María Teresa García

Vibrio tapetis sp. nov., the Causative Agent of the Brown Ring Disease Affecting Cultured Clams

A taxonomic characterization was carried out on strains of the bacteria that cause the brown ring disease of clams. On the basis of their phenotypic and genotypic characteristics, these strains can be considered to constitute a new taxonomic unit, distinct from other Vibrio species. The guanine-plus-cytosine content of the strains ranged between 42.9 and 45.5 mol% (43.2 mol% for the proposed type strain). DNA-DNA hybridization studies showed 100% intragroup relatedness, but levels of genetic relatedness to the reference strains of different Vibrio species tested ranged between 15 and 58%. The strains have all the properties characteristic of the genus Vibrio and can be clearly differentiated from other species of this genus by their growth at 4°C and their negative responses for growth at 30°C and in 6% NaCl, arginine dehydrolase, lysine decarboxylase, ornithine decarboxylase, and Voges-Proskauer reaction. The name Vibrio tapetis is proposed for the new species; strain B1090 (CECT 4600) is the type strain.

Chromohalobacter salexigens sp. nov., a moderately halophilic species that includes Halomonas elongata DSM 3043 and ATCC 33174.

Two strains that were originally isolated and characterized as members of the moderately halophilic species Halomonas elongata, strains DSM 3043 (= 1H11) and ATCC 33174 (= 1H15), were studied in detail. Their complete 16S rRNA sequences were determined and, when compared to sequences available from the databases, they showed a close phylogenetic relationship to Chromohalobacter marismortui. In addition, DNA-DNA hybridization experiments showed that both strains are members of the same species, but their DNA relatedness to the type strains of Halomonas elongata, ATCC 33173T, and Chromohalobacter marismortui, ATCC 17056T, is very low. Phenotypically, the two strains showed very similar features, related to those of Chromohalobacter, but clear differences were found between these two strains and Chromohalobacter marismortui. On the basis of these data, it is proposed that Halomonas elongata DSM 3043 and ATCC 33174 should be included in a new species of the genus Chromohalobacter, Chromohalobacter salexigens sp. nov. The type strain is DSM 3043T (= ATCC BAA-138T = CECT 5384T = CCM4921T = CIP106854T = NCIMB 13768T).

Classification of “Chromobacterium marismortui” in a New Genus, Chromohalobacter gen. nov., as Chromohalobacter marismortui comb. nov., nom. rev.

Phenotypic and chemotaxonomic characteristics of seven moderately halophilic isolates from a mediterranean saltern showed that they were very closely related to “Chromobacterium marismortui.” This species, which was originally described on the basis of strains isolated from the Dead Sea, was not included on the Approved Lists of Bacterial Names and is not accepted as a member of the genus Chromobacterium since it produces a pigment that is not violacein, does not possess the characteristic flagellar arrangement, and is not genotypically related to this genus as determined by deoxyribonucleic acid-ribosomal ribonucleic acid hybridization experiments. On the basis of the special features of these strains, we propose that they should be placed in a new genus, Chromohalobacter, which includes a single species, Chromohalobacter marismortui comb. nov., nom. rev. The deoxyribonucleic acid base composition is 62.1 to 64.9 mol% guanine plus cytosine. The type strain is strain ATCC 17056 (= CCM 3518).

Taxonomic Study and Amended Description of Vibrio costicola

A total of 54 moderately halophilic vibrios, which were isolated from several salterns located in different areas of Spain, were examined by using a wide range of morphological, physiological, biochemical, and nutritional tests. The resulting data, together with data for four reference Vibrio costicola strains including the type strain V. costicola NCMB 701, and other marine species that were similarly examined, were compared by using several numerical taxonomic methods. There was a strong similarity between the 54 isolates and four reference strains of V. costicola that were isolated from cured meats. On the basis of these and other molecular data, including guanine-plus-cytosine content of the deoxyribonucleic acid and the plasmid content, we propose an amended description of this species.

Transfer of Halomonas canadensis and Halomonas israelensis to the genus Chromohalobacter as Chromohalobacter canadensis comb. nov. and Chromohalobacter israelensis comb. nov.

16S rRNA gene sequence comparisons and DNA-DNA hybridization data support the conclusion that two species previously described as members of the genus Halomonas, Halomonas israelensis and Halomonas canadensis, should be placed in the genus Chromohalobacter. Both H. israelensis ATCC 43985T (= Ba1T) and H. canadensis ATCC 43984T (= NRCC 41227T) have been used extensively for physiological studies for many years; nevertheless, they were not named and classified taxonomically until quite recently. Their phenotypic resemblance (at least 65% Jaccard similarity) to some members of the genus Halomonas and the degree of DNA-DNA relatedness (lower than 60%) to other described species of this genus permitted the conclusion that they were distinct species belonging to the genus Halomonas. In this study, the 16S rDNA of both species has been sequenced completely and found to share higher similarity to the available sequences of the moderately halophilic bacterium Chromohalobacter marismortui than to sequences of members of the genus Halomonas. C. marismortui is the sole species of the genus Chromohalobacter, also included in the family Halomonadaceae, and shares many phenotypic features with H. canadensis and H. israelensis. It is proposed that the two species should be renamed as Chromohalobacter canadensis comb. nov. and Chromohalobacter israelensis comb. nov. An emended description of the genus Chromohalobacter is given in order to include the features of these two species.

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.

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

Background Among 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 Findings A 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. Conclusions In 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.

Bacillus halophilus sp. nov., a Moderately Halophilic Bacillus Species

Summary Bacillus halophilus, a new moderately halophilic (optimum growth at 15% salt) species, is described. This species was isolated from rotting wood from the Pacific Ocean. The G+C mol % (Tm) of its deoxyribonucleic acid is 51.5. The type strain is N23-2 (= ATCC 49085 = DSM 4771 = CCM 4074).

Characterization of Salicola sp. IC10, a lipase- and protease-producing extreme halophile.

In order to explore the diversity of extreme halophiles able to produce different hydrolytic enzymes (amylase, protease, lipase and DNAse) in hypersaline habitats of South Spain, a screening program was performed. A total of 43 extreme halophiles showing hydrolytic activities have been isolated and characterized. The isolated strains were able to grow optimally in media with 15-20% (w/v) total salts and in most cases, growth was detected up to 30% (w/v) total salts. Most hydrolase producers were assigned to the family Halobacteriaceae, belonging to the genera Halorubrum (22 strains), Haloarcula (nine strains) and Halobacterium (nine strains), and three isolates were characterized as extremely halophilic bacteria (genera Salicola, Salinibacter and Pseudomonas). An extremely halophilic isolate, strain IC10, showing lipase and protease activities and identified as a Salicola strain of potential biotechnological interest, was further studied. The optimum growth conditions for this strain were 15-20% (w/v) NaCl, pH 8.0, and 37 degrees C. Zymographic analysis of strain IC10 detected the lipolytic activity in the intracellular fraction, showing the highest activity against p-nitrophenyl-butyrate as a substrate in a colorimetric assay, whereas the proteolytic activity was detected in the extracellular fraction. This protease degraded casein, gelatin, bovine serum albumin and egg albumin.

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.