Chitinases in the tree of life Ecological, kinetic and structural studies of archaeal and marine bacterial chitinases

Chitin is after cellulose the second most abundant biopolymer on earth. It’s production is enormous, with estimates of up to 1011 tons for both the annual production and the steady-state amount. Chitin consists of β-1,4 glycosidic bonded N-acetyl-glucosamine subunits. It’s degradation is especially in the oceans an important step to ensure the continuous availability of carbon and nitrogen. Chitin is very resilient to physicochemical degradation due to its structure. It is mainly biodegraded by microorganisms. Until now three degradation pathways are know, utilising different enzymes. One very important enzyme found in the biodegradation pathways of bacteria, fungi and archaea is the chitinase. Chitinases hydrolyse the β-1,4 glycosidic bond between the N-acetyl-glucosamine subunits. This enzyme is used not only for the recovery of nutrients in microorganisms, it plays also a major role in moulting of arthropods and is utilised in defence mechanisms of higher organisms. This important enzyme, as proxy for chitinolytic activity, is detected with molecular methods and cultivation based approaches. Most of the studies detecting chitinases do either test for the genetic capability or the growth capability of the respective microorganisms on chitin. Moreover, direct proof of the the chitinolytic enzyme itself is lacking in many studies. Until now a more comprehensive chitinase test panel, combining cultivation and molecular screening of the cultivated strains for their genetic capabilities has not been implemented yet. Furthermore, the search for chitinolytic organisms was mainly focused on bacteria and fungi, but almost no chitin degrading archaea were detected until now. Within this study a novel three step chitinase test panel was established and tested, consisting of isolation and cultivation of microorganisms on chitin as sole carbon and nitrogen source, the molecular screening of the cultivated strains and the evaluation of the respective chitinase activity. This approach was used to investigate bacteria isolated from different marine microbial communities (Mediterranean Deep Sea sediments and Baltic Sea shrimp carapaces). In addition, bacterial strains (bryozoan derived isolates and actinomycetes) of the KiWiZ strain collection were also investigated. In total, 145 bacterial strains were investigated in this study. Furthermore, the first crenarchaeal chitinase gene from Sulfolobus tokodaii was detected, expressed in E. coli and the resulting chitinase was described. In addition, the chitinase gene of the halophilic euryarchaeon Halobacterium salinarum was expressed for the first time in E. coli

Chitinasen im Stammbaum des Lebens.Ökologische, kinetische und strukturelle Studien an archaeellen und marinen bakteriellen Chitinasen.

Chitin is after cellulose the second most abundant biopolymer on earth. It’s production is enormous, with estimates of up to 1011 tons for both the annual production and the steady-state amount. Chitin consists of β-1,4 glycosidic bonded N-acetyl-glucosamine subunits. It’s degradation is especially in the oceans an important step to ensure the continuous availability of carbon and nitrogen. Chitin is very resilient to physicochemical degradation due to its structure. It is mainly biodegraded by microorganisms. Until now three degradation pathways are know, utilising different enzymes. One very important enzyme found in the biodegradation pathways of bacteria, fungi and archaea is the chitinase. Chitinases hydrolyse the β-1,4 glycosidic bond between the N-acetyl-glucosamine subunits. This enzyme is used not only for the recovery of nutrients in microorganisms, it plays also a major role in moulting of arthropods and is utilised in defence mechanisms of higher organisms. This important enzyme, as proxy for chitinolytic activity, is detected with molecular methods and cultivation based approaches. Most of the studies detecting chitinases do either test for the genetic capability or the growth capability of the respective microorganisms on chitin. Moreover, direct proof of the the chitinolytic enzyme itself is lacking in many studies. Until now a more comprehensive chitinase test panel, combining cultivation and molecular screening of the cultivated strains for their genetic capabilities has not been implemented yet. Furthermore, the search for chitinolytic organisms was mainly focused on bacteria and fungi, but almost no chitin degrading archaea were detected until now. Within this study a novel three step chitinase test panel was established and tested, consisting of isolation and cultivation of microorganisms on chitin as sole carbon and nitrogen source, the molecular screening of the cultivated strains and the evaluation of the respective chitinase activity. This approach was used to investigate bacteria isolated from different marine microbial communities (Mediterranean Deep Sea sediments and Baltic Sea shrimp carapaces). In addition, bacterial strains (bryozoan derived isolates and actinomycetes) of the KiWiZ strain collection were also investigated. In total, 145 bacterial strains were investigated in this study. Furthermore, the first crenarchaeal chitinase gene from Sulfolobus tokodaii was detected, expressed in E. coli and the resulting chitinase was described. In addition, the chitinase gene of the halophilic euryarchaeon Halobacterium salinarum was expressed for the first time in E. coli.Chitin ist nach Zellulose das zweithäufigste Biopolymer auf der Erde. Sein Vorkommen ist enorm. Schätzungen gehen von einer Jahresproduktion von bis zu 1011 Tonnen aus. Chitin besteht aus N-Acetyl-Glukosaminuntereinheiten, die miteinander β-1,4-glykosidisch verknüpft sind. Der Abbau von Chitin ist vor allem in den Ozeanen ein sehr wichtiger Vorgang, um einen fortwährenden Nachschub an Kohlenstoff und Stickstoff sicher zu stellen. Aufgrund seiner Struktur ist Chitin sehr widerstandsfähig gegenüber dem physikochemischen Abbau. Es wird hauptsächlich von Mikroorganismen biologisch abgebaut. Bisher sind drei Abbauwege bekannt, die sich verschiedener Enzyme bedienen. Ein sehr wichtiges Enzym ist hierbei die Chitinase, die sich sowohl in Bakterien als auch in Pilzen und Archaea findet. Chitinasen hydrolisieren die β-1,4-glykosidische Verknüpfung zwischen den N-Acetyl-Glukosaminuntereinheiten. Sie werden nicht nur zur Nahrungsaufnahme, sondern auch beim Häuten von Arthropoden und bei der Immunabwehr höherer Organismen verwendet. Chitinasen werden in ökologischen Untersuchungen als Anzeiger für chitinolytische Mikroorganismen eingesetzt. Hierbei wird meistens molekularbiologisch nach dem genetischen Fingerabdruck von Chitinasen gesucht oder die jeweiligen Mikroorganismen werden auf Chitin kultiviert, um fest zu stellen, ob Chitin verwertet werden kann. Hierbei wurde bei den meisten Studien in der Vergangenheit nur eine der beiden Herangehensweisen verwendet. Auch der direkte Nachweis des Enzymes fehlt in den meisten Studien. Außerdem wurden bisherige Arbeiten vor allem auf Bakterien und Pilze fokussiert, wobei Archaea kaum berücksichtigt wurden. Um einen umfassenderen Ansatz zu finden, wurde in dieser Arbeit ein dreistufiges Screening Panel etabliert und getestet. Das Test-Panel besteht aus der Isolation und Kultivierung von Mikroorganismen auf Chitin als einziger Kohlenstoff- und Stickstoffquelle, dem molekularen Screening der kultivierten Isolate auf das Vorhandensein eines genetischen Chitinasemotivs, sowie der Evaluation der entsprechenden Chitinaseaktivität. Das etablierte Verfahren wurde genutzt, um verschiedene Lebensräume im Meer in Bezug auf die Chitin-abbauenden Bakterien zu vergleichen. Hierzu dienten Proben von Sedimenten aus dem Mittelmeer und der Oberfläche von Ostseegarnelen. Außerdem wurde Bakterien (Actinomyceten und Isolate von Bryozoen) aus der KiWiZ-Stammsammlung untersucht. Insgesamt wurden 145 Bakterienstämme in dieser Arbeit auf ihre chitinolytischen Eigenschaften hin untersucht. Weiterhin wurde in dieser Arbeit das erste crenarchaeelle Chitinase-Gen in Sulfolobus tokodaii identifiziert, in E. coli exprimiert und als aktive Chitinase erstmals beschrieben. Das Chitinase-Gen des Euryarchaeons Halobacterium salinarum wurde ebenfalls zum ersten Mal in E. coli exprimiert und das Genprodukt als aktive Chitinase charakterisiert

Combined genotyping, microbial diversity and metabolite profiling studies on farmed Mytilus spp. from Kiel Fjord

The blue mussel Mytilus is a popular food source with high economical value. Species of the M. edulis complex (M. edulis, M. galloprovincialis and M. trossulus) hybridise whenever their geographic ranges overlap posing difficulties to species discrimination, which is important for blue mussel aquaculture. The aim of this study was to determine the genetic structure of farmed blue mussels in Kiel Fjord. Microbial and metabolic profile patterns were studied to investigate a possible dependency on the genotype of the bivalves. Genotyping confirmed the complex genetic structure of the Baltic Sea hybrid zone and revealed an unexpected dominance of M. trossulus alleles being in contrast to the predominance of M. edulis alleles described for wild Baltic blue mussels. Culture-dependent and -independent microbial community analyses indicated the presence of a diverse Mytilus-associated microbiota, while an LC-MS/MS-based metabolome study identified 76 major compounds dominated by pigments, alkaloids and polyketides in the whole tissue extracts. Analysis of mussel microbiota and metabolome did not indicate genotypic dependence, but demonstrated high intraspecific variability of farmed mussel individuals. We hypothesise that individual differences in microbial and metabolite patterns may be caused by high individual plasticity and might be enhanced by e.g. nutritional condition, age and gender

First crenarchaeal chitinase found in Sulfolobus tokodaii

This is the first description of a functional chitinase gene within the crenarchaeotes. Here we report of the heterologues expression of the ORF BAB65950 from Sulfolobus tokodaii in E. coli. The resulting protein degraded chitin and was hence classified as chitinase (EC 3.2.4.14). The protein characterization revealed a specific activity of 75 mU/mg using colloidal chitin as substrate. The optimal activity of the enzyme was measured at pH 2.5 and 70 °C, respectively. A dimeric enzyme configuration is proposed. According to amino acid sequence similarities chitinases are attributed to the two glycoside hydrolase families 18 and 19. The derived amino acid sequence of the S. tokodaii gene differed from sequences of these two glycoside hydrolase families. However, within a phylogenetic tree of protein sequences, the crenarchaeal sequence of S. tokodaii clustered in close proximity to members of the glycoside hydrolase family 18

Diversity of Antibiotic-Active Bacteria Associated with the Brown Alga Laminaria saccharina from the Baltic Sea

Bacteria associated with the marine macroalga Laminaria saccharina, collected from the Kiel Fjord (Baltic Sea, Germany), were isolated and tested for antimicrobial activity. From a total of 210 isolates, 103 strains inhibited the growth of at least one microorganism from the test panel including Gram-negative and Gram-positive bacteria as well as a yeast. Most common profiles were the inhibition of Bacillus subtilis only (30%), B. subtilis and Staphylococcus lentus (25%), and B. subtilis, S. lentus, and Candida albicans (11%). In summary, the antibiotic-active isolates covered 15 different activity patterns suggesting various modes of action. On the basis of 16S rRNA gene sequence similarities >99%, 45 phylotypes were defined, which were classified into 21 genera belonging to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Phylogenetic analysis of 16S rRNA gene sequences revealed that four isolates possibly represent novel species or even genera. In conclusion, L. saccharina represents a promising source for the isolation of new bacterial taxa and antimicrobially active bacteria

Nachhaltige Aquakultur im Klimawandel

Die Herausforderung nachhaltiger Aquakultur besteht in dem Gewinn hoher Erträge auf geringer Fläche ohne Überlastung natürlicher Systeme. Veränderte Winter- und Sommertemperaturen durch den Klimawandel erfordern Anpassungsstrategien, vor allem in der Auswahl geeigneter Arten