Jens Harder

Introns in prokaryotes are classified as mobile genetic elements normally located within essential genes. Their RNA transcripts are functional if spliced by the intron´s ribozyme activity. The excised intron RNA is assumed to proliferate by leaving the host cell and being integrated into foreign genomes. Here we show the presence of a bacterial intron´s RNA in archaeal cells. The predatory ultramicrobial epibiont Candidatus Velamenicoccus archaeovorus contains a group I intron in its 23 S ribosomal RNA gene. In search for predation evidence, we visualized the intron RNA by in situ hybridization and observed it in dead cells of the filamentous archaeon Methanothrix soehngenii in a slowly growing methanogenic enrichment culture on limonene. Transcriptome analysis detected a ratio of one unspliced transcript or intron molecule per 20,000 mature 23 S rRNA molecules. Our study visualized that spliced intron RNA can leave the host and enter foreign cells. Spliced intron RNA adds to extracellular RNA, so far known to function as intercellular signaling molecules and metabolic inhibitors.

Limonene is a natural monoterpene omnipresent in human environments. It enters wastewater and is also metabolised in methanogenic digesters. A stable limonene‐degrading methanogenic enrichment culture was investigated by metagenomic, metatranscriptomic and metaproteomic data sets to characterise the microbial community and identify the limonene degradation pathway. Thirty‐two metagenome‐assembled genomes revealed a complex community of bacteria and methanogenic archaea dominated by Candidatus Velamenicoccus archaeovorus as the top predator, contributing two‐thirds of the reads in the metagenome. The presence of several fermenting bacteria ( Anaerolineaceae , Aminidesulfovibrio , Smithellaceae , Lentimicrobium ) indicated the recycling of necromass in a microbial loop. Only one hydrocarbon‐activating enzyme system was expressed, a member of the alkyl‐ and arylsuccinate synthase family which is a glycine radical enzyme that adds fumarate to hydrocarbons. The limonenylsuccinate synthase gene encodes a modified substrate binding pocket with two smaller amino acids, suggesting an adaptation for the larger structure of limonene. The limonenylsuccinate synthase operon and a ring cleavage operon, as well as genes for the final syntrophic fermentation to acetate, hydrogen and formate were encoded in a Syntrophobacteraceae genome. Almost all genes for this degradation pathway were highly transcribed and expressed, demonstrating a catalytic role for glycine radical enzymes in methanogenic systems degrading limonene.

Glycans shape microbiomes in the ocean and the gut, driving key steps in the global carbon cycle and human health. Yet, our ability to track microbial glycan turnover across microbiomes remains limited, as identifying active degraders without prior genomic knowledge remains a key challenge. Here, we introduce an activity-based fluorescence resonance energy transfer (FRET) probe that enables direct visualization and quantification of glycan metabolism in complex microbial communities. As a proof of concept, we investigated α-mannan degradation, a prominent glycan in algal blooms. Using automated glycan assembly, we synthesized a mannan hexasaccharide bearing a fluorescein–rhodamine FRET pair. The probe was validated using a recombinantly expressed endo-α-mannanase (GH76) from Salegentibacter sp. Hel_I_6. It was shown to function in cell lysates, pure cultures, and complex microbiomes (via plate assays and microscopy). This probe enabled spatiotemporal visualization of in situ α-mannan turnover in a marine microbiome. Glycan FRET probes are versatile tools for tracking glycan degradation across biological scales, from single enzymes to microbiomes.

Laminarin is a cytosolic storage polysaccharide of phytoplankton and macroalgae and accounts for over 10% of the world’s annually fixed carbon dioxide. Algal disruption, for example, by viral lysis releases laminarin. The soluble sugar is rapidly utilized by free-living planktonic bacteria, in which sugar transporters and the degrading enzymes are frequently encoded in polysaccharide utilization loci. The annotation of flavobacterial genomes failed to identify canonical laminarin utilization loci in several particle-associated bacteria, in particular in strains of Maribacter. In this study, we report in vivo utilization of laminarin by Maribacter forsetii accompanied by additional cell growth and proliferation. Laminarin utilization coincided with the induction of an extracellular endo-laminarinase, SusC/D outer membrane oligosaccharide transporters, and a periplasmic glycosyl hydrolase family 3 protein. An ABC transport system and sugar kinases were expressed. Endo-laminarinase activity was also observed in Maribacter sp. MAR_2009_72, Maribacter sp. Hel_I_7, and Maribacter dokdonensis MAR_2009_60. Maribacter dokdonensis MAR_2009_71 lacked the large endo-laminarinase gene in the genome and had no endo-laminarinase activity. In all genomes, genes of induced proteins were scattered across the genome rather than clustered in a laminarin utilization locus. These observations revealed that the Maribacter strains investigated in this study participate in laminarin utilization, but in contrast to many free-living bacteria, there is no co-localization of genes encoding the enzymatic machinery for laminarin utilization.

Arabinose and galactose are major, rapidly metabolized components of marine particulate and dissolved organic matter. In this study, we observed for the first time large microbiomes for the degradation of arabinogalactan and report a detailed investigation of arabinogalactan utilization by the flavobacterium Maribacter sp. MAR_2009_72. Cellular extracts hydrolysed arabinogalactan in vitro. Comparative proteomic analyses of cells grown on arabinogalactan, arabinose, galactose, and glucose revealed the expression of specific proteins in the presence of arabinogalactan, mainly glycoside hydrolases (GH). Extracellular glycan hydrolysis involved five alpha-l-arabinofuranosidases affiliating with glycoside hydrolase families 43 and 51, four unsaturated rhamnogalacturonylhydrolases (GH105) and a protein with a glycoside hydrolase family-like domain. We detected expression of three induced TonB-dependent SusC/D transporter systems, one SusC, and nine glycoside hydrolases with a predicted periplasmatic location. These are affiliated with the families GH3, GH10, GH29, GH31, GH67, GH78, and GH115. The genes are located outside of and within canonical polysaccharide utilization loci classified as specific for arabinogalactan, for galactose-containing glycans, and for arabinose-containing glycans. The breadth of enzymatic functions expressed in Maribacter sp. MAR_2009_72 as response to arabinogalactan from the terrestrial plant larch suggests that Flavobacteriia are main catalysts of the rapid turnover of arabinogalactans in the marine environment.

Planktonic particle-associated bacteria comprise particle-attached and motile free-living cells. These groups were obtained by settlement in Imhoff cones. Dilution plating on marine agar 2216 (ZoBell marine agar) and microscopic counts indicated a cultivability of 0.7% (0.4%–1.2%) of bacteria in coastal seawater collected at Helgoland Roads, North Sea. Particle-associated bacteria presented a minority population in seawater, but had a larger cultivability of 25% (0.9%–100%) for populations collected by settlement of particles and 5.7% (0.9%–24%) for populations collected by filtration. Partial 16S rRNA gene sequences indicated that 84% of the cultured taxa were either enriched in particle-associated microbiomes or only found in these microbiomes, including Sulfitobacter and other Rhodobacteraceae, Pseudoalteromonas, Psychromonas, Arcobacter and many Flavobacteriaceae. Illumina-based 16S rRNA V3V4 amplicon sequences of plate communities revealed that nearly all operational taxonomic units had a cultivated and described strain in close phylogenetic proximity. This suggested that decades of strain isolation from seawater on ZoBell marine agar had achieved a very good coverage of cultivable genera abundant in nature. The majority belonged to particle-associated bacteria, complementing observations that abundant free-living seawater bacteria often require cultivation conditions closer to their natural habitat like liquid cultivation in oligotrophic medium.

Epibiotic bacteria are known to live on and off bacterial cells. Here, we describe the ultramicrobacterial anaerobic epibiont OP3 LiM living on Archaea and Bacteria .

Suspended particulate matter, phytoplankton and bacteria can be exploited to form larger aggregates, so-called bio-flocs. These serve as feeds for cultured shrimps, govern inorganic nutrients and load bacteria including pathogens. The current study aimed to simulate aggregate formation from available particulate matter in shrimp pond water and investigate quality of aggregates as well as possible impact to the pond water. Molasses was added to cylindrical tanks containing shrimp pond waters, and the tanks were rolled for 48 h. Besides water quality (inorganic nutrients and physical parameters), the researchers investigated and separated bacterial community compositions (BCC) to free-living (FL) and bio-flocs/particle-attached (PA) bacteria via 16S rRNA amplicon sequencing, and measured macro-molecules contents (carbohydrates, lipids and proteins) in the bio-flocs. Molasses addition increased bacterial numbers in the bio-flocs to two-fold higher than the FL’s. Moreover, potential probiotics such as Halomonas, Psychrobacter, Mesonia and Chromohalobacter were detected associated to bio-flocs and dominated the BCC. In contrast, bio-flocs without molasses showed 4-fold less carbohydrates and harboured elevated potential pathogens such as Vibrio and Alteromonas. Results show that molasses (at C/N ratio 1:2) increases pH (to 8.2 ± 0.09 and 8.0 ± 0.04 after 24 h and 48 h, respectively) in pond water, improving beneficial biofloc formation. Molasses also increased carbohydrates and proteins in bio-flocs and maintained abundances of beneficial bacteria resulting in low inorganic nutrient concentrations. Thus, molasses is suitable for shrimp farming to improve rearing processes.

Marine heterotrophic microorganisms remineralize about half of the annual primary production, with the microbiomes on and around algae and particles having a major contribution. These microbiomes specifically include free-living chemotactic and particle-attached bacteria, which are often difficult to analyze individually, as the standard method of size-selective filtration only gives access to particle-attached bacteria. In this study, we demonstrated that particle collection in Imhoff sedimentation cones enriches microbiomes that included free-living chemotactic bacteria and were distinct from particle microbiomes obtained by filtration or centrifugation. Coastal seawater was collected during North Sea phytoplankton spring blooms, and the microbiomes were investigated using 16S rRNA amplicon sequencing and fluorescence microscopy. Enrichment factors of individual operational taxonomic units (OTUs) were calculated for comparison of fractionated communities after separation with unfractionated seawater communities. Filtration resulted in a loss of cells and yielded particle fractions including bacterial aggregates, filaments, and large cells. Centrifugation had the lowest separation capacity. Particles with a sinking rate of >2.4 m day^–1 were collected in sedimentation cones as a bottom fraction and enriched in free-living chemotactic bacteria, i.e., Sulfitobacter, Pseudoalteromonas, and Vibrio. Subfractions of these bottom fractions, obtained by centrifugation, showed enrichment of either free-living or particle-attached bacteria. We identified five distinct enrichment patterns across all separation techniques: mechano-sensitive and mechano-stable free-living bacteria and three groups of particle-attached bacteria. Simultaneous enrichment of particle-attached and chemotactic free-living bacteria in Imhoff sedimentation cones is a novel experimental access to these groups providing more insights into the diversity, structure, and function of particle-associated microbiomes, including members of the phycosphere.