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.

Impact of co-infection with Lepeophtheirus salmonis and Moritella viscosa on inflammatory and immune responses of Atlantic salmon (Salmo salar).

This study was conducted to determine the effects of a co-infection with Moritella viscosa at different exposure levels of sea lice Lepeophtheirus salmonis in Atlantic salmon (Salmo salar). M. viscosa (1.14 × 106  cfu/ml) was introduced to all experimental tanks at 10 days post-lice infection (dpLs). Mean lice counts decreased over time in both the medium lice co-infection (31.5 ± 19.0 at 7 dpLs; 16.9 ± 9.3 at 46 dpLs) and high lice co-infection (62.0 ± 10.8 at 7 dpLs; 37.6 ± 11.3 at 46 dpLs). There were significantly higher mortalities and more severe skin lesions in the high lice co-infected group compared to medium lice co-infected group or M. viscosa-only infection. Quantitative gene expression analysis detected a significant upregulation of genes in skin from the high lice co-infection group consistent with severe inflammation (il-8, mmp-9, hep, saa). Skin lesions retrieved throughout the study were positive for M. viscosa growth, but these were rarely located in regions associated with lice. These results suggest that while M. viscosa infection itself may induce skin lesion development in salmon, co-infection with high numbers of lice can enhance this impact and significantly reduce the ability of these lesions to resolve, resulting in increased mortality.

The transcriptional response of the Pacific oyster Crassostrea gigas under simultaneous bacterial and heat stresses.

Bacterial infection and heat stress are considered as two major environmental threats for the aquaculture industry of oyster Crassostrea gigas. In the present study, the expression profiles of mRNA transcripts in the hemocytes of oysters under bacterial challenge and heat stress were examined by next-generation sequencing. There were 21,095, 21,957 and 21,141 transcripts identified in the hemocytes of oysters from three groups, respectively, including control group (designated as Con group), Vibrio splendidus challenge group (Bac group), and bacterial and heat stress combined treatment group (BacHeat group). There were 4610, 5093 and 5149 differentially expressed transcripts (DTs) in the three pairwise comparisons Con/Bac, Con/BacHeat and Bac/BacHeat, respectively. The main enriched GO terms in biological process category of the DTs included the metabolic processes, cellular process, response to stimulus and immune system process. The expression patterns of DTs involved in pattern recognition, immune signal transduction and energy metabolic indicated that the immune response to bacterial challenge was disturbed under acute heat stress, which was also confirmed by quantitative real-time PCR. The neuroendocrine immunomodulation, especially the catecholaminergic regulation, played indispensable roles in stress response. The total energy reserves as well as cellular energy allocation (CEA) in hepatopancreas of oysters decreased remarkably especially in BacHeat group, while the energy consumption generally increased, suggesting that the immune defense against the simultaneous stimulation of pathogen and heat stress imposed greater costs on oyster's energy expenditure than a single stressor. These results above indicated that, the heat stress disturbed the normal expression of genes involved in immune response and energy metabolism, accelerated energy consumption and broke the metabolic balance, leading to a decline in resilience to infection and mass mortality of oyster in summer.

Moritella viscosa in lumpfish (Cyclopterus lumpus) and Atlantic salmon (Salmo salar).

Winter ulcer disease, caused by Moritella viscosa, is a significant problem in cold water salmonid farming, although the bacterium can infect and cause disease in a number of other fish species, such as lumpfish (Cyclopterus lumpus). Lumpfish are used as cleaner fish, to eat sea lice from Atlantic salmon (Salmo salar) in sea pens. It remains to be established whether M. viscosa can be transmitted between the fish species. In this study, we examined whether a salmon isolate of M. viscosa could infect and cause disease in lumpfish. We further examined whether a lumpfish isolate of M. viscosa could infect and cause disease in salmon. Finally, we examined whether vaccination of salmon with a salmon isolate of M. viscosa conferred protection against a lumpfish isolate. The data indicate that while lumpfish appeared to be resistant to a salmon isolate of M. viscosa, the salmon could be infected with a lumpfish isolate of M. viscosa. Vaccination protected the salmon against the salmon isolate of M. viscosa but did not confer sufficient protection to prevent infection with the lumpfish isolate.

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.

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.

Co-infection of Atlantic salmon (Salmo salar), by Moritella viscosa and Aliivibrio wodanis, development of disease and host colonization.

Two species of bacteria are repeatedly isolated from farmed fish with winter-ulcer disease. Moritella viscosa is the aetiological agent of the disease; the significance of Aliivibrio wodanis is uncertain but has not been related to the primary pathogenesis. A cell culture infection model showed that A. wodanis adhered to, but did not invade the fish cells. Exposure to culture supernatant of A. wodanis caused the fish cells to vacoulate, retract, round up and detach from the surface, and rearrange the actin filaments of the cytoskeleton. These observations suggest that the bacterium secretes toxins into the extracellular environment. Any pathologic effect of A. wodanis and the effect of co-culturing with M. viscosa was studied in Atlantic salmon (Salmo salar) bath challenged with; only M. viscosa or only A. wodanis or both bacteria together. Both M. viscosa and A. wodanis were re-isolated from external surfaces and internal organs from live and deceased co-infected fish. It is further hypothesized that A. wodanis colonization might influence the progression of a M. viscosa infection. This is to our knowledge the first study that reproduces field observations where both bacteria infect Atlantic salmon.

Moritella viscosa bypasses Atlantic salmon epidermal keratocyte clearing activity and might use skin surfaces as a port of infection.

Moritella viscosa is considered the main causative agent of winter ulcer disease in salmonid fish. In order to obtain more details on route of infection, we challenged Atlantic salmon (Salmo salar) epidermal keratocytes with M. viscosa and performed an Atlantic salmon immersion challenge. Although keratocytes were able to remove M. viscosa from surfaces, their engulfment capability appeared inefficient with reduced ability to reepithelialise superficial wounds (scale less skin surfaces) challenged with the bacterium. The immersion challenge revealed a significant connection between exposure area and mortality. Enhanced invasion ability and mortality was observed by M. viscosa exposure of the head and gill region compared to exposure of: the right side of the body; the left side of the body; or the body from pectoral to caudal fin (p=0.04). Ulcer development corresponded to area exposed (p=0.002), suggesting skin ulcer formation to result primarily from direct skin surface colonization. Ulceration of surfaces exposed to M. viscosa in parallel with occurrence of septicaemia suggests that both skin and gills may act as possible initiation sites for M. viscosa infections.

Inflammatory and regenerative responses in salmonids following mechanical tissue damage and natural infection.

Locale responses in muscle tissue against either a sterile tissue damage or infection were compared in salmonid fish in order to examine the inflammatory responses and regeneration of tissue. From higher vertebrates both damage and infection are known to cause inflammation since DAMPs released from injured cells as well as PAMPs from the surface of pathogens are immunogenic. To examine this in salmonid fishes, Atlantic salmon (Salmo salar) were infected with Moritella viscosus, the causative agent of winter ulcer. Muscle tissue was sampled from infected fish at 4, 7 and 14 days post infection. Samples were obtained from site of lesions and from locations without clinical signs of disease and lesions. The tissue damage was performed in rainbow trout (Oncorhynchus mykiss) by applying sterile needles to skin and muscle tissue to one side of the fish. Samples were taken 7, 14, 21, 28 and 42 days post injury from the injured side and non-injured site (internal control). From both infected and damaged fish, samples were subject to real-time RT-PCR for measuring the expression of IL-1beta, IL-8, IL-10, Hsp70, iNOS, TGF-beta, TLR-5m, TLR-9, TLR-22, TGF-beta, MMP-2, CTGF, myostatin-1alphabeta and collagen-1alpha which are coding for immunological factors and tissue regeneration. Locale, inflammatory responses were seen as strong up-regulation of IL-1beta and IL-8 in both groups of fish, but it was more pronounced in infected fish. Expression of the toll-like receptors showed induction of TLR-5m following infection, but TLR-9 and TLR-22 following damage. Further, in both studies the regenerative genes TGF-beta, MMP-2, CTGF, myostatin-1alphabeta were induced, but showed different kinetics. Collagen-1alpha was only induced in infected fish, probably due to heavier tissue damage in these.

The winter ulcer bacterium Moritella viscosa demonstrates adhesion and cytotoxicity in a fish cell model.

Moritella viscosa is considered the main aetiological agent of 'winter ulcer' disease in farmed salmonid fish. To further understand the pathogenesis of this disease, M. viscosa interaction with fish cells was studied using a Chinook salmon embryo cell line (CHSE-214). As winter ulcer appears exclusively at temperatures below 7-8 degrees C, we attempted to identify if this connection is explained by temperature regulated bacterial virulence. Therefore, infection studies were performed at a temperature range from 4 to 15 degrees C. At all temperatures, M. viscosa caused CHSE cells to retract and round up, lose their attachment abilities and finally disintegrate. The bacterium adhered to CHSE cells and caused changes to the cytoskeleton, however, it did not invade the cells. Increased adherence was demonstrated at 4 degrees C compared to adherence at higher temperatures. Extracellular proteins exerted rapid pore formation and lysis of CHSE cells at a temperature range from 4 to 22 degrees C. Furthermore, only small differences were found comparing extracellular proteomes of M. viscosa from 4 and 15 degrees C. We propose that the pathogenic mechanisms exerted by M. viscosa on CHSE cells are disruption of the cytoskeleton which affects cell rigidity and structure, followed by pore formation and lysis caused by secreted products from the bacterium. These processes can also occur at temperatures above those experienced from winter ulcer outbreaks. However, the adhesion mechanisms appear to be temperature regulated and may contribute to temperature dependent disease outbreaks.

Cloning and expression of lipP, a gene encoding a cold-adapted lipase from Moritella sp.2-5-10-1.

A gene (lipP, 837 bp in length) coding for a cold-adapted lipase of psychrophilic bacterium Moritella sp. 2-5-10-1 isolated from Antarctic region was cloned and sequenced in this study. The deduced amino acid sequence revealed a protein of 278 amino acid residues with a molecular mass of 30,521. The primary structure of the lipase deduced from the nucleotide sequence showed consensus pentapeptide containing the active serine [Gly-Trp-Ser-Leu-Gly] and a conserved His-Gly dipeptide in the N-terminal part of the enzyme. These sequences were involved in the lipase active site conformation. Structure factors that would allow proper enzyme flexibility at low temperatures were discussed. It was suggested that the changes in the primary structure of the psychrophilic lipases compared to the thermophilic ones could account for their ability to catalyze lipolysis at temperatures close to 0 degrees C. For expression, the sequence corresponding to the cold-adapted lipase of strain 2-5-10-1 was subcloned into the pET-28a expression vector to construct a recombinant lipase protein. Expression of the lipase by Escherichia coli BL21 (DE3) cells was observed as clear halos on 1% (vol/vol) tributyrin upon induction with IPTG at 25 degrees C.

Growth and lysis of the fish pathogen Moritella viscosa.

AIMS: The sensitivity to lysis is a profound bottleneck to studies of the fish pathogen Moritella viscosa. The aim of this study was to examine the growth and the lysis process of M. viscosa cells under different physical and chemical conditions. METHODS AND RESULTS: Growth and cell lysis were studied under different conditions. The growth rate was highest at 15 degrees C and lowest at 4 degrees C, but the cells reached a higher density at 4 degrees than at 15 degrees C and the cells were more stable. The presence of minerals reduced lysis. CONCLUSIONS: Premature lysis of the cells is dependent on environmental factors. Moritella viscosa should be cultivated and kept in media containing a certain set of minerals and at temperatures as low as 4 degrees C. Formalin favours the stability of cells. The instability of the M. viscosa cells at temperatures above 10 degrees C might be one of the factors responsible for their inability to infect fish at higher temperatures. The presence of DHA in the cell membranes is predicted to be responsible for the susceptibility of the cells to lysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The cultivation of M. viscosa cells is a key factor in studying the pathogenicity of the bacteria and in making an effective vaccine to prevent winter ulcers in farmed fish. The study provided recommendations on how to cultivate M. viscosa and how lysis of the cells can be minimized.

High pressure nmr study of dihydrofolate reductase from a deep-sea bacterium Moritella profunda.

We have investigated the effect of pressure and temperature on the structural and thermodynamic stability of a protein dihydrofolate reductase from a deep-sea bacterium Moritella profunda in its folate-bound form in the pressure range between 3 and 375 MPa and the temperature range between -5 and 30 degrees C. The on-line cell variable pressure 1H NMR spectroscopy has been used to analyze the chemical shift and signal intensity in one-dimensional 1H NMR spectra. Thermodynamic analysis based on signal intensities from protons in the core part indicates that the thermodynamic stability of Moritella profunda DHFR is relatively low over the temperature range between -5 and 30 degrees C (deltaG0=15.8 +/- 4.1 kJ/mol at 15 degrees C), but is well adapted to the living environment of the bacterium (2 degrees C and 28 MPa), with the maximum stability around 5 degrees C (at 0.1 MPa) and a relatively small volume change upon unfolding (deltaV= 66 +/- 19 ml/mol). Despite the relatively low overall stability, the conformation in the core part of the folded protein remains intact up to approximately 200 MPa, showing marked stability of the core of this protein.

Differences in malate dehydrogenases from the obligately piezophilic deep-sea bacterium Moritella sp. strain 2D2 and the psychrophilic bacterium Moritella sp. strain 5710.

The gene encoding malate dehydrogenase (MDH) of the obligately piezophilic deep-sea bacterium Moritella sp. strain 2D2 was cloned and sequenced. There were two positions [close to the active site (Ala-180) and in the subunit interaction site (His-229)] with 2D2-specific substitutions. The MDH genes of strain 2D2 and a psychrophilic bacterium Moritella sp. strain 5710 exhibiting the highest sequence similarity were overexpressed in Escherichia coli. The 2D2 MDH was more heat-stable than the 5710 MDH. The apparent Km value at 62.1 MPa for NADH of the 2D2 MDH was higher than that of the 5710 MDH. The 2D2 MDH in which a His-Gln substitution was introduced at position 229 decreased the thermal stability and Km value at 62.1 MPa. The 5710 MDH that was substituted Gln-229 with His increased the thermal stability and Km value at 62.1 MPa. These results indicate that the His residue at position 229 of the 2D2 MDH may play a role in the thermal stability and the MDH function at high pressure.

Metabolic enzymes from psychrophilic bacteria: challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi.

The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCase(Mab)), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase(Mab) displays maximal activity at rather low temperatures (23 to 25 degrees C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCase(Mab) displays higher K(m) values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue delta-N-phosphonoacetyl-L-ornithine (PALO). OTCase(Mab) differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high K(m) values and the lower sensitivity to PALO. The K(m) for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (k(cat)/K(m)) of OTCases adapted to different temperatures showed that OTCase(Mab) activity remains suboptimal at low temperature despite the 4.5-fold decrease in the K(m) value for ornithine observed when the temperature is brought from 20 to 5 degrees C. OTCase(Mab) adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.