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.

Growth condition optimization for docosahexaenoic acid (DHA) production by Moritella marina MP-1.

The marine organism Moritella marina MP-1 produces the polyunsaturated fatty acid docosahexaenoic acid (DHA). While the basic metabolic pathway for DHA production in this organism has been identified, the impact of growth conditions on DHA production is largely unknown. This study examines the effect of supplemental carbon, nitrogen and salts, growth temperature and media composition and pH on DHA and biomass production and the fatty acid profile. The addition of supplemental nitrogen significantly increased the overall DHA titer via an increase in biomass production. Supplemental glucose or glycerol increased biomass production, but decreased the amount of DHA per biomass, resulting in no net change in the DHA titer. Acidification of the baseline media pH to 6.0 increased DHA per biomass. Changes in growth temperature or provision of supplemental sodium or magnesium chloride did not increase DHA titer. This organism was also shown to grow on defined minimal media. For both media types, glycerol enabled more DHA production per biomass than glucose. Combination of these growth findings into marine broth supplemented with glycerol, yeast extract, and tryptone at pH 6.0 resulted in a final titer of 82±5 mg/L, a nearly eightfold increase relative to the titer of 11±1 mg/L seen in the unsupplemented marine broth. The relative distribution of other fatty acids was relatively robust to growth condition, but the presence of glycerol resulted in a significant increase in myristic acid (C14:0) and decrease in palmitic acid (C16:0). In summary, DHA production by M. marina MP-1 can be increased more than fivefold by changing the growth media. Metabolic engineering of this organism to increase the amount of DHA produced per biomass could result in additional increases in titer.

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.

Adaptive response to environmental changes in the fish pathogen Moritella viscosa.

The marine psychrophilic bacterium Moritella viscosa is the causative agent of winter ulcer in farmed Atlantic salmon and cod. In this study, the growth requirements of the pathogen were established. The effects of changes in salinity and temperature on growth, surface features and proteomic regulation were also investigated. The genome of this bacterium has not yet been sequenced; therefore, comparative two-dimensional gel electrophoresis (2-DE) was used, coupled with high performance tandem mass spectrometry (MS/MS), to perform cross-species protein identification. Results from this study establish that M. viscosa is a true marine psychrophilic bacterium capable of surviving and proliferating in an oligotrophic and cold environment. Low temperature combined with 3-4% NaCl resulted in significantly higher cell yields and stability compared to high temperature and 1% NaCl. Nine cytoplasmic proteins were shown to be regulated by temperature and 12 by salinity. Several of the regulated proteins indicated a stressful situation at 15 degrees C compared to 4 degrees C, consistent with the growth characteristics observed. Furthermore, temperature and salinity were demonstrated to be important determinants of motility and viscosity of M. viscosa.

Cultivation of microorganisms in the cultural medium made from squid internal organs and accumulation of polyunsaturated fatty acids in the cells.

The disposal and more efficient utilization of marine wastes is becoming increasingly serious. A culture media for microorganisms has been prepared from squid internal organs that are rich in polyunsaturated fatty acids (PUFAs). Both freshwater and marine bacteria grew well in this medium and some bacteria accumulated PUFAs in their lipids, suggesting uptake of exogenous PUFAs. Higher PUFA accumulations were observed in Escherichia coli mutant cells defective either in unsaturated fatty acid biosynthesis or fatty acid degradation, or both, compared to those without these mutations. Therefore, PUFA accumulation in cells can be improved by genetic modification of fatty acid metabolism in the bacteria. Squid internal organs would be a good source of medium, not only for marine bacteria but also for freshwater bacteria, and that this process may be advantageous to make efficient use of the fishery wastes and to produce PUFA-containing microbial cells and lipids.

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria.

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 microg cerulenin ml-1, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the content of DHA in the total fatty acids increased from 5.9-19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l-1 by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 microg cerulenin ml-1, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l-1. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.