Correction: A Straightforward Access to New Families of Lipophilic Polyphenols by Using Lipolytic Bacteria.

[This corrects the article DOI: 10.1371/journal.pone.0166561.].

Identification of an acetyl esterase in the supernatant of the environmental strain Bacillus sp. HR21-6.

Bacillus sp. HR21-6 is capable of the chemo- and regioselective synthesis of lipophilic partially acetylated phenolic compounds derived from olive polyphenols, which are powerful antioxidants important in the formulation of functional foods. In this work, an acetyl esterase was identified in the secretome of this strain by non-targeted proteomics, and classified in the GDSL family (superfamily SGNH). The recombinant protein was expressed and purified from Escherichia coli in the soluble form, and biochemically characterized. Site-directed mutagenesis was performed to understand the role of different amino acids that are conserved among GDSL superfamily of esterases. Mutation of Ser-10, Gly-45 or His-185 abolished the enzyme activity, while mutation of Asn-77 or Thr-184 altered the substrate specificity of the enzyme. This new enzyme is able to perform chemoselective conversions of olive phenolic compounds with great interest in the food industry, such as hydroxytyrosol, 3,4-dihydroxyphenylglycol, and oleuropein.

A Highly Sensitive Method for the Detection of Hydrolyzed Gluten in Beer Samples Using LFIA.

Most gluten analysis methods have been developed to detect intact gluten, but they have shown limitations in certain foods and beverages in which gluten proteins are hydrolyzed. Methods based on G12/A1 moAbs detect the sequences of gluten immunogenic peptides (GIP), which are the main contributors to the immune response of celiac disease (CD). Immunogenic sequences with tandem epitopes for G12/A1 have been found in beers with <20 mg/kg gluten, which could be consumed by CD patients according to the Codex Alimentarius. Therefore, an accurate method for the estimation of the immunogenicity of a beer is to use two moAbs that can recognize celiac T cell epitopes comprising most of the immunogenic response. Here, a specific and sensitive method based on G12/A1 LFIA was developed to detect GIP in beers labeled gluten-free or with low gluten content, with an LOD of 0.5 mg/kg. A total of 107 beers were analyzed, of those 6.5% showed levels higher than 20 mg/kg gluten and 29% showed levels above the LOD. In addition, G12/A1 LFIA detected gluten in 15 more beer samples than competitive ELISA with another antibody. Despite their labeling, these beers contained GIP which may cause symptoms and/or intestinal damage in CD patients.

An arsRB resistance operon confers tolerance to arsenite in the environmental isolate Terribacillus sp. AE2B 122.

Terribacillus sp. AE2B 122 is an environmental strain isolated from olive-oil agroindustry wastes. This strain displays resistance to arsenic, one of the most ubiquitous carcinogens found in nature. Terribacillus sp. AE2B 122 possesses an unusual ars operon, consisting of the transcriptional regulator (arsR) and arsenite efflux pump (arsB) but no adjacent arsenate reductase (arsC) locus. Expression of arsR and arsB was induced when Terribacillus was exposed to sub-lethal concentrations of arsenate. Heterologous expression of the arsB homologue in Escherichia coli∆arsRBC demonstrated that it conferred resistance to arsenite and reduced the accumulation of arsenic inside the cells. Two members of the arsC-like family (Te3384 and Te2854) found in the Terribacillus genome were not induced by arsenic, but their heterologous expression in E. coli ∆arsC and ∆arsRBC increased the accumulation of arsenic in both strains. We found that both Te3384 and Te2854 slightly increased resistance to arsenate in E. coli ∆arsC and ∆arsRBC, possibly by chelation of arsenic or by increasing the resistance to oxidative stress. Finally, arsenic speciation assays suggest that Terribacillus is incapable of arsenate reduction, in agreement with the lack of an arsC homologue in the genome.

A Straightforward Access to New Families of Lipophilic Polyphenols by Using Lipolytic Bacteria.

The chemical synthesis of new lipophilic polyphenols with improved properties presents technical difficulties. Here we describe the selection, isolation and identification of lipolytic bacteria from food-processing industrial wastes, and their use for tailoring a new set of compounds with great interest in the food industry. These bacteria were employed to produce lipolytic supernatants, which were applied without further purification as biocatalysts in the chemoselective and regioselective synthesis of lipophilic partially acetylated phenolic compounds derived from olive polyphenols. The chemoselectivity of polyphenols acylation/deacylation was analyzed, revealing the preference of the lipases for phenolic hydroxyl groups and phenolic esters. In addition, the alcoholysis of peracetylated 3,4-dihydroxyphenylglycol resulted in a series of lipophilic 2-alkoxy-2-(3,4-dihydroxyphenyl)ethyl acetate through an unexpected lipase-mediated etherification at the benzylic position. These new compounds are more lipophilic and retained their antioxidant properties. This approach can provide access to unprecedented derivatives of 3,4-dihydroxyphenylglycol with improved properties.

Phylogenetic Profiling and Diversity of Bacterial Communities in the Death Valley, an Extreme Habitat in the Atacama Desert.

The Atacama Desert, one of the driest deserts in the world, represents a unique extreme environmental ecosystem to explore the bacterial diversity as it is considered to be at the dry limit for life. A 16S rRNA gene (spanning the hyper variable V3 region) library was constructed from an alkaline sample of unvegetated soil at the hyperarid margin in the Atacama Desert. A total of 244 clone sequences were used for MOTHUR analysis, which revealed 20 unique phylotypes or operational taxonomic units (OTUs). V3 region amplicons of the 16S rRNA were suitable for distinguishing the bacterial community to the genus and specie level. We found that all OTUs were affiliated with taxa representative of the Firmicutes phylum. The extremely high abundance of Firmicutes indicated that most bacteria in the soil were spore-forming survivors. In this study we detected a narrower diversity as compared to other ecological studies performed in other areas of the Atacama Desert. The reported genera were Oceanobacillus (representing the 69.5 % of the clones sequenced), Bacillus, Thalassobacillus and Virgibacillus. The present work shows physical and chemical parameters have a prominent impact on the microbial community structure. It constitutes an example of the communities adapted to live in extreme conditions caused by dryness and metal concentrations .

Selection and characterization of biofuel-producing environmental bacteria isolated from vegetable oil-rich wastes.

Fossil fuels are consumed so rapidly that it is expected that the planet resources will be soon exhausted. Therefore, it is imperative to develop alternative and inexpensive new technologies to produce sustainable fuels, for example biodiesel. In addition to hydrolytic and esterification reactions, lipases are capable of performing transesterification reactions useful for the production of biodiesel. However selection of the lipases capable of performing transesterification reactions is not easy and consequently very few biodiesel producing lipases are currently available. In this work we first isolated 1,016 lipolytic microorganisms by a qualitative plate assay. In a second step, lipolytic bacteria were analyzed using a colorimetric assay to detect the transesterification activity. Thirty of the initial lipolytic strains were selected for further characterization. Phylogenetic analysis revealed that 23 of the bacterial isolates were Gram negative and 7 were Gram positive, belonging to different clades. Biofuel production was analyzed and quantified by gas chromatography and revealed that 5 of the isolates produced biofuel with yields higher than 80% at benchtop scale. Chemical and viscosity analysis of the produced biofuel revealed that it differed from biodiesel. This bacterial-derived biofuel does not require any further downstream processing and it can be used directly in engines. The freeze-dried bacterial culture supernatants could be used at least five times for biofuel production without diminishing their activity. Therefore, these 5 isolates represent excellent candidates for testing biofuel production at industrial scale.

Phenol degradation by halophilic bacteria isolated from hypersaline environments.

Phenol is a toxic aromatic compound used or produced in many industries and as a result a common component of industrial wastewaters. Phenol containing waste streams are frequently hypersaline and therefore require halophilic microorganisms for efficient biotreatment without dilution. In this study three halophilic bacteria isolated from different saline environments and identified as Halomonas organivorans, Arhodomonas aquaeolei and Modicisalibacter tunisiensis were shown to be able to grow on phenol in hypersaline media containing 100 g/L of total salts at a concentration of 3 mM (280 mg/L), well above the concentration found in most waste streams. Genes encoding the aromatic dioxygenase enzymes catechol 1,2 dioxygenase and protocatechuate 3,4-dioxygenase were present in all strains as determined by PCR amplification using primers specific for highly conserved regions of the genes. The gene for protocatechuate 3,4-dioxygenase was cloned from the isolated H. organivorans and the translated protein was evaluated by comparative protein sequence analysis with protocatechuate 3,4-dioxygenase proteins from other microorganisms. Although the analysis revealed a wide range of sequence divergence among the protocatechuate 3,4-dioxygenase family, all of the conserved domain amino acid structures identified for this enzyme family are identical or conservatively substituted in the H. organivorans enzyme.

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 ß-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 ß-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.

Carotenoids' production from halophilic bacteria.

Carotenoids have received considerable attention due to their interesting industrial applications and, more importantly, their potential beneficial effects on human health. Halophiles comprise a heterogeneous group of microorganisms that need salts for optimal growth. The pigments produced by these halophilic organisms comprise phytoene, ß-carotene, lycopene, derivatives of bacterioruberin, and salinixanthin. Here, we describe the procedure to obtain salinixanthin from the extremely halophilic bacterium Salinibacter ruber. Additionally, we describe the expression of the ß-carotene biosynthetic genes crtE, crtY, crtI, and crtB from Pantoea agglomerans in the moderately halophilic bacterium Halomonas elongata obtaining a strain able to produce practically pure ß-carotene. Thus, the use of these halophilic microorganisms as a source of carotenoids constitutes an important commercial alternative in the production of carotenoids from biological sources.

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 ß-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.

Cloning, characterization and analysis of cat and ben genes from the phenol degrading halophilic bacterium Halomonas organivorans.

BACKGROUND: Extensive use of phenolic compounds in industry has resulted in the generation of saline wastewaters that produce significant environmental contamination; however, little information is available on the degradation of phenolic compounds in saline conditions. Halomonas organivorans G-16.1 (CECT 5995(T)) is a moderately halophilic bacterium that we isolated in a previous work from saline environments of South Spain by enrichment for growth in different pollutants, including phenolic compounds. PCR amplification with degenerate primers revealed the presence of genes encoding ring-cleaving enzymes of the ß-ketoadipate pathway for aromatic catabolism in H. organivorans. FINDINGS: The gene cluster catRBCA, involved in catechol degradation, was isolated from H. organivorans. The genes catA, catB, catC and the divergently transcribed catR code for catechol 1,2-dioxygenase (1,2-CTD), cis,cis-muconate cycloisomerase, muconolactone delta-isomerase and a LysR-type transcriptional regulator, respectively. The benzoate catabolic genes (benA and benB) are located flanking the cat genes. The expression of cat and ben genes by phenol and benzoic acid was shown by RT-PCR analysis. The induction of catA gene by phenol and benzoic acid was also probed by the measurement of 1,2-CTD activity in H. organivorans growth in presence of these inducers. 16S rRNA and catA gene-based phylogenies were established among different degrading bacteria showing no phylogenetic correlation between both genes. CONCLUSIONS/SIGNIFICANCE: In this work, we isolated and determined the sequence of a gene cluster from a moderately halophilic bacterium encoding ortho-pathway genes involved in the catabolic metabolism of phenol and analyzed the gene organization, constituting the first report characterizing catabolic genes involved in the degradation of phenol in moderate halophiles, providing an ideal model system to investigate the potential use of this group of extremophiles in the decontamination of saline environments.

Biodegradation of aromatic hydrocarbons by Haloarchaea and their use for the reduction of the chemical oxygen demand of hypersaline petroleum produced water.

Ten halophilic Archaea (Haloarchaea) strains able to degrade aromatic compounds were isolated from five hypersaline locations; salt marshes in the Uyuni salt flats in Bolivia, crystallizer ponds in Chile and Cabo Rojo (Puerto Rico), and sabkhas (salt flats) in the Persian Gulf (Saudi Arabia) and the Dead Sea (Israel and Jordan). Phylogenetic identification of the isolates was determined by 16S rRNA gene sequence analysis. The isolated Haloarchaea strains were able to grow on a mixture of benzoic acid, p-hydroxybenzoic acid, and salicylic acid (1.5mM each) and a mixture of the polycyclic aromatic hydrocarbons, naphthalene, anthracene, phenanthrene, pyrene and benzo[a]anthracene (0.3mM each). Evaluation of the extent of degradation of the mixed aromatic hydrocarbons demonstrated that the isolates could degrade these compounds in hypersaline media containing 20% NaCl. The strains were shown to reduce the COD of hypersaline crude oil reservoir produced waters significantly beyond that achieved using standard hydrogen peroxide treatment alone.

The extremely halophilic bacterium Salicola marasensis IC10 accumulates the compatible solute betaine.

The extremely halophilic bacterium strain IC10 was isolated from a solar saltern on Isla Cristina (southern Spain). Phylogenetic, genotypic and phenotypic data supported the inclusion of this strain in the species Salicola marasensis. An analysis of intracellular organic osmotic solutes showed glycine betaine to be present, contributing to the overall osmotic balance, and this was the only compatible solute accumulated when S. marasensis IC10 was grown over a wide range of external NaCl concentrations (10-25%, w/v).

The haloprotease CPI produced by the moderately halophilic bacterium Pseudoalteromonas ruthenica is secreted by the type II secretion pathway.

The gene (cpo) encoding the extracellular protease CPI produced by the moderately halophilic bacterium Pseudoalteromonas ruthenica CP76 was cloned, and its nucleotide sequence was analyzed. The cpo gene encodes a 733-residue protein showing sequence similarity to metalloproteases of the M4 family. The type II secretion apparatus was shown to be responsible for secretion of the haloprotease CPI.

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.

Engineering the halophilic bacterium Halomonas elongata to produce beta-carotene.

Engineering halophilic bacteria to produce carotenoids is a subject of great scientific and commercial interest, as carotenoids are desirable products used as additives and colorants in the food industry, with beta-carotene the most prominent. With this target, we expressed the beta-carotene biosynthetic genes crtE, crtY, crtI, and crtB from Pantoea agglomerans and the cDNA encoding isopentenyl pyrophosphate isomerase from Haematococcus pluvialis in the halophilic bacterium Halomonas elongata obtaining a strain able to produce practically pure beta-carotene. Reverse transcription-polymerase chain reaction analysis showed crtY, crtI, and crtB heterologous expression in a selected exconjugant of H. elongata. Biosynthesis of beta-carotene was dependent on NaCl concentration in the culture medium, with the highest production (560 microg per g of dry weight) in 2% NaCl. On the contrary, no beta-carotene was detected in 15% NaCl. Successful construction of the beta-carotene biosynthetic pathway in H. elongata opens the possibility of engineering halophilic bacteria for carotenoid production.

Catabolic versatility of aromatic compound-degrading halophilic bacteria.

There is growing interest in the development and optimization of bioremediation processes to deal with environments with high salinity that are contaminated with aromatic compounds. To estimate the diversity of moderately halophilic bacteria that could be used in such processes, enrichments were performed based on growth with a variety of aromatic compounds including phenol as a model pollutant. A group of bacteria that were able to grow over a wide range of salt concentrations were isolated, with the majority of these assigned to the genus Halomonas using phenotypic features and 16S rRNA sequences comparison. PCR amplification with degenerate primers revealed the presence in these isolates of genes encoding ring-cleaving enzymes in the beta-ketoadipate pathway for aromatic catabolism: catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase. Furthermore, the activity of these two enzymes was detected in the newly described species Halomonas organivorans. Together, these studies indicate that moderately halophilic bacteria have the potential to catabolize aromatic compounds in environments with high salinity.

Thalassobacillus devorans gen. nov., sp. nov., a moderately halophilic, phenol-degrading, Gram-positive bacterium.

A novel moderately halophilic bacterium, strain G-19.1(T), has been isolated from a phenol enrichment of samples collected in hypersaline habitats of southern Spain. This enrichment culture was a part of a screening programme to isolate halophilic bacteria able to degrade various aromatic compounds. Strain G-19.1(T) has been characterized as a potential phenol-degrader over a wide range of saline conditions. Strain G-19.1(T) was found to be an aerobic, Gram-positive, endospore-forming, non-pigmented, moderately halophilic rod that grew optimally in media containing 7.5-10% NaCl at pH 7.0. The DNA G+C content was 42.4 mol%. Phylogenetic analysis based on comparison of 16S rRNA gene sequences indicated that the closest relatives were Halobacillus species (96.2-97.0%), although this novel isolate constitutes a separate line of descent within the radiation of Gram-positive rods. The cell-wall peptidoglycan contained meso-diaminopimelic acid, indicating that this strain does not share the main characteristic that differentiates members of the genus Halobacillus (which contain Orn-D-Asp) from other related genera. The predominant cellular fatty acids were anteiso-C(15:0), iso-C(16:0) and iso-C(15:0). On the basis of phenotypic, genotypic and phylogenetic analyses, this isolate should be classified in a novel genus and species, for which the name Thalassobacillus devorans gen. nov., sp. nov. is proposed. The type strain is strain G-19.1(T) (=DSM 16966(T)=CECT 7046(T)=CCM 7282(T)).