A rigorous assessment and comparison of enumeration methods for environmental viruses.

Determining exact viral titers in a given sample is essential for many environmental and clinical applications, e.g., for studying viral ecology or application of bacteriophages for food safety. However, virus quantification is not a simple task, especially for complex environmental samples. While clonal viral isolates can be quantified with relative high accuracy using virus-specific methods, i.e., plaque assay or quantitative real-time PCR, these methods are not valid for complex and diverse environmental samples. Moreover, it is not yet known how precisely laser-based methods, i.e., epifluorescence microscopy, flow cytometry, and nanoparticle tracking analysis, quantify environmental viruses. In the present study, we compared five state-of-the-art viral quantification methods by enumerating four model viral isolates of different genome and size characteristics as well as four different environmental water samples. Although Nanoparticle tracking analysis combined with gentle staining at 30 °C could be confirmed by this study to be a reliable quantification technique for tested environmental samples, environmental samples still lack an universally applicable and accurate quantification method. Special attention has to be put on optimal sample concentrations as well as optimized sample preparations, which are specific for each method. As our results show the inefficiency when enumerating small, or single-stranded DNA or RNA viruses, the global population of viruses is presumably higher than expected.

2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide.

For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon-embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a "speed protocol" for immunolabeling by reducing antibody incubation times. Finally, we present our results on three-dimensional immunogold labeling.

Archaeal Cell Walls.

The cell wall of archaea, as of any other prokaryote, is surrounding the cell outside the cytoplasmic membrane and is mediating the interaction with the environment. In this regard, it can be involved in cell shape maintenance, protection against virus, heat, acidity or alkalinity. Throughout the formation of pore like structures, it can resemble a micro sieve and thereby enable or disable transport processes. In some cases, cell wall components can make up more than 10% of the whole cellular protein. So far, a great variety of different cell envelope structures and compounds have be found and described in detail. From all archaeal cell walls described so far, the most common structure is the S-layer. Other archaeal cell wall structures are pseudomurein, methanochondroitin, glutaminylglycan, sulfated heteropolysaccharides and protein sheaths and they are sometimes associated with additional proteins and protein complexes like the STABLE protease or the bindosome. Recent advances in electron microscopy also illustrated the presence of an outer(most) cellular membrane within several archaeal groups, comparable to the Gram-negative cell wall within bacteria. Each new cell wall structure that can be investigated in detail and that can be assigned with a specific function helps us to understand, how the earliest cells on earth might have looked like.

A Complex Endomembrane System in the Archaeon Ignicoccus hospitalis Tapped by Nanoarchaeum equitans.

Based on serial sectioning, focused ion beam scanning electron microscopy (FIB/SEM), and electron tomography, we depict in detail the highly unusual anatomy of the marine hyperthermophilic crenarchaeon, Ignicoccus hospitalis. Our data support a complex and dynamic endomembrane system consisting of cytoplasmic protrusions, and with secretory function. Moreover, we reveal that the cytoplasm of the putative archaeal ectoparasite Nanoarchaeum equitans can get in direct contact with this endomembrane system, complementing and explaining recent proteomic, transcriptomic and metabolomic data on this inter-archaeal relationship. In addition, we identified a matrix of filamentous structures and/or tethers in the voluminous inter-membrane compartment (IMC) of I. hospitalis, which might be responsible for membrane dynamics. Overall, this unusual cellular compartmentalization, ultrastructure and dynamics in an archaeon that belongs to the recently proposed TACK superphylum prompts speculation that the eukaryotic endomembrane system might originate from Archaea.

Rectinema cohabitans gen. nov., sp. nov., a rod-shaped spirochaete isolated from an anaerobic naphthalene-degrading enrichment culture.

The anaerobic, non-motile strain HMT was isolated from the naphthalene-degrading, sulfate-reducing enrichment culture N47. For 20 years, strain HMT has been a stable member of culture N47 although it is neither able to degrade naphthalene nor able to reduce sulfate in pure culture. The highest similarity of the 16S rRNA gene sequence of strain HMT (89 %) is with a cultivated member of the family Spirochaetaceae, Treponema caldariumstrain H1T (=DSM 7334T), an obligately anaerobic, thermophilic spirochaete isolated from cyanobacterial mat samples collected at a freshwater hot spring in Oregon, USA. In contrast to this strain and the majority of spirochaete species described, strain HMT showed a rod-shaped morphology. Growth occurred at temperatures between 12 and 50 °C (optimum 37 °C) but the isolate was not able to grow at 60 °C. The strain fermented various sugars including d-glucose, d-fructose, lactose and sucrose. Addition of 0.1 % (w/v) yeast extract or 0.1 % (w/v) tryptone to the culture medium was essential for growth and could not be replaced by either the vitamin solutions tested or by 0.1 % (w/v) peptone or 0.1 % (w/v) casamino acids. The DNA G+C content of the isolate was 51.5 mol%. The major fatty acids were C14 : 0, C18 : 1ω13c, C16 : 1ω9t, C16 : 1ω11c and C16 : 1ω9c. Based on the unique morphology and the phylogenetic distance from the closest cultivated relative, a novel genus and species, Rectinema cohabitans gen. nov., sp. nov., is proposed. The type strain is strain HMT (=DSM 100378T=JCM 30982T).