The microbiome of Greenland Ice Sheet supraglacial habitats is still underinvestigated, and as a result there is a lack of representative genomes from these environments. In this study, we investigated the supraglacial microbiome through a combination of culturing-dependent and -independent approaches. We explored ice, cryoconite, biofilm, and snow biodiversity to answer: (1) how microbial diversity differs between supraglacial habitats, (2) if obtained bacterial genomes reflect dominant community members, and (3) how culturing versus high throughput sequencing changes our observations of microbial diversity in supraglacial habitats. Genomes acquired through metagenomic sequencing (133 high-quality MAGs) and whole genome sequencing (73 bacterial isolates) were compared to the metagenome assemblies to investigate abundance within the total environmental DNA. Isolates obtained in this study were not dominant taxa in the habitat they were sampled from, in contrast to the obtained MAGs. We demonstrate here the advantages of using metagenome SSU rRNA genes to reflect whole-community diversity. Additionally, we demonstrate a proof-of-concept of the application of in situ culturing in a supraglacial setting.

FimH-mediated adhesion of Escherichia coli to bladder epithelium is a prerequisite for urinary tract infections. FimH is also essential for blood-borne bacterial dissemination, but the mechanisms are poorly understood. The purpose of this study was to assess the influence of different FimH mutations on bacterial adhesion using a novel adhesion assay, which models the physiological flow conditions bacteria are exposed to. We introduced 12 different point mutations in the mannose binding pocket of FimH in an E. coli strain expressing type 1 fimbriae only (MSC95-FimH). We compared the bacterial adhesion of each mutant across several commonly used adhesion assays, including agglutination of yeast, adhesion to mono- and tri-mannosylated substrates, and static adhesion to bladder epithelial and endothelial cells. We performed a comparison of these assays to a novel method that we developed to study bacterial adhesion to mammalian cells under flow conditions. We showed that E. coli MSC95-FimH adheres more efficiently to microvascular endothelium than to bladder epithelium, and that only endothelium supports adhesion at physiological shear stress. The results confirmed that mannose binding pocket mutations abrogated adhesion. We demonstrated that FimH residues E50 and T53 are crucial for adhesion under flow conditions. The coating of endothelial cells on biochips and modelling of physiological flow conditions enabled us to identify FimH residues crucial for adhesion. These results provide novel insights into screening methods to determine the effect of FimH mutants and potentially FimH antagonists.

It has previously been reported that some strains of the marine bacterium Pseudoalteromonas luteoviolacea produce the purple bioactive pigment violacein as well as the antibiotic compound indolmycin, hitherto only found in Streptomyces. The purpose of the present study was to determine the relative role of each of these two compounds as antibacterial compounds in P. luteoviolacea S4054. Using Tn10 transposon mutagenesis, a mutant strain that was significantly reduced in violacein production in mannose-containing substrates was created. Full genome analyses revealed that the vio-biosynthetic gene cluster was not interrupted by the transposon; instead the insertion was located to the maeA gene encoding the malic enzyme. Supernatant of the mutant strain inhibited Vibrio anguillarum and Staphylococcus aureus in well diffusion assays and in MIC assays at the same level or even more pronounced as the wild type strain. The mutant strain killed V. anguillarum in co-culture experiments as efficiently as the wild type. Using UHPLC-UV/Vis analyses, we quantified violacein and indolmycin, and the mutant strain only produced 7-10% the amount of violacein compared to the wildtype strain. In contrast, the amount of indolmycin produced by the mutant strain was about 300% that of the wildtype. Since inhibition of V. anguillarum and S. aureus by the mutant strain was similar to that of the wild type, it is concluded that violacein is not the major antibacterial compound in P. luteoviolacea. We furthermore propose that production of violacein and indolmycin may be metabolically linked and that yet unidentified antibacterial compound(s) may be play a role in the antibacterial activity of P. luteoviolacea.

Microbial communities from extreme environments are largely understudied, but are essential as producers of metabolites, including enzymes, for industrial processes. As cultivation of most microorganisms remains a challenge, culture‐independent approaches for enzyme discovery in the form of metagenomics to analyse the genetic potential of a community are rapidly becoming the way forward. This study focused on analysing a metagenome from the cold and alkaline ikaite columns in Greenland, identifying 282 open reading frames (ORFs) that encoded putative carbohydrate‐modifying enzymes with potential applications in, for example detergents and other processes where activity at low temperature and high pH is desired. Seventeen selected ORFs, representing eight enzyme families were synthesized and expressed in two host organisms, Escherichia coli and Aliivibrio wodanis . Aliivibrio wodanis demonstrated expression of a more diverse range of enzyme classes compared to E. coli , emphasizing the importance of alternative expression systems for enzymes from extremophilic microorganisms. To demonstrate the validity of the screening strategy, we chose a recombinantly expressed cellulolytic enzyme from the metagenome for further characterization. The enzyme, Cel240, exhibited close to 40% of its relative activity at low temperatures (4°C) and demonstrated endoglucanase characteristics, with a preference for cellulose substrates. Despite low sequence similarity with known enzymes, computational analysis and structural modelling confirmed its cellulase‐family affiliation. Cel240 displayed activity at low temperatures and good stability at 25°C, activity at alkaline pH and increased activity in the presence of CaCl ₂ , making it a promising candidate for detergent and washing industry applications.

Thermal springs are excellent locations for discovery of thermostable microorganisms and enzymes. In this study, we identify a novel thermotolerant bacterial strain related to Paenibacillus dendritiformis, denoted Paenibacillus sp. 3179, which was isolated from a thermal spring in East Greenland. A functional expression library of the strain was constructed, and the library screened for β‐d‐galactosidase and α‐l‐fucosidase activities on chromogenic substrates. This identified two genes encoding a β‐d‐galactosidase and an α‐l‐fucosidase, respectively. The enzymes were recombinantly expressed, purified, and characterized using oNPG (2‐nitrophenyl‐β‐d‐galactopyranoside) and pNP‐fucose (4‐nitrophenyl‐α‐l‐fucopyranoside), respectively. The enzymes were shown to have optimal activity at 50°C and pH 7–8, and they were able to hydrolyze as well as transglycosylate natural carbohydrates. The transglycosylation activities were investigated using TLC and HPLC, and the β‐d‐galactosidase was shown to produce the galactooligosaccharides (GOS) 6'‐O‐galactosyllactose and 3'‐O‐galactosyllactose using lactose as substrate, whereas the α‐l‐fucosidase was able to transfer the fucose moiety from pNP‐fuc to lactose, thereby forming 2'‐O‐fucosyllactose. Since enzymes that are able to transglycosylate carbohydrates at elevated temperature are desirable in many industrial processes, including food and dairy production, we foresee the potential use of enzymes from Paenibacillus sp. 3179 in the production of, for example, instant formula.

The ikaite columns in the Ikka Fjord, SW Greenland, represent a permanently cold and alkaline environment known to contain a rich bacterial diversity. 16S and 18S rRNA gene amplicon and metagenomic sequencing was used to investigate the microbial diversity in the columns and for the first time, the eukaryotic and archaeal diversity in ikaite columns were analyzed. The results showed a rich prokaryotic diversity that varied across columns as well as within each column. Seven different archaeal phyla were documented in multiple locations inside the columns. The columns also contained a rich eukaryotic diversity with 27 phyla representing microalgae, protists, fungi, and small animals. Based on metagenomic sequencing, 25 high-quality MAGs were assembled and analyzed for the presence of genes involved in cycling of nitrogen, sulfur, and phosphorous as well as genes encoding carbohydrate-active enzymes (CAZymes), showing a potentially very bioactive microbial community.