Significance and taxonomic value of iso and anteiso monoenoic fatty acids and branded beta-hydroxy acids in Desulfovibrio desulfuricans.

The fatty acids obtained from extractable lipids of the anaerobic sulfate bacterium Desulfovibrio desulfuricans were identified. Saturated and monoenoic iso (C15-C19) and anteiso (C15, C17) fatty acids and saturated normal (C14-C18) and monoenoic normal (C16, C18) fatty acids were shown to be shown to be present by capillary gas chromatography-mass spectrometry. Iso and anteiso beta-hydroxy fatty acids were analyzed as trimethylsilyl ethers in the same way. The position of methyl branches in the monoenoic fatty acids was determined from characteristic fragment ions in the mass spectra of their methyl esters. Disilyloxy methyl esters, prepared by derivatization of the mono unsaturated methyl esters and analyzed by capillary gas chromatography-mass spectrometry, provided the position of double bonds. The monoenoic fatty acids identified in this way were normal (delta7-C16:1, delta9-C16:1, delta9-C18:1, delta11-C18:1), iso (delta7-C15:1, delta9-C16:1, delta9-C17:1, delta11-C18:1, delta11-C19:1), and anteiso (delta7-C15:1, delta9-C17:1). Iso delta9-C17:1 fatty acid is present as the major component. The occurrence of these monoenoic fatty acids in this bacterium is of taxonomical importance.     

Fatty Acid and Polar Lipid Composition in the Classification of Kurthia

Strains of Kurthia zopfii were degraded by acid methanolysis and the non-hydroxylated fatty acid esters so released were examined by gas liquid chromatography. The major fatty acid types were straight-chain, anteiso - and iso -methyl branched-chain acids. Monounsaturated fatty acids were not detected. The major fatty acid in five of the six strains examined consisted of 12-methyltetra-decanoic ( anteiso -C₁₅) acid. The other strain possessed major amounts of both 13-methyltetradecanoic ( iso -C₁₅) and anteiso -C₁₅ acids. Polar lipids of all the strains were examined by two-dimensional thin-layer chromatography. All possessed a very simple polar lipid composition consisting of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine.     

Isolation and characterization of a C12-lipopeptide produced by Bacillus subtilis HSO 121.

A new lipopeptide with C12 fatty acid has been isolated from the cell broth of Bacillus subtilis HSO121 by chromatographic methods, which is believed to be the homologue of lipopeptides. The fatty acid portion was methylated and analyzed by GC/MS, ESI Q-TOF MS and 1H-NMR. The peptide portion, of which the amino acid composition was obtained by HPLC combined with a phenyl isothiocyanate (PITC) derivatization methods, was analyzed by ESI Q-TOF MS. Comparing the obtained results with surfactin C13 showed that the new lipopeptide has a peptide moiety similar to that of surfactin and the difference exists in the fatty acid portion, which is an iso-C12 beta-hydroxy fatty acid. The critical micelle concentration (CMC) of this new homologue is estimated to be 6.27 x 10(-5) mol/l in 10 mmol/l phosphate buffer solution (PBS, pH 8.0) at 30 degrees C, and the surface tension at CMC (gamma CMC) achieved is as little as 27.71 mN/m. The hemolytic activities of the C12-lipopeptide on 2% human erythrocytes showed a HC50 of 26.5 micromol/l.     

Importance of 3-hydroxy fatty acid composition of lipopeptides for biosurfactant activity

Biosurfactant production may be an economic approach to improving oil recovery. To obtain candidates most suitable for oil recovery, 207 strains, mostly belonging to the genus Bacillus, were tested for growth and biosurfactant production in medium with 5% NaCl under aerobic and anaerobic conditions. All strains grew aerobically with 5% NaCl, and 147 strains produced a biosurfactant. Thirty-five strains grew anaerobically with 5% NaCl, and two produced a biosurfactant. In order to relate structural differences to activity, eight lipopeptide biosurfactants with different specific activities produced by various Bacillus species were purified by a new protocol. The amino acid compositions of the eight lipopeptides were the same (Glu/Gln:Asp/Asn:Val:Leu, 1:1:1:4), but the fatty acid compositions differed. Multiple regression analysis showed that the specific biosurfactant activity depended on the ratios of both iso to normal even-numbered fatty acids and anteiso to iso odd-numbered fatty acids. A multiple regression model accurately predicted the specific biosurfactant activities of four newly purified biosurfactants (r2= 0.91). The fatty acid composition of the biosurfactant produced by Bacillus subtilis subsp. subtilis strain T89-42 was altered by the addition of branched-chain amino acids to the growth medium. The specific activities of biosurfactants produced in cultures with different amino acid additions were accurately predicted by the multiple regression model derived from the fatty acid compositions (r2= 0.95). Our work shows that many strains of Bacillus mojavensis and Bacillus subtilis produce biosurfactants and that the fatty acid composition is important for biosurfactant activity.     

Identification of Fatty Acids and Aliphatic Hydrocarbons in Sarcina lutea by Gas Chromatography and Combined Gas Chromatography-Mass Spectrometry

The composition and nature of the fatty acids and hydrocarbons of Sarcina lutea were elucidated by gas chromatography and by combined gas chromatography-mass spectrometry. The distribution of fatty acids found in S. lutea showed two families of pairs, or dyads, of saturated monocarboxylic acids (C12-C18) with and without methyl branching. These pairs of fatty acids showed a pattern of iso and anteiso structures for C13, C15, and C17, and iso and normal structures for C12, C14, and C16. Only the C18 showed unsaturation. The distribution of hydrocarbons in the range C22-C29 showed two families of tetrads of unsaturated aliphatic hydrocarbons all showing methyl branching. Each tetrad was composed of four isomers identified as two iso olefins and two anteiso olefins. The only difference between the tetrads pertaining to different families was found in the relative gas chromatographic retention times of the last two components of each group.     

Exogenous isoleucine and fatty acid shortening ensure the high content of anteiso-C15:0 fatty acid required for low-temperature growth of Listeria monocytogenes

Previous studies have demonstrated that the branched-chain fatty acid anteiso-C15:0 plays a critical role in the growth of Listeria monocytogenes at low temperatures by ensuring sufficient membrane fluidity. Studies utilizing a chemically defined minimal medium revealed that the anteiso fatty acid precursor isoleucine largely determined the fatty acid profile and fatty acid response of the organism to lowered growth temperature. When isoleucine was sufficient, the fatty acid profile was very uniform, with anteiso fatty acids comprising up to 95% of total fatty acid, and the major fatty acid adjustment to low temperature was fatty acid chain shortening, which resulted in an increase of anteiso-C15:0 solely at the expense of anteiso-C17:0. When isoleucine was not supplied, the fatty acid profile became more complex and was readily modified by leucine, which resulted in a significant increase of corresponding iso fatty acids and an inability to grow at 10 degrees C. Under this condition, the increase of anteiso-C15:0 at low temperature resulted from the combined effect of increasing the anteiso:iso ratio and chain shortening. A branched-chain alpha-keto acid dehydrogenase-defective strain largely lost the ability to increase the anteiso:iso ratio. Cerulenin, an inhibitor of beta-ketoacyl-acyl carrier protein synthase (FabF), induced a similar fatty acid chain shortening as low temperature did. We propose that the anteiso precursor preferences of enzymes in the branched-chain fatty acid biosynthesis pathway ensure a high production of anteiso fatty acids, and cold-regulated chain shortening results in a further increase of anteiso-C15:0 at the expense of anteiso-C17:0.     

Cellular envelope phospholipids from Legionella lytica

The composition of phospholipids from the cellular envelope of Legionella lytica grown on artificial medium was determined by two-dimensional thin-layer chromatography. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidyl-N-monomethylethanolamine were the predominant phospholipids, while diphosphatidylglycerol, phosphatidylglycerol, and phosphatidyl-N,N-dimethylethanolamine were present at low concentrations. A trace amount of lipids carrying glycosyl residues was also observed. The fatty acids and their distribution in individual phospholipids were characterized using liquid chromatography/mass spectrometry (LC/MS), matrix-assisted laser desorption ionization-time of flight, and gas chromatography/MS methods. The characteristic feature of L. lytica phospholipids was the presence of an unbranched chain (which differentiates this bacterium from Legionella pneumophila) and branched iso and anteiso fatty acids as well as cis-9,10-methylenehexadecanoic acid. According to spectroscopic LC/MS data, the localization of saturated and unsaturated fatty acid residues on phosphorylglycerol was determined. Some aspects of the significance of phosphatidylcholine, one of the main phospholipids in L. lytica, are addressed and taxonomic implications of the data are discussed.     

Exogenous Isoleucine and Fatty Acid Shortening Ensure the High Content of Anteiso-C15:0 Fatty Acid Required for Low-Temperature Growth of Listeria monocytogenes

Previous studies have demonstrated that the branched-chain fatty acid anteiso-C_15:0 plays a critical role in the growth of Listeria monocytogenes at low temperatures by ensuring sufficient membrane fluidity. Studies utilizing a chemically defined minimal medium revealed that the anteiso fatty acid precursor isoleucine largely determined the fatty acid profile and fatty acid response of the organism to lowered growth temperature. When isoleucine was sufficient, the fatty acid profile was very uniform, with anteiso fatty acids comprising up to 95% of total fatty acid, and the major fatty acid adjustment to low temperature was fatty acid chain shortening, which resulted in an increase of anteiso-C_15:0 solely at the expense of anteiso-C_17:0. When isoleucine was not supplied, the fatty acid profile became more complex and was readily modified by leucine, which resulted in a significant increase of corresponding iso fatty acids and an inability to grow at 10°C. Under this condition, the increase of anteiso-C_15:0 at low temperature resulted from the combined effect of increasing the anteiso:iso ratio and chain shortening. A branched-chain α-keto acid dehydrogenase-defective strain largely lost the ability to increase the anteiso:iso ratio. Cerulenin, an inhibitor of β-ketoacyl-acyl carrier protein synthase (FabF), induced a similar fatty acid chain shortening as low temperature did. We propose that the anteiso precursor preferences of enzymes in the branched-chain fatty acid biosynthesis pathway ensure a high production of anteiso fatty acids, and cold-regulated chain shortening results in a further increase of anteiso-C_15:0 at the expense of anteiso-C_17:0.     

Effects of pH and Temperature on the Fatty Acid Composition of Bacillus acidocaldarius

The fatty acid composition of lipid extracts from cells of Bacillus acidocaldarius grown at temperatures of 50 to 70 C and pH values of 2 to 5 was determined by gas chromatography of the methyl esters. The most abundant fatty acids are 11-cyclohexylundecanoic and 13-cyclohexyltridecanoic, followed by anteiso- and iso-heptadecanoic; unsaturated acids are absent. Highly aerated cultures produce more of the iso and anteiso acids and less of the cyclohexyl acids. The effects of temperature and pH are interdependent; at lower pH, increasing temperature raises the proportion of the iso and anteiso acids, but at higher pH the effect of increasing temperature is reversed and the proportion of the cyclohexyl acids is increased.     

Comparative investigation of phospholipids and fatty acids of freshwater molluscs from the Volga river basin.

1. Four Gastropoda species and two Bivalvia species from the Volga river basin were examined. 2. Distribution of phospholipids in the molluscs was studied by qualitative and quantitative micro thin-layer chromatography. 3. Major phospholipid classes, phosphatidylethanolamine and phosphatidylcholine, were found to contain plasmalogens. 4. One mollusc species notably contained 67 fatty acids including 25 saturated (both iso and anteiso), 24 monoenoic, five dienoic, four trienoic and eight polyenoic compounds identified by capillary gas chromatography; fatty acid contents in the other studied species were considerably lower. 5. Relatively high concentrations of nonmethylene-interrupted fatty acids were detected in certain examined species.     

Surfactin isoforms from Bacillus coagulans

Bacillus coagulans has been found to produce several surfactins that are powerful lipopeptide surfactants. Four main components with molecular weights 1007, 1021 and 1035 Da were separated. Their structures have been confirmed by spectrometric and spectroscopic studies and by acid hydrolysis. The compounds were found to represent two pairs of surfactin isoforms in which beta-hydroxy-iso-C14 or anteiso-C15 fatty acids are linked to the [Leu7] or [Val7] heptapeptide moiety by both an amide group and a lactone bond.     

Fatty acid, isoprenoid quinone and polar lipid composition in the classification of Renibacterium salmoninarum

Twenty-one strains of Renibacterium salmoninarum were degraded by acid methanolysis and the non-hydroxylated fatty acid esters released examined by thin-layer and gas chromatography. The fatty acid profiles were composed almost exclusively of methyl-branched fatty acids with 12-methyltetradecanoic ( anteiso -C₁₅), 13-methyltetradecanoic ( iso -C₁₅) and 14-methylhexadecanoic ( anteiso -C₁₇) as major components. Polar lipids of the test strains were examined by two-dimensional thin-layer chromatography. All of the organisms possessed very characteristic polar lipid patterns consisting of diphosphatidylglycerol, two major and six or seven minor glycolipids, and two unidentified minor phospholipids. In all cases the major menaquinone components consisted of unsaturated menaquinones with nine isoprene units. The lipid data support the integrity of the genus Renibacterium and can be used to separate it from Corynebacterium and from coryneform bacteria which also contain lysine in the wall peptidoglycan.     

Determination of the structure of the fatty acid chain in a cyclic lipopeptide using GC-MS

A GC-EIMS method to determine the structure of the fatty acid chains in cyclic lipopeptides is described. The structure of the fatty acid chains can be determined by the characteristic peaks of the MS spectrogram according to the fact that the alpha cleavage predominates the MS of a fatty acid with amino and hydroxy groups, while the McLafferty rearrangement predominates the MS of one without amino or hydroxy group. The characteristics of the strongest peaks of 103 and 102 in MS spectrograms due to alpha cleavage represent the beta-hydroxy-fatty acid and the beta-amino fatty acid, respectively; the strongest peak of 117 due to alpha cleavage and the relatively weak peak of 88 due to McLafferty rearrangement indicate the beta-hydroxy-fatty acid with a branched methyl group at its alpha position. The strongest peak of 74 due to McLafferty suggests the fatty acid without hydroxy or amino group. The ratio of relative intensity (I(43)/I(57)) characterizes the branches of alkyl chains. The greater I(43)/I(57) corresponds to an iso alkyl, and the smaller I(43)/I(57) corresponds to an anteiso alkyl. This method can be used to determine the full structure of the fatty acid chains in lipopeptides.     

Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oil-emulsifying activity

Bacillus amyloliquefaciens strain LP03 isolated from soil, produced an antagonistic compound that strongly inhibited the growth of plant-pathogenic fungi and a lipopeptide biosurfactant. Also, isolated strain LP03 had a marked crude oil-emulsifying activity as it developed a clear zone around the colony after incubation for 24 h at 37°C. LP03 was identified as Bacillus amyloliquefaciens by analysis of partial 16 S rRNA gene and partial gyrA gene sequence. The lipopeptide was purified by acid precipitation of cell-free culture broth, extraction of the precipitates with methanol, silica gel column chromatography, and reverse-phase, high-pressure liquid chromatography. The purified biosurfactant was analyzed biochemical structure by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). The masses of the two peaks were observed by HPLC chromatography. Their masses were determined to be 1,044 and 1,058 m/z with MALDI-TOF mass spectrometry. As constituents of the peptide and lipophilic part of the m/z 1,022.6, seven amino acids (Glu-Leu-Met-Leu-Pro-Leu-Leu) and β-hydroxy-C13 fatty acid were determined by ESI-MS/MS. The lipopeptide of 1,022.6 Da differed from surfactins in the substitution of leucine, valine and aspartic acid in positions 3, 4, and 5 by methionine, leucine, and proline, respectively. Novel lipopeptide was designated as bamylocin A.     

Fatty Acid and Polar Lipid Composition in the Classification of Cellulomonas, Oerskovia and Related Taxa

Strains representing the taxa Cellulomonas, Oerskovia, Brevibacterium fermentans, Corynebacterium manihot and Nocardia cellulans were degraded by acid methanolysis and the non-hydroxylated fatty acid esters released examined by thin-layer and gas chromatography. The major fatty acid in all strains was 12-methyltetradecanoic acid ( anteiso C₁₅) which occurred together with other anteiso acids, iso and straight-chain acids. The fatty acid profiles of the cellulomonads were distinguished by the presence of 13-carbon acids and significantly higher proportions of straight-chain acids than found in the other test strains whose profiles were closely similar to one another. Two-dimensional thin-layer chromatography showed that almost identical and very characteristic polar lipid patterns were given by all the organisms under study: the only major components were diphosphatidylglycerol, phosphatidylinositol and two phospho-glycolipids chromatographing similarly to, but distinguishable from, the mono- and diacyl phosphatidylinositol dimannosides characteristic of Nocardia and other actinomycetes. The accumulated lipid data support the reclassification of B. fermentans, Cor. manihot and N. cellulans in the genus Oerskovia.     

Identification of a gene cluster responsible for the biosynthesis of cyclic lipopeptide verlamelin

Only limited studies are available on the molecular-level biosynthesis of cyclic lipopeptides (cyclic and hybrid molecules consisting of peptide and fatty acid moieties) in filamentous fungi. Here, we identified and characterized biosynthetic genes of the cyclic lipopeptides, known as verlamelins. Only four genes, coding for non-ribosomal peptide synthetase (NRPS), fatty acid hydroxylase, thioesterase, and AMP-dependent ligase, were found to be involved in verlamelin biosynthesis by the analysis of corresponding gene knockouts. Surprisingly, no gene(s) coding for fatty acid synthase or polyketide synthase was present in the cluster, while verlamelin A/B contained a 5-hydroxytetradecanoic acid moiety. Precursor feeding experiment indicated that both fatty acid hydroxylase and thioesterase are involved to supply 5-hydroxytetradecanoic acid. The results suggested that 5-hydroxytetradecanoic acid was supplied from primary metabolism via fatty acid hydroxylase and loaded onto NRPS. Elongation of the peptide and final cyclization were accomplished by NRPS. The knowledge obtained through this study should provide new insight into fungal lipopeptide biosynthesis.     

Effect of Growth Temperature on Fatty Acid Composition of Ten Thermus Strains

The fatty acid composition of Thermus spp., including T. aquaticus ATCC 25104, T. thermophilus DSM 579, T. flavus DSM 674, and seven wild strains was examined. Organisms were tested at a minimum of either 35, 40, or 45°C and at an optimum of 60 or 70°C. Total fatty acid content per dry weight of cells varied between 1.2 and 3.7%, and the quantity of fatty acids was higher at the high temperature range in the majority of strains. At the optimum temperature, strains could be assigned to three chemotaxonomic groups with reference to the ratio of iso C_15:0/iso C_17:0. In six of the strains the ratio of iso C_15:0/iso C_17:0 remained unchanged at the minimum temperature, whereas in four strains the ratio was reversed. The proportion of the C_15:0 and C_17:0 isobranched acids was decreased and the proportion of anteisobranched fatty acids, namely anteiso C_15:0, anteiso C_17:0, and anteiso C_17:1, was increased at the lower temperature range. Some changes were seen in the levels of the n-C_16:0 and iso C_16:0 acids, but these were strain specific.     

Simultaneous lipidation of a characterized peptide mixture by chemoselective ligation

The modification of a peptide antigen by a fatty acid such as palmitic acid is now recognized as a mean to induce cellular responses. Mixtures of lipopeptides, obtained by combining individually synthesized compounds, were shown to be promising synthetic vaccine candidates. Usually, in lipopeptide synthesis, the fatty acyl moiety is introduced on the crude peptide chain using solid-phase methods. The separation of the target compound from impurities by RP-HPLC is often complicated by the amphiphilic properties of lipopeptides and results in low overall yields. To overcome the difficulties associated with lipopeptide synthesis and mixture preparation, we have developed a method where the fatty acyl moiety is site-specifically and collectively introduced in solution onto a mixture of individually prepurified peptides. The lipidation is based on the quasistoichiometric and high-yielding ligation of a glyoxylyl lipid with hydrazinoacetyl peptides. The hydrazone constructs were prepared in a salt-free medium and could be isolated by direct lyophilization of the reaction mixture. This process is compatible with cysteinyl peptides, and no aggregation nor degradation could be observed.     

Cyclic lipopeptide profile of the plant-beneficial endophytic bacterium Bacillus subtilis HC8

In a previous study (Malfanova et al. in Microbial Biotech 4:523–532, 2011), we described the isolation and partial characterization of the biocontrol endophytic bacterium B. subtilis HC8. Using thin-layer chromatography, we have detected several bioactive antifungal compounds in the methanolic extract from the acid-precipitated supernatant of HC8. In the present study, we have further analyzed this methanolic extract using liquid chromatography-mass spectrometry. Based on the comparison of retention times and molecular masses with those of known antifungal compounds, we identified three families of lipopeptide antibiotics. These include four iturins A having fatty acyl chain lengths of C14 to C17, eight fengycins A (from C14 to C18 and from C15 to C17 containing a double bond in the acyl chain), four fengycins B (C15 to C18), and five surfactins (C12 to C16). Evaluation of the antifungal activity of the isolated lipopeptides showed that fengycins are the most active ones. To our knowledge, this is the first report of an endophytic Bacillus subtilis producing all three major families of lipopeptide antibiotics containing a very heterogeneous mixture of homologues. The questions remain open which of these lipopeptides (1) are being produced during interaction with the plant and (2) are contributing to the biocontrol activity of HC8.     

Lipids from the guinea pig Harderian gland: use of picolinyl and other pyridine-containing derivatives to investigate the structures of novel branched-chain fatty acids and glycerol ethers

The lipid extracted from guinea pig Harderian glands was hydrolysed and the constituents were examined as trimethylsilyl (TMS), (2H9)TMS, methyl ester/TMS, acetonide/TMS, nicotinate/TMS, picolinyl/TMS and nicotinylidene/TMS derivatives by capillary gas-liquid chromatography and gas chromatography/mass spectrometry. Over 70 compounds amounting to over 93% of the extract were identified. These consisted of 1-O-alkyl glycerols (glycerol ethers) with alkyl chains containing from 17 to 21 carbon atoms and fatty acids ranging from 14 to 26 carbon atoms. The alkyl chains in the glycerol ethers were straight, mono- and dimethyl-branched with the major site of branching being at C-14. All straight-chain acids from C14 to C26 were present, with the most abundant being n-24:0. Again mono- and dimethyl branched structures comprised the bulk of the remaining acids. Methyl groups tended to be towards the middle of the chain rather than in the more usual omega-1 (iso) and omega-2 (anteiso) positions, with C-14 again being a major site. The shorter-chain acids tended to have methyl groups closer to the acid group, with several of the short-chain compounds being substituted at C-2. Structural information on the acids was provided by the picolinyl derivatives and the sample provided an opportunity to evaluate these derivatives with branched acids other than the iso and anteiso compounds studied previously. They were found to be satisfactory for analysis of both mono- and dimethyl branched acids with the possible exception of compounds containing a methyl branch at C-4. However, in this case, structural information was provided by the methyl ester.(ABSTRACT TRUNCATED AT 250 WORDS)