Purification and characterization of antibacterial substances produced by a marine bacterium Pseudoalteromonas haloplanktis strain

Aims: To purify and characterize compounds with antimicrobial activity from Pseudoalteromonas haloplanktis inhibition (INH) strain. Methods and Results: The P. haloplanktis isolated from a scallop hatchery was used to analyse antibacterial activities. Crude extracts were obtained with ethyl acetate of the cultured broth, after separation of bacterial cells, and assays against six strains of marine bacteria and nine clinically important pathogenic bacteria. The active compounds were purified from ethyl acetate extracts, by a combination of SiO₂ column and thin layer chromatography. Two active fractions were isolated, and chemical structures of two products from the major one were unambiguously identified as isovaleric acid (3-methylbutanoic acid) and 2-methylbutyric acid (2-methylbutanoic acid), by comparing their mass spectra and ¹H- and ¹³C-nuclear magnetic resonance spectra to those of authentic compounds. Conclusions: In the antibacterial activity of P. haloplanktis INH strain, extra cell compounds are involucred, mainly isovaleric and 2-methylbutyric acids. Significance and Impact of the Study: Production of antimicrobial compounds by marine micro-organisms has been widely reported; however, the efforts not always are conducted to purification and applications of these active compounds. This study is a significant contribution to the knowledge of compounds unique from marine bacteria as potential sources of new drugs in the pharmacological industry.     

Characterization and anti-biofilm activity of extracellular polymeric substances produced by the marine biofilm-forming bacterium Pseudoalteromonas ulvae strain TC14

This study investigated soluble (Sol-EPS), loosely bound (LB-EPS), and tightly bound extracellular polymeric substances (TB-EPS) harvested from biofilm and planktonic cultures of the marine bacterium Pseudoalteromonas ulvae TC14. The aim of the characterization (colorimetric methods, FTIR, GC-MS, NMR, HPGPC, and AFM analyses) was to identify new anti-biofilm compounds; activity was assessed using the BioFilm Ring Test®. A step-wise separation of EPS was designed, based on differences in water-solubility and acidity. An acidic fraction was isolated from TB-EPS, which strongly inhibited biofilm formation by marine bacterial strains in a concentration-dependent manner. The main constituents of this fraction were characterized as two glucan-like polysaccharides. An active poly(glutamyl-glutamate) fraction was also recovered from TB-EPS. The distribution of these key EPS components in Sol-EPS, LB-EPS, and TB-EPS was distinct and differed quantitatively in biofilm vs planktonic cultures. The anti-biofilm potential of the fractions emphasizes the putative antifouling role of EPS in the environment.     

Inhibition of bacterial attachment by pulsed Nd:YAG laser irradiations: an in vitro study using marine biofilm-forming bacterium Pseudoalteromonas carrageenovora

The effect of low mean power laser irradiations with short pulse duration from an Nd:YAG (neodymium-doped yttrium aluminium garnet) laser on a marine biofilm-forming bacterium, Pseudoalteromonas carrageenovora, was investigated in the laboratory. Laser-irradiated bacteria were tested for their ability to attach on nontoxic titanium nitride (TiN) coupons with nonirradiated bacteria as the reference. Two durations of irradiation were tested, 10 and 15 min. Bacterial attachment was monitored after 20 min, 40 min, and 1 h of irradiation. The average laser fluence used for this study was 0.1 J/cm(2). The area of attachment of the irradiated bacteria was significantly less than the reference for both durations of irradiation. The growth of irradiated bacteria showed a longer lag phase than the nonirradiated sample, mainly due to mortality in the former. The bacterial mortality observed was 23.4 +/- 0.71 and 48.6 +/- 6.5% for 10- and 15-min irradiations, respectively. Thus, the results show that low-power pulsed laser irradiations resulted in a significant bacterial mortality and a reduced bacterial attachment on nontoxic hard surfaces.     

Production of exopolysaccharides by Antarctic marine bacterial isolates

AIMS: This study was undertaken to examine and characterize Antarctic marine bacterial isolates and the exopolysaccharides (EPS) they produce in laboratory culture. METHODS AND RESULTS: Two EPS-producing bacterial strains CAM025 and CAM036 were isolated from particulate material sampled from seawater and sea ice in the southern ocean. Analyses of 16S rDNA sequences placed these isolates in the genus Pseudoalteromonas. In batch culture, both strains produced EPS. The yield of EPS produced by CAM025 was 30-fold higher at -2 and 10 degrees C than at 20 degrees C. Crude chemical analyses showed that these EPS were composed primarily of neutral sugars and uronic acids with sulphates. Gas chromatographic analysis of monosaccharides confirmed these gross compositional findings and molar ratios of monosaccharides revealed differences between the two EPS. CONCLUSIONS: The EPS produced by Antarctic bacterial isolates examined in this study appeared to be polyanionic and, therefore, 'sticky' with respect to cations such as trace metals. SIGNIFICANCE AND IMPACT OF THE STUDY: As the availability of iron as a trace metal is of critical importance in the southern ocean where it is know to limit primary production, the role of these bacterial EPS in the Antarctic marine environment has important ecological implications.     

Cryoprotective properties and preliminary characterization of exopolysaccharide (P-Arcpo 15) produced by the Arctic bacterium Pseudoalteromonas elyakovii Arcpo 15

Twenty-two bacterial strains that secrete exopolysaccharides (EPS) were isolated from marine samples obtained from the Chukchi Sea in the Arctic Ocean; of these, seven strains were found to be capable of producing cryoprotective EPS. The ArcPo 15 strain was isolated based on its ability to secrete large amounts of EPS, and was identified as Pseudoalteromonas elyakovii based on 16S rDNA analysis. The EPS, P-ArcPo 15, was purified by protease treatment and gel filtration chromatography. The purified EPS (P-ArcPo 15) had a molecular mass of 1.7 × 10⁷ Da, and its infrared spectrum showed absorption bands of hydroxyl and carboxyl groups. The principal sugar components of P-ArcPo 15 were determined to be mannose and galacturonic acid, in the ratio of 3.3:1.0. The cryoprotective properties of P-ArcPo 15 were characterized by an Escherichia coli viability test. In the presence of 0.5% (w/v) EPS, the survival percentage of E. coli cells was as high as 94.19 ± 7.81% over five repeated freeze–thaw cycles. These biochemical characteristics suggest that the EPS P-ArcPo 15 may be useful in the development of cryoprotectants for biotechnological purposes, and we therefore assessed the utility of this novel cryoprotective EPS.     

Inhibition of algal spore germination by the marine bacterium Pseudoalteromonas tunicata

A collection of 56 bacteria isolated from different surfaces in the marine environment were assayed for their effects on the germination of spores from the common green alga Ulva lactuca. Thirteen bacterial isolates were shown to inhibit spore germination. Of these bacteria, Pseudoalteromonas tunicata displayed the most pronounced effects against algal spores. Further characterisation of the anti-algal activity of P. tunicata was performed and it was found that this bacterium produces an extracellular component with specific activity toward algal spores that is heat-sensitive, polar and between 3 and 10 kDa in size. This biologically active compound was also found to prevent the germination of spores from the red alga Polysiphonia sp. and, given the widespread occurrence of P. tunicata in a range of marine habitats, this may suggest that it is effective against a variety of marine algae.     

Isolation and characterization of <Emphasis Type="Italic">Pseudoalteromonas ruthenica</Emphasis> (SBT033), an EPS-producing biofilm bacterium from the seawater intake point of a tropical power station

Biofilm formation in bacteria is closely linked with production of exopolysaccharides (EPS). This study examined the quantitative variations in EPS production and biofilm-forming ability among bacteria isolated from the seawater intake point of a power station located on the east coast of India. Of the 233 isolates obtained from the intake site, 71 bacterial isolates displayed different colony morphological characteristics. Thirteen isolates that produced wide and thick mucoid colonies were further tested for their ability to attach and form biofilms by microtitre plate assay and confocal microscopy. EPS production among the selected bacterial isolates ranged from 826 to 1838 μg ml⁻¹. Strain SBT033, which produced the maximum amount of EPS also displayed the maximum biofilm-forming ability among the 13 isolates. This strain was selected for further characterization using biochemical and molecular methods. The pale orange-pigmented isolate was a Gram negative, aerobic, short rod-shaped and grew well only in the presence of 2% NaCl. On the basis of phenotypic characteristics the isolate SBT033 is shown to belong to the genus Pseudoalteromonas. Analysis of 16S rRNA of the isolate revealed 99% homology with Pseudoalteromonas ruthenica.     

Influence of subtilisin on the adhesion of a marine bacterium which produces mainly proteins as extracellular polymers

Aims:  The nature of exopolymers involved in the adhesion of a marine biofilm-forming bacterium Pseudoalteromonas sp. D41 was investigated to evaluate and understand the antifouling potential of subtilisin.
Methods and Results:  The exopolymers of D41 produced by fermentation were analysed by FTIR and SDS-PAGE showing the presence of polysaccharides, glycoproteins and proteins. A high content of proteins was detected both in soluble and capsular fractions. The microscopic observations of fluorescamine and calcofluor stained adhered D41 indicated mainly the presence of proteins in exopolymers produced during adhesion. Subtilisin, the broad spectrum protease, tested in natural sea water and in polystyrene microplates showed that antifouling activity was higher in the prevention of bacterial adhesion than in the detachment of adhered D41 cells.
Conclusions:  Overall, these results demonstrate the involvement of proteins in Pseudoalteromonas sp . D41 adhesion and confirm the high antifouling potential of subtilisin.
Significance and Impact of the Study:  This study emphasizes the major role of proteins instead of polysaccharides, thus extending our knowledge regarding the nature of extracellular polymers involved in bacterial adhesion. Furthermore, the high antifouling potential of subtilisin evaluated in the very first stages of fouling, bacterial adhesion, could lead to less toxic compounds than organometallic compounds in antifouling paint.     

Engineered marine Antarctic bacterium Pseudoalteromonas haloplanktis TAC125: a promising micro-organism for the bioremediation of aromatic compounds

Aims:  The recombinant Antarctic Pseudoalteromonas haloplanktis TAC125 ( P. haloplanktis TAC/ tou ) expressing toluene- o- xylene monooxygenase (ToMO) can efficiently convert several aromatic compounds into their corresponding catechols in a broad range of temperature. When the genome of P. haloplanktis TAC125 was analysed in silico , the presence of a DNA sequence coding for a putative laccase-like protein was revealed. It is well known that bacterial laccases are able to oxidize dioxygenated aromatic compounds such as catechols.
Methods and Results:  We analysed the catabolic features, conferred by recombinant ToMO activity and the endogenous laccase enzymatic activity, of P. haloplanktis TAC/ tou engineered strain and its ability to grow on aromatic compounds as sole carbon and energy sources.
Conclusions:  Results presented highlight the broad potentiality of P. haloplanktis TAC/ tou cells expressing recombinant ToMO in bioremediation and suggest the use of this engineered Antarctic bacterium in the bioremediation of chemically contaminated marine environments and/or cold effluents.
Significance and Impact of the Study:  This paper demonstrates the possibility to confer new and specific degradative capabilities to a bacterium isolated from an unpolluted environment (Antarctic seawater) transforming it into a bacterium able to grow on phenol as sole carbon and energy source.     

Structure of an acidic polysaccharide from the marine bacterium Pseudoalteromonas flavipulchra NCIMB 2033(T)

An acidic polysaccharide was isolated from Pseudoalteromonas flavipulchra type strain NCIMB 2033(T) and found to consist of 6-deoxy-L-talose (L-6dTal), D-galactose and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The identities of the monosaccharides were ascertained by sugar analysis and 1D 1H and 13C NMR spectroscopy in conjunction with 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments, which enabled determination of the following structure of the trisaccharide repeating unit of the polysaccharide:-->3)-alpha-L-6dTalp4Ac-(1-->3)-beta-D-Galp-(1-->7)-alpha-Kdop-(2-->.     

Partial characterization of an extracellular polysaccharide produced by the moderately halophilic bacterium Halomonas xianhensis SUR308

A moderately halophilic bacterium, Halomonas xianhensis SUR308 (Genbank Accession No. KJ933394) was isolated from a multi-pond solar saltern at Surala, Ganjam district, Odisha, India. The isolate produced a significant amount (7.87 g l^?1) of extracellular polysaccharides (EPS) when grown in malt extract–yeast extract medium supplemented with 2.5% NaCl, 0.5% casein hydrolysate and 3% glucose. The EPS was isolated and purified following the conventional method of precipitation and dialysis. Chromatographic analysis (paper, GC and GC-MS) of the hydrolyzed EPS confirmed its heteropolymeric nature and showed that it is composed mainly of glucose (45.74 mol%), galactose (33.67 mol %) and mannose (17.83 mol%). Fourier-transform infrared spectroscopy indicated the presence of methylene and carboxyl groups as characteristic functional groups. In addition, its proton nuclear magnetic resonance spectrum revealed functional groups specific for extracellular polysaccharides. X-ray diffraction analysis revealed the amorphous nature (CI_xrd, 0.56) of the EPS. It was thermostable up to 250°C and displayed pseudoplastic rheology and remarkable stability against pH and salts. These unique properties of the EPS produced by H. xianhensis indicate its potential to act as an agent for detoxification, emulsification and diverse biological activities.     

Partial purification and chemical characterization of a glycoprotein (putative hydrocolloid) emulsifier produced by a marine bacterium Antarctobacter

During screening for novel emulsifiers and surfactants, a marine alphaproteobacterium, Antarctobacter sp. TG22, was isolated and selected for its production of an extracellular emulsifying agent, AE22. This emulsifier was produced optimally in a low-nutrient seawater medium supplemented with glucose and was extractable by cold ethanol precipitation of the high-molecular-weight fraction (>100 kDa). Production of AE22 commenced towards the late exponential phase of growth, with maximum emulsifying activity detected after approximately 4 days of the cells entering the death phase. Chemical, chromatographic and nuclear magnetic resonance spectroscopic analysis confirmed AE22 to be a high-molecular-weight (>2,000 kDa) glycoprotein with high uronic acids content, thus denoting an apparent polyanionic structure. Functional characterization showed this polymer to compare well to xanthan gum and gum arabic as an emulsion-stabilizing agent for a range of different food oils. However, AE22 exhibited better stabilizing than emulsifying properties, which could be conferred by its viscosifying effect in solution or from certain chemical groups found on the polysaccharide or protein moieties of the polymer. This new high-molecular-weight glycoprotein exhibits interesting functional qualities that are comparable to other biopolymers of this type and shows particular promise as an emulsion-stabilizing agent in biotechnological applications.     

Isolation and characterization of a marine algicidal bacterium against the toxic dinoflagellate Alexandrium tamarense

Interactions between bacteria and harmful algal bloom (HAB) species have been acknowledged as an important factor regulating both the population dynamics and toxin production of these algae. A marine bacterium SP48 with algicidal activity to the toxic dinoflagellate, Alexandrium tamarense, was isolated from the Donghai Sea area, China. Genetic identification was achieved by polymerase chain reaction amplification and sequence analysis of 16S rDNA. Sequence analysis showed that the most probable affiliation of SP48 was to the γ-proteobacteria subclass and the genus Pseudoalteromonas. Bacterial isolate SP48 showed algicidal activity through an indirect attack. Additional organic nutrients but not algal-derived DOM was necessary for the synthesis of unidentified algicidal compounds but β-glucosidase was not responsible for the algicidal activity. The algicidal compounds produced by bacterium SP48 were heat tolerant, unstable in acidic condition and could be easily synthesized regardless of variation in temperature, salinity or initial pH for bacterial growth. This is the first report of a bacterium algicidal to the toxic dinoflagellate A. tamarense and the findings increase our knowledge of bacterial–algal interactions and the role of bacteria during the population dynamics of HABs.     

Isolation and characterization of a high molecular weight antibiotic produced by a marine bacterium

Many marine bacteria demonstrate antibiotic activity against organisms of terrestrial origin. Low molecular weight antibiotics have been extracted and, in some cases, purified, but few attempts have been made to isolate high molecular weight antibiotics produced by marine bacteria. In the study reported here, a high molecular weight antibiotic was extracted from whole cells ofAlteromonas strain P18 (NCMB 1890) grown on 2216E medium. Purification included ammonium sulfate precipitation, ultracentrifugation, chromatography on DEAE cellulose, and gel filtration on Ultrogel. A rapid method for measuring specific activity of the antibiotic was developed.     

Cryoprotective properties of exopolysaccharide (P-21653) produced by the Antarctic bacterium, Pseudoalteromonas arctica KOPRI 21653

Twenty-five bacterial strains that secrete mucous materials were isolated from sediment obtained from King George Island, Antarctica. Seven of these strains proved capable of producing cryoprotective exopolysaccharides. The strain KOPRI 21653 was selected for the further study of an anti-ice-nucleating polysaccharide (ANP), which originated from a polar region. KOPRI 21653 was identified as Pseudoalteromonas arctica as the result of 16S rRNA analysis. The exopolysaccharide, P-21653, was purified completely from the KOPRI 21653 cell culture via column chromatography and protease treatment. The principal sugar components of P-21653 were determined to be galactose and glucose, at a ratio of 1:1.5, via GC-MS analysis. The cryoprotective activity of P-21653 was characterized via an E. coli viability test. In the presence of 0.1% (w/v) P-21653, the survival ratio of E. coli cells was as high as 82.6% over three repeated freeze-thaw cycles. The survival ratio decreased drastically to 71.5 and 48.1%, respectively, in five and seven repeated cycle conditions; however, the survival ratios were greater over three (96.6-92.1%) to seven (100.5-91.6%) freeze-thaw cycles in the presence of 0.5 and 1.0% (w/v) P-21653. In addition, at much lower concentrations (0.1-1.0%), P-21653 resulted in survival ratios (83.1-98.4%) similar to those of two commercially available cryoprotectants (VEG plus X-1000, 92.9% and VM3, 95.3%), which were utilized at the recommended concentrations (90%). The biochemical characteristics of exopolysaccharide P-21653 reflect that this compound may be developed as a useful cryoprotectant for use in medical applications and in the food industry.     

Anti-oxidation of agar oligosaccharides produced by agarase from a marine bacterium

In order to prepare the active agar oligosaccharide, agarase extracted from a strain of unidentified marine bacterium from the South China Sea coast was selected for the agar depolymerization. The optimum decomposing conditions were determined to be pH 7.0, 35 °C and halophilic properties 2%. Three main degraded products, AOS-1, AOS-2 and AOS-3, were separated by ethanol fractionation and anion exchange chromatography. The molecular mass was analyzed by MALDI-TOF-MS. The agar oligosaccharides exhibited antioxidative activities in scavenging hydroxyl free radical, scavenging superoxide anion radical and inhibiting lipid peroxidation. The fragment with the sulfate group showed stronger antioxidative activities than that without the sulfate group. Higher antioxidative activities were found when the molecular mass was increased. The results indicated that the antioxidative activities were closely related to the molecular mass of the agar oligosaccharides and the substitute groups binding the carbohydrate ring.     

Production and characterization of exopolysaccharides excreted by thermophilic bacteria from shallow,marine hydrothermal vents of Flegrean Ares (Italy)

Thermophilic microorganisms (4001-4014), described as aerobic or facultatively anaerobic, endospore forming with growth optima temperatures in the range of 60 to 80 degrees C, have been isolated from hot marine springs around Ischia and from hydrothermal vents in the gulf of Naples. Mucous colonies are been selected for the recovery of new strains producing exopolysaccharides (EPS). To induce the biosynthesis of new exopolysaccharides, different sugars were tested as carbon sources in the media. The production of EPS in the strain 4009 reached 60 mg/l using trehalose as carbon source, increasing the yield of about 1000 fold. The 4001-EPS was a mannan with a molecular weight of 380.000 D and with a complex primary structure. In fact, the analysis of the permethylated polysaccharide in GC-MS, showed the presence of mannose, glucose, galactose, mannosamine in the relative ratio of 1:0.1:tr :tr, respectively. Nuclear magnetic resonance spectrum of the exopolysaccharide confirmed the presence of a repetitive unity formed by seven monosaccharides, six with alpha gluco/galacto configuration and one residue with beta conformation.     

Effects of ammonium feeding on the production of bioactive metabolites (cordycepin and exopolysaccharides) in mycelial culture of a Cordyceps sinensis fungus

AIMS: To examine the effects of ammonium feeding on the production of cordycepin (3'-deoxyadenosine, a nucleoside analogue) and exopolysaccharides (EPS) in mycelial culture of a new Cordyceps sinensis fungus Cs-HK1. METHODS AND RESULTS: Cs-HK1 fungus was cultivated in a liquid medium containing glucose, yeast extract, peptone and a few major inorganic salts. NH(4)Cl was fed to the mycelial culture at various concentrations from 5 to 40 mmol l(-1) on day 3 (during exponential phase). NH(4)Cl, fed at 10 mmol l(-1), stimulated the cordycepin production most significantly, with nearly fourfold increase in the cordycepin content of mycelia (from 28.5 to 117 microg g(-1)), and also increased the EPS production by 40% (from 2.6 to 3.7 g l(-1)). The ammonium feeding had a slightly positive effect at 5-10 mmol l(-1), but a negative effect at higher concentrations on the mycelium growth. Ammonium feeding also caused a sharp drop of the medium pH, owing perhaps to the uptake of NH(3) and the release of H(+) by the fungal cells. CONCLUSIONS: Ammonium feeding to the mycelial culture of Cs-HK1 fungus enhanced the intracellular cordycepin accumulation and the EPS production. The enhanced cordycepin production may be attributed to the uptake of ammonia for nucleoside synthesis, and the enhanced EPS to the increased uptake of glucose for EPS biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: It is useful for the production of bioactive metabolites and for understanding ammonium metabolism and its relationship to the biosynthesis of nucleosides in a precious medicinal fungus.