Seasonal Variation in the Body Composition, Carcass Composition, and Offal Quality in the Wild Fallow Deer (Dama dama L.).

The goal of this study was to examine the seasonal variation in the body composition, carcass composition, and quality of edible internal organs from the fallow deer hunt-harvested in the summer (n = 9) and the winter (n = 10) seasons. The weight and proportion of the mesenteric and omental fat were greater for the animals harvested in the winter (1.12 kg and 2.75%) compared to those from the summer season (0.43 kg and 1.02%). The winter-harvested animals had more perinephric fat (0.75 kg and 1.84%) than those hunted in summer (0.26 kg and 1.84%). The gastrointestinal tract of the fallow deer hunted in summer was more filled with feed and therefore heavier (7.92 kg) compared to those from the winter season (5.16 kg). The proportion of fat was significantly greater in the carcasses obtained in winter compared to the summer season (6.55% vs. 3.79%). No seasonal variety was found in the physicochemical characteristics of the edible offal, but the content of extractable fat was significantly affected by the season. In conclusion, the effect of the season on the slaughter value of the hunt-harvested fallow deer was limited to a variation in the proportion of some of the internal organs and affected the fat deposition in the body of the examined animals. The season significantly affected the fat content in the carcass and the extractable fat content in the examined offal.

A Single Biosynthetic Gene Cluster Is Responsible for the Production of Bagremycin Antibiotics and Ferroverdin Iron Chelators.

Biosynthetic gene clusters (BGCs) are organized groups of genes involved in the production of specialized metabolites. Typically, one BGC is responsible for the production of one or several similar compounds with bioactivities that usually only vary in terms of strength and/or specificity. Here we show that the previously described ferroverdins and bagremycins, which are families of metabolites with different bioactivities, are produced from the same BGC, whereby the fate of the biosynthetic pathway depends on iron availability. Under conditions of iron depletion, the monomeric bagremycins are formed, representing amino-aromatic antibiotics resulting from the condensation of 3-amino-4-hydroxybenzoic acid with p-vinylphenol. Conversely, when iron is abundantly available, the biosynthetic pathway additionally produces a molecule based on p-vinylphenyl-3-nitroso-4-hydroxybenzoate, which complexes iron to form the trimeric ferroverdins that have anticholesterol activity. Thus, our work shows a unique exception to the concept that BGCs should only produce a single family of molecules with one type of bioactivity and that in fact different bioactive molecules may be produced depending on the environmental conditions.IMPORTANCE Access to whole-genome sequences has exposed the general incidence of the so-called cryptic biosynthetic gene clusters (BGCs), thereby renewing their interest for natural product discovery. As a consequence, genome mining is the often first approach implemented to assess the potential of a microorganism for producing novel bioactive metabolites. By revealing a new level of complexity of natural product biosynthesis, we further illustrate the difficulty of estimation of the panel of molecules associated with a BGC based on genomic information alone. Indeed, we found that the same gene cluster is responsible for the production of compounds which differ in terms of structure and bioactivity. The production of these different compounds responds to different environmental triggers, which suggests that multiplication of culture conditions is essential for revealing the entire panel of molecules made by a single BGC.

Influence of the method of storage on the quality of venison from wild fallow deer (Dama dama).

The quality of two muscles (musculus longissimus lumborum and musculus semimembranosus) was studied in a group of 15 wild fallow deer does hunted in January. The aim of the research was to analyse the influence of the method of storage on the quality of venison. The pH value after chilled storage and after freezer storage ranged from 5.64 to 5.70, indicating high meat quality of meat. The freezer storage caused a decrease in the redness and chroma in the longissimus lumborum muscle (P < 0.0001 and P = 0.001). The frozen and thawed venison characterised with a higher percentage of free water (P = 0.001), drip loss (P = 0.033) and lower plasticity (P = 0.001) compared to the meat stored under chilled conditions. The instrumental measures of tenderness were affected by the storage. The results indicated a lower technological quality of venison after freezer storage compared to meat stored under chilled conditions.

The seasonal variation in the quality of venison from wild fallow deer (Dama dama) - A pilot study.

The studied material included 19 fallow deer does hunt-harvested in two seasons, summer and winter. The post-mortem body weight of analysed animals was not affected by the season (P = 0.762). The pH value measured 24 h post-mortem in the M. longissimus lumborum was significantly higher in the summer compared to the winter season. The venison obtained in the winter season characterized with higher L* (P < 0.0001) and b* (P = 0.002) and lower a* (P < .0001) compared to meat from the summer season. There was also a seasonal variation in the purge in vacuum bags (P = 0.001), water compartments and cooking loss (P < 0.0001). Considering the proximal chemical composition, the extractable fat content was higher in winter (P < 0.0001) compared to the summer season. On the basis of the research results, one can conclude about greater usability for processing and storage of venison obtained in the winter season.

Complete Genome Sequence of Streptomyces lunaelactis MM109T, Isolated from Cave Moonmilk Deposits.

Streptomyces lunaelactis MM109T is a ferroverdin A (anticholesterol) producer isolated from cave moonmilk deposits. The complete genome sequence of MM109T was obtained by combining Oxford Nanopore MinION and Illumina HiSeq and MiSeq technologies, revealing an 8.4-Mb linear chromosome and two plasmids, pSLUN1 (127,264 bp, linear) and pSLUN2 (46,827 bp, circular).

High-Throughput Sequencing Analysis of the Actinobacterial Spatial Diversity in Moonmilk Deposits.

Moonmilk are cave carbonate deposits that host a rich microbiome, including antibiotic-producing Actinobacteria, making these speleothems appealing for bioprospecting. Here, we investigated the taxonomic profile of the actinobacterial community of three moonmilk deposits of the cave "Grotte des Collemboles" via high-throughput sequencing of 16S rRNA amplicons. Actinobacteria was the most common phylum after Proteobacteria, ranging from 9% to 23% of the total bacterial population. Next to actinobacterial operational taxonomic units (OTUs) attributed to uncultured organisms at the genus level (~44%), we identified 47 actinobacterial genera with Rhodoccocus (4 OTUs, 17%) and Pseudonocardia (9 OTUs, ~16%) as the most abundant in terms of the absolute number of sequences. Streptomycetes presented the highest diversity (19 OTUs, 3%), with most of the OTUs unlinked to the culturable Streptomyces strains that were previously isolated from the same deposits. Furthermore, 43% of the OTUs were shared between the three studied collection points, while 34% were exclusive to one deposit, indicating that distinct speleothems host their own population, despite their nearby localization. This important spatial diversity suggests that prospecting within different moonmilk deposits should result in the isolation of unique and novel Actinobacteria. These speleothems also host a wide range of non-streptomycetes antibiotic-producing genera, and should therefore be subjected to methodologies for isolating rare Actinobacteria.

Isolation, Characterization, and Antibacterial Activity of Hard-to-Culture Actinobacteria from Cave Moonmilk Deposits.

Cave moonmilk deposits host an abundant and diverse actinobacterial population that has a great potential for producing novel natural bioactive compounds. In our previous attempt to isolate culturable moonmilk-dwelling Actinobacteria, only Streptomyces species were recovered, whereas a metagenetic study of the same deposits revealed a complex actinobacterial community including 46 actinobacterial genera in addition to streptomycetes. In this work, we applied the rehydration-centrifugation method to lessen the occurrence of filamentous species and tested a series of strategies to achieve the isolation of hard-to-culture and rare Actinobacteria from the moonmilk deposits of the cave "Grotte des Collemboles". From the "tips and tricks" that were tested, separate autoclaving of the components of the International Streptomyces Project (ISP) medium number 5 (ISP5) medium, prolonged incubation time, and dilution of the moonmilk suspension were found to most effectively improve colony forming units. Taxonomic analyses of the 40 isolates revealed new representatives of the Agromyces, Amycolatopsis, Kocuria, Micrococcus, Micromonospora, Nocardia, and Rhodococcus species, as well as additional new streptomycetes. The applied methodologies allowed the isolation of strains associated with both the least and most abundant moonmilk-dwelling actinobacterial operational taxonomic units. Finally, bioactivity screenings revealed that some isolates displayed high antibacterial activities, and genome mining uncovered a strong potential for the production of natural compounds.

Assessment of the Potential Role of Streptomyces in Cave Moonmilk Formation.

Moonmilk is a karstic speleothem mainly composed of fine calcium carbonate crystals (CaCO3) with different textures ranging from pasty to hard, in which the contribution of biotic rock-building processes is presumed to involve indigenous microorganisms. The real microbial input in the genesis of moonmilk is difficult to assess leading to controversial hypotheses explaining the origins and the mechanisms (biotic vs. abiotic) involved. In this work, we undertook a comprehensive approach in order to assess the potential role of filamentous bacteria, particularly a collection of moonmilk-originating Streptomyces, in the genesis of this speleothem. Scanning electron microscopy (SEM) confirmed that indigenous filamentous bacteria could indeed participate in moonmilk development by serving as nucleation sites for CaCO3 deposition. The metabolic activities involved in CaCO3 transformation were furthermore assessed in vitro among the collection of moonmilk Streptomyces, which revealed that peptides/amino acids ammonification, and to a lesser extend ureolysis, could be privileged metabolic pathways participating in carbonate precipitation by increasing the pH of the bacterial environment. Additionally, in silico search for the genes involved in biomineralization processes including ureolysis, dissimilatory nitrate reduction to ammonia, active calcium ion transport, and reversible hydration of CO2 allowed to identify genetic predispositions for carbonate precipitation in Streptomyces. Finally, their biomineralization abilities were confirmed by environmental SEM, which allowed to visualize the formation of abundant mineral deposits under laboratory conditions. Overall, our study provides novel evidences that filamentous Actinobacteria could be key protagonists in the genesis of moonmilk through a wide spectrum of biomineralization processes.

A Phenotypic and Genotypic Analysis of the Antimicrobial Potential of Cultivable Streptomyces Isolated from Cave Moonmilk Deposits.

Moonmilk speleothems of limestone caves host a rich microbiome, among which Actinobacteria represent one of the most abundant phyla. Ancient medical texts reported that moonmilk had therapeutical properties, thereby suggesting that its filamentous endemic actinobacterial population might be a source of natural products useful in human treatment. In this work, a screening approach was undertaken in order to isolate cultivable Actinobacteria from moonmilk of the Grotte des Collemboles in Belgium, to evaluate their taxonomic profile, and to assess their potential in biosynthesis of antimicrobials. Phylogenetic analysis revealed that all 78 isolates were exclusively affiliated to the genus Streptomyces and clustered into 31 distinct phylotypes displaying various pigmentation patterns and morphological features. Phylotype representatives were tested for antibacterial and antifungal activities and their genomes were mined for secondary metabolite biosynthetic genes coding for non-ribosomal peptide synthetases (NRPSs), and polyketide synthases (PKS). The moonmilk Streptomyces collection was found to display strong inhibitory activities against a wide range of reference organisms, as 94, 71, and 94% of the isolates inhibited or impaired the growth of Gram-positive, Gram-negative bacteria, and fungi, respectively. Interestingly, 90% of the cave strains induced strong growth suppression against the multi-drug resistant Rasamsonia argillacea, a causative agent of invasive mycosis in cystic fibrosis and chronic granulomatous diseases. No correlation was observed between the global antimicrobial activity of an individual strain and the number of NRPS and PKS genes predicted in its genome, suggesting that approaches for awakening cryptic metabolites biosynthesis should be applied to isolates with no antimicrobial phenotype. Overall, our work supports the common belief that moonmilk might effectively treat various infectious diseases thanks to the presence of a highly diverse population of prolific antimicrobial producing Streptomyces, and thus may indeed constitute a promising reservoir of potentially novel active natural compounds.

Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 µM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 µM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins.

Streptomyces lunaelactis sp. nov., a novel ferroverdin A-producing Streptomyces species isolated from a moonmilk speleothem.

A novel actinobacterium, designated MM109(T), was isolated from a moonmilk deposit collected from the cave 'Grotte des Collemboles' located in Comblain-au-Pont, Belgium. Based on a polyphasic taxonomic approach comprising chemotaxonomic, phylogenetic, morphological, and physiological characterization, the isolate has been affiliated to the genus Streptomyces. Multilocus sequence analysis based on the 16S rRNA gene and five other house-keeping genes (atpD, gyrB, rpoB, recA and trpB) showed that the MM109(T) isolate is sufficiently distinct from its closest relative, Streptomyces peucetius strain AS 4.1799(T), as to represent a novel species. The phylogenetic distinctiveness of the taxon represented by isolate MM109(T) was supported by the isolation and identification of additional twelve moonmilk-derived isolates, which according to multilocus sequence analysis were clustered along with MM109(T). Scanning electron microscopy observations revealed complex and diversified structures within a MM109(T) colony, made from branching vegetative mycelia. The spore chains of the MM109(T) isolate undergo complete septation at the late stages of the morphological differentiation process, leading to the formation of packs of smooth cylindrical-shaped spores. Isolate MM109(T) produces several intracellular and diffusible pigments, particularly an intracellular green-pigmented secondary metabolite, which was identified through UPLC-ESI-MS analysis as ferroverdin A, an iron-chelating molecule formerly extracted and characterized from Streptomyces sp. strain WK-5344. The isolate MM109(T) is thus considered to represent a novel species of Streptomyces, for which the name Streptomyces lunaelactis sp. nov. is proposed with the type strain MM109(T) (=DSM 42149(T) = BCCM/LMG 28326(T)).

Altered desferrioxamine-mediated iron utilization is a common trait of bald mutants of Streptomyces coelicolor.

Streptomyces coelicolor is an important model organism for developmental studies of filamentous GC-rich actinobacteria. The genetic characterization of mutants of S. coelicolor blocked at the vegetative mycelium stage, the so-called bald (bld) mutants that are unable to erect spore-forming aerial hyphae, has opened the way to discovering the molecular basis of development in actinomycetes. Desferrioxamine (DFO) production and import of ferrioxamines (FO; iron-complexed DFO) are key to triggering morphogenesis of S. coelicolor and we show here that growth of S. coelicolor on the reference medium for Streptomyces developmental studies is fully dependent on DFO biosynthesis. UPLC-ESI-MS analysis revealed that all bld mutants tested are affected in DFO biosynthesis, with bldA, bldJ, and ptsH mutants severely impaired in DFO production, while bldF, bldK, crr and ptsI mutants overproduce DFO. Morphogenesis of bldK and bldJ mutants was recovered by supplying exogenous iron. Transcript analysis showed that the bldJ mutant is impaired in expression of genes involved in the uptake of FO, whereas transcription of genes involved in both DFO biosynthesis and FO uptake is increased in bldK mutants. Our study allows proposing altered DFO production and/or FO uptake as a novel phenotypic marker of many S. coelicolor bld mutants, and strengthens the role of siderophores and iron acquisition in morphological development of actinomycetes.

Unsuspected control of siderophore production by N-acetylglucosamine in streptomycetes.

Iron is one of the most abundant elements on earth but is found in poorly soluble forms hardly accessible to microorganisms. To subsist, they have developed iron-chelating molecules called siderophores that capture this element in the environment and the resulting complexes are internalized by specific uptake systems. While biosynthesis of siderophores in many bacteria is regulated by iron availability and oxidative stress, we describe here a new type of regulation of siderophore production. We show that in Streptomyces coelicolor, their production is also controlled by N-acetylglucosamine (GlcNAc) via the direct transcriptional repression of the iron utilization repressor dmdR1 by DasR, the GlcNAc utilization regulator. This regulatory nutrient-metal relationship is conserved among streptomycetes, which indicates that the link between GlcNAc utilization and iron uptake repression, however unsuspected, is the consequence of a successful evolutionary process. We describe here the molecular basis of a novel inhibitory mechanism of siderophore production that is independent of iron availability. We speculate that the regulatory connection between GlcNAc and siderophores might be associated with the competition for iron between streptomycetes and their fungal soil competitors, whose cell walls are built from the GlcNAc-containing polymer chitin. Alternatively, GlcNAc could emanate from streptomycetes' own peptidoglycan that goes through intense remodelling throughout their life cycle, thereby modulating the iron supply according to specific needs at different stages of their developmental programme.

(AC)n microsatellite polymorphism and 14-nucleotide deletion in exon 42 ankyrin-1 gene in several families with hereditary spherocytosis in a population of South-Western Poland.

Defects in ankyrin-1 have been implicated in approximately half of all patients with hereditary spherocytosis. However, not all polymorphisms in this gene lead to the changes in expressed protein or to the changes of the level of its expression. In this study, we report on several cases of the (AC)n microsatellite polymorphism in 3' untranslated region of ANK1 gene found in nine families (19 patients) with hereditary spherocytosis (HS) and also in ten healthy individuals from the same territory. We also found that 14-nucleotide deletion in this region of ANK1 which was shifted by five nucleotides in relation to another 14-nucleotide deletion listed in Single Nucleotide Polymorphism National Center for Biotechnology Information (SNP NCBI) database. This deletion seems to be present only in individuals with 11/14 and 13/14 AC repeats what would be an interesting correlation between these two features. However, comparison of the data obtained for HS patients and healthy individuals indicates that both polymorphisms are not connected to the pathology of hereditary spherocytosis.