X-ray mosaic nanotomography of large microorganisms.

Full-field X-ray microscopy is a valuable tool for 3D observation of biological systems. In the soft X-ray domain organelles can be visualized in individual cells while hard X-ray microscopes excel in imaging of larger complex biological tissue. The field of view of these instruments is typically 10(3) times the spatial resolution. We exploit the assets of the hard X-ray sub-micrometer imaging and extend the standard approach by widening the effective field of view to match the size of the sample. We show that global tomography of biological systems exceeding several times the field of view is feasible also at the nanoscale with moderate radiation dose. We address the performance issues and limitations of the TOMCAT full-field microscope and more generally for Zernike phase contrast imaging. Two biologically relevant systems were investigated. The first being the largest known bacteria (Thiomargarita namibiensis), the second is a small myriapod species (Pauropoda sp.). Both examples illustrate the capacity of the unique, structured condenser based broad-band full-field microscope to access the 3D structural details of biological systems at the nanoscale while avoiding complicated sample preparation, or even keeping the sample environment close to the natural state.

Experimental taphonomy of giant sulphur bacteria: implications for the interpretation of the embryo-like Ediacaran Doushantuo fossils.

The Ediacaran Doushantuo biota has yielded fossils interpreted as eukaryotic organisms, either animal embryos or eukaryotes basal or distantly related to Metazoa. However, the fossils have been interpreted alternatively as giant sulphur bacteria similar to the extant Thiomargarita. To test this hypothesis, living and decayed Thiomargarita were compared with Doushantuo fossils and experimental taphonomic pathways were compared with modern embryos. In the fossils, as in eukaryotic cells, subcellular structures are distributed throughout cell volume; in Thiomargarita, a central vacuole encompasses approximately 98 per cent cell volume. Key features of the fossils, including putative lipid vesicles and nuclei, complex envelope ornament, and ornate outer vesicles are incompatible with living and decay morphologies observed in Thiomargarita. Microbial taphonomy of Thiomargarita also differed from that of embryos. Embryo tissues can be consumed and replaced by bacteria, forming a replica composed of a three-dimensional biofilm, a stable fabric for potential fossilization. Vacuolated Thiomargarita cells collapse easily and do not provide an internal substrate for bacteria. The findings do not support the hypothesis that giant sulphur bacteria are an appropriate interpretative model for the embryo-like Doushantuo fossils. However, sulphur bacteria may have mediated fossil mineralization and may provide a potential bacterial analogue for other macroscopic Precambrian remains.

A bacterium that is not a microbe.

A new discovery challenges the prevailing view of the boundaries of bacterial cell size.

A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles.

Cells of most bacterial species are around 2 micrometers in length, with some of the largest specimens reaching 750 micrometers. Using fluorescence, x-ray, and electron microscopy in conjunction with genome sequencing, we characterized Candidatus (Ca.) Thiomargarita magnifica, a bacterium that has an average cell length greater than 9000 micrometers and is visible to the naked eye. These cells grow orders of magnitude over theoretical limits for bacterial cell size, display unprecedented polyploidy of more than half a million copies of a very large genome, and undergo a dimorphic life cycle with asymmetric segregation of chromosomes into daughter cells. These features, along with compartmentalization of genomic material and ribosomes in translationally active organelles bound by bioenergetic membranes, indicate gain of complexity in the Thiomargarita lineage and challenge traditional concepts of bacterial cells.

Mammoth mangrove bacterium has complex cell.

"Eye-opening" discovery challenges evolutionary thinking on microbes.

Imaging of Cellular Oxidoreductase Activity Suggests Mixotrophic Metabolisms in Thiomargarita spp.

The largest known bacteria, Thiomargarita spp., have yet to be isolated in pure culture, but their large size allows for individual cells to be monitored in time course experiments or to be individually sorted for omics-based investigations. Here we investigated the metabolism of individual cells of Thiomargarita spp. by using a novel application of a tetrazolium-based dye that measures oxidoreductase activity. When coupled with microscopy, staining of the cells with a tetrazolium-formazan dye allows metabolic responses in Thiomargarita spp. to be to be tracked in the absence of observable cell division. Additionally, the metabolic activity of Thiomargarita sp. cells can be differentiated from the metabolism of other microbes in specimens that contain adherent bacteria. The results of our redox dye-based assay suggest that Thiomargarita is the most metabolically versatile under anoxic conditions, where it appears to express cellular oxidoreductase activity in response to the electron donors succinate, acetate, citrate, formate, thiosulfate, H2, and H2S. Under hypoxic conditions, formazan staining results suggest the metabolism of succinate and likely acetate, citrate, and H2S. Cells incubated under oxic conditions showed the weakest formazan staining response, and then only to H2S, citrate, and perhaps succinate. These results provide experimental validation of recent genomic studies of Candidatus Thiomargarita nelsonii that suggest metabolic plasticity and mixotrophic metabolism. The cellular oxidoreductase response of bacteria attached to the exterior of Thiomargarita also supports the possibility of trophic interactions between these largest of known bacteria and attached epibionts.IMPORTANCE The metabolic potential of many microorganisms that cannot be grown in the laboratory is known only from genomic data. Genomes of Thiomargarita spp. suggest that these largest of known bacteria are mixotrophs, combining lithotrophic metabolism with organic carbon degradation. Our use of a redox-sensitive tetrazolium dye to query the metabolism of these bacteria provides an independent line of evidence that corroborates the apparent metabolic plasticity of Thiomargarita observed in recently produced genomes. Finding new cultivation-independent means of testing genomic results is critical to testing genome-derived hypotheses on the metabolic potentials of uncultivated microorganisms.

Microbial Community Associated with Thioploca sp. Sheaths in the Area of the Posolski Bank Methane Seep, Southern Baikal.

Bacterial mats formed by a colorless sulfur bacterium Thioploca sp. in the area of the Posolski Bank cold methane seep (southern Baikal) were -studied using electron microscopy and phylogenetic analysis. Morphologically the bacteria were identified as Thioploca ingrica.- Confocal microscopy of DAPI-stained samples revealed numerous rod-shaped, filamentous, and spiral microorganisms in the sheaths, as well as in- side and between the trichomes. Transmission electron microscopy revealed nonvacuolated bacteria and small cells-without cell envelopes within the sheath. Bacteria with pronounced intracytoplasmic membranes characteristic; of type I methanotrophs were observed at the outer side of the sheath. Based on analysis of the 16S rRNA gene sequences, the following phyla were idenified in the sheath community: Bacteroidetes, Nitro- spira, Chloroflexi, Planctomycetes, Verrucomicrobia,'y-, and 6-Proteobacteria, Euryarchaeota, Crenarchaeota, and Thaumarchaeota, as well as anammox bacteria. A hypothetical scheme of matter flows in the Lake Baikal bacterial mats was proposed based on the data on metabolism of the cultured homologues.

New communities of large filamentous sulfur bacteria in the eastern South Pacific.

New complex communities of morphologically diverse and sometimes abundant large, multicellular, filamentous bacteria were discovered in the oxygen-deficient, organically laden, shelf sediments under the oxygen minimum zone off the coast of the eastern Pacific, i.e., off the coasts of central and northern Chile; central and northern Perú; Roca Redonda, Galápagos Archipielago, Ecuador; and off the Pacific coasts of Panamá and Costa Rica. Similar microbial communities were also observed in the reduced layer of a muddy-sand beach adjacent to a mangrove swamp on Coiba Island, Pacific Panamá, and in the organically laden bottom underneath a salmon culture pen in southern Chile (region X). Of varying morphology, the diameters of the bacteria range from 1 to 10 mum, and their lengths from around 10 mum to usually several hundreds but at times several thousands of micrometers. The new filamentous bacterial component is at least one order of magnitude smaller than the also multicellular "megabacteria" Thioploca spp. and Beggiatoa spp., and is collectively referred to as "macrobacteria". A recent review only mentioned a few of these free-living filamentous bacteria, remarking on their scarcity despite the obvious advantages of a large size. This prokaryote size-window has been rarely investigated optically by researchers; thus, assemblages that appear to have had world-wide distribution probably since pre-Cambrian times have been overlooked.

Novel vacuolate sulfur bacteria from the Gulf of Mexico reproduce by reductive division in three dimensions.

Large spherical sulfur bacteria, 180-375 microm in diameter, were found regularly and in abundance in surface sediments collected from hydrocarbon seeps (water depth 525-640 m) in the Gulf of Mexico. These bacteria were characterized by a thin 'shell' of sulfur globule-filled cytoplasm that surrounded a central vacuole (roughly 80% of biovolume) containing high concentrations of nitrate (average 460 mM). Approximately 800 base pairs of 16S rRNA gene sequence data, linked to this bacterium by fluorescent in situ hybridization, showed 99% identity with Thiomargarita namibiensis, previously described only from sediments collected off the coast of Namibia (Western Africa). Unlike T. namibiensis, where cells form a linear chain within a common sheath, the Gulf of Mexico strain occurred as single cells and clusters of two, four and eight cells, which were clearly the product of division in one to three planes. In sediment cores maintained at 4 degrees C, which undoubtedly experienced a diminishing flux of hydrogen sulfide over time, the Thiomargarita-like bacterium remained viable for up to 2 years. During that long period, each cell appeared to undergo (as judged by change in biovolume) one to three reductive divisions, perhaps as a dispersal strategy in the face of diminished availability of its putative electron donor.

Incidence of Simonsiella in the oral cavity of dogs.

The gliding bacterium Simonsiella (Cytophagales, Simonsiellaceae) was found in palate samples from 66 out of 67 dogs. It is considered a common resident in the oral cavities of dogs.

The Santa Barbara Basin is a symbiosis oasis.

It is generally agreed that the origin and initial diversification of Eucarya occurred in the late Archaean or Proterozoic Eons when atmospheric oxygen levels were low and the risk of DNA damage due to ultraviolet radiation was high. Because deep water provides refuge against ultraviolet radiation and early eukaryotes may have been aerotolerant anaerobes, deep-water dysoxic environments are likely settings for primeval eukaryotic diversification. Fossil evidence shows that deep-sea microbial mats, possibly of sulphur bacteria similar to Beggiatoa, existed during that time. Here we report on the eukaryotic community of a modern analogue, the Santa Barbara Basin (California, USA). The Beggiatoa mats of these severely dysoxic and sulphidic sediments support a surprisingly abundant protistan and metazoan meiofaunal community, most members of which harbour prokaryotic symbionts. Many of these taxa are new to science, and both microaerophilic and anaerobic taxa appear to be represented. Compared with nearby aerated sites, the Santa Barbara Basin is a 'symbiosis oasis' offering a new source of organisms for testing symbiosis hypotheses of eukaryogenesis.

[Aquatic myxobacteria (Sporocytophaga cauliformis) and the order "Myxobacterales" (author's transl)]. / Wassermyxobakterien (Sporocytophaga cauliformis) und die Ordnung "Myxobacterales".

The indicator function of aquatic myxobacteria for the purpose of evaluating drinking water quality as well as their occurrence in sewage effluent make it desirable to describe these hitherto little known organisms in more detail. To this end, a comparative investigation of anaerobic myxobacteria of the genus Sphaerocytophaga, two typical representatives of the Order Myxobacterales (Myxococcus fulvus, Sporocytophage cauliformis), and a strain ov Vitrepscilla (Vitroescilla proteolytica) was undertaken. With respect to culture morphology, the migratory fringe surrounding colonies of Sphaerocytophaga similar to the other strains studies was a prominent characteristic. In particular, the similarity with colonies of Sporocytophaga cauliformis was apparent. The gliding motility typical of Sphaerocytophaga could be demonstrated in all of the strains investigated. Scanning electron micrographs revealed an amorphous layer of slime covering the cell surfaces in all strains compared in this study, thus excluding the existence of more rigid organelles of locomotion. Taxonomically, the anaerobec myxobacteria of the oral cavity (Sphaerocytophaga) belong to the Order "Myxobacterales" and not the "Eubacterales", i.e., to the genus Fusobacterium. This is clearly suggested by their motility lacking flagella and, above all, by their cell morphology which differs from the Eubacterales.

New communities of large filamentous sulfur bacteria in the eastern South Pacific

New complex communities of morphologically diverse and sometimes abundant large, multicellular, filamentous bacteria were discovered in the oxygen-deficient, organically laden, shelf sediments under the oxygen minimum zone off the coast of the eastern Pacific, i.e., off the coasts of central and northern Chile; central and northern Perú; Roca Redonda, Galápagos Archipielago, Ecuador; and off the Pacific coasts of Panamá and Costa Rica. Similar microbial communities were also observed in the reduced layer of a muddy-sand beach adjacent to a mangrove swamp on Coiba Island, Pacific Panamá, and in the organically laden bottom underneath a salmon culture pen in southern Chile (region X). Of varying morphology, the diameters of the bacteria range from 1 to 10 mum, and their lengths from around 10 mum to usually several hundreds but at times several thousands of micrometers. The new filamentous bacterial component is at least one order of magnitude smaller than the also multicellular «megabacteria» Thioploca spp. and Beggiatoa spp., and is collectively referred to as «macrobacteria». A recent review only mentioned a few of these free-living filamentous bacteria, remarking on their scarcity despite the obvious advantages of a large size. This prokaryote size-window has been rarely investigated optically by researchers; thus, assemblages that appear to have had world-wide distribution probably since pre-Cambrian times have been overlooked (AU)

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