by Christoph
Figure 1. Direct visualization of taxa on sand grains using CARD-FISH and confocal laser scanning microscopy. Targeted taxa are indicated in the individual panels b, d, and e (frontpage). DAPI signal (in blue) shows all cells not targeted by the probes. Images are composite micrographs of fluorescent signals and transmitted light of the sand grain's surface. Micrograph b is a superresolution structured illumination image (SR-SIM). If not otherwise indicated, scale bar refers to 10 μm. Source (Open Access PDF here)
What you see here in Figure 1d competes with a NASA photo from 2012 for the best shot of the Milky Way but – spoiler alert! – it's actually a close-up of some humble microbes gathered on a grain of sand (another one is on the frontpage ). I concluded the previous post about bacteria on sand grains with the question: and where are the archea ? The reason for this question wasn't sheer curiosity: based on a survey of prokaryotic diversity in the subsurface water of the Pacific Ocean, Karner et al. estimated that the global oceans are populated by 1.3 × 1028 archaea and 3.1 × 1028 bacteria (including an estimated 2.4 × 1028 SAR11/ Pelagibacter ubique cells ). No, I won't juggle with numbers here again. But if archaea do indeed represent ~1/3 of the prokaryotes in the oceans it would be surprising to not find them at least in the upper layers of the sediments, on sand grains for example. Well, here they are, see Figure 1b.
When Probandt et al. made their 'census' of microbes on single sand grains in a sample of North Sea sediment they found up to 105 microbial cells per grain, and these were, in their majority, members of 10 bacterial taxa. To also detect archaea they employed CARD-FISH, a technique that allows to detect microbes from phylogenetically diverse clades directly in their natural habitat (CARD-FISH is briefly described in the legend for Figure 2 ). According to their oligonucleotide probe, the single cells and small aggregates of archaea (3 – 10 cells; green dots in Figure 1b ) belong to the Thaumarcheota, a phylum of the archaea that was formerly 'lumped together' with the Crenarcheota, and whose known member species are chemolithoautotrophic ammonia-oxidizers. Suspiciously, these archaea were sometimes found in close neighborhood (0 – 10 µm ) of small aggregates of Nitrospirae bacteria (red dots ). 'Suspicously' because it is well known that archaeal ammonia oxidizers (AOA) and their bacterial counterparts from the phyla Nitrospirae and Betaproteobacteria (AOB) usually occur as densely clustered communities, for example in waste water treatment plants (with the archaea exceeding the bacteria in cell numbers by a factor of 10 ). The co-localization of archaeal and bacterial ammonia-oxidizers on the sand grains indicates that the entire nitrogen cycle can take place in the upper, permeable layer of seafloor sediments where metabolic intermediates can be transported advectively through the matrix.
Figure 2. Setup of a CARD-FISH ('Catalyzed Reporter Deposition Fluorescence in situ Hybridization') experiment. Prokaryotic cells in solution or in situ are permeabilized by treatment with lysozyme and/or achromopeptidase. Step 1: after inactivation of endogenous peroxidases, the permeabilized cells are incubated with rRNA-targeting oligonucleotides for hybridization; the oligonucleotides are coupled to horseradish peroxidase (HRP). Step 2: The cells are incubated with fluorophore-coupled tyramide in the presence of H2O2. HRP coupled to the hybridized oligonucleotides catalyzes locally a reaction that results in the production of hydroxyl radicals (·OH) from H2O2 in the presence of O2·–. The radicalized tyramine now binds covalently to nearby protein molecules, thereby amplifying the signal (up to 100-fold). The readout is done with a confocal laser microscope. Modified from Source
By combining metagenomics with cell identification by localization – thus allowing the reconstruction of microbial communities and their metabolic pathways – the study by Probandt et al. is an excellent example for the resolution power of the culture-independent characterization of a particular microbial habitat. Add to this metatranscriptomics (a wordmonster, meaning that regulation and expression profiles are generated in addition to and for projection onto metagenomic data ) and you can access not only questions like "who's there?", and "who's where, exactly?", but also "who's busy and thriving? who's chilling?". This doesn't sound a lot like an approach that could fit into the straightjacket of Koch's postulates but, hey, studying microbial consortia in the wild is more fun. Ah! Before I forget: there are also eukaryotic microbes, and they also populate the sand grains. Based on 16S rRNA sequences Probandt et al. classified them as algal chloroplasts, and based on morphology as (mostly ) diatoms with a high species diversity. They were also detected by chloroplast autofluorescence and a Eukarya-specific probe in CARD-FISH experiments.
An aside: sand grains had already attracted the ever-curious Antonie van Leeuwenhoek (1632–1723), and he included a drawing of sand grains as he saw them through his microscope in a letter to the Royal Society, 4th December 1703. No, he did not see his cherished animalcules on the sand grains.
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