Marinobacter Lipolyticus Research Articles

A Self‐Cleaning Mucus‐like and Hierarchical Ciliary Bionic Surface for Marine Antifouling

The flexible mucus film and hydrophilic hierarchical micro‐/nanoprotuberance structure on the skin surfaces of marine organisms contribute to an excellent intrinsic antifouling performance. Inspired by this fact, a self‐cleaning mucus‐like and hierarchical ciliary bionic antifouling surface (SMCAS) is designed for marine antifouling based on the electrostatic flocking technology. The results of scanning electron microscopy (SEM) show that the bioinspired SMCAS has a hierarchical micro‐/nanociliary structure and the micrometer array of polyamide (PA) microfibers is covered with the nanometer carbon nanotube/polyvinyl alcohol (CNT/PVA) hydrogel particles. The biomasses of the two marine microoganisms, Nitzschia closterium f. minutissima and Marinobacter lipolyticus SM19 (T), attached on the SMCAS are investigated using a UV–visible spectrophotometer (UV–vis) and SEM. The results reveal that the microoganisms attached on the SMCAS are significantly lower number in comparison with the organic silicon coating. In addition, the SMCAS has a greater ability of stain removal in the dyed petroleum ether washing test. Therefore, the SMCAS demonstrates an extraordinary antifouling performance due to the synergetic effect of the mucus‐like hydrogel film and the hierarchical micro‐/nanostructured micropillars.

Alleviation of Salinity Stress on Some Growth Parameters of Wheat by Exopolysaccharide-Producing Bacteria

After isolation of halophilic bacteria from saline soils, the effects of two superior isolates producing exopolysaccharide TP7 and TP5 was investigated on some wheat growth parameters such as shoot and root dry weight and concentration of K+, Ca2+, Na+ and K+/Na+, Ca2+/Na+ ratios under saline stress. Inoculation with bacteria (TP7, TP5 and both of strains) had a positive effect on shoot weight (74.71, 57.52 and 82.45%) at EC 8 dS m−1 and (45.26, 60.95 and 71.28%) at EC 16 dS m−1. Also, TP7 and TP5 isolates can increase plant resistance to salt and drought stresses by decreasing Na+ uptake and in 16 dS m−1 led to an increase in dry weight of root in comparison with control by 97.95, 128.29 and 193.53%, respectively. In higher-salinity treatments, all of treatments (TP5, TP7 and both of strains) had a significant effect on K+ concentration, especially in EC 16 dS m−1 by 29.58, 45.94 and 53.56% and 35.50, 52.37 and 58.65% in water stress and non-water stress, respectively. The determination of 16S rRNA gene sequences of isolates TP7 and TP5 showed that they are mostly close to Bacillus subtilis susp. inaquosorum (98.49% sequence similarity) and Marinobacter lipolyticus SM19 (T) (97.69% sequence similarity), respectively. Generally, results showed that these isolates can be used as a tool for reducing adverse effects of salinity and drought stresses in wheat plant.

Marinobacter profundi sp. nov., a slightly halophilic bacterium isolated from a deep-sea sediment sample of the New Britain Trench

A piezotolerant, cold-adapted, slightly halophilic bacterium, designated strain PWS21T, was isolated from a deep-sea sediment sample collected from the New Britain Trench. Cells were observed to be Gram-stain negative, rod-shaped, oxidase- and catalase-positive. Growth of the strain was observed at 4-45°C (optimum 37°C), at pH 5.0-9.0 (optimum 7.0) and in 0.5-20% (w/v) NaCl (optimum 3-4%). The optimum pressure for growth was 0.1MPa (megapascal) with tolerance up to 70MPa. 16S rRNA gene sequence analysis showed that strain PWS21T is closely related to Marinobacter guineae M3BT (98.4%) and Marinobacter lipolyticus SM19T (98.2%). Multilocus sequence analysis (MLSA) based on sequences of housekeeping genes gyrB, recA, atpD, rpoB and rpoD indicates that strain PWS21T represents a distinct evolutionary lineage within the genus Marinobacter. Furthermore, strain PWS21T showed low ANI and diDDH values to the closely related species. The principal fatty acids were identified as C12:0, C12:0 3-OH, C16:1ω9c, C16:0 and C18:1ω9c. Ubiquinone-9 was identified as the major respiratory quinone. The polar lipids were identified as phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), aminophospholipid (APL), two unidentified lipids and an unidentified phospholipid (PL). The G + C content of the genomic DNA was determined to be 60.3mol%. On the basis of phenotypic, chemotaxonomic and molecular data, we conclude that strain PWS21T represents a novel species of the genus Marinobacter, for which the name Marinobacter profundi sp. nov. is proposed (type strain PWS21T = KCTC 52990T = MCCC 1K03345T).

Structural characterization and biological evaluation of Staphylosan (dimannooleate), a new glycolipid surfactant produced by a marine Staphylococcus saprophyticus SBPS-15

Marine microbes have gained significant attention as potential biofactories for broad spectrum bioactive compounds. In the recent years, bioactive biosurfactants have warranted renewed interest from both environmental and clinical sectors as anti-biofilm agents due to their excellent properties of dispersing microbial biofilms. The present study explores a new glycolipid biosurfactant produced by a marine Staphylococcus saprophyticus exhibiting interesting biological activities. This glycolipid biosurfactant was purified and identified as Mannose-Mannose-Oleic acid (named as Staphylosan) based on the results of NMR, GC, GC–MS, MALDI-TOF-MS and tandem MS analysis. The surface tension and critical micelle concentration of purified Staphylosan was 30.9 mN m−1 and 24 mg L−1. Further, it showed promising biofilm inhibition and dislodging activities against a panel of profuse biofilm forming bacteria at both single and multi-species level which were isolated from boat hull biofilm environment such as Bacillus subtilis BHKH-7, Acinetobacter beijerinckii BHKH-11, Pseudomonas aeruginosa BHKH-19, Serratia liquefaciens BHKH-23, Marinobacter lipolyticus BHKH-31 and Micrococcus luteus BHKH-39. Moreover, it exhibited anionic charge and revealed non-toxicity towards brine shrimps, suggesting its environmental safety. This is a first report on Staphylosan, a multifunctional glycolipid surfactant from a marine Staphylococcus saprophyticus SBPS-15, exhibiting promising anti-biofilm activities and non-toxic in nature and thus finds possible potential use in many environmental applications especially under marine conditions.

Characterization of Organic Solvent-Tolerant Lipolytic Enzyme from Marinobacter lipolyticus Isolated from the Antarctic Ocean.

The Antarctic marine environment provides a good source of novel lipolytic enzymes that possess beneficial properties, i.e., resistance to extreme physical and chemical conditions. We found a lipolytic Escherichia coli colony that was transformed using genomic DNA from Marinobacter lipolyticus 27-A9 isolated from the Antarctic Ross Sea. DNA sequence analysis revealed an open reading frame of lipolytic enzyme gene. The gene translates a protein (LipA9) of 404 amino acids with molecular mass of 45,247Da. Recombinant LipA9 was expressed in E. coli BL21 (DE3) cells and purified by anion exchange and gel filtration chromatography. The kcat/Km of LipA9 was 175s-1μM-1, and the optimum temperature and pH were 70°C and pH 8.0, respectively. LipA9 had quite high organic solvent stability; it was stable toward several common organic solvents up to 50% concentration. Substrate specificity studies showed that LipA9 preferred a short acyl chain length of p-nitrophenyl ester and triglyceride. Sequence analysis showed that LipA9 contained catalytic Ser72 and Lys75 in S-x-x-K motif, like family VIII esterases. Homology modeling and site-directed mutagenesis studies revealed that Tyr141 and Tyr188 residues were located near the conserved motif and played an important role in catalytic activity.

Glycerol and nitrate utilisation by marine microalgae Nannochloropsis salina and Chlorella sp. and associated bacteria during mixotrophic and heterotrophic growth

Mixotrophic growth is an ability of microalgae to concurrently assimilate CO2 and organic carbon. In the present study, the use of glycerol to enhance microalgal biomass and lipid productivities was investigated in relation to nitrate availability. Under nitrate sufficient conditions, the biomass productivities of Nannochloropsis salina (CCMP 1776) and a marine Chlorella sp. were 1.7 and 1.9 times higher in mixotrophic culture than under strictly photoautotrophic conditions, respectively. Both algae required light to assimilate glycerol. No significant algae growth was observed under heterotrophic conditions, despite apparent utilisation of both nitrate and glycerol. Under nitrate deplete conditions both species took up only minimal amounts of glycerol thereby indicating the importance of the combined effects of carbon and nitrogen. Additionally, a carbon mass balance was conducted based on glycerol utilisation and biomass production in N. salina cultures, which revealed a net release of CO2 during mixotrophic growth. In the presence of glycerol, the proliferation of bacteria was consistently observable in these non-axenic cultures. 16S rRNA sequencing confirmed the presence of two phylogenetically distinct Gram-negative bacterial isolates belonging to α- and γ-subclass of Proteobacteria (Paracoccus sp. MBIC4036, and Marinobacter alkaliphilus NBSL03 and Marinobacter lipolyticus SM-19, respectively) in the cultures of N. salina. Among these strains, M. alkaliphilus was shown to have denitrifying capabilities, which allowed it to oxidise glycerol using nitrate as a terminal electron acceptor. This provides an explanation for the utilisation of nitrate and glycerol that was observed in heterotrophic cultures despite the absence of microalgal growth. These results provide a better understanding of the interactions between bacteria and algae in mixotrophic growth that may enable the development of strategies to better utilise nitrogen and carbon for algae production. The present study has demonstrated the possibility of reutilisation of glycerol, a biodiesel by-product, to promote algal growth and lipid accumulation.

Marinobacter salinus sp. nov., a moderately halophilic bacterium isolated from a tidal flat environment.

Two Gram-stain-negative, aerobic, motile, halophilic, rod-shaped bacteria, designated Hb8T and Hb20, were isolated from a tidal flat environment located on the South-West Korean peninsula. The isolates grew at 10-37 °C, at pH 5.0-9.0 and in NaCl concentrations of 0.5-15 % (w/v; optimum, 3.0-6.0 %). Sequence analysis of the 16S rRNA indicated that the isolates belong to the genus Marinobacter and are most closely related to Marinobacter sediminumR65T (98.3 %), followed by Marinobacter lipolyticus SM19T, Marinobacter salsuginis SD-14BT and Marinobacter similis A3d10T. The overall 16S rRNA gene sequence similarity with these species was 97.9 %, but Hb8T and Hb20 showed 100 % sequence similarity with each other. DNA-DNA relatedness values of H8T and Hb20 suggested that these isolates represent a single species, while DNA-DNA relatedness values of the two novel isolates with M. sediminum DSM 27079T and M. similis DSM 15400T were only 21.3 and 22.9 %, respectively. The major fatty acids present in strain Hb8T were identified as C16 : 0, C16 : 1ω9c, C18 : 1ω9c, C18 : 0 3-OH and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). Ubiquinone-9 was the main respiratory quinone in both the novel strains. The polar lipids found to be present included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four unidentified phospholipids and five unidentified lipids. The genomic DNA G+C content of Hb8T and Hb20 was 54.5 mol%. Polyphasic analysis indicated that the two isolates are representatives of a novel species of the genus Marinobacte, for which the name Marinobacter salinus sp. nov. is proposed. The type strain is Hb8T (=KCTC 52255T=JCM 31416T).

Marinobacter halotolerans sp. nov., a halophilic bacterium isolated from a saltern crystallizing pond.

A Gram-stain-negative, moderately halophilic, motile bacterium, designated strain CP12T, was isolated from a crystallizing pond of a saltern of the Yellow Sea in Korea. Cells of strain CP12T were non-spore-forming rods and produced whitish-yellow colonies. Growth was observed at 10-37 °C (optimum 37 °C), at pH 6.0-9.0 (optimum pH 7.0), and in the presence of 0.5-20 % (w/v) NaCl (optimum 3 %). Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain CP12T was closely related to Marinobacter flavimaris SW-145T (98.4 % 16S rRNA gene sequence similarity), Marinobacter algicola DG893T (98.2 %), Marinobacter adhaerens HP15T (98.2 %), Marinobacter salsuginis SD-14BT (97.9 %), Marinobacter salarius R9SW1T (97.6 %) and Marinobacter lipolyticus SM19T (97.1 %). DNA-DNA hybridization studies showed values lower than 18.6 % between strain CP12T and any of these species. The predominant respiratory isoprenoid quinone was ubiquinone-9 and the major cellular fatty acids of strain CP12T were C16 : 0, C12 : 0 3-OH, C12 : 0, Summed feature 3, C16 : 0 10-methyl and C18 : 1ω9c. On the basis of phenotypic properties, and phylogenetic and chemotaxonomic data, it is evident that strain CP12T represents a novel species of the genus Marinobacter, for which the name Marinobacter halotolerans sp. nov. is proposed. The type strain is CP12T (=KACC 18381T=NBRC 110910T).

Marinobacter lipolyticus from Red Sea for lipase production and modulation of silver nanomaterials for anti-candidal activities.

In this study, the bacterial strain CEES 33 was isolated from the coastal area of the Red Sea, Jeddah, Kingdom of Saudi Arabia. The bacterium isolate was identified and characterized by using biochemical and molecular methods. The isolate CEES 33 has been identified as Gram-negative rod shaped and cream pigmented spherical colonies. It also demonstrated a positive result for nitrate reduction, oxidase, catalase, citrate utilization, lipase and exopolysaccharide production. Strain CEES 33 was characterized at the molecular level by partial 16S rRNA sequencing and it has been identified as Marinobacter lipolyticus (EMBL|LN835275.1). The lipolytic activity of the isolate was also observed 2.105 nkatml-1. Furthermore, the bacterial aqueous extract was used for green synthesis of silver nanoparticles (AgNPs), which was further confirmed by UV-visible spectra (430 nm), XRD and SEM analysis. Moreover, the biological functional group that involved in AgNPs synthesis was confirmed by FTIR spectra. The biological activities of AgNPs were also investigated, which showed a significant growth inhibition of Candida albicans with 16 ± 2 mm zone of inhibition at 10 μg dose/wells. Therefore, bacterium Marinobacter lipolyticus might be used in future for lipase production and nanoparticles fabrication for biomedical application, to control fungal diseases caused by C. albicans.

Marinobacter aromaticivorans sp. nov., a polycyclic aromatic hydrocarbon-degrading bacterium isolated from sea sediment.

A rod-shaped, Gram-stain-negative, slightly halotolerant bacterium, designated strain D15-8PT, was isolated from a sediment sample from the South China Sea. The strain could grow in NaCl concentrations ranging from 0.5 % to 10 % (w/v) (optimum 0.5-1.5 %), and could be cultivated at 10-40 °C (optimum 25 °C) and pH 5.5-9.5 (optimum pH 7.0-8.0). The strain was positive for catalase, oxidase, and hydrolysis of Tween 80, but negative for hydrolysis of DNA and gelatin, nitrite reduction, indole production, Voges-Proskauer reaction, and methyl red test. Strain D15-8PT could biodegrade naphthalene, phenanthrene, and anthracene. The major respiratory quinone was Q-9. The main cellular fatty acids were C12 : 0 (11.5 %), C14 : 0 3-methyl (22.0 %), C16 : 0 (19.2 %), C16 : 1ω9c (22.9 %), and C18 : 1ω9c (6.7 %). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid and an unidentified phospholipid. The DNA G+C content was 56.8 mol%. Phylogenetic analyses based on 16S rRNA genes showed that strain D15-8PT was most closely related to Marinobacter maritimus JCM 12521T (98.5 % 16S rRNA gene sequence similarity), Marinobacter antarcticus CGMCC 1.10835T (98.1 %), Marinobacter lipolyticus DSM 15157T (97.1 %), and Marinobacter guineae CECT 7243T (97.0 %). Results of the gyrB gene analysis and DNA-DNA hybridization were both less than the cut-off values (90 % for gyrB gene sequence similarity and 70 % for DNA-DNA hybridization). On the basis of this taxonomic study using a polyphasic approach, strain D15-8PT represents a novel species of the genus Marinobacter, for which the name Marinobacter aromaticivorans sp. nov. is proposed. The type strain is D15-8PT ( = CGMCC 1.11015T = KCTC 23781T).

Draft Genome Sequence of the Moderately Halophilic Bacterium Marinobacter lipolyticus Strain SM19.

Marinobacter lipolyticus strain SM19, isolated from saline soil in Spain, is a moderately halophilic bacterium belonging to the class Gammaproteobacteria. Here, we report the draft genome sequence of this strain, which consists of a 4.0-Mb chromosome and which is able to produce the halophilic enzyme lipase LipBL.

Halophilic bacteria as a source of novel hydrolytic enzymes.

Hydrolases constitute a class of enzymes widely distributed in nature from bacteria to higher eukaryotes. The halotolerance of many enzymes derived from halophilic bacteria can be exploited wherever enzymatic transformations are required to function under physical and chemical conditions, such as in the presence of organic solvents and extremes in temperature and salt content. In recent years, different screening programs have been performed in saline habitats in order to isolate and characterize novel enzymatic activities with different properties to those of conventional enzymes. Several halophilic hydrolases have been described, including amylases, lipases and proteases, and then used for biotechnological applications. Moreover, the discovery of biopolymer-degrading enzymes offers a new solution for the treatment of oilfield waste, where high temperature and salinity are typically found, while providing valuable information about heterotrophic processes in saline environments. In this work, we describe the results obtained in different screening programs specially focused on the diversity of halophiles showing hydrolytic activities in saline and hypersaline habitats, including the description of enzymes with special biochemical properties. The intracellular lipolytic enzyme LipBL, produced by the moderately halophilic bacterium Marinobacter lipolyticus, showed advantages over other lipases, being an enzyme active over a wide range of pH values and temperatures. The immobilized LipBL derivatives obtained and tested in regio- and enantioselective reactions, showed an excellent behavior in the production of free polyunsaturated fatty acids (PUFAs). On the other hand, the extremely halophilic bacterium, Salicola marasensis sp. IC10 showing lipase and protease activities, was studied for its ability to produce promising enzymes in terms of its resistance to temperature and salinity.

Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus

The lipolytic enzyme family VIII currently includes only seven members but represents a group of lipolytic enzymes with interesting properties. Recently, we identified a gene encoding the family VIII lipase LipBL from the halophilic bacterium Marinobacter lipolyticus. This enzyme, like most lipolytic enzymes from family VIII, possesses two possible nucleophilic serines located in an S-X-X-K β-lactamase motif and a G-X-S-X-G lipase motif. The serine in the S-X-X-K motif is a catalytic residue, but the role of serine within the common lipase consensus sequence G-X-S-X-G has not yet been systematically studied. Here, the previously reported time-intensive procedure for purification of recombinant LipBL was replaced by one-step metal-affinity chromatography purification in the presence of ATP. Heterologous co-expression of His(6)-tagged LipBL with the cytoplasmic molecular chaperones GroEL/GroES was necessary to obtain catalytically active LipBL. Site-directed mutagenesis performed to map the active site of LipBL revealed that mutation of serine and lysine in the β-lactamase motif (S(72)-M-T-K(75)) to alanine abolished the enzyme activity of LipBL, in contrast to mutation of the serine in the lipase consensus motif (S321A). Furthermore, mutagenesis was performed to understand the role of the G-X-S-X-G motif and other amino acids that are conserved among family VIII esterases. We describe how mutations in the conserved G-X-S-X-G motif altered the biochemical properties and substrate specificity of LipBL. Molecular modelling results indicate the location of the G-X-S(321)-X-G motif in a loop close to the catalytic centre of LipBL, presumably representing a substrate-binding site of LipBL.

A Novel Halophilic Lipase, LipBL, Showing High Efficiency in the Production of Eicosapentaenoic Acid (EPA)

BackgroundAmong extremophiles, halophiles are defined as microorganisms adapted to live and thrive in diverse extreme saline environments. These extremophilic microorganisms constitute the source of a number of hydrolases with great biotechnological applications. The interest to use extremozymes from halophiles in industrial applications is their resistance to organic solvents and extreme temperatures. Marinobacter lipolyticus SM19 is a moderately halophilic bacterium, isolated previously from a saline habitat in South Spain, showing lipolytic activity.Methods and FindingsA lipolytic enzyme from the halophilic bacterium Marinobacter lipolyticus SM19 was isolated. This enzyme, designated LipBL, was expressed in Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C β-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80°C and the pH optimum determined at 25°C was 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited high activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. For improving the stability and to be used in industrial processes LipBL was immobilized in different supports. The immobilized derivatives CNBr-activated Sepharose were highly selective towards the release of EPA versus DHA. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested.ConclusionsIn this study we isolated, purified, biochemically characterized and immobilized a lipolytic enzyme from a halophilic bacterium M. lipolyticus, which constitutes an enzyme with excellent properties to be used in the food industry, in the enrichment in omega-3 PUFAs.

Marinobacter adhaerens sp. nov., isolated from marine aggregates formed with the diatom Thalassiosira weissflogii

A Gram-negative, motile, rod-shaped bacterial strain, HP15(T), was isolated from aggregates taken from surface waters of the German Wadden Sea (German Bight). Of 82 marine isolates, HP15(T) was chosen for further study because of its high potential to induce production of transparent exopolymeric particles and aggregate formation while interacting with the diatom Thalassiosira weissflogii. HP15(T) grew optimally at 34-38 °C and pH 7.0-8.5, and was able to tolerate salt concentrations of 0.5-20% (w/v) NaCl. HP15(T) was characterized chemotaxonomically by possessing ubiquinone-9 as the major respiratory lipoquinone, as well as C(16:0), C(18:1)ω9c and C(16:1)ω7c/iso-C(15:0) 2-OH as the predominant fatty acids. The DNA G+C content of strain HP15(T) was 56.9 mol%. The closest relative based on 16S rRNA gene sequence analysis was the type strain of Marinobacter flavimaris, with 99% similarity. Whole-genome relatedness values of HP15(T) to the type strains of M. flavimaris, Marinobacter salsuginis, Marinobacter lipolyticus and Marinobacter algicola were less than 70%, as determined by DNA-DNA hybridization. On the basis of phenotypic and chemotaxonomic properties as well as phylogenetic analyses, the isolate represents a novel species, Marinobacter adhaerens sp. nov.; the type strain is HP15(T) (=DSM 23420(T)=CIP 110141(T)).

Marinobacter goseongensis sp. nov., from seawater

A Gram-negative marine bacterium, designated strain En6(T), was isolated from seawater of the East Sea of Korea. The organism grew in 1-25 % (w/v) NaCl and at 10-37 degrees C and pH 5.3-9.3, with optimal growth occurring in 4-5 % NaCl and at 25-30 degrees C and pH 7.5. Phylogenetic analysis of the 16S rRNA gene sequence of strain En6(T) placed this bacterium in the clade Marinobacter within the class Gammaproteobacteria. The 16S rRNA gene sequence similarity between strain En6(T) and Marinobacter lipolyticus SM19(T), the most closely related species, was 98.4 %, and the level of DNA-DNA relatedness between the two strains was 22 %. On the basis of the phylogenetic analysis and phenotypic and chemotaxonomic data, strain En6(T) is considered to represent a novel species of the genus Marinobacter. The name Marinobacter goseongensis sp. nov. is proposed, with strain En6(T) (=KCTC 12515(T)=DSM 19471(T)) as the type strain.

Marinobacter guineae sp. nov., a novel moderately halophilic bacterium from an Antarctic environment

Two Gram-negative, cold-adapted, moderately halophilic, aerobic bacteria, designated strains M3B(T) and M3T, were isolated from marine sediment collected from the South Shetland Islands, Antarctica. The organisms were rod-shaped, catalase- and oxidase-positive, and motile by means of polar flagella. These two psychrotolerant strains required Na(+) and grew at NaCl concentrations of 1-15 % and temperatures between 4 and 42 degrees C. 16S rRNA gene sequence analysis placed strains M3B(T) and M3T within the genus Marinobacter. DNA-DNA hybridization experiments between the Antarctic isolate M3B(T) and type strains of phylogenetically related species, namely Marinobacter lipolyticus, Marinobacter flavimaris, Marinobacter sediminum, Marinobacter algicola, Marinobacter maritimus and Marinobacter koreensis, revealed levels of relatedness lower than 32 %. Strain M3T showed 99 % DNA relatedness to strain M3B(T). The DNA G+C contents of M3B(T) and M3T were 57.1 and 57.4 mol%, respectively, and their major isoprenoid quinone was ubiquinone-9. Several phenotypic characteristics, together with data on cellular fatty acid composition, served to differentiate strains M3B(T) and M3T from strains of related Marinobacter species. On the basis of the polyphasic taxonomic evidence presented in this study, it can be concluded that strains M3B(T) and M3T belong to the same genospecies and represent a novel species of the genus Marinobacter, for which the name Marinobacter guineae sp. nov. is proposed. The type strain is M3B(T) (=LMG 24048(T)=CECT 7243(T)).

Marinobacter gudaonensis sp. nov., isolated from an oil-polluted saline soil in a Chinese oilfield

Two novel strains, SL014B61A(T) and SL014B11A, were isolated from an oil-polluted saline soil from Gudao in the coastal Shengli Oilfield, eastern China. Cells of strains SL014B61A(T) and SL014B11A were motile, Gram-negative and rod-shaped. Growth occurred at NaCl concentrations of between 0 and 15 % and at temperatures of between 10 and 45 degrees C. Strain SL014B61A(T) had Q9 as the major respiratory quinone and C16 : 0 (21.2 %), C18 : 1omega9c (20.3 %), C16 : 1omega7c (7.3 %) and C16 : 1omega9c (6.4 %) as predominant fatty acids. The G+C content of the DNA was 57.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SL014B61A(T) belonged to the genus Marinobacter in the class Gammaproteobacteria. Strain SL014B61A(T) showed the highest 16S rRNA gene sequence similarity with Marinobacter bryozoorum (97.9 %) and showed 97.8 % sequence similarity to Marinobacter lipolyticus. DNA-DNA relatedness to the reference strains Marinobacter bryozoorum and Marinobacter lipolyticus was 35.5 % and 33.8 %, respectively. On the basis of these data, it is proposed that strains SL014B61A(T) and SL014B11A represent a novel species, Marinobacter gudaonensis sp. nov. The type strain is strain SL014B61A(T) (=DSM 18066(T)=LMG 23509(T)=CGMCC 1.6294(T)).

Marinobacter koreensis sp. nov., isolated from sea sand in Korea

A marine, Gram-negative, aerobic, motile, straight-rod-shaped, moderately halophilic bacterium, designated strain DD-M3T, was isolated from sea sand in Pohang, Korea. A phylogenetic tree based on 16S rRNA gene sequences showed that the strain fell within the evolutionary radiation encompassed by the genus Marinobacter. The levels of 16S rRNA gene sequence similarity between the novel strain and the type strains of recognized Marinobacter species ranged from 94.2 to 97.6%, the highest values being with Marinobacter flavimaris SW-145T (97.6%) and Marinobacter lipolyticus SM19T (96.8%). The values for DNA-DNA relatedness between isolate DD-M3T and the type strains of the most closely related species, M. flavimaris and M. lipolyticus, were 41 and 36%, respectively. Strain DD-M3T was characterized as having Q-9 as the predominant respiratory quinone and 16:0, summed feature 3 and 18:1omega9c as the main fatty acids. The DNA G+C content was 54.1 mol%. On the basis of its phenotypic and genotypic characteristics, DD-M3T represents a novel species of the genus Marinobacter, for which the name Marinobacter koreensis sp. nov. is proposed, with DD-M3T (=KACC 11513T=DSM 17924T) as the type strain.

Marinobacter maritimus sp. nov., a psychrotolerant strain isolated from sea water off the subantarctic Kerguelen islands

A psychrotolerant, Gram-negative, motile bacterium, designated CK 47(T), was isolated from sea water off the subantarctic Kerguelen islands (50 degrees 40' S 68 degrees 25' E). The isolate grew optimally at 22 degrees C and minimum and maximum temperature of growth were 4 and 37 degrees C, respectively. It required Na(+) for growth and exhibited optimum growth at pH 8.5 and 4% NaCl. It utilized hexane, heptane and petroleum ether as sole sources of carbon. Strain CK 47(T) had Q9 as the major respiratory quinone and C(16 : 0) (21.7%), C(17:0) (21.3%), C(18:0) (5.7%), C(18:1)omega7c (9.0%) and C(18:1)omega9c (31.4%) as predominant fatty acids. The G+C content of the DNA was 58 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that CK 47(T) formed a coherent cluster within the genus Marinobacter. It exhibited highest 16S rRNA gene sequence similarity of 96.8% with Marinobacter lipolyticus. However, the level of DNA-DNA relatedness between strain CK47(T) and M. lipolyticus was only 55%. On the basis of phenotypic characteristics, and phylogenetic and genotypic distinctiveness, strain CK 47(T) is considered to represent a novel species of the genus Marinobacter. The name Marinobacter maritimus sp. nov. is proposed, with CK 47(T) (=JCM 12521(T)=MTCC 6519(T)) as the type strain.