A comparative analysis of alternative splicing patterns in Atlantic salmon (Salmo salar) in response to Moritella viscosa and sea lice (Lepeophtheirus salmonis) infection.

Moritella viscosa (M. viscosa) and sea lice (Lepeophtheirus salmonis) are severe pathogens that primarily infect the skin of Atlantic salmon (Salmo salar), which cause significant economic losses in the farming industry. However, the pathogenesis and molecular mechanisms underlying the host's immune defence at the post-transcriptional level remain unclear. Alternative splicing (AS) is an evolutionarily conserved post-transcriptional mechanism that can greatly increase the richness of the transcriptome and proteome. In this study, transcriptomic data derived from skin tissues of Atlantic salmon after M. viscosa and sea lice infections were used to examine the AS profiles and their differential expression patterns. In total, we identified 33,044 AS events (involving 13,718 genes) in the control (CON) group, 35,147 AS events (involving 14,340 genes) in the M. viscosa infection (MV) group, and 30,364 AS events (involving 13,142 genes) in the sea lice infection (LC) group, respectively. Among the five types of AS identified in our study (i.e., SE, A5SS, A3SS, MXE, and RI), SE was the most prevalent type in all three groups (i.e., CON, MV, and LC groups). Decreased percent-spliced-in (PSI) levels were observed in SE events under both MV- and LC-infected conditions, suggesting that MV or LC infection elevated exon-skipping isoforms and promoted the selection of shorter transcripts in numerous DAS genes. In addition, most of the differential AS genes were found to be associated with pathways related to mRNA regulation, epithelial or muscle development, and immune response. These findings provide novel insights into the role of AS in host-pathogen interactions and represent the first comparative analysis of AS in response to bacterial and parasitic infections in fish.

Differential expression of immune-related biomarkers in primary cultures from Atlantic salmon (Salmo salar) exposed to processed Paecilomyces variotii with or without inactivated Moritella viscosa.

Paecilomyces variotii (a filamentous fungus), is a promising novel protein source in fish feeds due to its high nutritional value. Also, P. variotii has Microbial-Associated Molecular Patterns (MAMPs) such as glucans and nucleic acids that could modulate the host's immune response. To understand the potential bioactive properties of this fungus in Atlantic salmon (Salmo salar), our study was conducted to evaluate the gene expression of immune-related biomarkers (e.g., cytokines, effector molecules and receptors) on primary cultures from salmon head kidney (HKLs) and spleen leukocytes (SLs) exposed to either UV inactivated or fractions from P. variotii with or without inactivated Moritella viscosa (a skin pathogen in salmonids). Moreover, the effect of the fermentation conditions and down-stream processing on the physical ultrastructure and cell wall glucan content of P. variotii was characterized. The results showed that drying had a significant effect on the cell wall ultrastructure of the fungi and the choice of fermentation has a significant effect on the quantity of ß-glucans in P. variotii. Furthermore, stimulating Atlantic salmon HKLs and SLs with P. variotii and its fractions induced gene expression related to pro-inflammatory (tnfα, il1ß) and antimicrobial response (cath2) in HKLs, while response in SLs was related to both pro-inflammatory and regulatory response (tnfα, il6 and il10). Similarly, the stimulation with inactivated M. viscosa alone led to an up-regulation of genes related to pro-inflammatory (tnfα, il1ß, il6) antimicrobial response (cath2), intra-cellular signalling and recognition of M. viscosa (sclra, sclrb) and a suppression of regulatory response (il10) in both HKLs and SLs. Interestingly, the co-stimulation of cells with P. variotii and M. viscosa induced immune homeostasis (il6, tgfß) and antimicrobial response (cath2) in SLs at 48h. Thus, P. variotii induces immune activation and cellular communication in Atlantic salmon HKLs and SLs and modulates M. viscosa induced pro-inflammatory responses in SLs. Taken together, the results from physical and chemical characterization of the fungi, along with the differential gene expression of key immune biomarkers, provides a theoretical basis for designing feeding trials and optimize diets with P. variotii as a functional novel feed ingredient for Atlantic salmon.

Antigenic similarities and clinical cross-protection between variant and classic non-viscous strains of Moritella viscosa in Atlantic salmon in Norway.

Moritella viscosa (M. viscosa) is one of the major etiological agents of winter-ulcers in Atlantic salmon (Salmo salar) in Norway. Outbreaks of ulcerative disease in farmed fish occur across the North Atlantic region, causing reduced animal welfare and economical challenges, and are of hindrance for sustainable growth within the industry. Commercially available multivalent core vaccines containing inactivated bacterin of M. viscosa reduce mortality and clinical signs related to winter ulcer disease. It has previously been described two major genetic clades within M. viscosa, typical (hereafter referred to as classic) and variant, based on gyrB sequencing. In addition, there are phenotypical traits such as viscosity that may differ between different types of isolates. Western blot using salmon plasma showed that classic non-viscous strains are antigenically different from the classic viscous type included in core vaccines. Further, Western blot also showed that there are similarities in binding patterns between Norwegian variant and classic non-viscous isolates, indicating they may be antigenically related. Vaccination-challenge trials using Norwegian gyrB-classic non-viscous isolates of M. viscosa, demonstrate that the isolates from the classic clade that are included in current commercial multivalent core vaccines, provide limited cross protection against the emerging non-viscous strains. However, a vaccine recently approved for marketing authorization in Norway, containing inactivated antigen of a variant M. viscosa strain, demonstrates reduced mortality as well as clinical signs caused by infections with the classic non-viscous M. viscosa isolated from outbreaks in Norwegian salmon farms. The study shows that there are antigenic similarities between variant and classic non-viscous types of M. viscosa, and these similarities are mirrored in the observed cross-protection in vaccination-challenge trials.

New vaccination strategies are required for effective control of winter ulcer disease caused by emerging variant strains of Moritella viscosa in Atlantic salmon.

Moritella viscosa is one on the major etiological agents of winter-ulcers in Atlantic salmon (Salmo salar) in Norway. Outbreaks of ulcerative disease in farmed fish occurs across the North Atlantic region and is an impeding factor for sustainable growth within the industry. Commercially available multivalent core vaccines containing inactivated bacterin of M. viscosa reduce mortality and clinical signs related to winter ulcer disease. Two major genetic clades within M. viscosa have previously been described based on gyrB sequencing, namely typical (hereafter referred to as classic) and variant. Vaccination-challenge trials using vaccines including either variant and or classic isolates of M. viscosa show that classic clade isolates included in current commercial multivalent core vaccines provide poor cross-protection against emerging variant strains, while variant strains confer high level of protection against variant M. viscosa but to a lesser extent to classic clade isolates. This demonstrates that future vaccine regimens should include a combination of strains from both clades.

Global gene expression responses of Atlantic salmon skin to Moritella viscosa.

Moritella viscosa is a Gram-negative pathogen that causes large, chronic ulcers, known as winter-ulcer disease, in the skin of several fish species including Atlantic salmon. We used a bath challenge approach to profile the transcriptome responses of M. viscosa-infected Atlantic salmon skin at the lesion (Mv-At) and away from the lesion (Mv-Aw) sites. M. viscosa infection was confirmed through RNA-based qPCR assays. RNA-Seq identified 5212 and 2911 transcripts differentially expressed in the Mv-At compared to no-infection control and Mv-Aw groups, respectively. Also, there were 563 differentially expressed transcripts when comparing the Mv-Aw to control samples. Our results suggest that M. viscosa caused massive and strong, but largely infection site-focused, transcriptome dysregulations in Atlantic salmon skin, and its effects beyond the skin lesion site were comparably subtle. The M. viscosa-induced transcripts of Atlantic salmon were mainly involved in innate and adaptive immune response-related pathways, whereas the suppressed transcripts by this pathogen were largely connected to developmental and cellular processes. As validated by qPCR, M. viscosa dysregulated transcripts encoding receptors, signal transducers, transcription factors and immune effectors playing roles in TLR- and IFN-dependent pathways as well as immunoregulation, antigen presentation and T-cell development. This study broadened the current understanding of molecular pathways underlying M. viscosa-triggered responses of Atlantic salmon, and identified biomarkers that may assist to diagnose and combat this pathogen.

Comparative genomic analysis of obligately piezophilic Moritella yayanosii DB21MT-5 reveals bacterial adaptation to the Challenger Deep, Mariana Trench.

Hadal trenches are the deepest but underexplored ecosystems on the Earth. Inhabiting the trench bottom is a group of micro-organisms termed obligate piezophiles that grow exclusively under high hydrostatic pressures (HHP). To reveal the genetic and physiological characteristics of their peculiar lifestyles and microbial adaptation to extreme high pressures, we sequenced the complete genome of the obligately piezophilic bacterium Moritella yayanosii DB21MT-5 isolated from the deepest oceanic sediment at the Challenger Deep, Mariana Trench. Through comparative analysis against pressure sensitive and deep-sea piezophilic Moritella strains, we identified over a hundred genes that present exclusively in hadal strain DB21MT-5. The hadal strain encodes fewer signal transduction proteins and secreted polysaccharases, but has more abundant metal ion transporters and the potential to utilize plant-derived saccharides. Instead of producing osmolyte betaine from choline as other Moritella strains, strain DB21MT-5 ferments on choline within a dedicated bacterial microcompartment organelle. Furthermore, the defence systems possessed by DB21MT-5 are distinct from other Moritella strains but resemble those in obligate piezophiles obtained from the same geographical setting. Collectively, the intensive comparative genomic analysis of an obligately piezophilic strain Moritella yayanosii DB21MT-5 demonstrates a depth-dependent distribution of energy metabolic pathways, compartmentalization of important metabolism and use of distinct defence systems, which likely contribute to microbial adaptation to the bottom of hadal trench.

Absolute quantification of priority bacteria in aquaculture using digital PCR.

Modern aquaculture systems are designed for intensive rearing of fish or other species. Both land-based and offshore systems typically contain high loads of biomass and the water quality in these systems is of paramount importance for fish health and production. Microorganisms play a crucial role in removal of organic matter and nitrogen-recycling, production of toxic hydrogen sulfide (H2S), and can affect fish health directly if pathogenic for fish or exerting probiotic properties. Methods currently used in aquaculture for monitoring certain bacteria species numbers still have typically low precision, specificity, sensitivity and are time-consuming. Here, we demonstrate the use of Digital PCR as a powerful tool for absolute quantification of sulfate-reducing bacteria (SRB) and major pathogens in salmon aquaculture, Moritella viscosa, Yersinia ruckeri and Flavobacterium psychrophilum. In addition, an assay for quantification of Listeria monocytogenes, which is a human pathogen bacterium and relevant target associated with salmonid cultivation in recirculating systems and salmon processing, has been assessed. Sudden mass mortality incidents caused by H2S produced by SRB have become of major concern in closed aquaculture systems. An ultra-sensitive assay for quantification of SRB has been established using Desulfovibrio desulfuricans as reference strain. The use of TaqMan® probe technology allowed for the development of multi-plex assays capable of simultaneous quantification of these aquaculture priority bacteria. In single-plex assays, limit of detection was found to be at around 20 fg DNA for M. viscosa, Y. ruckeri and F. psychrophilum, and as low as 2 fg DNA for L. monocytogenes and D. desulfuricans.

Structure and Mechanism of the Ketosynthase-Chain Length Factor Didomain from a Prototypical Polyunsaturated Fatty Acid Synthase.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential ingredients of the human diet. They are synthesized by LC-PUFA synthases (PFASs) expressed in marine bacteria and other organisms. PFASs are large enzyme complexes that are homologous to mammalian fatty acid synthases and microbial polyketide synthases. One subunit of each PFAS harbors consecutive ketosynthase (KSc) and chain length factor (CLF) domains that collectively catalyze the elongation of a nascent fatty acyl chain via iterative carbon-carbon bond formation. We report the X-ray crystal structure of the KS-CLF didomain from a well-studied PFAS in Moritella marina. Our structure, in combination with biochemical analysis, provides a foundation for understanding the mechanism of substrate recognition and chain length control by the KS-CLF didomain as well as its interaction with a cognate acyl carrier protein partner.

Sequence analysis of nonulosonic acid biosynthetic gene clusters in Vibrionaceae and Moritella viscosa.

Nonulosonic acid (NulO) biosynthesis in bacteria is directed by nab gene clusters that can lead to neuraminic, legionaminic or pseudaminic acids. Analysis of the gene content from a set mainly composed of Aliivibrio salmonicida and Moritella viscosa strains reveals the existence of several unique nab clusters, for which the NulO products were predicted. This prediction method can be used to guide tandem mass spectrometry studies in order to verify the products of previously undescribed nab clusters and identify new members of the NulOs family.

Function of three RuBisCO enzymes under different CO2 conditions in Hydrogenovibrio marinus.

The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110 has three ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isoenzymes, designated CbbLS-1, CbbLS-2, and CbbM, which are encoded by the cbbL1S1, cbbL2S2, and cbbM genes, respectively. Functions of these isoenzymes at different CO2 concentrations were investigated using deletion mutants of their genes. Deletion of cbbL1 had no effect on cell growth under any of the test growth conditions. The cbbL2 mutant was unable to grow under lower (≤0.15%) CO2 conditions, though it grew normally under higher (≥2%) CO2 conditions. Growth of the cbbM mutant was retarded under higher CO2 conditions but was not affected by lower CO2 conditions. These results indicate that CbbLS-2 and CbbM specifically function under lower and higher CO2 conditions, respectively. The growth retardation of the cbbL2 and cbbM mutants was not restored by complementation with plasmids carrying the cbbL2S2 and cbbM genes, respectively. The cbbL2S2 and cbbM genes are followed by the carboxysome genes and the cbbQmOm genes, respectively. Co-expression of these downstream genes was probably necessary for the in vivo function of CbbLS-2 and CbbM. CbbLS-1 was upregulated in the cbbL2 and cbbM mutants under the lower and higher CO2 conditions, respectively, indicating that the expression of cbbL1S1 was controlled to compensate the deficiency of the other RuBisCO isoenzymes.

Draft Genome Sequence of the Deep-Sea Bacterium Moritella sp. JT01 and Identification of Biotechnologically Relevant Genes.

Deep-sea bacteria can produce various biotechnologically relevant enzymes due to their adaptations to high pressures and low temperatures. To identify such enzymes, we have sequenced the genome of the polycaprolactone-degrading bacterium Moritella sp. JT01, isolated from sediment samples from Japan Trench (6957 m depth), using a Illumina HiSeq2000 sequencer (12.1 million paired-end reads) and CLC Genomics Workbench (version 6.5.1) for the assembly, resulting in a 4.83-Mb genome (42 scaffolds). The genome was annotated using Rapid Annotation using Subsystem Technology (RAST), Protein Homology/analogY Recognition Engine V 2.0 (PHYRE2), and BLAST2Go, revealing 4439 protein coding sequences and 101 RNAs. Gene products with industrial relevance, such as lipases (three) and esterases (four), were identified and are related to bacterium's ability to degrade polycaprolactone. The annotation revealed proteins related to deep-sea survival, such as cold-shock proteins (six) and desaturases (three). The presence of secondary metabolite biosynthetic gene clusters suggests that this bacterium could produce nonribosomal peptides, polyunsaturated fatty acids, and bacteriocins. To demonstrate the potential of this genome, a lipase was cloned an introduced into Escherichia coli. The lipase was purified and characterized, showing activity over a wide temperature range (over 50% at 20-60 °C) and pH range (over 80% at pH 6.3 to 9). This enzyme has tolerance to the surfactant action of sodium dodecyl sulfate and shows 30% increased activity when subjected to a working pressure of 200 MPa. The genomic characterization of Moritella sp. JT01 reveals traits associated with survival in the deep-sea and their potential uses in biotechnology, as exemplified by the characterized lipase.

Cold Adaptation of Triosephosphate Isomerase.

The main problem for enzymes from psychrophilic species, which need to work near the freezing point of liquid water, is the exponential decay of reaction rates as the temperature is decreased. Cold-adapted enzymes have solved this problem by shifting the activation enthalpy-entropy balance for the catalyzed reaction compared to those of their mesophilic orthologs. To understand the structural basis of this universal feature, it is necessary to examine pairs of such orthologous enzymes, with known three-dimensional structures, at the microscopic level. Here, we use molecular dynamics free energy calculations in combination with the empirical valence bond method to evaluate the temperature dependence of the activation free energy for differently adapted triosephosphate isomerases. The results show that the enzyme from the psychrophilic bacterium Vibrio marinus indeed displays the characteristic shift in enthalpy-entropy balance, compared to that of the yeast ortholog. The origin of this effect is found to be located in a few surface-exposed protein loops that show differential mobilities in the two enzymes. Key mutations render these loops more mobile in the cold-adapted triosephosphate isomerase, which explains both the reduced activation enthalpy contribution from the protein surface and the lower thermostability.

Pan genome and CRISPR analyses of the bacterial fish pathogen Moritella viscosa.

BACKGROUND: Winter-ulcer Moritella viscosa infections continue to be a significant burden in Atlantic salmon (Salmo salar L.) farming. M. viscosa comprises two main clusters that differ in genetic variation and phenotypes including virulence. Horizontal gene transfer through acquisition and loss of mobile genetic elements (MGEs) is a major driving force of bacterial diversification. To gain insight into genomic traits that could affect sublineage evolution within this bacterium we examined the genome sequences of twelve M. viscosa strains. Matches between M. viscosa clustered, regularly interspaced, short palindromic, repeats and associated cas genes (CRISPR-Cas) were analysed to correlate CRISPR-Cas with adaptive immunity against MGEs. RESULTS: The comparative genomic analysis of M. viscosa isolates from across the North Atlantic region and from different fish species support delineation of M. viscosa into four phylogenetic lineages. The results showed that M. viscosa carries two distinct variants of the CRISPR-Cas subtype I-F systems and that CRISPR features follow the phylogenetic lineages. A subset of the spacer content match prophage and plasmid genes dispersed among the M. viscosa strains. Further analysis revealed that prophage and plasmid-like element distribution were reflected in the content of the CRISPR-spacer profiles. CONCLUSIONS: Our data suggests that CRISPR-Cas mediated interactions with MGEs impact genome properties among M. viscosa, and that patterns in spacer and MGE distributions are linked to strain relationships.

Co-cultivation and transcriptome sequencing of two co-existing fish pathogens Moritella viscosa and Aliivibrio wodanis.

BACKGROUND: Aliivibrio wodanis and Moritella viscosa have often been isolated concurrently from fish with winter-ulcer disease. Little is known about the interaction between the two bacterial species and how the presence of one bacterial species affects the behaviour of the other. RESULTS: The impact on bacterial growth in co-culture was investigated in vitro, and the presence of A. wodanis has an inhibitorial effect on M. viscosa. Further, we have sequenced the complete genomes of these two marine Gram-negative species, and have performed transcriptome analysis of the bacterial gene expression levels from in vivo samples. Using bacterial implants in the fish abdomen, we demonstrate that the presence of A. wodanis is altering the gene expression levels of M. viscosa compared to when the bacteria are implanted separately. CONCLUSIONS: From expression profiling of the transcriptomes, it is evident that the presence of A. wodanis is altering the global gene expression of M. viscosa. Co-cultivation studies showed that A. wodanis is impeding the growth of M. viscosa, and that the inhibitorial effect is not contact-dependent.

Host specificity and clade dependent distribution of putative virulence genes in Moritella viscosa.

Moritella viscosa is the aetiological agent of winter-ulcer disease in farmed salmonids in the North Atlantic. Previously, two major (typical and variant) genetic clades have been demonstrated within this bacterial species, one of which is almost solely related to disease in Atlantic salmon (Salmo salar). In the present study infection trials demonstrated that 'typical' M. viscosa isolated from Norwegian Atlantic salmon was highly virulent in this fish species but resulted in lower levels of mortality in rainbow trout. 'Variant' M. viscosa isolated from rainbow trout resulted in modest mortality levels in both Atlantic salmon and rainbow trout. To investigate the possible genetic background for inter-strain virulence differences, 38 M. viscosa isolates of diverse geographical origin and host species and a number of other Moritella spp. were investigated for the presence/absence of putative virulence related homologs. All isolates were positive for DNA sequences coding for; the Type VI secretion ATPase (clpV), hemolysin co-regulated protein (hcp), bacterioferritins (bfrA and bfrB), lectin (hemG), phospholipase D (pld), multifunctional autoprocessing repeats-in-toxin (martxA), aerolysin (aer), invasin (inv), and cytotoxic necrotizing factor (cnf), with the exception of one isolate in which cnf could not be confirmed. The product of an ABC transporter metal-binding lipoprotein (mat) was consistently detected although 11 isolates, all phylogenetically related, appear to produce a truncated version. A putative insecticidal toxin complex (mitABC) was detected almost exclusively in 'typical' Atlantic salmon isolates, and our data indicate that this complex of genes is expressed and co-transcribed. Transmission electron microscopy investigation revealed pili and flagella surface structures on nine M. viscosa representing both typical and variant isolates. Our results provide strong support for the existence of host specificity/high virulence in 'typical' M. viscosa related to Atlantic salmon. The gene distribution also provides further support for the genetic division within M. viscosa, and constitutes a basis for further study of the importance of the mitABC complex in winter-ulcer pathogenesis.

Crystal structures of substrate-bound chitinase from the psychrophilic bacterium Moritella marina and its structure in solution.

The four-domain structure of chitinase 60 from Moritella marina (MmChi60) is outstanding in its complexity. Many glycoside hydrolases, such as chitinases and cellulases, have multi-domain structures, but only a few have been solved. The flexibility of the hinge regions between the domains apparently makes these proteins difficult to crystallize. The analysis of an active-site mutant of MmChi60 in an unliganded form and in complex with the substrates NAG4 and NAG5 revealed significant differences in the substrate-binding site compared with the previously determined complexes of most studied chitinases. A SAXS experiment demonstrated that in addition to the elongated state found in the crystal, the protein can adapt other conformations in solution ranging from fully extended to compact.

Genome sequence of the psychrophilic deep-sea bacterium Moritella marina MP-1 (ATCC 15381).

Moritella marina MP-1 is a bacterial species known for its production of docosahexaenoic acid. We present the draft genome sequence of the type strain Moritella marina MP-1 (ATCC 15381), having 4,636,778 bp with a G+C content of 40.5% and consisting of 83 contigs.

Draft genome sequence of Moritella dasanensis strain ArB 0140, a psychrophilic bacterium isolated from the Arctic Ocean.

The psychrophilic bacterium Moritella dasanensis strain ArB 0140 was isolated near a glacier in Kongsfjorden, Svalbard Archipelago, Norway. Here we report a 4.89-Mb draft genome sequence of Moritella dasanensis ArB 0140, which could provide comprehensive information on a psychrophilic mechanism in extreme environments.

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.

Reduced susceptibility of Moritella profunda dihydrofolate reductase to trimethoprim is not due to glutamate 28.

The E28D variant of dihydrofolate reductase from Moritella profunda was generated and found to have the same K (i) (within error) for the competitive inhibitor trimethoprim as the wild type enzyme. Contrary to a previous claim in the literature, Glu 28 is therefore not the cause of the reduced affinity for trimethoprim relative to dihydrofolate reductase from Escherichia coli.