Effect of exogenous fatty acids on the growth and production of exopolysaccharides of obligately methylotrophic bacterium Methylophilus quaylei

Accelerating growth and increasing exopolysaccharide production in obligate methylotrophic bacterium Methylophilus quaylei were observed in the presence of C12-C18 fatty acids added to the growth media. Sodium oleate was the best growth factor. Based on data on the composition of the free fatty acids fraction in the cells and the values of the zeta-potential and fluorescence anisotropy of whole cells, we suggested that fatty acids were incorporated in the outer membrane of M. quaylei.     

Methylophilus quaylei sp. nov., a new aerobic obligately methylotrophic bacterium

A new obligately methylotrophic bacterium (strain MTT) with the ribulose monophosphate pathway of carbon assimilation is described. The isolate, utilizing only methanol, is an aerobic, Gram-negative, asporogenous, non-motile short rod multiplying by binary fission. Its cellular fatty acids profile consists primarily of straight-chain saturated C16:0 and unsaturated C16:l acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. Diphosphatidylglycerol (cardiolipin) is absent. Optimal growth conditions are 25-29 degree C, pH 6.5 - 7.5, 0.5% CH3OH and 0.05% NaCl. Strain MTT lacks alpha-ketoglutarate dehydrogenase, the glyoxylate shunt enzymes, and glutamate dehydrogenase. Ammonium is assimilated by the operation of the glutamate cycle enzymes: glutamine synthetase and glutamate synthase. An exopolysaccharide consisting of rhamnose, glucose and galactose is formed under nitrogen limitation. The G + C content of the DNA is 54.0 mol%. Based on 16S rDNA sequence analysis and DNA-DNA relatedness (29-34%) with type strains of the genus Methylophilus, the novel isolate was classified as a new species of this genus and named Methylophilus quaylei MTT (VKM B-2338T, DSMZ, etc.).     

Stearic acid methyl ester: A new extracellular metabolite of the obligate methylotrophic bacterium Methylophilus quaylei

Methyl esters of fatty acids, free fatty acids, and hydrocarbons were found in the culture liquid and in the cellular lipids of the obligate methylotrophic bacterium Methylophilus quaylei under optimal growth conditions and osmotic stress. The main extracellular hydrophobic metabolite was methyl stearate. Exogenous free fatty acids C₁₆–C₁₈ and their methyl esters stimulated the M. quaylei growth and survivability, as well as production of exopolysaccharide under osmotic and oxidative stress, playing the role of growth factors and adaptogens. The order of hydrophobic supplements according to the ability to stimulate bacterial growth is C_{18: 1} > C_{18: 0} > C_{16: 0} > methyl oleate > methyl stearate > no supplements > C_{14: 0} > C_{12: 0}. The mechanism underlying the protective action of fatty acids and their methyl esters is discussed.     

The terminal respiratory chain of the methylotrophic bacterium Methylophilus methylotrophus

Cytochrome oxidase o has been isolated from the obligately aerobic, methylotrophic bacterium Methylophilus methylotrophus in the form of a cytochrome cL-o complex. The latter is comprised of cytochrome cL (Mr 21 000) and cytochrome o (Mr 29 000) in a 1-2:1 ratio, possibly in association with one or more minor polypeptides; the complex exhibits a high ascorbate-TMPD oxidase activity which is inhibited non-competitively by cyanide (Ki approximately 2 microM). In contrast, the oxidation of methanol by whole cells is inhibited uncompetitively by cyanide (Ki approximately 4 microM), thus indicating the involvement in methanol oxidation of cytochrome oxidase aa3 rather than o.     

Energy conservation in whole cells of the methylotrophic bacterium Methylophilus methylotrophus

Respiratory chain phosphorylation has been investigated in the methylotrophic bacterium Methylophilus methylotrophus following the addition of oxidisable substrates to aerobic, whole cell suspensions. Initial-rate experiments showed that ATP synthesis occurred at the overall expense of AMP and inorganic phosphate via the sequential action of the ATP phosphohydrolase and adenylate kinase; some of the nascent ATP was rapidly used to synthesis nonadenine nucleoside triphosphates. After being corrected for ATP turnover, Pi/O quotients of 0.46 to 0.54, 0.77 and 1.37 nmol/ng-atom O were obtained for the oxidation of methanol dehydrogenase-linked substrates (methanol, ethanol and acetaldehyde), duroquinol and formate (NAD⁺-linked) respectively. These values were proportional to the H⁺/O and/or K⁺/O quotients exhibited by these substrates, and yielded an average H⁺/ATP (H⁺/Pi) quotient of 4.2 ng-ion H⁺/nmol. Steady-state experiments showed that the extent of cellular energisation varied with the respiration rate but was always in the order methanol > duroquinol > acetaldehyde, thus indicating that under these longer-term conditions methanol was completely oxidised to yield PQQH₂ and 2NAD(P)H. These results are discussed in terms of the various reactions which lead to the generation or utilisation of the protonmotive force in this organism.Abbreviations FCCP carbonylcyanide p-trifluoromethyxyphenyl-hydrazone - bulk phase, transmembrane electrochemical potential difference of protons ( ) - pH bulk phase, transmembrane pH difference (pH_in–pH_out) - bulk phase, transmembrane electrical potential difference (_in - _out) - [P] concentration of anhydride phosphate bonds in adenine nucleotides (2[ATP]+[ADP]) - FPLC fast protein liquid chromatography - PQQ pyrroloquinoline quinone - Gp phosphorylation potential     

Deuterium oxide as a stress factor to the methylotrophic bacterium Methylophilus sp

The adaptation of the methylotrophic bacterium Methylophilus sp. B-7741 to growth in highly deuterated media was studied. For the first time, we showed the cross adaptation of bacterial cells to deuterated media and oxidative and osmotic stresses. The activity at catalase in deuterated cells was higher than in the control cells. Deuterated cell-free culture liquids showed protective effects on the growth of Methylophilus sp. B-7741 in deuterated media, which was manifested as an increase in the deuterated biomass yield. These data and the data available in the literature suggest that the mechanisms of bacterial cell adaptation to heavy water and to oxidative and osmotic stresses are similar.     

乳杆菌胞外多糖对人体粪便菌群及其产生短链脂肪酸的影响

目的在体外利用7名志愿者的新鲜粪便作为来源,选择菊粉作为对照,研究干酪乳杆菌LC2W与鼠李糖乳杆菌Y37胞外多糖对人体肠道菌群的影响。方法利用PCR-DGGE(变性梯度凝胶电泳)方法分析样品中菌群组成,采用高效液相色谱的方法分析短链脂肪酸组成。结果与菊粉相比,粪便菌群在添加乳杆菌胞外多糖培养后,可以促进短链脂肪酸的生成,保持肠道菌群的多样性。结论干酪乳杆菌LC2W与鼠李糖乳杆菌Y37的胞外多糖在体外有调节粪便菌群的作用。     

The apparent oxidation of NADH by whole cells of the methylotrophic bacterium Methylophilus methylotrophus

Previous reports that whole cells of Methylophilus methylotrophus oxidase exogenous NADH have been investigated. Essentially identical rates of oxygen consumption were observed following the addition of methanol or NADH to whole cells. Both activities were inhibited by EDTA and hydroxylamine, but not by HQNO, and exhibited similar pH optima. Analyses of the reaction stoichiometry with NADH as substrate showed that the expected amount of oxygen was consumed, but also revealed acidification (instead of alkalinisation) and no oxidation of NADH. Further studies showed that commerical NADH is contaminated with ethanol which is oxidised to acetic acid by the low specificity methanol oxidase system present in this organism. The oxidation of exogenous NADH by whole cells of M. methylotrophus reported previously is therefore spurious.Abbreviations EDTA = ethylenediaminetetracetate - HQNO = 2–n-heptyl-4-hydroxy-quinoline-N-oxide - DEAE = diethylaminoethyl     

Effect of growth conditions on the activities of methylotrophic enzymes in Methylophilus W3A1

Abstract The specific activities of four methylotrophic enzymes were measured in batch and chemostat cultures of Methylophilus W3A1. Methanol dehydrogenase was a constitutive enzyme. Methylamine dehydrogenase was regulated by an induction/repression mechanism. Trimethylamine dehydrogenase and dimethylamine monooxygenase were induced co-ordinately, probably by dimethylamine. Methanol completely prevented synthesis of the methyl-amine oxidising enzymes when ammonium was also present; the absence of ammonium relieved this repression. The significance of the results for the evolution of methylotrophic pathways in restricted facultative methylotrophs is discussed.     

Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi.

Bioluminescent bacteria require myristic acid (C14:0) to produce the myristaldehyde substrate of the light-emitting luciferase reaction. Since both endogenous and exogenous C14:0 can be used for this purpose, the metabolism of exogenous fatty acids by luminescent bacteria has been investigated. Both Vibrio harveyi and Vibrio fischeri incorporated label from [1-14C]myristic acid (C14:0) into phospholipid acyl chains as well as into CO2. In contrast, Photobacterium phosphoreum did not exhibit phospholipid acylation or beta-oxidation using exogenous fatty acids. Unlike Escherichia coli, the two Vibrio species can directly elongate fatty acids such as octanoic (C8:0), lauric (C12:0), and myristic acid, as demonstrated by radio-gas liquid chromatography. The induction of bioluminescence in late exponential growth had little effect on the ability of V. harveyi to elongate fatty acids, but it did increase the amount of C14:0 relative to C16:0 labeled from [14C]C8:0. This was not observed in a dark mutant of V. harveyi that is incapable of supplying endogenous C14:0 for luminescence. Cerulenin preferentially decreased the labeling of C16:0 and of unsaturated fatty acids from all 14C-labeled fatty acid precursors as well as from [14C]acetate, suggesting that common mechanisms may be involved in elongation of fatty acids from endogenous and exogenous sources. Fatty acylation of the luminescence-related synthetase and reductase enzymes responsible for aldehyde synthesis exhibited a chain-length preference for C14:0, which also was indicated by reverse-phase thin-layer chromatography of the acyl groups attached to these enzymes. The ability of V. harveyi to activate and elongate exogenous fatty acids may be related to an adaptive requirement to metabolize intracellular C14:0 generated by the luciferase reaction during luminescence development.     

The effect of EDTA and related chelating agents on the oxidation of methanol by the methylotrophic bacterium, Methylophilus methylotrophus

The effects of EDTA and related metal-chelating agents on the respiratory system of the methylotrophic bacterium Methylophilus methylotrophus have been investigated. EDTA completely inhibited whole cell methanol oxidase activity and concomitantly decreased the aerobic steady-state reduction level of cytochrome c, but only partially inhibited methanol dehydrogenase activity. Analysis of the inhibition kinetics of EDTA and other chelating agents on methanol oxidase activity indicated that they were effective in the order CDTA greater than EDTA greater than HEDTA greater than EGTA greater than EDDA, and that they inhibited in an uncompetitive manner. Inhibition by EDTA varied as a function of the ambient cell mass in the assay, and could be partly reversed by the addition of divalent cations. EDTA had no effect on the activity of solubilised methanol dehydrogenase. These and other results indicate that EDTA binds a divalent metal ion, probably Mg2+, which is involved in the functional association of methanol dehydrogenase with the respiratory membrane. This concept is discussed in terms of the electron transfer properties and spatial organisation of the terminal respiratory chain of M. methylotrophus.     

Effect of exogenous fatty acids on growth,membrane fluidity,and phospholipid fatty acid composition in yeast

The growth response of a double-mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis-Δ¹¹- eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity. There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 1 5:0, and 1 6:0. We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism.     

Lipid compositional manipulation in Acholeplasma laidlawii B. Effect of exogenous fatty acids on fatty acid composition and cell growth when endogenous fatty acid production is inhibited

A variety of potential inhibitors of de novo fatty acid biosynthesis have been tested for activity in Acholeplasma laidlawii B. Two compounds, avidin and N,N-dimethyl-4-oxo-2trans-dodecenamide (CM-55), an antimicrobial fatty amide, strongly inhibit de novo biosynthesis without nonspecific toxic effects at moderate dosages. Avidin is the more potent inhibitor, abolishing de novo fatty acid synthesis and greatly reducing the chain elongation of exogenous fatty acids at level of 25 U/l. CM-55 gives complete inhibition of de novo biosynthesis only at low temperatures and inhibits exogenous fatty acid elongation to a variable extent. However, CM-55 is still a more potent antilipogenic agent in this organism than is the fungal antibiotic cerulenin. Cells cultured with avidin grow only when one or more exogenous medium- or long-chain fatty acids are added to the growth medium. The extent of cell growth under these conditions depends primarily on the physical properties of the exogenous fatty acid(s). In general, fatty acids giving diacylglycerolipids of very high or very low fluidity are unsuitable growth substrates, while those whose diacylglycerol derivatives are of intermediate fluidity support fair to good cell growth.     

Statistical media optimization for growth and PHB production from methanol by a methylotrophic bacterium

Media components were optimized by statistical design for cell growth and PHB production of Methylobacterium extorquens DSMZ 1340. Four important components of growth media were optimized by central composite design. The growth increased from an OD = 1.35 for Choi medium as control to an OD = 2.15 for optimal medium. Then media components for PHB production were optimized. Optimization of five important factors was conducted by response surface method. The optimal composition of PHB production medium was found to be at 7.8 (g/L) Na₂HPO₄ · 12H₂O, and surprisingly at zero concentration of (NH₄)₂SO₄, KH₂PO₄, MgSO₄ and MnSO₄. The PHB production was found to be 2.95 (g/L) at this medium. RSM results indicated that a deficiency of nitrogen and magnesium is crucial for PHB accumulation in this microorganism. Also, PHB production was carried out in a 5 L fermentor at the optimum condition which resulted in 9.5 g/L PHB and 15.4 g/L cell dry weight with 62.3% polymer content.     

Anaerobic oxidation of fatty acids by Clostridium bryantii sp. nov., a sporeforming,obligately syntrophic bacterium

From marine and freshwater mud samples strictly anaerobic, Gram-positive, sporeforming bacteria were isolated which oxidized fatty acids in obligately syntrophic association with H₂-utilizing bacteria. Even-numbered fatty acids with up to 10 carbon atoms were degraded to acetate and H₂, odd-numbered fatty acids with up to 11 carbon atoms including 2-methylbutyrate were degraded to acetate, propionate and H₂. Neither fumarate, sulfate, thiosulfate, sullur, nor nitrate were reduced. A marine isolate, strain CuCal, is described as type strain of a new species, Clostridium bryantii sp. nov.     

The stress-shock response of the bacterium Methylophilus methylotrophus

A sudden increase in the growth temperature of Methylophilus methylotrophus results in the synthesis of a number of unique proteins. The major heat-shock proteins have apparent molecular masses of 83, 78, 63, 60, 16 and 14 kDa. Other stress conditions elicit a similar response, although there are significant differences in the sets of proteins produced under the various conditions. Addition of methanol induces proteins identical in size to the heat-shock 83, 79, 63 and 14 kDa proteins and also induces unique 94, 36 and 29 kDa species. Addition of ethanol induces proteins identical in size to the 78 and 20 kDa heat-shock proteins and the 94 and 36 kDa methanol-induced proteins and an apparently unique 13 kDa species. Simultaneous exposure to elevated temperature and either methanol or ethanol resulted in the synthesis of all of the proteins induced by the separate treatments. The stress-shock proteins are differentially located in cytoplasmic, periplasmic and membrane fractions.     

Effect of fatty acids on aflatoxin production byAspergillus parasiticus

The effect of saturated and unsaturated fatty acids on aflatoxin production was studied in a synthetic medium. The aflatoxin production decreased (10-75%) in the presence of lauric acid and palmitic acid but the addition of behenic and sebacic acid stimulated aflatoxin production by 125-541%. Linolenic and linoleic acids effected aflatoxin production and mycelium growth. An 34-fold increase in aflatoxin production was observed with 50 mM linoleic acid. An inverse relationship was observed between aflatoxin production and mycelium mass, irrespective of the nature of the fatty acid.     

Stimulation of the growth and respiration of a methylotrophic bacterium by morphine.

The growth of a pseudomonad on methanol was stimulated by the presence of morphine (or codeine) in the medium. The drug appeared to influence the amount of growth rather than its rate. Respiration of resting cells on a variety of substrates was stimulated by adding morphine. This report appears to be the first case of a microorganism whose growth and respiration is stimulated by an opiate.