reduced metabolism in human skin by toluene.

PURPOSE: To measure penetration and metabolic effects of ion-stabilized, polar, 15 nm gold nanoparticles in aqueous solution (AuNP-Aq) and sterically stabilized, non-polar, 6 nm gold nanoparticles in toluene (AuNP-TOL) on excised human skin. METHODS: Gold nanoparticles were characterized with dynamic light scattering and transmission electron microscopy (TEM). Skin penetration studies were done on frozen or fresh excised skin using static Franz diffusion cells. Viable treated skin was assessed by dermoscopy, reflectance confocal microscopy (RCM), multiphoton tomography (MPT) with fluorescence lifetime imaging microscopy (FLIM), and TEM. RESULTS: Dermoscopy and RCM showed large aggregates in the furrows of AuNP-Aq-treated skin. Treatment of thawed and viable skin only showed enhanced permeability to nanoparticles in the AuNP-TOL group with MPT and FLIM imaging to stratum spinosum of epidermis. TEM analysis revealed gold nanoparticles within AuNP-treated stratum corneum. FLIM analysis of NAD(P)H showed a significant decrease in total NAD(P)H in all toluene-treated groups. CONCLUSIONS: Gold nanoparticles, 15 nm, in aqueous solution aggregated on the skin surface. Toluene treatment eliminated skin metabolism; skin treated with toluene/gold nanoparticles (6 nm) for 24 h, but not at 4 h, showed increased nanoparticle permeability. These results are of value to nanotoxicology.

Time-correlated single photon counting for simultaneous monitoring of zinc oxide nanoparticles and NAD(P)H in intact and barrier-disrupted volunteer skin.

PURPOSE: There is a lack of relevant, non-animal alternatives for assessing exposure and toxicity of nanoparticle-containing cosmetics, e.g. sunscreens. Our goal was to evaluate timecorrelated single photon counting (TCSPC) for simultaneous monitoring of zinc oxide nanoparticles (ZnO-NP) and the metabolic state of volunteer skin. METHODS: We separated the fluorescence lifetime signatures of endogenous fluorophore signals (i.e. nicotinamide adenine dinucleotide phosphate, NAD(P)H and keratin) and the ZnO-NP signal using advanced TCSPC to simultaneously determine ZnO-NP penetration profiles and NAD(P)H changes in subjects with altered barrier function, including tape-stripped skin and in psoriasis or atopic dermatitis lesions. RESULTS: We detected no ZnO-NP penetration into viable human skin in any group. ZnO-NP signal was significantly increased (p < 0.01) on the surface of tape-stripped and lesional skin after 4 and 2 h of treatment, respectively. Free NAD(P)H signal significantly increased in tape-stripped viable epidermis treated for 4 h of ZnO-NP compared to vehicle control. No significant NAD(P)H changes were noted in the lesional study. CONCLUSION: TCSPC techniques enabled simultaneous, real-time quantification of ZnO-NP concentration and NAD(P)H via non-invasive imaging in the stratum corneum and viable epidermis of volunteers.

pmoA Primers for detection of anaerobic methanotrophs.

Published pmoA primers do not match the pmoA sequence of "Candidatus Methylomirabilis oxyfera," a bacterium that performs nitrite-dependent anaerobic methane oxidation. Therefore, new pmoA primers for the detection of "Ca. Methylomirabilis oxyfera"-like methanotrophs were developed and successfully tested on freshwater samples from different habitats. These primers expand existing molecular tools for the study of methanotrophs in the environment.

Physiological role of the respiratory quinol oxidase in the anaerobic nitrite-reducing methanotroph 'Candidatus Methylomirabilis oxyfera'.

The anaerobic nitrite-reducing methanotroph 'Candidatus Methylomirabilis oxyfera' ('Ca. M. oxyfera') produces oxygen from nitrite by a novel pathway. The major part of the O(2) is used for methane activation and oxidation, which proceeds by the route well known for aerobic methanotrophs. Residual oxygen may serve other purposes, such as respiration. We have found that the genome of 'Ca. M. oxyfera' harbours four sets of genes encoding terminal respiratory oxidases: two cytochrome c oxidases, a third putative bo-type ubiquinol oxidase, and a cyanide-insensitive alternative oxidase. Illumina sequencing of reverse-transcribed total community RNA and quantitative real-time RT-PCR showed that all four sets of genes were transcribed, albeit at low levels. Oxygen-uptake and inhibition experiments, UV-visible absorption spectral characteristics and EPR spectroscopy of solubilized membranes showed that only one of the four oxidases is functionally produced by 'Ca. M. oxyfera', notably the membrane-bound bo-type terminal oxidase. These findings open a new role for terminal respiratory oxidases in anaerobic systems, and are an additional indication of the flexibility of terminal oxidases, of which the distribution among anaerobic micro-organisms may be largely underestimated.

Nitrite-driven anaerobic methane oxidation by oxygenic bacteria.

Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named 'Candidatus Methylomirabilis oxyfera', was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that 'M. oxyfera' bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis.

Close relationship of RNase P RNA in Gemmata and anammox planctomycete bacteria.

The relationship of RNase P RNA from anammox bacteria 'Candidatus Brocadia anammoxidans' and 'Candidatus Kuenenia stuttgartiensis' with that from other Planctomycetes was investigated. Newly identified rnpB gene sequences were aligned against existing planctomycete RNase P RNA sequences and secondary structures deduced by a comparative approach. Deduced secondary structures were similar in both anammox bacteria and both possessed an insert within the P13 helix analogous to that present in all Gemmata isolates. Phylogenetic analysis also revealed a possible relationship between the RNase P RNA molecules of the two anammox organisms and the genus Gemmata.

Comparative analysis of ribonuclease P RNA of the planctomycetes.

The planctomycetes, order Planctomycetales, are a distinct phylum of domain Bacteria. Genes encoding the RNA portion of ribonuclease P (RNase P) of some planctomycete members were sequenced and compared with existing database planctomycete sequences. rnpB gene sequences encoding RNase P RNA were generated by a conserved primer PCR strategy for Planctomyces brasiliensis, Planctomyces limnophilus, Pirellula marina, Pirellula staleyi strain ATCC 35122, Isosphaera pallida, one other Isosphaera strain, Gemmata obscuriglobus and three other strains of the Gemmata group. These sequences were aligned against reference bacterial sequences and secondary structures of corresponding RNase P RNAs deduced by a comparative approach. P12 helices were found to be highly variable in length, as were helices P16.1 and P19, when present. RNase P RNA secondary structures of Gemmata isolates were found to have unusual features relative to other planctomycetes, including a long P9 helix and an insert in the P13 helix not found in any other member of domain Bacteria. These unique features are consistent with other unusual properties of this genus, distinguishing it from other bacteria. Phylogenetic analyses indicate that relationships between planctomycetes derived from RNase P RNA are consistent with 16S rRNA-based analyses.

Molecular and ultrastructural confirmation of classification of ATCC 35122 as a strain of Pirellula staleyi.

A freshwater isolate from Campus Lake, Baton Rouge, LA, USA, strain ATCC 35122 (= ICPB 4362 = Schmidt CLPM White = Tekniepe BT2 white), which had been proposed as a putative reference strain for 'Planctomyces staleyi' (later reclassified as Pirellula staleyi), has been re-examined to establish its relationship to the type strain of Pirellula staleyi, ATCC 27377T. 165 rRNA sequencing confirms its very close relationship to ATCC 27377T and its membership of the order Planctomycetales. Ultrastructural characteristics are also consistent with its membership of the planctomycetes and of the genus Pirellula. These characteristics include polar crateriform structures and the occurrence of the unique internal, single-membrane-bounded compartment enclosing the nucleoid and ribosome-like particles, the pirellulosome, and a polar cap region. Cells of strain ATCC 35122 often displayed pointed, hump-like protrusions opposite each other on the cell, constituting prosthecae, and these were also found to be present on cells of strain ATCC 27377T. The original identification of ATCC 35122 as a strain of Pirellula staleyi is confirmed on both molecular and phenotypic grounds.