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.     

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.     

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.     

Lipid and protein composition of membranes of Bacillus megaterium variants in the temperature range 5 to 70 degrees C.

Membranes were prepared from four temperature range variants of Bacillus megaterium: one obligate thermophile, one facultative thermophile, one mesophile, and one facultative psychrophile, covering the temperature interval between 5 and 70 degrees C. The following changes in membrane composition were apparent with increasing growth temperatures: (i) the relative amount of iso fatty acids increased and that of anteiso acids decreased, the ratio of iso acids to anteiso acids being 0.34 at 5 degrees C and 3.95 at 70 degrees C, and the pair iso/anteiso acids thus seemed to parallel the pair saturated/unsaturated acids in their ability to regulate membrane fluidity; (ii) the relative/unsaturated acids in their ability to regulate membrane fluidity; (ii) the relative amount of long-chain acids (C16 to C18) increased fivefold over that of short-chain acids (C14 and C15) between 5 and 70 degrees C; (iii) the relative amount of phosphatidylethanolamine increased, and this phospholipid accordingly dominated in the thermophilic strains, whereas diphosphatidylglycerol was predominant in the two other strains; and (iv) the ratio of micromoles of phospholipid to milligrams of membrane protein increased three-fold between 5 and 70 degrees C. Moreover, a quantitative variation in membrane proteins was evident between the different strains. Briefly, membrane phospholipids with higher melting points and packing densities appeared to be synthesized at elevated growth temperatures.     

Adaptation of the psychrotroph Arthrobacter chlorophenolicus A6 to growth temperature and the presence of phenols by changes in the anteiso/iso ratio of branched fatty acids

Arthrobacter chlorophenolicus is a previously described Gram-positive bacterium capable of degrading high concentrations of several phenolic compounds under optimal mesophilic (28 degrees C) as well as psychrophilic (5 degrees C) conditions. However, the exact mechanisms by which this organism is able to tolerate such extremes in temperature and high levels of toxic compounds are currently not known. In this study, we monitored changes in the fatty acid composition of the cell membrane under different extreme growth conditions. Arthrobacter chlorophenolicus adapts to differences in temperature and phenol concentrations by altering the anteiso/iso ratio of fatty acids in the cell membrane to different extents. According to the different physico-chemical properties of those two species of branched fatty acids, the bacteria showed an increased amount of anteiso fatty acids when grown under psychrophilic conditions to decrease the viscosity of their membranes. On the other hand, at higher growth temperatures as well as in the presence of toxic concentrations of phenol, 4-chlorophenol and 4-nitrophenol, the cells adapted their membrane by a dose-dependent decrease in the anteiso/iso ratio, leading to a more rigid membrane and counteracting the fluidity increase caused by the higher temperature and the organic solvents.     

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.     

Alterations in growth temperature range and fatty acid composition of Thermus as a result of plasmid elimination

Elimination of plasmids from Thermus flavus, T. thermophilus and three wild Thermus strains caused alterations in growth temperature range, pigmentation and membrane fatty acids without affecting viability. Following plasmid elimination all Thermus strains lost their ability to grow above 70°C. In addition, the minimum growth temperature was lowered by 5–10°C. Fatty acids were reduced by an average of approximately 35%. In addition, the contribution of iso- and anteisobranched fatty acids were altered in four of the five strains. The iso C_15:0/iso C_17:0 ratio approached 1.0 in all strains, whereas the anteiso C_15:0/anteiso C_17:0 was reduced to 0.2. The iso C_16:0/normal-C_16:0 ratio increased in all strains due to an increase in iso C_16:0 in four strains and a reduction in normal-C_16:0 relative to iso C_16:0 in one strain. However, it was evident that the plasmid-free strains were able to compensate for these alterations in membrane fluidity to a certain extent by reducing the average chain length of isobranched acids. Altered fatty acid metabolism at the level of precursors may have influenced membrane composition and consequently growth temperature range.     

Acyl chains of phospholipids and their variation with age inStreptomyces griseus

Total and individual phospholipids ofStreptomyces griseus exhibit a predominance of iso and anteiso fatty acids. The ratio of saturated straight chain fatty acids to branched chain plus unsaturated fatty acids remained unchanged during growth.     

Structural characterization of eight cyclic lipopeptides produced by Bacillus subtilis HSO121

Lipopeptides are amphiphilic compounds which contain both hydrophobic fatty acid moieties and amphiphilic peptide moieties. From the cell-free broth of Bacillus subtilis HSO121, eight cyclic lipopeptides were isolated by reversed-phase high performance liquid chromatography (RP-HPLC). The peptide part of each lipopeptide was elucidated according to electrospray ionization quadruple-time-of-flight mass spectrometry (ESI Q-TOF MS) and the fatty acid part was analyzed by electroionization gas chromatography/mass spectrometry (EI GC/MS). It showed that fractions 1-8 had molecular masses of 1007, 1021, 1021, 1035, 1035, 1035, 1063, and 1049, respectively. Analysis of hydrolyzed lipopeptides revealed that they had invariant amino acid compositions. The differences in molecular weights represent changes in the number of methylene groups and different types of branched chains in fatty acids. Peptide sequences of two of the eight lipopeptides appeared to be N-Asp-Leu-Leu-Val-Glu-Leu-Leu-C, which was different from previously reported lipopeptides. The remaining six had an identical peptide sequence of N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C. The fatty acid parts were found to be mixtures of iso C(12), iso C(13), anteiso C(13), iso C(14), n C(14), iso C(15), anteiso C(15), n C(15), anteiso C(16) and anteiso C(17) beta-hydroxy fatty acids. The structure of each lipopeptide was determined to be the beta-hydroxy fatty acid bonded to the peptide chain.     

Effect of non-target cells on the sensitivity of the PCR for Escherichia coli O157 : H7

The fatty acid composition from mycelia of Streptomyces hygroscopicus strains was studied. A significant proportion of C_{18 : 2} was found in cultures. High levels of C_{16 : 0}, iso-C_{16 : 0} and C_{18 : 1} were also detected in all S. hygroscopicus strains. The different representatives of S. hygroscopicus had almost the same proportion of unsaturated fatty acids. Certain shifts in the amount of iso, anteiso and straight-chain fatty acids in some cultures were revealed. This might be explained by the adaptation capability of strains belonging to one species to form a variety of available fatty acids determined by particular cell membrane composition favouring certain antibiotic biosynthesis.     

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.     

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.     

Composition of the saturated and monounsaturated fatty acids of Mycobacterium phlei

Combined gas-liquid chromatography-mass spectrometry has been used to identify unusual fatty acids of Mycobacterium phlei. In addition to many normal saturated and monounsaturated fatty acids, series of iso, anteiso, 10-methyl, and (n-8)-methyl substituted fatty acids have been found and identified. These mid-chain branched acids may arise by methylation of monounsaturated acids followed (if necessary) by chain elongation.     

Cellular fatty acid composition of rod and coccus forms of Arthrobacter globiformis, A.crystallopoietes and A.nicotianae isolated from the water fern Azolla

Seven strains of Arthrobacter globiformis , and one each of A. crystallopoietes and A. nicotianae , isolated from the water fern Azolla, were cultured for 2–3 d at 30 °C on Pseudomonas Agar F to obtain rods and for 5 d to obtain cocci, then analysed for total cellular fatty acids. In rods, saturated iso - and anteiso -branched 13–19 carbon fatty acids averaged 85% of the total in A. globiformis , 81% in A. crystallopoietes , and 97% in A. nicotianae . Minor components included unsaturated branched chains, hydroxy and cyclopropane fatty acids. Fatty acid composition of A. globiformis was similar to that of A. crystallopoietes but different from that of A. nicotianae . Saturated straight chains in A. nicotianae averaged 1·5% of the total compared with 14–16% in A. globiformis/crystallopoietes , and anteiso -15:0 constituted 73% of the total in A. nicotianae compared with 18–19% in the other species. Composition of cocci was different from that of rods in A. globiformis and A. crystallopoietes. As cells changed from rods to cocci, percentages of saturated and unsaturated straight chains decreased and saturated iso - and anteiso -branched chains increased. In A. nicotianae there were no significant differences in the fatty acids between rods and cocci.     

Fatty Acid Composition of the Complex Lipids of Staphylococcus aureus During the Formation of the Membrane-bound Electron Transport System

In Staphylococcus aureus, 64 fatty acids could be separated by gas-liquid chromatography. The fatty acids consisted of normal, iso, and anteiso saturated fatty acids of from 10 to 21 carbon atoms. Of the total fatty acids, 2 to 4% were normal, iso, and anteiso monoenoic fatty acids. Positional isomers of the normal monoenoic fatty acids could be detected. The fatty acids could be extracted, leaving 1 to 2% of the total fatty acids in the residue. The proportions of the fatty acids in the residue and the total lipids differed significantly. The lipid extract contained less than 0.12% free fatty acid. Between 5 and 10% of the lipid fatty acids were associated with neutral lipids. The majority of the fatty acids were associated with the complex lipids: mono- and diglucosyl diglyceride, phosphatidyl glycerol, lysyl phosphatidyl glycerol, and cardiolipin. The proportions of the fatty acids changed markedly between bacteria grown anaerobically (no membrane-bound electron transport system) and those grown aerobically (containing a functional electron transport system). In each of the complex lipids, the proportions of the fatty acids, as well as the magnitude and direction of change in the molar quantity of the fatty acids per bacterium, changed dramatically between these growth conditions. Since the glucosyl diglycerides and phospholipids were formed from the same pool of diglyceride intermediates, the marked differences in fatty acids indicate that acyl transferase activities must be an important part of complex lipid metabolism in S. aureus.     

Stability of the fatty acid composition of actinomycetes

The stability of fatty acid composition of total extractable lipids was studied in Streptomyces cultures. The type of fatty acid composition typical of the Streptomyces genus remains stable when the actinomycetes were grown as submerged cultures in various synthetic media: saturated fatty acids with methyl branching in the chain predominated in all of the cases, and fatty acids with an uneven number of carbon atoms in the chain prevailed in most of the cases. Fatty acids with the anteiso structure predominated among the acids with a branched chain, amounting to more than a half of the latter and reaching sometimes 50% of the total fatty acid content. Methyl branchings were located in the anteiso position in fatty acids with an uneven number of carbon atoms, and in the iso position in fatty acids with an even number of carbons. Unsaturated fatty acids were found as a minor component.     

Effect of Exogenous Fatty Acids on the Cellular Fatty Acid Composition and Neomycin Formation in a Mutant Strain of Streptomyces fradiae

A mutant of Streptomyces fradiae which requires oleic acid for neomycin formation was isolated and the effects of exogenous fatty acids and other additives on the formation of neomycin were studied. Palmitic acid and high concentration of sodium ions could replace oleic acid in neomycin formation. The fatty acid spectrum of the mutant strain ST–5B was quite different from that of the parent strain 3123. The major fatty acid components of the mutant and the parent were anteiso 15:0 and iso 16: 0, respectively. However the fatty acid composition of the mutant was changed from the anteiso 15: 0-type to the parental iso 16: 0-type by the supplement of oleic acid or high concentration of sodium ions in the medium. In the case of palmitic acid, the major fatty acid component of the mutant cells was changed from anteriso 15: 0 to normal 16:0. The role of these additives in neomycin formation by the mutant is discussed.     

Identification and characterization of thermophilic bacteria isolated from hot springs in Turkey

The present study was conducted to identify and characterize the thermophilic bacteria isolated from various hot springs in Turkey by using phenotypic and genotypic methods including fatty acid methyl ester and rep-PCR profilings, and 16S rRNA sequencing. The data of fatty acid analysis showed the presence of 17 different fatty acids in 15 bacterial strains examined in this study. Six fatty acids, 15:0 iso, 15:0 anteiso, 16:0, 16:0 iso, 17:0 iso, and 17:0 anteiso, were present in all strains. The bacterial strains were classified into three phenotypic groups based on fatty acid profiles which were confirmed by genotypic methods such as 16S rRNA sequence analysis and rep-PCR genomic fingerprint profiles. After evaluating several primer sets targeting the repetitive DNA elements of REP, ERIC, BOX and (GTG)₅, the (GTG)₅ and BOXA1R primers were found to be the most reliable technique for identification and taxonomic characterization of thermophilic bacteria in the genera of Geobacillus, Anoxybacillus and Bacillus spp. Therefore, rep-PCR fingerprinting using the (GTG)₅ and BOXA1R primers can be considered as a promising genotypic tool for the identification and characterization of thermophilic bacteria from species to strain level.     

Physiological and biochemical studies on psychrotolerance in Listeria monocytogenes

Studies were undertaken to explain the ability of Listeria monocytogenes to grow at low temperatures in a chemostat. It was found that when grown in continuous culture at a dilution rate of 0·02 h⁻¹ L. monocytogenes had a lower proportion of anteiso -17:0, and a higher proportion of anteiso -15:0, and smaller chain fatty acids when grown at 10°C compared to 30°C. A previously unreported glycolipid was only seen after growth at low temperature. Growth temperature had no effect on the rate of glucose uptake.     

Confirmation of the Identification of the Major C-29 Hydrocarbons of Sarcina lutea

Although there is agreement on the fact that Sarcina lutea strain FD-533 has branched C-29 monoalkenes as major hydrocarbon components, there is disagreement in the literature as to the nature of the branching. This has been resolved by analyses of the fatty acids produced by permanganate-periodate treatment of each of the resolvable hydrocarbon fractions making up the C-29 complex. The three major components are identified as doubly branched, Delta(13) species with two iso terminations, one iso and one anteiso termination, and two anteiso terminations.