Böhnke, Stefanie, Adam-Beyer, Nicole, Bährle, Rebecca Juliana and Perner, Mirjam (2022) Diving into the ocean’s microbial dark matter: from sensor triggered sampling and activity-based enzyme searches. [Poster] In: GRC Marine Microbes 2022. , 29.05.-03.06.2022, Les Disablerets, Switzerland .

Abstract

Most of the microbes assumed to colonize the seafloor and its subsurface resist cultivation. Thus, their catalytic
capabilities and contribution to element cycling remain enigmatic. In marine sediments, it is estimated that
microbial dark matter accounts for up to 91%. Although sequence-based approaches allow culture-independent
insights into taxonomic diversity, and metabolic potential, they rely on database comparisons. Therefore, sequence
data can only reflect what is already known. Indeed, for more than one-third of the genes found in genomes from
uncultured bacteria, no predicted function can be assigned. Besides the lack of homologous genes, a further
challenge when investigating metatranscriptomes is the community bias caused by sample processing delays.
Hence, the need for in situ fixation of RNA, particularly for samples from greater water depths and longer sample
transport times, is a prerequisite to obtain unbiased metatranscriptomic data reflecting gene expression profiles in
its original state. Although activity-based searches for novel enzymes by means of metagenomic fosmid libraries
allow to link previously not allocated genes to a function, they are hampered by expression limitations in the
surrogate host. In situ monitoring of chemical compounds can provide rates, but do not allow assignment of
responsible organisms and may also overlook possible intermediates and reactions.
Here we present a holistic approach that combines in situ incubation and rate measurements at the seafloor of
Boknis Eck (BE, Eckernfoerde Bay, south-western Baltic Sea) with activity-based metagenomic screening and
metatranscriptomics. At BE, the seasonal stratification of the water column causes pronounced hypoxia,
sporadically even anoxia. Once a month we deploy a mini chamber lander system for short-term in situ incubations.
Embedded sensor technology allows monitoring of chemical dynamics (e.g., oxygen, sulphide) and sampling under
pre-defined chemical conditions (e.g. if oxygen drops during incubation under a specific threshold value).
Metagenomic and metatranscriptomic data will be used to determine the abundance of identified novel enzymes
with specific functions (CO2 reduction, H2 conversion, etc.) that have been detected by means of activity-based
searches. This comparison can give an estimate of how important the novel enzyme and its function might actually
be for the investigated microbial community and thus for ecosystem functioning. Our results suggest that such
combined approaches have the potential to open a window into the metabolic network of uncultured microbes and
their catalytic ability in the biogeochemical cycling of key elements.

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