Anammox bacteria wield an energy-efficient nitrogen metabolism enveloped in anammoxosome organelle composed of unique ladderane lipids. Thus, waste anammox biomass seems to be an attractive target for the isolation of ladderanes and subsequent production of artificial vesicles for drug delivery. This study proposed a novel method to isolate ladderane-rich anammoxosomes from aggregate mixed culture of Ca. Brocadia sapporoensis. Compared to conventional isolation protocols, the protocol was simplified by omitting the prepurification of anammox cells, replacing Percoll® with a sucrose gradient and prolonging the application of EDTA. This enhanced and simplified procedure efficiently removed EPS and other debris, thus yielding the layer of anammoxosomes as confirmed by control experiments and TEM. For the first time, the resulting ladderane isolates were used for the preparation of liposomes, both with and without the addition of pure dipalmitoylphosphatidylcholine (DPPC). Vesicles were successfully created, characterised by TEM and DLS, and anammox-based ladderanes were incorporated into their walls. These liposomes had interesting functional properties such as increased colloid stability at elevated concentrations, meaning a reduced tendency to form aggregates compared to model liposomes made solely of DPPC. Overall, this study offers insights into converting waste anammox biomass into a valuable resource for drug delivery.

• Keywords
• Ca. Brocadia sapporoensis, anammox bacteria, anammoxosomes, artificial liposomes, ladderanes,
• MeSH
• Liposomes * chemistry   metabolism   isolation & purification   MeSH
• Planctomycetes * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Liposomes * MeSH

L-Asparaginases are enzymes known for decades due to their use in medicine for the treatment of acute lymphoblastic leukemia. Recently, they have also found application in the food industry, and other possibilities are emerging in the treatment of infectious diseases or in the design of biosensors. For this reason, an ongoing effort has been made to find and characterize new enzymes with properties suitable for these specific applications. In this work, L-asparaginase from Paenibacillus thiaminolyticus (isoenzyme 1) belonging to the least explored group of L-asparaginases derived from L-asparaginase from Rhizobium etli was recombinantly produced with high yields (335 mg per L of culture medium) in E. coli cells and characterized: KM = (26 ± 8) mmol L-1, pHopt = 10.3, without L-glutaminase and urease activity. A probable oligomeric structure (homodimer) was derived by computer modeling and confirmed by gel chromatography experiments. The results of this work extend the current limited knowledge about the poorly described class of R. etli L-asparaginases. Moreover, this L-asparaginase exhibits suitable properties for use in biosensor construction because of the high yields during recombinant production, KM value, stability, and absence of L-glutaminase activity.

• Keywords
• Paenibacillus thiaminolyticus, Acrylamide mitigation, Acute lymphoblastic leukemia, Asparaginase, Biosensors, Food industry,
• Publication type
• Journal Article MeSH

Ladderane lipids synthesised by anammox bacteria hold significant potential for applications in jet fuel, drug delivery, and optoelectronics. Despite the widespread use of anammox bacteria in nitrogen removal from wastewater, the optimal conditions for maximising ladderane production remain unclear, limiting their broader application. To address this, we operated a fed-batch bioreactor with anammox bacteria, gradually adjusting the pH from 6.5 to 7.5 while regularly sampling for microbial community composition (Illumina sequencing), proteins, and ladderane lipids (UHPLC-HRMS). Our findings reveal that ladderane production positively correlates with rising pH increasing nearly fivefold as pH rose from 6.5 to 7.5, with a notable shift towards lipids containing two ladderane alkyl chains at higher pH. However, the conditions at an alkaline pH range also induced mild stress in anammox bacteria, as evidenced by our proteomic and microbial community data. Therefore, we propose maintaining a pH above 7.5 to enrich ladderane-rich anammox biomass but emphasise the need for gradual adaptation. This approach could optimise anammox installations for producing high-value ladderane lipids from wastewater.

• Keywords
• Alkalinity, Anammox bacteria, Biorefinery, Ladderane lipids, Wastewater treatment, pH,
• MeSH
• Bacteria * metabolism   MeSH
• Bioreactors microbiology   MeSH
• Nitrogen metabolism   MeSH
• Hydrogen-Ion Concentration MeSH
• Lipids * MeSH
• Wastewater MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nitrogen MeSH
• Lipids * MeSH
• Wastewater MeSH

An aerobic, Gram-stain-positive and non-spore-forming strain, designated C1-1T, was isolated from a fellfield soil sample collected from frost-sorted polygons on Jane Col, Signy Island, Maritime Antarctic. Cells with a size of 0.65-0.9×1.2-1.7 µm have a flagellar motile apparatus and exhibit a rod-coccus growth cycle. Optimal growth conditions were observed at 15-20 °C, pH 7.0 and NaCl concentration up to 0.5 % (w/v) in the medium. The 16S rRNA gene sequence of C1-1T showed the highest pairwise similarity of 98.77 % to Arthrobacter glacialis NBRC 113092T. Phylogenetic trees based on the 16S rRNA and whole-genome sequences revealed that strain C1-1T belongs to the genus Arthrobacter and is most closely related to members of the 'Arthrobacter psychrolactophilus group'. The G+C content of genomic DNA was 58.95 mol%. The original and orthologous average nucleotide identities between strain C1-1T and A. glacialis NBRC 113092T were 77.15 % and 77.38 %, respectively. The digital DNA-DNA relatedness values between strain C1-1T and A. glacialis NBRC 113092T was 21.6 %. The polar lipid profile was composed mainly of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. The predominant cellular fatty acids were anteiso-C15 : 0 (75 %) and anteiso-C17 : 0 (15.2 %). Menaquinone MK-9(H2) (86.4 %) was the major respiratory quinone in strain C1-1T. The peptidoglycan type was determined as A3α (l-Lys-l-Ala3; A11.6). Based on all described phylogenetic, physiological and chemotaxonomic characteristics, we propose that strain C1-1T (=DSM 112353T=CCM 9148T) is the type strain of a novel species Arthrobacter polaris sp. nov.

• Keywords
• Antarctica, Arthrobacter, Micrococcaceae, cold-adapted microorganisms, whole-genome sequencing,
• MeSH
• Arthrobacter * MeSH
• Sodium Chloride MeSH
• DNA, Bacterial genetics   MeSH
• Phosphatidylinositols MeSH
• Phospholipids chemistry   MeSH
• Phylogeny MeSH
• Glycolipids chemistry   MeSH
• Nucleic Acid Hybridization MeSH
• Cardiolipins MeSH
• Fatty Acids chemistry   MeSH
• Micrococcaceae * MeSH
• Nucleotides MeSH
• Peptidoglycan chemistry   MeSH
• Soil MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Sequence Analysis, DNA MeSH
• Bacterial Typing Techniques MeSH
• Vitamin K 2 chemistry   MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Antarctic Regions MeSH
• Names of Substances
• Sodium Chloride MeSH
• DNA, Bacterial MeSH
• Phosphatidylinositols MeSH
• Phospholipids MeSH
• Glycolipids MeSH
• Cardiolipins MeSH
• Fatty Acids MeSH
• menaquinone 9 MeSH Browser
• Nucleotides MeSH
• Peptidoglycan MeSH
• Soil MeSH
• RNA, Ribosomal, 16S MeSH
• Vitamin K 2 MeSH

Fucosylated compounds are abundantly present in nature and are associated with many biological processes, therefore carrying great potential for use in medicine and biotechnology. Efficient ways to modify fucosylated compounds are still being developed. Promising results are provided by glycosyl hydrolases with transglycosylating activities, such as α-l-fucosidase isoenzyme 2 from Paenibacillus thiaminolyticus (family GH151 of Carbohydrate-Active enZYmes). Currently, there is no 3D structure representing this glycoside hydrolase family and only a few members have been investigated. Here, we present the first structure-function study of a GH151 member, providing the key insights into its specific oligomerization and active site properties. According to the crystal structure, small-angle X-ray scattering data and catalytic investigation, this enzyme functions as a tetramer of a new type and represents the second known case of active site complementation among all α-l-fucosidases. Mutation of the active site-complementing residue histidine 503 to alanine confirmed its influence on α-l-fucosidase activity and, specifically, on substrate binding. Several unique features of GH151 family α-l-fucosidases were revealed, including the oligomerization pattern, active site accessibility and complementation, and substrate selectivity. Some common properties of GH151 glycosyl hydrolases then would be the overall three-domain structure and conservation of the central domain loop 2 function, including its complementation role and the formation of the carbohydrate-binding platform in the active site vicinity.

• Keywords
• GH151, active site complementation, crystal structure, α-l-fucosidase,
• MeSH
• alpha-L-Fucosidase * chemistry   genetics   metabolism   MeSH
• Catalytic Domain MeSH
• Catalysis MeSH
• Carbohydrates * MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• alpha-L-Fucosidase * MeSH
• Carbohydrates * MeSH

The adaptation of bacteria involved in anaerobic ammonium oxidation (anammox) to low temperatures will enable more efficient removal of nitrogen from sewage across seasons. At lower temperatures, bacteria typically tune the synthesis of their membrane lipids to promote membrane fluidity. However, such adaptation of anammox bacteria lipids, including unique ladderane phospholipids and especially shorter ladderanes with absent phosphatidyl headgroup, is yet to be described in detail. We investigated the membrane lipids composition (UPLC-HRMS/MS) and dominant anammox populations (16S rRNA gene amplicon sequencing, Fluorescence in situ hybridization) in 14 anammox enrichments cultivated at 10-37 °C. "Candidatus Brocadia" appeared to be the dominant organism in all but two laboratory enrichments of "Ca. Scalindua" and "Ca. Kuenenia". At lower temperatures, the membranes of all anammox populations were composed of shorter [5]-ladderane ester (reduced chain length demonstrated by decreased fraction of C20/(C18 + C20)). This confirmed the previous preliminary evidence on the prominent role of this ladderane fatty acid in low-temperature adaptation. "Ca. Scalindua" and "Ca. Kuenenia" had distinct profile of ladderane lipids compared to "Ca. Brocadia" biomasses with potential implications for adaptability to low temperatures. "Ca. Brocadia" membranes contained a much lower amount of C18 [5]-ladderane esters than reported in the literature for "Ca. Scalindua" at similar temperature and measured here, suggesting that this could be one of the reasons for the dominance of "Ca. Scalindua" in cold marine environments. Furthermore, we propose additional and yet unreported mechanisms for low-temperature adaptation of anammox bacteria, one of which involves ladderanes with absent phosphatidyl headgroup. In sum, we deepen the understanding of cold anammox physiology by providing for the first time a consistent comparison of anammox-based communities across multiple environments.

• Keywords
• Anaerobic ammonium oxidation, Candidatus Brocadia, Candidatus Scalindua, Effect of temperature, Ladderane phospholipids,
• MeSH
• Anaerobiosis MeSH
• Anaerobic Ammonia Oxidation * MeSH
• Bacteria * MeSH
• In Situ Hybridization, Fluorescence MeSH
• Membrane Lipids MeSH
• Oxidation-Reduction MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Membrane Lipids MeSH
• RNA, Ribosomal, 16S MeSH

Natural compounds isolated from macroalgae are promising, ecofriendly, and multifunctional bioinoculants, which have been tested and used in agriculture. Ulvans, for instance, one of the major polysaccharides present in Ulva spp. cell walls, have been tested for their plant growth-promoting properties as well as their ability to activate plant immune defense, on a large variety of crops. Recently, we have characterized for the first time an arabinogalactan protein-like (AGP-like) from Ulva lactuca, which exhibits several features associated to land plant AGPs. In land plant, AGPs were shown to play a role in several plant biological functions, including cell morphogenesis, reproduction, and plant-microbe interactions. Thus, isolated AGP-like proteins may be good candidates for either the plant growth-promoting properties or the activation of plant immune defense. Here, we have isolated an AGP-like enriched fraction from Ulva lactuca and we have evaluated its ability to (i) protect oilseed rape (Brassica napus) cotyledons against Leptosphaeria maculans, and (ii) its ability to activate immune responses. Preventive application of the Ulva AGP-like enriched fraction on oilseed rape, followed by cotyledon inoculation with the fungal hemibiotroph L. maculans, resulted in a major reduction of infection propagation. The noticed reduction correlated with an accumulation of H2O2 in treated cotyledons and with the activation of SA and ET signaling pathways in oilseed rape cotyledons. In parallel, an ulvan was also isolated from Ulva lactuca. Preventive application of ulvan also enhanced plant resistance against L. maculans. Surprisingly, reduction of infection severity was only observed at high concentration of ulvan. Here, no such significant changes in gene expression and H2O2 production were observed. Together, this study indicates that U. lactuca AGP-like glycoproteins exhibit promising elicitor activity and that plant eliciting properties of Ulva extract, might result not only from an ulvan-originated eliciting activities, but also AGP-like originated.

• Keywords
• Arabinogalactan proteins, Ulva lactuca, elicitor, hemibiotrophic fungus, plant defense, plant immunity,
• Publication type
• Journal Article MeSH

UNLABELLED: The ability to predict the transglycosylation activity of glycosidases by in silico analysis was investigated. The transglycosylation abilities of 7 different β-d-galactosidases from GH family 2 were tested experimentally using 7 different acceptors and p-nitrophenyl-β-d-galactopyranoside as a donor of galactosyl moiety. Similar transglycosylation abilities were confirmed for all enzymes originating from bacteria belonging to Enterobacteriaceae, which were able to use all tested acceptor molecules. Higher acceptor selectivity was observed for all others used bacterial strains. Structure models of all enzymes were constructed using homology modeling. Ligand-docking method was used for enzymes-transglycosylation products models construction and evaluation. Results obtained by in silico analysis were compared with results arisen out of experimental testing. The experiments confirmed that significant differences in transglycosylation abilities are caused by small differences in active sites composition of analyzed enzymes. According to obtained result, it is possible to conclude that homology modeling may serve as a quick starting point for detection or exclusion of enzymes with defined transglycosylation abilities, which can be used for subsequent synthesis of e.g., pharmaceutically interesting glycosides. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02715-w.

• Keywords
• Carbohydrate family, Catalysis, Homology modeling, Hydrolases, Ligand-docking,
• Publication type
• Journal Article MeSH

Arabinogalactan proteins (AGPs) encompass a diverse group of plant cell wall proteoglycans, which play an essential role in plant development, signaling, plant-microbe interactions, and many others. Although they are widely distributed throughout the plant kingdom and extensively studied, they remain largely unexplored in the lower plants, especially in seaweeds. Ulva species have high economic potential since various applications were previously described including bioremediation, biofuel production, and as a source of bioactive compounds. This article presents the first experimental confirmation of AGP-like glycoproteins in Ulva species and provides a simple extraction protocol of Ulva lactuca AGP-like glycoproteins, their partial characterization and unique comparison to scarcely described Solanum lycopersicum AGPs. The reactivity with primary anti-AGP antibodies as well as Yariv reagent showed a great variety between Ulva lactuca and Solanum lycopersicum AGP-like glycoproteins. While the amino acid analysis of the AGP-like glycoproteins purified by the β-d-glucosyl Yariv reagent showed a similarity between algal and land plant AGP-like glycoproteins, neutral saccharide analysis revealed unique glycosylation of the Ulva lactuca AGP-like glycoproteins. Surprisingly, arabinose and galactose were not the most prevalent monosaccharides and the most outstanding was the presence of 3-O-methyl-hexose, which has never been described in the AGPs. The exceptional structure of the Ulva lactuca AGP-like glycoproteins implies a specialized adaptation to the marine environment and might bring new insight into the evolution of the plant cell wall.

• Keywords
• Ulva lactuca, AGP-like glycoproteins, cell wall, extraction, green algae, methylated hexose,
• MeSH
• Chlorophyta * MeSH
• Galactans MeSH
• Glycoproteins MeSH
• Mucoproteins MeSH
• Plant Proteins MeSH
• Ulva * MeSH
• Embryophyta * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• arabinogalactan proteins MeSH Browser
• arabinogalactan MeSH Browser
• Galactans MeSH
• Glycoproteins MeSH
• Mucoproteins MeSH
• Plant Proteins MeSH

α-l-Fucosidase isoenzyme 1 from bacterium Paenibacillus thiaminolyticus is a member of the glycoside hydrolase family GH29 capable of cleaving l-fucose from nonreducing termini of oligosaccharides and glycoconjugates. Here we present the first crystal structure of this protein revealing a novel quaternary state within this family. The protein is in a unique hexameric assembly revealing the first observed case of active site complementation by a residue from an adjacent monomer in this family. Mutation of the complementing tryptophan residue caused changes in the catalytic properties including a shift of the pH optimum, a change of affinity to an artificial chromogenic substrate and a decreased reaction rate for a natural substrate. The wild-type enzyme was active on most of the tested naturally occurring oligosaccharides and capable of transglycosylation on a variety of acceptor molecules, including saccharides, alcohols or chromogenic substrates. Mutation of the complementing residue changed neither substrate specificity nor the preference for the type of transglycosylation acceptor molecule; however, the yields of the reactions were lower in both cases. Maltose molecules bound to the enzyme in the crystal structure identified surface carbohydrate-binding sites, possibly participating in binding of larger oligosaccharides.

• MeSH
• alpha-L-Fucosidase chemistry   genetics   MeSH
• Bacterial Proteins chemistry   genetics   MeSH
• Catalytic Domain MeSH
• Crystallography, X-Ray MeSH
• Mutation MeSH
• Paenibacillus enzymology   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• alpha-L-Fucosidase MeSH
• Bacterial Proteins MeSH