Whole genome sequencing and comparative genomic analyses of Planococcus alpniumensis MSAK28401T, a new species isolated from Antarctic krill.

BACKGROUND: Extremophiles have attracted much attention in the last few decades, as they possess different properties by producing certain useful metabolites. However, the secondary metabolism of the extremophiles of Antarctic krill has received little attention. RESULTS: In this study, a new bacterial strain MSAK28401T from Antarctic krill was isolated and identified. The results of analysis on phenotypic, chemotaxonomic, and genomic characteristics showed that the strain MSAK28401T belongs to the genus Planococcus. Cells of this strain were coccoid (0.89-1.05 µm) and aerobic. The majority of the fatty acid content was C15:0 anteiso (37.67 ± 0.90%) followed by C16:1 ω7c alcohol (10.37 ± 1.22%) and C16:0 iso (9.36 ± 0.71%). The calculated average nucleotide identity and DNA-DNA hybridization values between the strain MSAK28401T and type strains P. citreus DSM 20549T and P. rifietoensis M8T were lower than 91 and 70%, respectively. The strain MSAK28401T (=KCTC 43283T and MCCC 1k05448T) represented a new member of the genus Planococcus and was named P. alpniumensis sp. nov. Moreover, genes involved in the degradation of aromatic compounds (e.g., salicylate, gentisate, and quinate) were found in the genome, implying that strain MSAK28401T has an aromatic compound as its potential metabolite. This work will help us understand the genomic characteristics and potential metabolic pathway of Planococcus from Antarctic krill. CONCLUSIONS: This study reported the genomic information and phenotypic characteristics of the new strain P. alpniumensis MSAK28401T isolated from Antarctic krill, and provided the genome information of Planococcus strains for further studying the function roles in aromatic compound metabolism.

Diversity and function of the Antarctic krill microorganisms from Euphausia superba.

The diversity and ecological function of microorganisms associated with Euphausia superba, still remain unknown. This study identified 75 microbial isolates from E. superba, that is 42 fungi and 33 bacteria including eight actinobacteria. And all the isolates showed NaF tolerance in conformity with the nature of the fluoride krill. The maximum concentration was 10%, 3% and 0.5% NaF for actinobacteria, bacteria and fungi, respectively. The results demonstrated that 82.4% bacteria, 81.3% actinobacteria and 12.3% fungi produced antibacterial metabolites against pathogenic bacteria without NaF; the MIC value reached to 3.9 µg/mL. In addition, more than 60% fungi produced cytotoxic metabolites against A549, MCF-7 or K562 cell lines. The presence of NaF led to a reduction in the producing antimicrobial compounds, but stimulated the production of cytotoxic compounds. Furthermore, seven cytotoxic compounds were identified from the metabolites of Penicillium citrinum OUCMDZ4136 under 0.5% NaF, with the IC50 values of 3.6-13.1 µM for MCF-7, 2.2-19.8 µM for A549 and 5.4-15.4 µM for K562, respectively. These results indicated that the krill microbes exert their chemical defense by producing cytotoxic compounds to the mammalians and antibacterial compounds to inhibiting the pathogenic bacteria.

Characterization of a salt-activated protease with temperature-dependent secretion in Stenotrophomonas maltophilia FF11 isolated from frozen Antarctic krill.

Seafood is sometimes wasted due to the growth of psychrotolerant microbes which secrete proteases and break down proteins. Stenotrophomonas maltophilia FF11, isolated from frozen Antarctic krill, grows at a wide range of temperatures and secretes more proteases at low temperatures. According to zymogram analysis, two kinds of proteases were produced from this strain. A major protease was produced largely at 15 °C, but not at 37 °C. The temperature-dependent secreted protease was purified to homogeneity. Its molecular mass was determined at 37.4 kDa and its amino acid sequence was also obtained. This protease is a member of the subtilase group according to the NCBI blast analysis. The enzyme was highly stable at high salt concentration (4 M). Interestingly, its activity increased about 1.6-fold under high salt condition. The enzyme remains active and stable in different organic solvents (50 %, v/v) such as dimethylsulfoxide, dimethyl formamide, dioxane and acetone. These properties may provide potential applications in quality control for sea foods, in protein degradation at high salt concentration, in biocatalysis and biotransformation within non-aqueous media, such as detergent and transesterification.

Histophagous ciliate Pseudocollinia brintoni and bacterial assemblage interaction with krill Nyctiphanes simplex. I. Transmission process.

Histophagous ciliates of the genus Pseudocollinia cause epizootic events that kill adult female krill (Euphausiacea), but their mode of transmission is unknown. We compared 16S rRNA sequences of bacterial strains isolated from stomachs of healthy krill Nyctiphanes simplex specimens with sequences of bacterial isolates and sequences of natural bacterial communities from the hemocoel of N. simplex specimens infected with P. brintoni to determine possible transmission pathways. All P. brintoni endoparasitic life stages and the transmission tomite stage (outside the host) were associated with bacterial assemblages. 16S rRNA sequences from isolated bacterial strains showed that Photobacterium spp. and Pseudoalteromonas spp. were dominant members of the bacterial assemblages during all life phases of P. brintoni and potential pathobionts. They were apparently unaffected by the krill's immune system or the histophagous activity of P. brintoni. However, other bacterial strains were found only in certain P. brintoni life phases, indicating that as the infection progressed, microhabitat conditions and microbial interactions may have become unfavorable for some strains of bacteria. Trophic infection is the most parsimonious explanation for how P. brintoni infects krill. We estimated N. simplex vulnerability to P. brintoni infection during more than three-fourths of their life span, infecting mostly adult females. The ciliates have relatively high prevalence levels (albeit at <10% of sampled stations) and a short life cycle (estimated <7 d). Histophagous ciliate-krill interactions may occur in other krill species, particularly those that form dense swarms and attain high population densities that potentially enhance trophic transmission and allow completion of the Pseudocollinia spp. life cycle.

Histophagous ciliate Pseudocollinia brintoni and bacterial assemblage interaction with krill Nyctiphanes simplex. II. Host responses.

Unlike decapod crustaceans of commercial interest, the krill defense system and its response to parasites and pathogens is virtually unknown. Histophagous ciliates of the genus Pseudocollinia interact with at least 7 krill species in the northeastern Pacific. Although they can cause epizootic events, the physiology of the histophagous ciliate-host interaction and krill (host) defenses remain unknown. From 1 oceanographic survey along the southwestern coast of the Baja California Peninsula near Bahía Magdalena and 2 in the Gulf of California, we investigated parasitoid-host physiological responses (fatty acid and oxidative stress indicators) of the subtropical krill Nyctiphanes simplex infected with the ciliate P. brintoni. All life stages of P. brintoni were associated with opportunistic bacterial assemblages that have not been explicitly investigated in other Pseudocollinia species (P. beringensis, P. oregonensis, and P. similis). Parasitoid ciliates exclusively infected adult females, which showed increased lipid content during gonad development. As the infection progressed, omega-3 eicosapentaenoic and docosahexaenoic fatty acids, which may act as energy sources to produce high numbers of ciliate transmission stages, were quickly depleted. Antioxidant enzymes, components of the crustacean defense system, varied throughout infection, but without inhibiting Pseudocollinia infection, i.e. higher levels of lipid oxidative damage were detected in late stages of infection. The ineffective response of the krill antioxidant defense system against histophagous ciliates and the bacteria associated with the ciliates suggests that Pseudocollinia ciliates are functionally analogous to krill predators and may have a strong influence on the population dynamics of krill.

Purification and characterization of cellulase from North Pacific krill (Euphausia pacifica). Analysis of cleavage specificity of the enzyme.

Krill are filter feeders that consume algae, plankton and detritus, indicating that krill possess an adequate cellulose digesting system. However, less is known about the enzymatic properties of crustacean cellulases compared to termite cellulases. In the present study, 48 kDa-cellulase was highly purified from krill (Euphausia pacifica) in an effort to determine the cleavage specificity of the enzyme. The most notable characteristic of the enzyme was its high activity against both lichenan and carboxymethyl cellulose. The enzyme hydrolyzed internal ß-1,4 glycosidic bonds within lichenan as well as carboxymethyl cellulose to release oligosaccharides and glucose. The effects of pH and temperature on the activity and stability of both enzyme activities were almost identical. Cello-oligosaccharides with a degree of polymerization of 4-6 were hydrolyzed by the enzyme, and the same endo-products, cellotriose, cellobiose and glucose, were produced from these oligosaccharides. Neither cellotriose nor cellobiose was hydrolyzed by the enzyme. The enzyme digested filter paper and sea lettuce to produce cellobiose, cellotriose and glucose as major products. Although amino acid sequence homology of the enzyme with termite cellulases and the presence of oligosaccharides in the enzyme suggested that the enzyme is produced by krill itself, further analysis is necessary.

Molecular characterization of a cryptic plasmid from the psychrotrophic antarctic bacterium Pseudoalteromonas sp. 643A.

We report the identification and nucleotide sequence analysis of pKW1, a plasmid of the psychrotrophic bacterium Pseudoalteromonas sp. 643A isolated from the stomach of Antarctic krill Euphasia superba. pKW1 consists of 4583 bp, has a G+C content of 43% and seven putative open reading frames (ORFs). The deduced amino acid sequence from ORF-1 shared significant similarity with the plasmid replicase protein of Psychrobacter cryohalolentis, strain K5. The DNA region immediately downstream of the ORF-1 showed some homology with the Rep-binding sequence of the theta-replicating ColE2-type plasmids. The ORF-3 amino acid sequence revealed amino acid sequence homology with the mobilization protein of Psychrobacter sp. PRwf-1 and Moraxella catarrhalis, with identities of 28% and 25%, respectively. The ORF-4 showed 46% amino acid sequence homology with the putative relaxase/mobilization nuclease MobA of Hafnia alvei and 44% homology with the putative mobilization protein A of Pasterulla multocida. The copy number of pKW1 in Pseudoalteromonas sp. 643A was estimated of 15 copies per chromosome.

Histopathology of Antarctic krill, Euphausia superba, bearing black spots.

Small black spots have been noticed on the cephalothorax of Antarctic krill, Euphausia superba, since January, 2001. To study the nature of the black spots, the krill were sampled in the winter of 2003, 2006, and 2007 in the South Georgia region, the Antarctic Ocean. Histological observations revealed that the black spots were melanized nodules that were composed of hemocytes surrounding either bacteria or amorphous material. In the 2007 samples, 42% of the krill had melanized nodules. Most of the nodules had an opening on the body surface of the krill. A single melanized nodule often contained more than one type of morphologically distinct bacterial cell. Three bacteria were isolated from these black spots, and classified into either Psychrobacter or Pseudoalteromonas based on the sequences of 16S rRNA genes. More than three bacterial species or strains were also confirmed by in situ hybridization for 16S rRNA. The melanized nodules were almost always accompanied by a mass of atypical, large heteromorphic cells, which were not observed in apparently healthy krill. Unidentified parasites were observed in some of the krill that had melanized nodules. These parasites were directly surrounded by the large heteromorphic cells. Histological observations suggested that these heteromorphic cells were attacking the parasites. These results suggest the possibility that the krill had been initially affected by parasite infections, and the parasitized spots were secondary infected by environmental bacteria after the parasites had escaped from the host body.

A cold-adapted esterase from psychrotrophic Pseudoalteromas sp. strain 643A.

A psychrotrophic bacterium producing a cold-adapted esterase upon growth at low temperatures was isolated from the alimentary tract of Antarctic krill Euphasia superba Dana, and classified as Pseudoalteromonas sp. strain 643A. A genomic DNA library of strain 643A was introduced into Escherichia coli TOP10F', and screening on tributyrin-containing agar plates led to the isolation of esterase gene. The esterase gene (estA, 621 bp) encoded a protein (EstA) of 207 amino acid residues with molecular mass of 23,036 Da. Analysis of the amino acid sequence of EstA suggests that it is a member of the GDSL-lipolytic enzymes family. The purification and characterization of native EstA esterase were performed. The enzyme displayed 20-50% of maximum activity at 0-20 degrees C. The optimal temperature for EstA was 35 degrees C. EstA was stable between pH 9 and 11.5. The enzyme showed activity for esters of short- to medium-chain (C(4) and C(10)) fatty acids, and exhibited no activity for long-chain fatty acid esters like that of palmitate and stearate. EstA was strongly inhibited by phenylmethylsulfonyl fluoride, 2-mercaptoethanol, dithiothreitol and glutathione. Addition of selected divalent ions e.g. Mg(2+), Co(2+) and Cu(2+) led to the reduction of enzymatic activity and the enzyme was slightly activated ( approximately 30%) by Ca(2+) ions.

Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted beta-galactosidase.

The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 beta-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase, which efficiently hydrolyzes lactose at 0-20 degrees C, as indicated by its specific activity of 21-67 U mg(-1) of protein (11-35% of maximum activity) in this temperature range, as well as k(cat) of 157 s(-1), and k(cat)/K(m) of 47.5 mM(-1) s(-1) at 20 degrees C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 degrees C, thus below the optimum growth temperature of the bacterium (15 degrees C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40 degrees C (190 U mg(-1) of protein) and pH 6.0-8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The Pseudoalteromonas sp. 22b beta-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K(+), Na(+), Mn(2+)-40% increase, Mg(2+)-15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu(2+). Noteworthy, Ca(2+) ions do not affect the enzyme activity, and the homogeneous protein is stable at 4 degrees C for at least 30 days without any stabilizers.