Determination of fatty acid compositions of Bacillus cereus and related bacteria: a rapid gas chromatographic method using a glass capillary column.

A rapid gas chromatographic method for the determination of fatty acid compositions of Bacillus cereus and related bacteria is presented. By the use of a free fatty acid phase-coated glass capillary column, the complete separation of fatty acids, including the branched ones, was achieved. The method enables a more distinct differentiation of Bacillus species than can be obtained with packed columns.     

Fatty acid composition and primer specificity of de novo fatty acid synthetase in Bacillus globispores, Bacillus insolitus, and Bacillus psychrophilus

The fatty acid compositions of three psychrophilic species of Bacillus were determined by gas--liquid chromatography. The proportions of straight-chain fatty acids, branched-chain fatty acids, and unsaturated fatty acids were found to be 13.3, 86.7, and 26.1% of the total cellular fatty acids for Bacillus globispores, 36.6, 63.4, and 25.1% for Bacillus insolitus, and 6.9, 93.1, and 18.4% for Bacillus psychrophilus, respectively. In all three organisms the de novo fatty acid synthetase specificity towards acyl-CoA primers was butyryl-CoA greater than propionyl-CoA much greater than acetyl-CoA. This shows that B. insolitus, which has an unusually large proportion of straight-chain fatty acids for Bacillus, does not possess a different de novo fatty acid synthetase than the other two organisms. Therefore, the greater proportion of straight-chain fatty acids in B. insolitus may be explained by a large supply of straight-chain primer.     

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.     

Cellular Fatty Acid Analysis of Isolates of Bacillus thuringiensis Serovar kurstaki,Strain HD-1

Whole-cell cellular fatty acid (CFA) composition was utilized to determine if Bacillus thuringiensis serovar kurstaki (HD-1) larvicides produced by different manufacturers could be distinguished from each other and to determine whether these larvicides were distinguishable from isolates deposited in public collections. This study analyzed Biobit, Dipel, Foray, Thuricide, and the non HD-1 larvicides Delfin and Javelin, as well as the 1971 and 1980 Standards of HD-1. Isolates of HD-1 deposited in the collections of the American Type Culture Collection, Bacillus Genetic Stock Center, Pasteur Institute, and United States Department of Agriculture (USDA) were also analyzed. The data were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages (UPGMA). Samples that linked at a Euclidean distance ≤2.0 units were considered to belong to the same fatty acid strain. Isolates of HD-1 from commercial products and deposits of HD-1 in the public collections and Standards were polytypic; 22 separate fatty acid strains were identified in the 1971 and 1980 Standards and 35 fatty acid strains were identified in the public collections. The type strain for Btk contained multiple fatty acid strains; three fatty acid strains were present in both the Bacillus Genetic Stock Center and the Pasteur Institute collections. In contrast, the type strain for serovar kurstaki in the USDA collection (HD-73) was monotypic and its fatty acid strain did not occur in other collections. We could distinguish between HD-1 and non-HD-1 larvicides using CFA composition. We conclude that CFA analysis may be used to identify commercial products.     

Type selective inhibition of microbial fatty acid synthases by thiolactomycin

The antibiotic, thiolactomycin, is known to selectively inhibit the Type II straight-chain fatty acid synthase (monofunctional enzyme system, e.g. Escherichia coli enzyme) but not Type I straight-chain fatty acid synthase (multifunctional enzyme system, e.g. Saccharomyces cerevisiae enzyme). We have studied the effect of thiolactomycin on the branched-chain fatty acid synthases from Bacillus subtilis, Bacillus cereus, and Bacillus insolitus. Fatty acid synthase from all three Bacilli was not inhibited or only slightly inhibited by thiolactomycin. E. coli synthase, as expected, was strongly inhibited by thiolactomycin. Branched-chain fatty acid synthase from Bacillus species is a monofunctional enzyme system but, unlike Type II E. coli synthase, it is largely insensitive to thiolactomycin.     

Nutritional Alteration of the Fatty Acid Composition of a Thermophilic Bacillus Species

The fatty acid composition of a thermophilic Bacillus sp. was altered by the addition of isobutyrate, isovalerate, alpha-methylbutyrate, leucine, and isoleucine to the growth medium. With isobutyrate, 81% of the fatty acids had 16 carbon atoms and 79% were iso-fatty acids with an even number of carbon atoms. With leucine, 58% of the fatty acids had 15 carbon atoms and 86% were iso-fatty acids with an odd number of carbon atoms. With isoleucine, 72% of the fatty acids had 17 carbon atoms and 88% were anteiso-fatty acids with an odd number of carbon atoms. Thus, by altering the composition of the growth medium, cells were produced in which the majority of the fatty acids had either 15, 16, or 17 carbons and belonged to each of the three groups of branched-chain fatty acids. The wide variation observed in the fatty acid composition makes it unlikely that any specific branched-chain fatty acid is required for vital functions.     

Microbial metabolism of alkylbenzene sulphonates Enzyme system of a Bacillus species responsible for Β-oxidation of the alkyl side chain of alkylbenzene sulphonates

A Bacillus species was isolated from sewage capable of utilising alkylbenzene sulphonates (ABS) as the sole source of carbon and sulphur. The enzymic mechanism involved in alkyl-side-chain biodegradation of various ABS detergent isomers by the Bacillus species was demonstrated to involve the classical-Β-oxidation equence characteristic of long-chain fatty acid oxidation, by appropriate enzyme inductions. The combined results from both enzyme induction studies and molecular separation of induced enzymes by gel-filtration indicated a single set of enzymes to be responsible for the Β-oxidation of both ABS isomers and long-chain fatty acid isomers in the Bacillus species. The substrate specificity of partially purified enzymes after growth on appropriate substrates confirmed the feasibility of a single Β-oxidation pathway in this microorganism capable of catalising the oxidation of a wide range of different synthetic and naturally occurring chemicals and biochemicals containing alkyl side chains. This work was supported at Newcastle by grants from the Science Research Council and The Royal Society.     

Activation and inhibition of Candida rugosa and Bacillus-related lipases by saturated fatty acids, evaluated by a new colorimetric microassay

Research on lipase inhibitors could help in the therapy of diseases caused by lipase-producing microorganisms and in the design of novel lipase substrate specificities for biotechnology. Here we report a fast and sensitive colorimetric microassay that is low-cost and suitable for high-throughput experiments for the evaluation of lipase activity and inhibition. Comparison of Candida rugosa activity and inhibition with previous HPLC results validated the method, and revealed the importance of the reaction mixture composition. The assay was used to evaluate the effect of saturated fatty acids on Bacillus-related lipases. Cell-bound esterases were strongly inhibited by fatty acids, suggesting a negative feedback regulation by product, and a role of these enzymes in cell membrane turnover. Bacillus subtilis LipA was moderately activated by low concentrations of fatty acids and was inhibited at greater concentrations. LipB-like esterases were highly activated by myristic and lauric acids and were only slightly inhibited by high capric acid concentrations. Such an activation, reported here for the first time in bacterial lipases, seems to be part of a regulatory system evolved to ensure a high use of carbon sources, and could be related to the successful adaptation of Bacillus strains to nutrient-rich environments with strong microbial competition.     

Characterization of Bacillus strains of marine origin.

A total of twenty aerobic endospore-forming bacilli, isolated from marine invertebrates and sea water of different areas of the Pacific Ocean, were taxonomically characterized. Most of the bacilli (11 strains) of marine origin belonged to the species Bacillus subtilis, according to their phenotypic characteristics, antibiotic susceptibility profiles, and fatty acids patterns. A group of four alkaliphilic strains formed a separate cluster that was tentatively classified as B. horti. One isolate, KMM 1717, associated with a sponge from the Coral Sea was identified as B. pumilus. Two strains, Bacillus KMM 1916 and KMM 1918, showed antibiotic sensitivity profiles similar to B. licheniformis, but they had a distinct fatty acid composition and peculiar phenotypic traits. The taxonomic affiliation of KMM 1810 and KMM 1763 remained unclear since their fatty acid composition and antibiotic sensitivity patterns were not resembled with none of these obtained for Bacillus strains.     

Fatty Acids in the Genus Bacillus I. Iso- and Anteiso-Fatty Acids as Characteristic Constituents of Lipids in 10 Species

Fatty acids produced by 22 strains of 10 species of the genus Bacillus were analyzed on a very efficient and selective gas-liquid chromatographic column. All of the 10 species, alvei, brevis, cereus, circulans, licheniformis, macerans, megaterium, polymyxa, pumilus, and subtilis, produced eight fatty acids, six branched (anteiso-C(15), anteiso-C(17), iso-C(14), iso-C(15), iso-C(16), and iso-C(17)) and two normal (n-C(14) and n-C(16)). In all cases, the six branched-chain fatty acids made up over 60% of the total fatty acids. In addition to the eight fatty acids, B. cereus produced four extra fatty acids, three branched (anteiso-C(13), iso-C(12), and iso-C(13)) and one monoenoic-n-C(16). Furthermore, there were distinct differences in the relative amounts of fatty acids produced between B. cereus and the remaining nine species. B. cereus produced iso-C(15) fatty acid in the largest amount on a glucose-yeast extract medium as well as on Pennassay Broth. On the other hand, for the remaining nine species, anteiso-C(15) fatty acid was the major fatty acid from the glucose-yeast extract medium, whereas the amount of iso-C(15) fatty acid from Penassay Broth became comparable to that of anteiso-C(15) fatty acid. Mechanisms and various factors affecting the fatty acid distribution pattern in the 10 Bacillus species are discussed.     

Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases

The gene coding for an extracellular lipase of Bacillus licheniformis was cloned using PCR techniques. The sequence corresponding to the mature lipase was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal. High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30 degrees C. A one step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified enzyme was 130 units/mg with p-nitrophenyl-palmitate as substrate. The enzyme showed maximum activity at pH 10-11.5 and was remarkably stable at alkaline pH values up to 12. The enzyme was active toward p-nitrophenyl esters of short to long chains fatty acids but with a marked preference for esters with C(6) and C(8) acyl groups. The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus. Based on the amino acid identity and biochemical characteristics, we propose that Bacillus lipases be classified into two distinct subfamilies of their own.     

Studies on the biosynthesis of iturin, an antibiotic of Bacillus subtilis, and a lipopeptide containing beta-hydroxy fatty acids

The biosynthesis of iturin, an antibiotic containing a beta-amino fatty acid, was studied by incubating Bacillus subtilis in the presence of various 14C-labelled precursors. Sodium acetate or palmitic acid were incorporated into the beta-amino acids of iturin. Among the alpha-amino acids (asparagine, glutamine, serine, proline and tyrosine) in the peptidic part of iturin, asparagine appears to be the best precursor. In the presence of sodium [14C]acetate or [14C]asparagine, there was a synthesis of radioactive compound (compound X) before the synthesis of radioactive iturin. Compound X contained asparagine and/or aspartic acid, glutamine and/or glutamic acid and beta-hydroxy fatty acids.     

Stable-isotope-based labeling of styrene-degrading microorganisms in biofilters

Deuterated styrene ([(2)H(8)]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [(2)H(8)]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [(2)H(8)]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.     

beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis

A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes. We examined the biochemical basis for the production of branched-chain fatty acids by gram-positive bacteria. Two genes that were predicted to encode homologs of the beta-ketoacyl-acyl carrier protein synthase III of Escherichia coli (eFabH) were identified in the Bacillus subtilis genome. Their protein products were expressed, purified, and biochemically characterized. Both B. subtilis FabH homologs, bFabH1 and bFabH2, carried out the initial condensation reaction of fatty acid biosynthesis with acetyl-coenzyme A (acetyl-CoA) as a primer, although they possessed lower specific activities than eFabH. bFabH1 and bFabH2 also utilized iso- and anteiso-branched-chain acyl-CoA primers as substrates. eFabH was not able to accept these CoA thioesters. Reconstitution of a complete round of fatty acid synthesis in vitro with purified E. coli proteins showed that eFabH was the only E. coli enzyme incapable of using branched-chain substrates. Expression of either bFabH1 or bFabH2 in E. coli resulted in the appearance of a branched-chain 17-carbon fatty acid. Thus, the substrate specificity of FabH is an important determinant of branched-chain fatty acid production.     

Unsaturated and branched chain-fatty acids in temperature adaptation of Bacillus subtilis and Bacillus megaterium.

The effect of growth temperature on the cellular fatty acid profiles of Bacillus subtilis and Bacillus megaterium was studied over a temperature range from 40 to 10 degrees C. As the growth temperature of B. subtilis was reduced, the lower-melting point anteiso-acids increased, while the higher-melting point iso-acids decreased. Consequently the ratio of branched- to straight-chain acids was unaffected by temperature, although changes in the position of fatty acid branching and the degree of unsaturated branched-chain fatty acids occurred. In B. megaterium a more complicated, biphasic behaviour was observed. Saturated, straight-chain and iso-branched acids decreased only from 40 degrees C down to 20-26 degrees C, and anteiso-acids decreased only from 20-26 degrees C to 10 degrees C, while unsaturated acids increased over the whole temperature range studied. Thus, in B. megaterium total branched-chain acids decreased and straight-chain acids increased as temperature decreased. However, the overall cellular content of lower-melting point fatty acids increased with decreasing temperature in both bacilli, and unsaturated fatty acids appeared to be essential components in the adaptation of the microbes to changes in temperatures. Since changes in the relative amounts of branched- and straight-chain fatty acid biosynthesis are known to reflect differences in fatty acid primers, temperature seems to affect not only the activity of the fatty acid desaturases but also the formation or availability of these primers. The results indicate, however, that notable species-specific regulatory features exist in this genus of bacteria.     

Mutants of Bacillus species isolated on the basis of protonophore resistance are deficient in fatty acid desaturase activity.

The fatty acid desaturase activity in cell extracts of Bacillus subtilis was characterized and found to be O2 dependent, NADH dependent, and cyanide sensitive. In cell fractionation studies, only 10% of the desaturase activity was recovered in the membrane fraction; the addition of cytosolic factors, which by themselves were devoid of activity, restored membrane activity to the level found in the unfractionated cell extracts. NADH was preferred over NADPH as an electron donor, and palmitoyl-coenzyme A was used preferentially over stearoyl-coenzyme A as the straight-chain fatty acid substrate. An increase in desaturase activity was observed when either the growth or the assay temperature was lowered from 37 to 20 degrees C, although the assay temperature appeared to be the more important parameter. Three protonophore-resistant mutants of B. subtilis and a comparable mutant of Bacillus megaterium had been found to possess reduced levels of unsaturated fatty acids in their membrane phospholipids; their protonophore resistance was abolished when grown in the presence of an unsaturated fatty acid supplement. All of these strains were found to be either significantly deficient in or totally lacking desaturase activity in comparison with their wild-type parent strains. Full, protonophore-sensitive revertants of the mutants had levels of desaturase activity comparable to those of the wild-type. Temperature-sensitive revertants of two of the mutants, which grew at 32 degrees C but not at 26 degrees C in the presence of protonophore, exhibited desaturase activity comparable to that of the wild-type at 26 degrees C but lacked activity at 32 degrees C. These results indicate that the biochemical basis for protonophore resistance in these Bacillus mutants is a fatty acid desaturase deficiency.     

Production of lipids from a thermoacidophilic bacillus strain. I. Lipids from bacillus acidocaldarius ZIMET 11274

A new isolated Bacillus strain was cultivated continuously at 68°C and pH 3 for a long time. Extractable lipids represent about 3.6% of cell dry weight and are made up of 18% neutral lipids, 39.3% glycolipids, and 42% acidic lipids. Main components of the fatty acid fraction are ω-cyclohexylundecanoic acid and ω-cyclohexyltridecanoic acid, respectively. MK-7 and hop-22(29)-ene are components of the unsaponifiable fraction. The glycolipid fraction contains the pentacyclic triterpenoid tetrahydroxybacteriohopane. Based on the thermoacidophilic growth conditions, the morphologic and physiological properties, and the nature of lipids, it can assumed that the new isolated strain belongs to the species Bacillus acidocaldarius.     

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.     

Cellular fatty acid analysis as a potential tool for predicting mosquitocidal activity of Bacillus sphaericus strains.

Gas-liquid chromatography of fatty acid methyl esters and numerical analysis were carried out with 114 Bacillus sphaericus strains. Since only two clusters harbored mosquitocidal strains, this technique could be developed in screening programs to limit bioassays on mosquito larvae. It also allows differentiation of highly homologous strains.