Marine Pseudoalteromonas Research Articles

Characterization of β-N-acetylglucosaminidase from a marine Pseudoalteromonas sp. for application in N-acetyl-glucosamine production

ABSTRACTThe psychrotolerant Pseudoalteromonas issachenkonii PAMC 22718 was isolated for its high exo-acting chitinase activity in the Kara Sea, Arctic. An exo-acting chitinase (W-Chi22718) was homogeneously purified from the culture supernatant of PAMC 22718, the molecular weight of which was estimated to be approximately 112 kDa. Due to its β-N-acetylglucosaminidase activity, W-Chi22718 was able to produce N-acetyl-D-glucosamine (GlcNAc) monomers from chitin oligosaccharide substrates. W-Chi22718 displayed chitinase activity from 0 to 37°C (optimal temperature of 30°C) and maintained activity from pH 6.0 to 9.0 (optimal pH of 7.6). W-Chi22718 exhibited a relative activity of 13 and 35% of maximal activity at 0 and 10°C, respectively, which is comparable to the activities of previously characterized, cold-adapted bacterial chitinases. W-Chi22718 activity was enhanced by K+, Ca2+, and Fe2+, but completely inhibited by Cu2+ and SDS. We found that W-Chi22718 can produce much more (GlcNAcs) from colloidal chitin, working together with previously characterized cold-active endochitinase W-Chi21702. Genome sequencing revealed that the corresponding gene (chi22718_IV) was 2,856 bp encoding a 951 amino acid protein with a calculated molecular weight of approximately 102 kDa.

Screening of bromotyramine analogues as antifouling compounds against marine bacteria

Rapid and efficient synthesis of 23 analogues inspired by bromotyramine derivatives, marine natural products, by means of CuSO4-catalysed [3+2] alkyne–azide cycloaddition is described. The final target was then assayed for anti-biofilm activity against three Gram-negative marine bacteria, Pseudoalteromonas ulvae (TC14), Pseudoalteromonas lipolytica (TC8) and Paracoccus sp. (4M6). Most of the synthesised bromotyramine/triazole derivatives are more active than the parent natural products Moloka’iamine (A) and 3,5-dibromo-4-methoxy-β-phenethylamine (B) against biofilm formation by the three bacterial strains. Some of these compounds were shown to act as non-toxic inhibitors of biofilm development with EC50 < 200 μM without any effect on bacterial growth even at high concentrations (200 μM).

Characterization of a New Cold-Adapted and Salt-Activated Polysaccharide Lyase Family 7 Alginate Lyase from Pseudoalteromonas sp. SM0524.

Marine bacterial alginate lyases play a role in marine alginate degradation and carbon cycling. Although a large number of alginate lyases have been characterized, reports on alginate lyases with special characteristics are still rather less. Here, a gene alyPM encoding an alginate lyase of polysaccharide lyase family 7 (PL7) was cloned from marine Pseudoalteromonas sp. SM0524 and expressed in Escherichia coli. AlyPM shows 41% sequence identity to characterized alginate lyases, indicating that AlyPM is a new PL7 enzyme. The optimal pH for AlyPM activity was 8.5. AlyPM showed the highest activity at 30°C and remained 19% of the highest activity at 5°C. AlyPM was unstable at temperatures above 30°C and had a low Tm of 37°C. These data indicate that AlyPM is a cold-adapted enzyme. Moreover, AlyPM is a salt-activated enzyme. AlyPM activity in 0.5–1.2 M NaCl was sixfolds higher than that in 0 M NaCl, probably caused by a significant increase in substrate affinity, because the Km of AlyPM in 0.5 M NaCl decreased more than 20-folds than that in 0 M NaCl. AlyPM preferably degraded polymannuronate and mainly released dimers and trimers. These data indicate that AlyPM is a new PL7 endo-alginate lyase with special characteristics.

극지해양 Pseudoalteromonas 유래의 소형 플라스미드에 기반한 Pseudoalteromonas - Escherichia coli 셔틀벡터 제작

A small plasmid (pDK4) from the Antarctic marine organism Pseudoalteromonas sp. PAMC 21150, was purified, sequenced and analyzed. pDK4 was determined to be 3,480 bp in length with a G+C content of 41.64% and contains three open reading frames encoding a replication initiation protein (RepA), a conjugative mobilization protein (Mob) and a hypothetical protein. PCR-amplified pDK4 was cloned in high-copy pUC19 to yield the fusion vector pDOC153. The chloramphenicol resistance gene was inserted into pDOC153 to give an ampicillin and chloramphenicol-resistant, Pseudoalteromonas – Escherichia coli shuttle vector (7,216 bp; pDOC155). The TonB-dependent receptor (chi22718_IV ) and exochitinase (chi22718_III ) genes from Arctic marine P. issachenkonii PAMC 22718 were cloned into pDOC155 to produce pDOC158 and pDOC165, respectively. Both vector derivatives were transferred into plasmid-free Pseudoalteromonas sp. PAMC 22137 by the triparental mating method. PCR experiments showed that the genes were stably maintained both in Pseudoalteromonas sp. PAMC 22137 and E. coli DH5α cells, indicating the potential use of pDOC155 as a new gene transfer system into marine Pseudoalteromonas spp.

Exopolysaccharide production by a marine Pseudoalteromonas sp. strain isolated from Madeira Archipelago ocean sediments

Exopolysaccharides (EPS) are polymers excreted by some microorganisms with interesting properties and used in many industrial applications. A new Pseudoalteromonas sp. strain, MD12-642, was isolated from marine sediments and cultivated in bioreactor in saline culture medium containing glucose as carbon source. Its ability to produce EPS under saline conditions was demonstrated reaching an EPS production of 4.4g/L within 17hours of cultivation, corresponding to a volumetric productivity of 0.25g/Lh, the highest value so far obtained for Pseudoalteromonas sp. strains. The compositional analysis of the EPS revealed the presence of galacturonic acid (41-42mol%), glucuronic acid (25-26mol%), rhamnose (16-22mol%) and glucosamine (12-16mol%) sugar residues. The polymer presents a high molecular weight (above 1000kDa). These results encourage the biotechnological exploitation of strain MD12-642 for the production of valuable EPS with unique composition, using saline by-products/wastes as feedstocks.

제주 조간대로부터 단백질 가수분해효소를 생산하는 세균의 분리 및 동정

Eleven protease-producing bacteria were isolated from the organisms’ external shells and the inorganic materials collected from intertidal zone of Jeju Island, Republic of Korea. The samples were diluted serially, inoculated on Zobell agar plates with 1% skim milk and incubated at 20°C. Clear zone forming colonies were selected as protease-producing bacteria and each strain was identified based on the phylogenetic analysis with their 16S rDNA sequences. Strains JJM125, JJM129, YG47 and YG49 belong to the marine bacterial genus Pseudoalteromonas; strain JJM122 belong to the genus Microbulbifer; strains YG51, YG52, YG62 and YG63 belong to the genus Vibrio; and strain YG65 belong to genus Bacillus. Hence, the present study suggests that these protease producing bacteria could be further used to develop new varieties of protease with various biotechnological applications.

Marine bacterial transparent exopolymer particles (TEP) and TEP precursors: Characterization and RO fouling potential

This paper investigated the characteristics and membrane fouling potential of bacterial transparent exopolymer particles (TEP)/TEP precursors released from two marine bacteria, Pseudidiomarina homiensis (P. homiensis) and Pseudoalteromonas atlantica (P. atlantica), isolated from the Red Sea. Results showed that both bacteria grew at the similar rate, but the production of TEP/TEP precursors from P. atlantica was higher than that from P. homiensis. During the 168h of incubation time, production rates of TEP/TEP precursors from P. atlantica and P. homiensis were 0.30 and 0.08 xanthan gum eq. mg/L-h, respectively. Isolated bacterial TEP precursors were mainly biopolymer, and P. atlantica produced a significantly higher concentration of biopolymer than that produced by P. homiensis. TEP/TEP precursors from both marine bacteria possessed protein-like material and were very similar in composition to previously reported foulants isolated from a fouled reverse osmosis (RO) membrane. Bacterial TEP/TEP precursors mostly consisted of aliphatic hydrocarbon from amino acids and amide group carbon of proteins (around 55%). Bacterial TEP precursors caused obvious fouling on RO membranes, which may create an ideal environment for bacteria attachment and promote to biofouling.

Life-style and genome structure of marine Pseudoalteromonas siphovirus B8b isolated from the northwestern Mediterranean Sea.

Marine viruses (phages) alter bacterial diversity and evolution with impacts on marine biogeochemical cycles, and yet few well-developed model systems limit opportunities for hypothesis testing. Here we isolate phage B8b from the Mediterranean Sea using Pseudoalteromonas sp. QC-44 as a host and characterize it using myriad techniques. Morphologically, phage B8b was classified as a member of the Siphoviridae family. One-step growth analyses showed that this siphovirus had a latent period of 70 min and released 172 new viral particles per cell. Host range analysis against 89 bacterial host strains revealed that phage B8b infected 3 Pseudoalteromonas strains (52 tested, >99.9% 16S rRNA gene nucleotide identity) and 1 non-Pseudoaltermonas strain belonging to Alteromonas sp. (37 strains from 6 genera tested), which helps bound the phylogenetic distance possible in a phage-mediated horizontal gene transfer event. The Pseudoalteromonas phage B8b genome size was 42.7 kb, with clear structural and replication modules where the former were delineated leveraging identification of 16 structural genes by virion structural proteomics, only 4 of which had any similarity to known structural proteins. In nature, this phage was common in coastal marine environments in both photic and aphotic layers (found in 26.5% of available viral metagenomes), but not abundant in any sample (average per sample abundance was 0.65% of the reads). Together these data improve our understanding of siphoviruses in nature, and provide foundational information for a new ‘rare virosphere’ phage–host model system.

A novel family 19 chitinase from the marine-derived Pseudoalteromonas tunicata CCUG 44952T: Heterologous expression, characterization and antifungal activity

The Ptchi19 gene of the marine Pseudoalteromonas tunicata CCUG 44952T was cloned and expressed in Escherichia coli. The recombinant chitinase PtChi19p of 483 amino acids has a molecular weight of 53.5kDa and a multi-domain structure characteristic of family 19 chitinases. The relevant constituents of this multi-domain structure are the domain (D132–A155) where the active site is located, and the domain (A437–W479) that includes a C-terminal carbohydrate-binding module 5. The purified protein was active in the temperature range of 20–50°C and at pH values of 6–9.5, maintaining a high stability under suboptimal conditions and in the presence of different metal ions. The recombinant enzyme hydrolyzed colloidal and crystalline chitin, as well as p-NP N-acetyl-β-d-glucosaminide. As PtChi19p exhibited antifungal activity against phytopathogenic and human pathogenic fungi, it could be used as an alternative biofungicide.

Effect of marine Pseudoalteromonas sp. on the microstructure and corrosion behaviour of 2205 duplex stainless steel

The effects of isolated marine Pseudoalteromonas sp. on the microstructure and corrosion behaviour of 2205 duplex stainless steel were investigated using electrochemical and surface analysis methods. Electrochemical studies demonstrated a negative shift in corrosion potential and an increase in corrosion current density in the presence of bacteria. EDS results showed a high concentration of chloride ions in the biofilm structure and a decrease in Cr content beneath the biofilm layer and near cracks. These results could lead to localised corrosion on metal surfaces. FESEM images illustrated the process of bacterial attachment on the metal surfaces at different exposure times.

Flavonoids from the halophyte Apocynum venetum and their antifouling activities against marine biofilm-derived bacteria

Eleven flavonoids were isolated from the leaves of the halophyte Apocynum venetum. Among them, the isolation of plumbocatechin A (1), 8-O-methylretusin (2) and kaempferol 3-O-(6″-O-acetyl)-β-d-galactopyranoside (7) was reported for the first time from this plant. Their structures were identified by using spectral methods, including 2D NMR experiments, and confirmed by comparing with the literature data. In addition, the antifouling activities of these compounds against the marine fouling bacteria, Bacillus thuringiensis, Pseudoalteromonas elyakovii and Pseudomonas aeruginosa, have been evaluated in this article.

Efficient preparation of pseudoalteromone A from marine Pseudoalteromonas rubra QD1-2 by combination of response surface methodology and high-speed counter-current chromatography: a comparison with high-performance liquid chromatography

Pseudoalteromone A (PA) is a cytotoxic and anti-inflammatory ubiquinone discovered recently from a marine bacterium Pseudoalteromonas sp. CGH2XX. In order to meet its sample supply for further in vivo pharmacological investigation, an efficient method was developed for the preparation of PA by combination of response surface methodology (RSM) and high-speed counter-current chromatography (HSCCC) from marine bacterium P. rubra QD1-2. First, optimization of culture conditions was studied by the RSM to enhance PA production. The results indicated that the optimal cultivation condition was peptone (2.21 g/l), yeast extract (3.125 g/l), glucose (0.125 g/l), KBr (0.02 g/l), inoculum size (6.5 %), medium volume (595 ml), initial pH value (7.0), temperature (28 °C). Under the optimized fermentation condition, PA production was 1.04 mg/l with 14.8-fold increase comparing to 0.07 mg/l under original standard fermentation condition. The PA production was further investigated using a 14-l jar fermenter. Compared to the flask culture, P. rubra QD1-2 offered 45 % increase of PA production at 1.51 mg/l. Then, a rapid and efficient method for the separation and purification of PA from crude culture extract was developed using HSCCC. The two-phase solvent system used for HSCCC separation was composed of n-hexane-ethyl acetate-methanol-water (5:5:9:5, v/v/v/v). The isolation was accomplished within 100 min, and the purity of PA was over 95 %. The recovery of the process was 93 %.

An Extra Peptide within the Catalytic Module of a β-Agarase Affects the Agarose Degradation Pattern

Agarase hydrolyzes agarose into a series of oligosaccharides with repeating disaccharide units. The glycoside hydrolase (GH) module of agarase is known to be responsible for its catalytic activity. However, variations in the composition of the GH module and its effects on enzymatic functions have been minimally elucidated. The agaG4 gene, cloned from the genome of the agarolytic Flammeovirga strain MY04, encodes a 503-amino acid protein, AgaG4. Compared with elucidated agarases, AgaG4 contains an extra peptide (Asn(246)-Gly(302)) within its GH module. Heterologously expressed AgaG4 (recombinant AgaG4; rAgaG4) was determined to be an endo-type β-agarase. The protein degraded agarose into neoagarotetraose and neoagarohexaose at a final molar ratio of 1.5:1. Neoagarooctaose was the smallest substrate for rAgaG4, whereas neoagarotetraose was the minimal degradation product. Removing the extra fragment from the GH module led to the inability of the mutant (rAgaG4-T57) to degrade neoagarooctaose, and the final degradation products of agarose by the truncated protein were neoagarotetraose, neoagarohexaose, and neoagarooctaose at a final molar ratio of 2.7:2.8:1. The optimal temperature for agarose degradation also decreased to 40 °C for this mutant. Bioinformatic analysis suggested that tyrosine 276 within the extra fragment was a candidate active site residue for the enzymatic activity. Site-swapping experiments of Tyr(276) to 19 various other amino acids demonstrated that the characteristics of this residue were crucial for the AgaG4 degradation of agarose and the cleavage pattern of substrate.

Analysis of Extracellular Alginate Lyase (alyA) Expression and its Regulatory Region in a Marine Bacterial Strain, Pseudoalteromonas atlantica AR06, Using a gfp Gene Reporter System

Extracellular alginate lyase (alyA) has an important role in the use of alginate in the marine bacterial strain Pseudoalteromonas atlantica AR06. Green fluorescent protein (GFP) is a convenient, useful tool for visualization of gene expression, and here we introduced the gfp gene at the end of alyA by homologous recombination in AR06. The gfp gene was expressed as a polycistronic transcript in reporter strain ARAG (AR06 alyA-gfp). The analysis of GFP fluorescence of ARAG in minimal medium containing a carbon source (sucrose, mannitol, glucose, or glucuronate) revealed that the alyA was induced by alginate and regulated by carbon catabolite repression by the coexistence of each carbon substrate (sucrose, mannitol, and glucose). The transcription start site of alyA was identified 192bp upstream from translation start site of alyA and the 10-bp sequence was a palindrome in which the transcription start site [G] was at the end. Thirteen kinds of plasmids were constructed for promoter analysis of alyA, which contains between 215 and 618bp of upstream sequence from the translation start and gfp. Longer sequences than 339bp were capable to increase the GFP fluorescence responding to alginate in the homologous recombination alyA deletion mutant strain ARAΔ (AR06 ΔalyA). The 339-bp upstream sequence was proved sufficiency for reproduction of the catabolite repression of alyA. These results indicate that (a) transcription of alyA is probably regulated by substrate recognition systems driven by multiple factors and (b) regulation of alyA requires a cis element on the upstream region of alyA.

Genome Sequences of Type Strains of Seven Species of the Marine Bacterium Pseudoalteromonas

There are over 30 species in the marine bacterial genus Pseudoalteromonas. However, our knowledge about this genus is still limited. We sequenced the genomes of type strains of seven species in the genus, facilitating the study of the physiology, adaptation, and evolution of this genus.

The anti-biofilm activity secreted by a marine Pseudoalteromonas strain

Bacterial biofilms occur on all submerged structures in marine environments. The authors previously reported that the marine bacterium Pseudoalteromonas sp. 3J6 secretes antibiofilm activity. Here, it was discovered that another Pseudoalteromonas sp. strain, D41, inhibited the development of strain 3J6 in mixed biofilms. Confocal laser scanning microscope observations revealed that the culture supernatant of strain D41 impaired biofilm formation of strain 3J6 and another marine bacterium. A microtiter plate assay of the antibiofilm activity was set up and validated with culture supernatants of Pseudoalteromonas sp. 3J6. This assay was used to determine the spectra of action of strains D41 and 3J6. Each culture supernatant impaired the biofilm development of 13 marine bacteria out of 18. However, differences in the spectra of action and the physical behaviours of the antibiofilm molecules suggest that the latter are not identical. They nevertheless share the originality of being devoid of antibacterial activity against planktonic bacteria.

Monitoring of microbial changes in salted cabbage (Jeolimbaechu) during recycled brining operation

To investigate possible recycling of a brine solution for the production of salted cabbage (jeolimbaechu), bacterial cell counts in the brine solution and salted cabbages were measured after repeated salting processes. After a salting process with a 13% brine solution for 15 hr at 10°C, the salt concentration in brine and salted cabbage decreased to 9.0–9.9 and 1.5–3.2% (depending on parts), respectively. After 5 repetitions, the total microbial count in the brine solution and salted cabbage gradually increased to 8.9 and 8.7 log CFU/g, respectively. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCRDGGE) showed that the marine bacteria, Pseudoalteromonas sp. and Halomonas sp. appeared from the 3rd batch; their intensities increased steadily and dominated the recycled brine solution. Meanwhile, Lactobacillus sakei found in the cabbage during the first salting process decreased with successive repetitions. These results show a high risk of marine microbial contamination in salted cabbage when the brine solution is re-used. Therefore, to re-use brine solution, application of proper inhibitory methods to prevent microbial cell growth in the brine solution is required.

Highly Brominated Antimicrobial Metabolites from a MarinePseudoalteromonassp.

Extracts of a marine Pseudoalteromonas sp. (CMMED 290) isolated from the surface of a nudibranch collected in Kaneohe Bay, Oahu, displayed significant antimicrobial activity against methicillin-resistant Staphylococcus aureus. Bioassay-guided fractionation of the lipophilic extract led to the isolation and structure elucidation of two new highly brominated compounds, 2,3,5,7-tetrabromobenzofuro[3,2-b]pyrrole (1) and 4,4',6-tribromo-2,2'-biphenol (2). In addition, we have identified the known compounds pentabromopseudilin and bromophene. We describe the isolation and structure elucidation of the compounds 1 and 2 together with their antimicrobial activities against methicillin-resistant Staphylococcus aureus.

Ecogenomics and genome landscapes of marine Pseudoalteromonas phage H105/1

Marine phages have an astounding global abundance and ecological impact. However, little knowledge is derived from phage genomes, as most of the open reading frames in their small genomes are unknown, novel proteins. To infer potential functional and ecological relevance of sequenced marine Pseudoalteromonas phage H105/1, two strategies were used. First, similarity searches were extended to include six viral and bacterial metagenomes paired with their respective environmental contextual data. This approach revealed 'ecogenomic' patterns of Pseudoalteromonas phage H105/1, such as its estuarine origin. Second, intrinsic genome signatures (phylogenetic, codon adaptation and tetranucleotide (tetra) frequencies) were evaluated on a resolved intra-genomic level to shed light on the evolution of phage functional modules. On the basis of differential codon adaptation of Phage H105/1 proteins to the sequenced Pseudoalteromonas spp., regions of the phage genome with the most 'host'-adapted proteins also have the strongest bacterial tetra signature, whereas the least 'host'-adapted proteins have the strongest phage tetra signature. Such a pattern may reflect the evolutionary history of the respective phage proteins and functional modules. Finally, analysis of the structural proteome identified seven proteins that make up the mature virion, four of which were previously unknown. This integrated approach combines both novel and classical strategies and serves as a model to elucidate ecological inferences and evolutionary relationships from phage genomes that typically abound with unknown gene content.

ChemInform Abstract: The Absolute Configuration and Total Synthesis of Korormicin.

The marine antibiotic korormicin, isolated from the culture filtrate of marine bacterial strain Pseudoalteromonas sp. F-420, specifically inhibits the growth of marine Gram-negative bacteria without affecting terrestrial species. The absolute configuration of korormicin was determined by the combination of a CD exciton chirality method and chemical degradation. Convergent total synthesis of koromicin has been also achieved.