Biosurfactants produced by Serratia species: Classification, biosynthesis, production and application.

Biosurfactants are surface-active molecules that are synthesised non-ribosomally by a wide range of microorganisms including bacteria, yeast and filamentous fungi. The bacterial genus Serratia is gaining international interest, as biosurfactants produced by this genus have emerged as a promising source of antimicrobial, antifouling and antitumour compounds that possess emulsification and surface activity. Various species of Serratia have been identified as biosurfactant producers, including Serratia marcescens, Serratia rubidaea and Serratia surfactantfaciens. Members of the Serratia genus have been reported to principally produce two classes of biosurfactants, namely lipopeptides and glycolipids. Lipopeptides produced by Serratia species include serrawettins and stephensiolides, while identified glycolipids include rubiwettins and rhamnolipids. This review will primarily focus on the classification of biosurfactants produced by Serratia species and the genes and mechanisms involved in the biosynthesis of these biosurfactant compounds. Thereafter, an indication of the primary growth conditions and nutrient composition required for the optimum production of biosurfactants by this genus will be outlined. An overview of the latest advances and potential applications of the biosurfactants produced by Serratia in the medical, pharmaceutical, agricultural and petroleum industries is also provided.

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2013-2014.

This review is the eighth update of the original article published in 1999 on the application of Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2014. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, and arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly- saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. © 2018 Wiley Periodicals, Inc. Mass Spec Rev 37:353-491, 2018.

Rhamnolipid CMC prediction.

Relationships between the purity, pH, hydrophobicity (logKow) of the carbon substrate, and the critical micelle concentration (CMC) of rhamnolipid type biosurfactants (RL) were investigated using a quantitative structure-property relationship (QSPR) approach and are presented here for the first time. Measured and literature CMC values of 97 RLs, representing biosurfactants at different stages of purification, were considered. An arbitrary scale for RLs purity was proposed and used in the modelling. A modified evolutionary algorithm was used to create clusters of equations to optimally describe the relationship between CMC and logKow, pH and purity (the optimal equation had an R2 of 0.8366). It was found that hydrophobicity of the carbon substrate used for the biosynthesis of the RL had the most significant influence on the final CMC of the RL. Purity of the RLs was also found to have a significant impact, where generally the less pure the RL the higher the CMC. These results were in accordance with our experimental data. Therefore, our model equation may be used for controlling the biosynthesis of biosurfactants with properties targeted for specific applications.

Membrane glycerolipidome of soybean root hairs and its response to nitrogen and phosphate availability.

Root hairs are tubular extensions of specific root epidermal cells important in plant nutrition and water absorption. To determine membrane glycerolipids in root hairs and roots may differ, as well as their respective response to nutrient availability, this study analyzed the membrane glycerolipid species in soybean root hairs and in roots stripped of root hairs, and their response to nitrogen (N) and phosphate (Pi) supplementation. The ratio of phospholipids to galactolipids was 1.5 fold higher in root hairs than in stripped roots. Under Pi deficiency, the ratio of phospholipids to galactolipids in stripped roots decreased with the greatest decrease found in the level of phosphatidylethanolamine (PE) in root hairs and stripped roots, and root hairs had an increased level of phosphatidic acid (PA). When seedlings were not supplied with N, the level of the N-containing lipids PE and phosphatidylserine in root hairs decreased whereas the level of non-N-containing lipids galactolipids and PA increased compared to N-supplied conditions. In stripped roots, the level of major membrane lipids was not different between N-sufficient and -deficient conditions. The results indicate that the membrane glycerolipidomes in root hairs are more responsive to nutrient availability than are the rest of roots.

LipidBlast templates as flexible tools for creating new in-silico tandem mass spectral libraries.

Tandem mass spectral libraries (MS/MS) are usually built by acquiring experimentally measured mass spectra from chemical reference compounds. We here show the versatility of in-silico or computer generated tandem mass spectra that are directly obtained from compound structures. We use the freely available LipidBlast development software to generate 15 000 MS/MS spectra of the glucuronosyldiacylglycerol (GlcADG) lipid class, recently discovered for the first time in plants. The generation of such an in-silico MS/MS library for positive and negative ionization mode took 5 h development time, including the validation of the obtained mass spectra. Such libraries allow for high-throughput annotations of previously unknown glycolipids. The publicly available LipidBlast templates are universally applicable for the development of MS/MS libraries for novel lipid classes.

Separation and classification of lipids using differential ion mobility spectrometry.

Correlations between the dimensions of a 2-D separation create trend lines that depend on structural or chemical characteristics of the compound class and thus facilitate classification of unknowns. This broadly applies to conventional ion mobility spectrometry (IMS)/mass spectrometry (MS), where the major biomolecular classes (e.g., lipids, peptides, nucleotides) occupy different trend line domains. However, strong correlation between the IMS and MS separations for ions of same charge has impeded finer distinctions. Differential IMS (or FAIMS) is generally less correlated to MS and thus could separate those domains better. We report the first observation of chemical class separation by trend lines using FAIMS, here for lipids. For lipids, FAIMS is indeed more independent of MS than conventional IMS, and subclasses (such as phospho-, glycero-, or sphingolipids) form distinct, often non-overlapping domains. Even finer categories with different functional groups or degrees of unsaturation are often separated. As expected, resolution improves in He-rich gases: at 70% He, glycerolipid isomers with different fatty acid positions can be resolved. These results open the door for application of FAIMS to lipids, particularly in shotgun lipidomics and targeted analyses of bioactive lipids.

Distribution of receptor glycolipids for Lactobacilli in murine digestive tract and production of antibodies cross-reactive with them by immunization of rabbits with Lactobacilli.

In the digestive tract of mice (HR-1 strain), glycolipids belonging to the ganglio-series were revealed to be expressed in region-specific manners, i.e. FGA1 and FGM1 in the stomach, GA1 in the small intestine, and FGA1 and sulphatides in the cecum. The amount of GA1 as a receptor glycolipid for Lactobacilli was especially higher in the small intestine than in the other regions, it comprising 1.6-2.8 microg/mg dry weight. On immunization of rabbits with Lactobacillus johnsonii and Lactobacillus intestinalis, both of which are murine intestinal bacteria, antibodies toward bacterial glycolipids, i.e. Galalpha1-2Glcalpha1-3DG, and tri- and tetrahexaosyl DGs, were effectively generated and, in addition, they were found to cross-react with GA1 and GalCer, but not with structurally related glycolipids such as Lc(4)Cer, nLc(4)Cer and IV(3)Galalpha-nLc(4)Cer, indicating that GA1 is a preferable antigen for anti-lactobacillus antisera and suggesting the presence of epitopes common to both Lactobacilli and the host. In fact, molecules reacting with anti-GA1 antibodies were detected among bacterial proteins on Western blotting, indicating a possible occurrence of the carbohydrate structure mimicking GA1 in bacterial proteins.

Characterization of new types of mannosylerythritol lipids as biosurfactants produced from soybean oil by a basidiomycetous yeast, Pseudozyma shanxiensis.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These show not only the excellent surface-active properties but also versatile biochemical actions. In course of MEL production from soybean oil by P. shanxiensis, new extracellular glycolipids (more hydrophilic than the previously reported MELs) were found in the culture medium. As a result of the structural characterization, the glycolipids were identified as a mixture of 4-O-[(2', 4'-di-O-acetyl-3'-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol and 4-O-[(4'-O-acetyl-3'-O-alka(e)noyl-2'-O-butanoyl)-beta-D-mannopyranosyl]-D-erythritol. Interestingly, the new MELs possessed a much shorter chain (C(2) or C(4)) at the C-2' position of the mannose moiety compared to the MELs hitherto reported, which mainly possess a medium-chain acid (C(10)) at the position. They would thus show higher hydrophilicity and/or water-solubility, and expand the development of the environmentally advanced yeast biosurfactants.

Rhamnolipids from the rhizosphere bacterium Pseudomonas sp. GRP(3) that reduces damping-off disease in Chilli and tomato nurseries.

A detailed screening of bacterial isolates from the Central Himalayan region for plant growth promotion and antimycelial activity against Pythium and Phytophthora strains afforded seven isolates, of which three were particularly effective against the incidence of damping-off in field trials on chilli and tomato. In this investigation an initial spectroscopic survey of the methanolic extracts of the seven bacterial isolates showed complex mixtures except for Pseudomonas sp. GRP3, one of the most promising isolates on the basis of field studies. Strain GRP3 was selected for structural characterization of its secondary metabolites, particularly glycolipids. The extracellular secondary metabolites were enriched by Amberlite XAD-16 adsorber resin followed by separation and structural analysis using TLC, LC-MS, MS-MS, and NMR spectroscopy. Acquired data show the presence of a number of mono- and dirhamnolipids and include rhamnose (Rha)-C8-C10, Rha-C10-C8, Rha-C10-C10, Rha-C10-C12:1, Rha-C10-C12, Rha-Rha-C8-C10, Rha-Rha-C10-C10, Rha-Rha-C10-C10:1, Rha-Rha-C10-C12, Rha-Rha-C10-C12:1, Rha-Rha-C12-C12:1, and Rha-Rha-C12-C12 in strain GRP3. Since rhamnolipids are involved in the lysis of the plasma membrane of zoospores of fungi, application of such rhamnolipid-producing rhizobacteria could facilitate control of damping-off plant pathogens.

Alterations in the molecular species of plasmalogen phospholipids and glycolipids due to peroxisomal dysfunction in Chinese hamster ovary-mutant Z65 cells by FABMS method.

Changes in the molecular species of lipids associated with Pex2 gene-mutation were investigated to elucidate the pathogeneses of peroxisome biogenesis disorders. Although no differences were observed in the concentrations of cholesterol and phosphatidyl choline between mutated Z65 and control CHO-K1 cells, the amounts of cholesterol esters and glycolipids in Z65 cells were twice those in CHO-K1 cells, but phosphatidyl ethanolamine (PE), particularly 1-O-octadec-1'-enyl-2-oleoyl PE, was absent in Z65 cells by FABMS. Enhanced synthesis of glycolipids in Z65 cells was associated with an abundance of lignoceric acid-containing ones, suggesting a role of glycolipids in the retention of longer saturated fatty acids.

Glycolipid class profiling by packed-column subcritical fluid chromatography.

The potential of packed-column subcritical fluid chromatography (SubFC) for the separation of lipid classes has been assessed in this study. Three polar stationary phases were checked: silica, diol, and poly(vinyl alcohol). Carbon dioxide (CO2) with methanol as modifier was used as mobile phase and detection performed by evaporative light scattering detection. The influence of methanol content, temperature, and pressure on the chromatographic behavior of sphingolipids and glycolipids were investigated. A complete separation of lipid classes from a crude wheat lipid extract was achieved using a modifier gradient from 10 to 40% methanol in carbon dioxide. Solute selectivity was improved using coupled silica and diol columns in series. Because the variation of eluotropic strength depending on the fluid density changes, a normalized separation factor product (NSP) was used to select the nature, the number and the order of the columns to reach the optimum glycolipid separation.

Lipase-mediated deacetylation and oligomerization of lactonic sophorolipids.

The direct enzymatic polymerization of lactonic sophorolipids (SLs) was investigated with four lipases, including porcine pancreatic lipase (PPL), immobilized Mucor miehei lipase (MML), lyophilized Candida antarctica lipase (Fraction B, CAL-B), and lyophilized Pseudomonas sp. lipase (PSL). Several organic solvents, covering a wide range of polarity, were compared for suitability as the reaction medium. Isopropyl ether and toluene were found most effective. According to the quantification and structure identification by HPLC and LC-MS, the reaction proceeded with the formation of monoacetylated lactonic SLs and the subsequent conversion of the intermediates to oligomers and polymers, presumably through ring-opening polymerization. Temperature was found to have significant effects on the reaction. Both the conversion of reactant SLs and the subsequent formation of oligomers and polymers from the intermediates were faster at 60 degrees C than at 50 degrees C. The substrate selectivity among the three dominant reactant SLs also differed with the temperature. The conversion rate increased with the ring size of the lactones at 60 degrees C, but it decreased with the size at 50 degrees C.

Asialo-GM1 and asialo-GM2 are putative adhesion molecules for Moraxella catarrhalis.

Moraxella catarrhalis is an important pathogen of respiratory and middle ear infections. We previously reported that the attachment of M. catarrhalis to pharyngeal epithelial cells is mediated by ganglioside M2 (GM2). Several sets of adhesins or receptors are involved in such attachment process. In this study, we used the same strains and similar bacterial culture conditions as those in our previous study, and demonstrated by thin layer chromatography that M. catarrhalis can also bind to asialo-GM1 (Gg4Cer) and asialo-GM2 (Gg3Cer). GalNAcbeta1-->4Galbeta1 is a common sequence in both Gg4Cer and Gg3Cer, and in many respiratory bacteria, this sequence acts as a receptor for attachment to host cells. Treatment of human pharyngeal epithelial cells with anti-GM2 and anti-Gg4Cer antibodies significantly decreased attachment of M. catarrhalis to these cells; however, treatment with anti-Gg3Cer antibody did not decrease M. catarrhalis attachment. Immunofluorescence microscopy revealed that human pharyngeal epithelial cells are positive for GM2 and Gg4Cer, but not for Gg3Cer. Our results indicate that Gg4Cer on human pharyngeal epithelial cells, and Gg3Cer,possibly on other cells, could serve as molecules for attachment of M. catarrhalis.

Analysis of molecular species of glycolipids in fruit pastes of red bell pepper (Capsicum annuum L.) by high-performance liquid chromatography-mass spectrometry.

Five major glycolipid classes (acylated steryl glucoside, steryl glucoside, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and glucocerebroside) from fruit pastes of red bell pepper were separated by silica gel column chromatography. The molecular species of each glycolipid were separated and characterized by reversed-phase high-performance liquid chromatography coupled with on-line mass spectrometry using atmospheric pressure chemical ionization. The molecular species of steryl glucoside were beta-sitosteryl and campesteryl glucosides, and those of the acylated steryl glucoside were their fatty acid esters. The dilinolenoyl species was predominant in monogalactosyldiacylglycerol in addition to small amounts of another five molecular species, whereas digalactosyldiacylglycerol consisted of seven molecular species varying in their degree of unsaturation. The glucocerebroside class contained at least seven molecular species, which were characterized by proton nuclear magnetic resonance spectroscopy.

Glycolipid antigen for use in diagnostic assays for bovine tuberculosis.

A glycolipid antigen, was isolated, purified and characterized from Mycobacterium bovis An5. Chemical analysis (thin-layer chromatography, nuclear magnetic resonance and infrared spectra) showed that this glycolipid was a 2,3-di-O-acyl trehalose (DAT), similar to the DAT of M. tuberculosis. This antigen was used to establish ELISA-based serodiagnostic tests for M. bovis-infected cattle. The sensitivity and specificity of the assay were investigated using sera of cattle from tuberculosis-free herds and from tuberculosis-infected herds. No correlation was found between DAT-ELISA and the skin test, nor between DAT-ELISA and interferon-gamma with bovine purified protein derivative. The antibody titres were not related to cell-mediated immunity. Although the antigen was highly specific (95.9%), the sensitivity of DAT-ELISA, as judged from assays in bacteriologically confirmed tuberculosis, was low (29 to 36.8%). The low sensitivity of ELISA might also be attributed to a reciprocal relationship between B-cell proliferation and T-cell protective immunity.

Complex polar lipids of a hot spring cyanobacterial mat and its cultivated inhabitants.

The complex polar lipids of the hot spring cyanobacterial mat in the 50 to 55 degrees C region of Octopus Spring, Yellowstone National Park, and of thermophilic bacteria cultivated from this or similar habitats, were compared in an attempt to understand the microbial sources of the major lipid biomarkers in this community. Intact complex lipids were analyzed directly by fast atom bombardment mass spectrometry (FAB-MS), two-dimensional thin-layer chromatography (TLC), and combined TLC-FAB-MS. FAB-MS and TLC gave qualitatively similar results, suggesting that the mat contains major lipids most like those of the cyanobacterial isolate we studied, Synechococcus sp. strain Y-7c-s. These include monoglycosyl, diglycosyl, and sulfoquinosovyl diglycerides (MG, DG, and SQ, respectively) and phosphatidyl glycerol (PG). Though Chloroflexus aurantiacus also contains MG, DG, and PG, the fatty acid chain lengths of mat MGs, DGs, and PGs resemble more those of cyanobacterial than green nonsulfur bacterial lipids. FAB-MS spectra of the lipids of nonphototrophic bacterial isolates were distinctively different from those of the mat and phototrophic isolates. The lipids of these nonphototrophic isolates were not detected in the mat, but most could be detected when added to mat samples. The mat also contains major glycolipids and aminophospholipids of unknown structure and origin. FAB-MS and TLC did not always give quantitatively similar results. In particular, PG and SQ may give disproportionately high FAB-MS responses.

Glycosphingolipid clusters and the sorting of GPI-anchored proteins in epithelial cells

We studied the role of the association between glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1-DAF, a GPI-anchored protein that is sorted differentially by three epithelial cell lines. Differently from MDCK cells, where both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-ConAr) gD1-DAF was mis-sorted to both surfaces but GlcCer was still targeted to the apical surface. In both MDCK and MDCK-ConAr cells, gD1-DAF became associated with TX-100 insoluble GSL clusters during transport to the cell surface. In contrast to MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. Both MDCK and FRT cells had the ability to assemble GSLs into TX-100-insoluble complexes, but, surprisingly, in FRT cells, gD1-DAF did not associate with GSLs and, therefore, remained completely soluble in TX100. This clustering defect in FRT cells correlated with the absence of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the TGN. This suggests that VIP21/caveolin may have an important role in recruiting GPI-anchored proteins into GSL complexes, necessary for their apical sorting. However,since MDCK-ConAr cells expressed caveolin and clustered GPI-anchored proteins normally, yet mis-sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting and that additional factors are required for the accurate apical sorting of GPI-anchored proteins

Structure and function of mycobacterial glycolipids and glycopeptidolipids.

Earlier work from this and other laboratories has revealed the presence within Mycobacterium spp. of three classes of glycolipid antigens which we have called the glycopeptidolipids, the lipooligosaccharides and the phenolic glycolipids. Representative structures of each from different species and sub-species have been proposed. More recently, new variants of these antigens and older structures have been analyzed by Fourier transform infrared, NMR, particularly at high temperatures, and, most notably, by fast atom bombardment and Californium desorption mass spectrometry. Extraordinary novelty and diversity were revealed, particularly at the distal non-reducing end of the oligosaccharide chains, marked by the presence of new branched-chain sugars, amino sugars and sugar acids. These epitopes and monoclonal antibodies to them have been used for the critical identification of mycobacteria. In addition, the pure antigens are the basis of specific serological tests for various mycobacterioses. The resurgence of interest in "atypical" mycobacteria stems from their occurrence as opportunistic pathogens in many patients with acquired immunodeficiency syndrome, although they have long been associated with pulmonary and other organ infections. Foremost among these mycobacteria are serovars of the Mycobacterium avium-Mycobacterium intracellulare complex (the M. avium complex). The surface antigens which differentiate these serovars are glycopeptidolipids, related to "mycoside C" and, accordingly, composed of a glycosylated lipopeptide "core", fatty acyl-D-Phe-D-alloThr-D-Ala-L-acanyl-O- (3,4-di-O-methyl-alpha-L-rhamnopyranoside), to which a haptenic oligosaccharide is linked at the threonine substituent; this oligoglycosyl unit is the source of type specificity.(ABSTRACT TRUNCATED AT 250 WORDS)