Identification and characterization of uncultivated archaeal virocells using correlative fluorescence and scanning electron microscopy

Abstract text (incl. figure legends and references)

Microbes and their interactions with viruses play important roles in biogeochemical cycles and shape Earth"s biomes. However, information on viral interactions has largely been extrapolated from a very small fraction of cultivated exemplars – the large majority of microbes remains uncultivated. Viral infections of key environmental microbes with their respective viruses forming so-called virocells, are hard to visualize and characterize in environmental samples¹. Emerging methods in fluorescence microscopy enlighten smaller and less frequent structures within microbial cells. For example, it is now possible to design fluorescent probes to target viruses infecting uncultivated hosts and identify different infection stages in environmental samples using virusFISH². Here we introduce the correlation of virusFISH with scanning electron microscopy for locating virocells and studying their morphology in environmental samples.

VirusFISH for identifying viral infections of Candidatus Altiarchaeum hamiconexum within environmental biofilms was performed as described previously², but modified specifically to carry out the correlation. After localizing virocells using fluorescence microscopy on a gridded coverslip, samples were processed for scanning electron microscopy using osmium tetroxide and an acetone dehydration series, followed by critical point drying and coating using Pt/Pd. The gridded coverslip facilitated the correlation of the micrographs using GIMP, showing that the biofilms stayed attached to the grid throughout sample preparation. Similar to cultivation-based studies, it was possible to identify morphological changes of virocells. However unlike other archaea, where a significant increase of cell size over several orders of magnitude was observed³, Candidatus Altiarchaeum hamiconexum only shows a slight, but significant increase of cell size by 10-20%. Within virocells a spatial separation of host DNA and viral reproduction was observed allowing conclusions regarding spatial and functional organization within virocells. Besides the study of virocells itself, this study also supports previous hypotheses that cell lysis due to viral infection fuels the growth of heterotrophic organisms in the deep subsurface.

We conclude that the correlation of virusFISH with electron microscopy enables the characterization of virus-host interactions in environmental samples. In the future, this approach will support hypothesizes based on in silico studies to shed like on a board range of interactions of uncultivated microorganisms.

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