Fatty Acids Associated with the Cell Wall in Film Strains of Saccharomyces

Fatty acid contents were estimated in the cell wall of Saccharomyces. The fatty acids responsible for cell wall hydrophobicity were classified by ease of extraction to ‘readily extractable’ and ‘bound’ acids. The readily extractable fatty acids were easily extracted with pentane and chloroform-methanol. The fatty acids extracted with chloroform-methanol were quite effective for cell wall hydrophobicity, but the fatty acids extracted with pentane were not. The bound fatty acids comprised in the phospholipids phosphatidylethanolamine and phosphatidylserine, which were rigidly associated with the cell wall. These phospholipids were not extractable until they were released from the cell wall by pronase. Chloroform-methanol extraction caused a reduction in cell wall phospholipid content, particularly after treatment with pronase. The fatty acid content of the resultant cell wall was lowered to below 7% of initial content. Phospholipids contained more saturated fatty acid than readily extractable lipids. Phospholipids greatly contributed to cell wall hydrophobicity of various film strains of Saccharomyces.     

Inhibition of Microbial Lipases by Fatty Acids

Addition of lard or sodium oleate to the medium used for lipase production by Pseudomonas fragi resulted in a decreased accumulation of lipase in the culture supernatant fluid without affecting cell growth. The production and activity of lipase was inhibited by lard, sodium oleate, and the salts of other unsaturated fatty acids. Some divalent cations, Tweens, lecithin, and bovine serum prevented oleate inhibition, but did not reverse it. Similar inhibitory actions were observed with Geotrichum candidum lipase, but not with a staphylococcal lipase or pancreatic lipase. A protective effect by protein in crude enzyme preparations is indicated. The ability of oleate to lower surface tension does not appear to be related to its ability to inhibit lipase.     

微生物多不饱和脂肪酸的研究进展

介绍了多不饱和脂肪酸(PUFAs)的微生物来源在微生物体内的代谢途径、分子生物学研究进展以及微生物的发酵生产状况。重点论述了微生物PUFAs的最新分子生物学研究进展。     

Stimulatory Effect of Inhibitors of Cell Wall Synthesis on Protein Production by Bacillus brevis

Mutants of Bacillus brevis No. 47 that grew in synthetic media containing a high concentration of ammonium sulfate were stable and had high protein production. Among various antibiotics tested, inhibitors of cell wall synthesis, such as bacitracin or β-lactam antibiotics, were effective in greatly increasing the accumulation of exoproteins.

When 60 µg/ml of bacitracin was added to the culture at the early logarithmic growth phase, about 9 mg/ml of proteins was produced. Such a protein yield was estimated to be nearly maximum from a given amount of glucose. Alterations in cell wall components were found in cells grown in the presence of bacitracin. Possible relationships between cell wall structure and protein production were discussed.     

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.     

Cell Wall Teichoic Acids of Two Brevibacterium Strains

Structurally identical teichoic acids were detected in cell walls of two soil isolates assigned to Brevibacterium linens based on phylogenetic data. Both cell walls contain unsubstituted 1,3-poly(glycerol phosphate) and poly(glycosylglycerol phosphate). Repeating units of the latter--alpha-D-GlcpNAc-(1-->4)-beta-D-Galp-(1-->1)-Gro--are bound by phosphodiester bonds including OH-3 of galactose and OH-3 of glycerol. Some of the N-acetylglucosamine residues have 4,6-pyruvic acid acetal, amounts of the latter in the two strains being unequal. Species-specificity of the structures of teichoic acids in the genus Brevibacterium is discussed.     

Synthesis of Very-Long-Chain Fatty Acids in the Epidermis Controls Plant Organ Growth by Restricting Cell Proliferation

Plant organ growth is controlled by inter-cell-layer communication, which thus determines the overall size of the organism. The epidermal layer interfaces with the environment and participates in both driving and restricting growth via inter-cell-layer communication. However, it remains unknown whether the epidermis can send signals to internal tissue to limit cell proliferation in determinate growth. Very-long-chain fatty acids (VLCFAs) are synthesized in the epidermis and used in the formation of cuticular wax. Here we found that VLCFA synthesis in the epidermis is essential for proper development of Arabidopsis thaliana. Wild-type plants treated with a VLCFA synthesis inhibitor and pasticcino mutants with defects in VLCFA synthesis exhibited overproliferation of cells in the vasculature or in the rib zone of shoot apices. The decrease of VLCFA content increased the expression of IPT3, a key determinant of cytokinin biosynthesis in the vasculature, and, indeed, elevated cytokinin levels. These phenotypes were suppressed in ipt3;5;7 triple mutants, and also by vasculature-specific expression of cytokinin oxidase, which degrades active forms of cytokinin. Our results imply that VLCFA synthesis in the epidermis is required to suppress cytokinin biosynthesis in the vasculature, thus fine-tuning cell division activity in internal tissue, and therefore that shoot growth is controlled by the interaction between the surface (epidermis) and the axis (vasculature) of the plant body.     

Microbial Assimilation of Hydrocarbons II. Fatty Acids Derived from 1-Alkenes

The utilization of 1-alkenes by Micrococcus cerificans was investigated with respect to characteristic fatty acid profiles resulting from growth at the expense of these substrates. Saturated fatty acids containing even numbers of carbon atoms were produced from 1-dodecene and 1-tetradecene. Unsaturated fatty acids related to the parent alkene were not detected. The fatty acid profile from 1-pentadecene utilization resulted in the identification of 14-pentadecenoic acid, indicating preferential methyl-group attack. Studies with 1-hexadecene and 1-octadecene indicated simultaneous methyl-group and double-bond attack. Omega-Unsaturated fatty acids related to carbon number of parent alkene and odd-carbon fatty acids one carbon less than the substrate molecule were identified. A mechanism involving double bond epoxidation and oxidative cleavage was supported by measuring the release of formaldehyde. It appears that a dichotomous mechanism is functional in the assimilation of higher carbon number alkenes.     

Plasma and Red Blood Cell Fatty Acids in Peroxisomal Disorders

The demonstration of abnormal levels of fatty acids or plasmalogens in plasma or red blood cells is key to the diagnosis of peroxisomal disorders. We report the levels of 62 fatty acids and plasmalogens in patients with X-linked adrenoleukodystrophy (X-ALD), Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), both at baseline and after dietary interventions. Lorenzo's Oil therapy in X-ALD normalizes the levels of saturated very long chain fatty acids in plasma, but leads to reduced levels of omega 6 and other omega 3 fatty acids, and requires monitoring and appropriate dietary supplements. Patients with ZS, NALD and IRD have reduced levels of docosahexaenoic acid (DHA) and arachidonic acid (AA) which can be normalized by the oral administration of microencapsulated DHA and AA.     

Microbial Communities Involved in Anaerobic Degradation of Unsaturated or Saturated Long-Chain Fatty Acids

Anaerobic long-chain fatty acid (LCFA)-degrading bacteria were identified by combining selective enrichment studies with molecular approaches. Two distinct enrichment cultures growing on unsaturated and saturated LCFAs were obtained by successive transfers in medium containing oleate and palmitate, respectively, as the sole carbon and energy sources. Changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing. A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in oleate and palmitate enrichment cultures clustered with fatty acid-oxidizing bacteria within Syntrophomonadaceae and Syntrophobacteraceae families. A low methane yield, corresponding to 9 to 18% of the theoretical value, was observed in the oleate enrichment, and acetate, produced according to the expected stoichiometry, was not further converted to methane. In the palmitate enrichment culture, the acetate produced was completely mineralized and a methane yield of 48 to 70% was achieved from palmitate degradation. Furthermore, the oleate enrichment culture was able to use palmitate without detectable changes in the DGGE profile. However, the palmitate-specialized consortia degraded oleate only after a lag phase of 3 months, after which the DGGE profile had changed. Two predominant bands appeared, and sequence analysis showed affiliation with the Syntrophomonas genus. These bands were also present in the oleate enrichment culture, suggesting that these bacteria are directly involved in oleate degradation, emphasizing possible differences between the degradation of unsaturated and saturated LCFAs.     

Effect of Inhibitors of Bacterial Cell Wall Synthesis on the Growth of Anabaena variabilis,a Blue-green Alga

The effects of penicillins and several other antibiotics (vancomycin, ristocetin, bacitracin, novobiocin, and d-cycloserine), all known as inhibitors of bacterial cell wall synthesis, were tested on the growth of Anabaena variabilis, a strain of blue-green alga.

All the antibiotics tested inhibited the growth of Anabaena variabilis at concentrations ranging from 10 µg to 1 mg per ml. However, penicillins and the other antibiotics tested did not inhibit growth of strains of green algae.     

Variation in Microbial Biomass and Community Structure in Sediments of Eutrophic Bays as Determined by Phospholipid Ester-Linked Fatty Acids

The distribution of phospholipid ester-linked fatty acids (PLFA) in sediments of eutrophic bays (Hiroshima Bay and Aki Nada) was studied to quantify the microbial biomass, community structure, and nutritional status. A total of 63 fatty acids in the range of C₁₀ to C₂₄ were determined. They consist of saturated fatty acids, branched fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids, and variation was revealed in the relative proportions of these fatty acids in sediments. On the basis of the PLFA concentration in sediments, the calculated microbial biomass showed variation (mean ± standard deviation = 0.70 × 10⁸ ± 0.53 × 10⁸ cells per g [dry weight] of sediment) in the eutrophic bays. In sediments, a higher amount of biomass was observed in the coastal area of Hiroshima Bay than that observed in the rest of the bay and adjacent Aki Nada. The microbial community structure of the present study area, as characterized by the PLFA profiles, showed very low percentages of polyunsaturated fatty acids and long-chain fatty acids characteristic of microeukary-otes and terrestrial input, respectively, and high percentages of fatty acids characteristic of bacteria. The distribution of PLFA profiles also showed the relative contribution of both aerobic and anaerobic bacteria, especially sulfate-reducing bacteria, in the study area. The relative proportions of PLFA revealed distinctive differences among the stations of the study area, as is evidenced from six clusters obtained for the PLFA profiles. The results of Tukey's honestly significant difference test further confirmed that the sediments in the coastal area of Hiroshima Bay were significantly enriched by a number of fatty acids when compared with other areas investigated where relatively few fatty acids were present in significant quantities. No marked variation in environmental parameters in the surface- and bottom-water samples was observed, indicating the absence of any water movement in the study area. Furthermore, low redox potential and the levels of sulfide in the sediment revealed the reduced condition of the sediment. The existing environmental conditions and pollution of the study area were attributed to the observed microbial community structure in the sediments.     

Omega-3 and Omega-6 Fatty Acids Act as Inhibitors of the Matrix Metalloproteinase-2 and Matrix Metalloproteinase-9 Activity

Polyunsaturated fatty acids have been reported to play a protective role in a wide range of diseases characterized by an increased metalloproteinases (MMPs) activity. The recent finding that omega-3 and omega-6 fatty acids exert an anti-inflammatory effect in periodontal diseases has stimulated the present study, designed to determine whether such properties derive from a direct inhibitory action of these compounds on the activity of MMPs. To this issue, we investigated the effect exerted by omega-3 and omega-6 fatty acids on the activity of MMP-2 and MMP-9, two enzymes that actively participate to the destruction of the organic matrix of dentin following demineralization operated by bacteria acids. Data obtained (both in vitro and on ex-vivo teeth) reveal that omega-3 and omega-6 fatty acids inhibit the proteolytic activity of MMP-2 and MMP-9, two enzymes present in dentin. This observation is of interest since it assigns to these compounds a key role as MMPs inhibitors, and stimulates further study to better define their therapeutic potentialities in carious decay.     

细胞膜脂肪酸组成改变促进乳腺癌细胞凋亡的研究

目的:研究n-6脂肪酸脱氢酶fat-1基因在人乳腺癌细胞内的表达,改变细胞膜脂肪酸组成,对乳腺癌细胞的凋亡作用.方法:构建含有fat-1基因的重组腺病毒载体(Ad.GFP.fat-1),通过包装细胞系(293)产生的腺病毒,感染人乳腺癌细胞MCF-7.提取细胞的总RNA,以fat-1的反义mRNA作探针,用Northern blot检测fat-1基因在MCF-7细胞内的表达.MTT法分析fat-1基因对MCF-7细胞增殖的影响,凋亡染色试剂盒检测细胞的凋亡.气相色谱仪分析对MCF-7细胞的n-6 PUFAs/n-3 PUFAs含量影响.结果:通过基因重组技术,得到预期的重组病毒;fat-1基因在人乳腺癌细胞MCF-7中能有效异源表达,2d后,可检测到fat-1 mRNA的条带.与对照细胞相比,fat-1基因有效地抑制了MCF-7细胞的增殖(23%,p＜0.05),促进了凋亡(增加35%);同时降低了人乳腺癌细胞MCF-7细胞膜n-6 PUFAs/n-3 PUFAs的比率.结论:腺病毒介导的fat-1基因能在人乳腺癌细胞MCF-7内有效异源表达,且抑制了MCF-7细胞的增殖.机理为降低了细胞膜的n-6 PUFAs/n-3 PUFAs的比率.     

Estimations of Uronic Acids as Quantitative Measures of Extracellular and Cell Wall Polysaccharide Polymers from Environmental Samples

The extracellular polysaccharide polymers can bind microbes to surfaces and can cause physical modification of the microenvironment. Since uronic acids appear to be the components of these extracellular films that are most concentrated in a location outside the cell membrane, a quantitative assay for uronic acids was developed. Polymers containing uronic acids are resistant to quantitative hydrolysis, and the uronic acids, once released, form lactones irreproducibly and are difficult to separate from the neutral sugars. These problems were obviated by the methylation of the uronic acids and their subsequent reduction with sodium borodeuteride to the corresponding alcohol while they were in the polymer and could not form lactones. This caused the polymers to lose the ability to adhere to their substrates, so they could be quantitatively recovered. The hydrolysis of the dideuterated sugars was reproducible and could be performed under conditions that were mild enough that other cellular and extracellular polymers were not affected. The resulting neutral sugars were readily derivatized and then were separated and assayed by glass capillary gas-liquid chromatography. The dideuterated portion of each pentose, hexose, or heptose, identified by combined capillary gas-liquid chromatography and mass spectrometry, accurately provided the proportion of each uronic acid in each carbohydrate of the polymer. Examples of the applications of this methodology include the composition of extracellular polymers in marine bacteria, invertebrate feeding tubes and fecal structures, and the microfouling films formed on titanium and aluminum surfaces exposed to seawater.     

Wall Teichoic Acid Protects Staphylococcus aureus against Antimicrobial Fatty Acids from Human Skin

Skin-colonizing gram-positive bacteria produce wall teichoic acids (WTAs) or related glycopolymers for unclear reasons. Using a WTA-deficient Staphylococcus aureus mutant, we demonstrated that WTA confers resistance to antimicrobial fatty acids from human sebaceous glands by preventing fatty acid binding. Thus, WTA is probably important for bacterial skin colonization.Bacterial life on mammalian skin depends on efficient adaptation strategies to cope with high salt concentrations and dryness. In addition, skin is protected by a variety of antibacterial molecules, such as antimicrobial peptides (16), bacteriolytic enzymes (14), and antibacterial fatty acids (AFAs) (6, 11, 23). The main source of free fatty acids is the sebum, produced by sebaceous glands, and differentiating keratinocytes of the stratum corneum, the outermost layer of the epidermis, which is composed of dead, keratin-filled cells. Sebaceous glands are found in nearly all mammals, and the composition of the sebum is remarkably species specific (12). Up to 47% of human sebum consists of free fatty acids with palmitoleic acid isomer (C_16:1Δ6) as the predominant monoene AFA. Lauric acid (C_12:0) is the most potent saturated AFA (23). Palmitic acid (C_16:0), stearic acid (C_18:0), oleic acid (C_18:1Δ9), and linoleic acid (C_18:2Δ9Δ12) are the main fatty acids in the stratum corneum (9, 23).While most skin-colonizing bacteria are harmless commensals, Staphylococcus aureus frequently causes endogenous infections, ranging from cutaneous infections to life-threatening sepsis and endocarditis (10). S. aureus has developed efficient strategies to survive in its natural niches, the human anterior nares and skin, and to evade the immune system (4, 8). However, only a few studies have previously addressed the molecular basis of staphylococcal resistance to AFA. The major surface protein expressed by S. aureus under iron-limited conditions, IsdA, has recently been shown to confer AFA resistance because it increases the bacterial surface hydrophilicity (2). In addition to proteins, cell wall glycopolymers such as the teichoic acids are thought to govern bacterial surface hydrophobicity. Such polymers are found in most gram-positive bacteria, forming a highly charged mesh within the cell wall (21). They often consist of alternating glycerolphosphate or ribitolphosphate units, which are partially substituted by d-alanine and various glycosyl residues (13, 21). Teichoic acids are anchored in the cytoplasmic membrane via a glycolipid (lipoteichoic acid) or in the peptidoglycan via a phosphodiester linkage (wall teichoic acid [WTA]). A variety of roles in bacterial cell envelope processes and integrity have been assigned to WTA but the major functions of WTA have still remained elusive (21). Our group has recently generated a WTA-deficient S. aureus mutant and demonstrated that WTA is crucial for S. aureus nasal colonization and endovascular infection (19, 20, 22). The tagO gene disrupted in this mutant encodes an N-acetylglucosamine-phosphate transferase catalyzing the first step of WTA biosynthesis (24). The tagO mutant shows a total loss of WTA but seems to be unaffected in growth behavior and susceptibility to different antimicrobial peptides (19). However, the mutant exhibits increased resistance to human beta-defensin 3 (7).In order to study the contribution of WTA to the surface hydrophobicity of S. aureus SA113, a frequently used laboratory strain (5, 19, 22), the affinities of the wild type and the tagO mutant for the hydrophobic solvent dodecan were compared by the microbial adhesion to hydrocarbon test (15). In fact, the hydrophilicity of the WTA-deficient mutant was considerably decreased compared to those of the parental and complemented mutant strains (Fig. ​(Fig.1),1), confirming the crucial impact of WTA on the physicochemical surface properties of S. aureus. Subsequently, the MICs of a variety of saturated and unsaturated fatty acids occurring in human sebum and stratum corneum were determined (Table ​(Table1).1). Twenty-four-well plates with 50%-concentrated Müller-Hinton broth (Sigma) containing increasing concentrations of AFAs were inoculated with the bacterial strains, and the optical density was measured after 48 h of growth at 37°C. The tagO mutant showed a profound increase in susceptibility to all tested AFAs compared to the parental strain and the complemented mutant. The strongest MIC reductions were found for palmitoleic acid (sixfold) and linoleic acid (26-fold). In order to compare potential differences in susceptibility to the bactericidal activities of AFAs, bacteria grown overnight in 50%-concentrated Müller-Hinton broth were resuspended in phosphate-buffered saline (PBS) at an optical density of 0.5 at 578 nm, and 1 ml of each suspension was shaken with increasing concentrations of AFAs at 37°C. Incubation was stopped at different time points by dilution with PBS, and numbers of surviving bacteria were determined by counting CFU. Palmitoleic acid exhibited dose-dependent bactericidal activity to SA113, with the tagO mutant having 26-fold reduced survival compared to that of the wild type at 1.25 mM after 10 min of incubation (Fig. ​(Fig.2A).2A). When different incubation times were used for a given concentration, the tagO mutant was much more rapidly killed than the parental strain, thereby confirming the crucial role of WTA in AFA resistance (Fig. ​(Fig.2B2B).Open in a separate windowFIG. 1.The WTA-deficient ΔtagO mutant has decreased surface hydrophilicity compared to the wild type and the complemented (compl.) mutant strain, as assessed by the microbial adhesion to hydrocarbon test. The percentages of bacteria associated with the hydrophilic phase are shown. Data represent means ± standard errors of the means from three independent experiments. ***, P < 0.001; ns, not significant (in comparison to the wild-type value).Open in a separate windowFIG. 2.The WTA-deficient tagO mutant is more susceptible to the bactericidal activity of AFAs than the wild-type strain. (A) Bacteria were exposed to the indicated concentrations of palmitoleic acid for 10 min. **, P < 0.005; ***, P < 0.001. (B) The wild type (▪) and the tagO mutant (▴) were exposed to lauric acid (5 mM), cis-6-hexadecenoic acid (5 mM), and palmitoleic acid (1.25 mM) for the indicated times. Data represent means ± standard errors of the means from at least three independent experiments.

TABLE 1.

WTA-deficient Sa113 tagO mutant is more susceptible to growth-inhibiting activity of AFAs than wild-type and complemented mutant strains

多不饱和脂肪酸对细胞膜功能影响的研究进展

多不饱和脂肪酸是细胞膜磷脂的重要组成成份,影响细胞膜的稳定性.它具有广泛的生物学功能,包括细胞内信号传导通路、基因表达和细胞凋亡的调控等.主要从细胞膜脂质的组成,细胞膜的流动性及膜脂质过氧化等方面对多不饱和脂肪酸对细胞膜功能的影响进行了综述.