Bryndís Björnsdóttir

Characterisation of an extracellular vibriolysin of the fish pathogen Moritella viscosa.

Moritella viscosa causes winter ulcer disease in salmonids. The aim of the present work was to isolate and partially characterise an extracellular peptidase from M. viscosa, and to study its role in virulence. The peptidase, termed MvP1, was a 38-kDa metallopeptidase produced in late exponential growth. The optimum temperature for MvP1 was 40 degrees C, but the enzyme was active over a broad range of temperatures. MvP1 was non-lethal to salmon at concentrations up to 0.22microg/g fish, but extracellular products were lethal to salmon. MvP1 degraded casein, gelatin and collagen from lumpfish skin. It caused considerable tissue necrosis and hemorrhages at the site of injection, and affected cell-cell adhesions in EPC and BF-2 cell lines, but was not highly cytotoxic. The peptidase partially degraded fish IgM heavy chain but was non-hemolytic. The mvp1 gene was sequenced and encoded a 734-aa polypeptide containing a signal sequence, an N-terminal propeptide, a mature peptidase domain and a C-terminal propeptide. The MvP1 propeptide undergoes both N-terminal and C-terminal processing and different C-terminal processing results in the formation of several active isoforms of the mature peptidase. The catalytic domain showed highest sequence similarity with several vibriolysins (EC 3.4.24.25) originating from Pseudoalteromonas strains, showing up to 80% aa identity. The results indicate that MvP1 is a previously unknown vibriolysin that might affect M. viscosa virulence by aiding in the invasion and dissemination of the bacterium in its host, by causing tissue destruction.

Virulence properties of Moritella viscosa extracellular products.

Moritella viscosa is the causative agent of winter ulcer disease of marine fish. Knowledge of its pathogenicity is limited and there are no reports comparing the virulence properties of a collection of bacterial isolates. The in vivo and in vitro virulence of the extracellular products (ECP) of 22 M. viscosa isolates was screened. Two non-virulent Canadian isolates and a Norwegian isolate with reduced virulence produced non-lethal ECP. Correlation was obtained between cytotoxin and haemolysin production of M. viscosa. Isolates from salmon produced ECP with lower cytotoxic and haemolytic activities than ECP of isolates originating from other hosts. Correlation was not found between lethality of ECPs in salmon and cytotoxic or haemolytic activities. All isolates secreted esterases and a metallopeptidase (MvP1), degraded starch and produced siderophores. Variable levels of ECP protein concentration, different enzymatic activities and siderophore production could not explain differences in virulence. The results show that virulent M. viscosa isolates secrete a lethal toxic factor of unknown nature and that cytotoxin production may reflect host adaptation. Cell-culture models may not be optimal for determining the virulence of M. viscosa, as no association between cytotoxicity and bacterial virulence was obtained. Non-virulent strains may be useful in future research on M. viscosa virulence, as construction of mutants has not been successful.

Identification of type VI secretion systems in Moritella viscosa

The study describes the identification of type VI secretion systems (T6SSs) in Moritella viscosa, the aetiological agent of winter ulcer disease. Despite the availability of commercial vaccines, M. viscosa causes significant financial losses in salmonid farming. The T6SS transports bacterial proteins from the cell into the environment or directly into host cells, and has been implicated with bacterial virulence. The aim of the study was to identify potential T6SSs in M. viscosa and to determine whether it possesses active T6S, providing further insight into the biology of the bacterium. The genome of M. viscosa 06/09/139 was screened for homology with known T6SS encoding genes. Two genetically distinct loci, termed Moritella Type Six Secretion 1 and 2 (mts1 and mts2), were identified as encoding putative T6SSs. Each locus contained known T6S core genes. The mts2 locus contained species specific genes, some of which have not previously been connected with T6S. The mts1 locus showed sequence homology and synteny to T6SSs of the fish pathogen Aliivibrio salmonicida and a non-pathogenic Moritella sp. PE36. The mts2 locus was more similar to a Vibrio parahaemolyticus T6SS. A functional T6SS was confirmed through identification of secreted Mts1-M, a hemolysin coregulated protein (Hcp) which is a part of the secretion system. Both virulent and avirulent M. viscosa isolates expressed two genes encoding Hcp, mts1-M and mts2-M. The results show that M. viscosa has a functional T6S, but the role of the secretion system and possible connections with virulence need further examination.

Biorefinery Approach to the Use of Macroalgae as Feedstock for Biofuels

Macroalgae (also called seaweeds) have gained attention in recent years as feedstock for the production of fuels and chemicals. This is due to their advantages over traditional terrestrial feedstocks for biorefinery: higher productivity cultivation (amount of biomass produced per unit of surface area) than terrestrial crops, no competition for arable land, lower fresh water consumption during cultivation, and no requirement for fertilizer (van den Burg et al. 2013). In addition, macroalgae have a distinctive chemical composition that differs from lignocelluloses and terrestrial crops, and some 104species are rich in carbohydrates, proteins, fatty acids, and/or bioactive components that make them very suitable for biorefinery (Kraan 2013, van den Burg et al., 2013). For the production of fuels, the most studied routes are the biological conversion of sugars into liquid fuels such as ethanol or butanol, the thermochemical conversion of macroalgae biomass into liquid fuel by hydrothermal liquefaction (HTL), the chemo-catalytic conversion of sugars into furans, and the anaerobic digestion of biomass into methane. Various reviews of the use of macroalgae for biofuels have appeared in recent years, including Chen et al. 2015, Jiang et al. 2016, Milledge et al. 2014, Suutari et al. 2015, Wei et al. 2013. Chen et al. (2015) concluded that biodiesel production from macroalgae seems less attractive than that from microalgae, given the low content of lipids in macroalgae. Therefore, biodiesel from macroalgal lipids was left outside the scope of this chapter.

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