Caulobacter crescentus fatty acid-dependent cell cycle mutant.

A fatty acid auxotroph of Caulobacter crescentus, AE6001, which displays a strict requirement for unsaturated fatty acids to grow on glucose as the carbon source has been isolated. Starvation of AE6001 for unsaturated fatty acids resulted in a block in the cell cycle. Starved cultures accumulated at the predivisional cell stage after a round of DNA replication had been completed and after a flagellum had been assembled at the pole of the cell. Cell division and cell growth failed to occur probably because the mutant was unable to synthesize a membrane. An analysis of double mutants containing the fatB503 allele and other mutations in membrane biogenesis demonstrated that the cell cycle of AE6001 blocked at a homeostatic state. The addition of oleic acid to starved cultures permitted cell division and the initiation of a new round of DNA replication. The coincident block in both the initiation of DNA replication and membrane assembly, exhibited by starved cultures of this mutant, suggests that the fatB503 gene product may be involved in the coordination of these events.     

Isolation and characterization of a glycerol auxotroph of Rhodopseudomonas capsulata: effect of lipid synthesis on the synthesis of photosynthetic pigments.

A glycerol auxotroph was isolated from Rhodopseudomonas capsulata for use as a system for studying membrane synthesis and function. When the mutant was deprived of glycerol, net phospholipid synthesis ceased immediately and a small amount of free fatty acids accumulated. A turnover of lipid occurred in both deprived and supplemented cultures. Deoxyribonucleic acid and protein synthesis continued for one doubling of cell massand then slowed down in deprived cells. Net ribonucleic acid synthesis slowed down more dramatically. Oxidative phosphorylation activity of membrane preparations from aerobically and semi-anaerobically grown cells appeared unaffected by glycerol deprivation, indicating that simultaneous lipid synthesis is not a requirement for new oxidative phosphorylating activity. In the absence of net phospholipid synthesis, bacteriochlorophyll and carotenoid syntheses were reduced to 30% of the activity of supplemented cultures. Delta-Aminolevulinic acid synthase, the first enzyme on the bacteriochlorophyll pathway that is subject to regulatory control, increased in activity in deprived cultures. Lascelles and Szilagyi (1965) showed an association between phospholipid synthesis and pigment production. They found an increased lipid content associated with pigmented cells. The present results indicate that not only is there an association between lipid and pigment synthesis, but also there is actually a dependence of bacteriochlorophyll synthesis on phospholipid synthesis.     

Identification of the plasma membrane H+-biotin symporter of Saccharomyces cerevisiae by rescue of a fatty acid-auxotrophic mutant.

Bakers' yeast is auxotrophic for biotin (vitamin H) and depends on the efficient uptake of this compound from the environment. A mutant strain with strongly reduced biotin uptake and with reduced levels of protein biotinylation was identified. The strain was auxotrophic for long-chain fatty acids, and this auxotrophy could be suppressed with high levels of biotin in the medium. After transformation of this mutant with a yeast genomic library, the unassigned open reading frame YGR065C was identified to complement this mutation. This gene codes for a protein with 593 amino acids and 12 putative transmembrane helices. Northern blot analysis revealed that, in wild-type cells, the corresponding mRNA levels were increased at low biotin concentrations. Likewise, cellular biotin uptake was increased with decreasing biotin availability. Expression of YGR065C under the control of the constitutive ADH1 promoter resulted in very high biotin transport rates across the plasma membrane that were no longer regulated by the biotin concentration in the growth medium. We conclude that YGR065C encodes the first biotin transporter identified for a non-mammalian organism and designate this gene VHT1 for vitamin H transporter 1.     

Caulobacter cresentus mutant defective in membrane phospholipid synthesis.

To study the relationship between phospholipid synthesis and organelle biogenesis in the dimorphic bacterium Caulobacter crescentus, auxotrophs have been isolated which require exogenous glycerol or glycerol 3-phosphate for growth when glucose is used as the carbon source. Upon glycerol deprivation, net phospholipid synthesis ceased immediately in a glycerol 3-phosphate auxotroph which was shown to have levels of biosynthetic sn-glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) activity 10 times lower than that of the wild type. In the absence of glycerol, the optical density of the culture continued to increase for the equivalent of one generation, although the cells did not divide. After the equivalent of one generation time, rapid cell death occurred. Cell death also occurred when phospholipid synthesis was inhibited by cerulenin. Although ribonucleic acid and protein syntheses continued at a reduced rate for the equivalent of one generation in mutant strains, a substantial decrease in the rate of deoxyribonucleic acid synthesis occurred immediately upon glycerol deprivation. Revertant strains had wild-type levels of glycerol 3-phosphate dehydrogenase activity and normal rates of phospholipid and macromolecular synthesis.     

Membranes of Rhodopseudomonas sphaeroides: effect of cerulenin on assembly of chromatophore membrane.

The effects of cerulenin were investigated in Rhodopseudomonas sphaeroides to elucidate further the mechanisms controlling the assembly of the chromatophore membrane. When this potent inhibitor of fatty acid biosynthesis was added to photosynthetically grown cultures, there was an immediate cessation of phospholipid, bacteriochlorophyll a, carotenoid, and ubiquinone formation. Concurrently, there was also a marked decrease in the rate of incorporation of protein into the chromatophore membrane. In contrast, only a small decrease in the rate of soluble and cell envelope protein synthesis was observed and, in chemotrophically grown cells, protein continued to be incorporated into both the cytoplasmic and outer membranes. The removal of delta-aminolaevulinate from mutant H-5 of R. sphaeroides, which requires this porphyrin precursor, was reexamined to determine whether cerulenin-induced cessation of chromatophore protein incorporation was due solely to blocked bacteriochlorophyll a synthesis. In the deprived H-5 cells, inhibition of [35S]methionine incorporation into chromatophores was confined mainly to apoproteins of bacteriochlorophyll a complexes. Other minor chromatophore proteins continued to be inserted to a greater extent than in cerulenin-treated wild type where phospholipid synthesis has also ceased. These results indicated that the assembly of the chromatophore membrane is under strict regulatory control involving concomitant phospholipid, pigment, and protein syntheses.     

Fatty acid degradation in Caulobacter crescentus.

Fatty acid degradation was investigated in Caulobacter crescentus, a bacterium that exhibits membrane-mediated differentiation events. Two strains of C. crescentus were shown to utilize oleic acid as sole carbon source. Five enzymes of the fatty acid beta-oxidation pathway, acyl-coenzyme A (CoA) synthase, crotonase, thiolase, beta-hydroxyacyl-CoA dehydrogenase, and acyl-CoA dehydrogenase, were identified. The activities of these enzymes were significantly higher in C. crescentus than the fully induced levels observed in Escherichia coli. Growth in glucose or glucose plus oleic acid decreased fatty acid uptake and lowered the specific activity of the enzymes involved in beta-oxidation by 2- to 3-fold, in contrast to the 50-fold glucose repression found in E. coli. The mild glucose repression of the acyl-CoA synthase was reversed by exogenous dibutyryl cyclic AMP. Acyl-CoA synthase activity was shown to be the same in oleic acid-grown cells and in cells grown in the presence of succinate, a carbon source not affected by catabolite repression. Thus, fatty acid degradation by the beta-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. Tn5 insertion mutants unable to form colonies when oleic acid was the sole carbon source were isolated. However, these mutants efficiently transported fatty acids and had beta-oxidation enzyme levels comparable with that of the wild type. Our inability to obtain fatty acid degradation mutants after a wide search, coupled with the high constitutive levels of the beta-oxidation enzymes, suggest that fatty acid turnover, as has proven to be the case fatty acid biosynthesis, might play an essential role in membrane biogenesis and cell cycle events in C. crescentus.     

Regulatory interactions between phospholipid synthesis and DNA replication in Caulobacter crescentus.

Several Caulobacter crescentus mutants with lesions in phospholipid biosynthesis have DNA replication phenotypes. A C. crescentus mutant deficient in glycerol 3-phosphate dehydrogenase activity (gpsA) blocks phospholipid synthesis, ceases DNA replication, and loses viability in the absence of a glycerol phosphate supplement. To investigate the interaction between membrane synthesis and DNA replication during a single cell cycle, we moved the gpsA mutation into a synchronizable, but otherwise wild-type, strain. The first effect of withholding supplement was the cessation of synthesis of phosphatidylglycerol, a major component of the C. crescentus membrane. In the absence of glycerol 3-phosphate, DNA replication was initiated in the stalked cell at the correct time in the cell cycle and at the correct site on the chromosome. However, after replication proceeded bidirectionally for a short time, DNA synthesis dropped to a low level. The cell cycle blocked at a distinct middivision stalked cell, and this was followed by cell death. The "glycerol-less" death of the gpsA mutant could be prevented if the cells were treated with novobiocin to prevent the initiation of DNA replication. Our observations suggest that the processivity of C. crescentus replication requires concomitant phospholipid synthesis and that cell death results from incomplete replication of the chromosome.     

Arachidonic acid stimulates glucose uptake in 3T3-L1 adipocytes by increasing GLUT1 and GLUT4 levels at the plasma membrane. Evidence for involvement of lipoxygenase metabolites and peroxisome proliferator-activated receptor gamma

Exposure of insulin-sensitive tissues to free fatty acids can impair glucose disposal through inhibition of carbohydrate oxidation and glucose transport. However, certain fatty acids and their derivatives can also act as endogenous ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor that positively modulates insulin sensitivity. To clarify the effects of externally delivered fatty acids on glucose uptake in an insulin-responsive cell type, we systematically examined the effects of a range of fatty acids on glucose uptake in 3T3-L1 adipocytes. Of the fatty acids examined, arachidonic acid (AA) had the greatest positive effects, significantly increasing basal and insulin-stimulated glucose uptake by 1.8- and 2-fold, respectively, with effects being maximal at 4 h at which time membrane phospholipid content of AA was markedly increased. The effects of AA were sensitive to the inhibition of protein synthesis but were unrelated to changes in membrane fluidity. AA had no effect on total cellular levels of glucose transporters, but significantly increased levels of GLUT1 and GLUT4 at the plasma membrane. While the effects of AA were insensitive to cyclooxygenase inhibition, the lipoxygenase inhibitor, nordihydroguaiaretic acid, substantially blocked the AA effect on basal glucose uptake. Furthermore, adenoviral expression of a dominant-negative PPARgamma mutant attenuated the AA potentiation of basal glucose uptake. Thus, AA potentiates basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes by a cyclooxygenase-independent mechanism that increases the levels of both GLUT1 and GLUT4 at the plasma membrane. These effects are at least partly dependent on de novo protein synthesis, an intact lipoxygenase pathway and the activation of PPARgamma with these pathways having a greater role in the absence than in the presence of insulin.     

l-Glutamic Acid Fermentation

It is well known that biotin has a marked effect on l-glutamic acid fermentation.

The authors have intended to find strains which are independent of the amounts of biotin in the culture medium. As a result, oleic acid-requiring mutants were obtained from a strain of Brevibacterium thiogenitalis which is an auxotroph for biotin. The growth of the mutant was remarkably stimulated by Tween 20, 40, 60, Ca ions and a small amount of corn steep liquor. And also, the mutant was found to have lost its requirement for biotin and showed growth response only to oleic acid or unsaturated fatty acids.

The effect of biotin, oleic acid and other unsaturated fatty acids on the production of l-glutamic acid was investigated by using an oleic acid-requiring mutant of Brevibacterium thiogenitalis No. 653. The results described in the present paper showed that the oleic acid-requiring mutant D-248 produced a large amount of l-glutamic acid in the excess biotin-contaming media, and that oleic acid seemed to be completely replaced by other unsaturated fatty acids such as palmitoleic acid and linoleic acid.     

Outer Membrane Changes in a Toluene-Sensitive Mutant of Toluene-Tolerant Pseudomonas putida IH-2000

We isolated a toluene-sensitive mutant, named mutant No. 32, which showed unchanged antibiotic resistance levels, from toluene-tolerant Pseudomonas putida IH-2000 by transposon mutagenesis with Tn5. The gene disrupted by insertion of Tn5 was identified as cyoC, which is one of the subunits of cytochrome o. The membrane protein, phospholipid, and lipopolysaccharide (LPS) of IH-2000 and that of mutant No. 32 were examined and compared. Some of the outer membrane proteins showed a decrease in mutant No. 32. The fatty acid components of LPS were found to be dodecanoic acid, 2-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, and 3-hydroxydecanoic acid in both IH-2000 and No. 32; however, the relative proportions of these components differed in the two strains. Furthermore, cell surface hydrophobicity was increased in No. 32. These data suggest that mutation of cyoC caused the decrease in outer membrane proteins and the changing fatty acid composition of LPS. These changes in the outer membrane would cause an increase in cell surface hydrophobicity, and mutant No. 32 is considered to be sensitive to toluene.     

Synthesis and utilization of fatty acids by wild-type and fatty acid auxotrophs of Caulobacter crescentus.

The fatty acid composition of the dimorphic bacterium Caulobacter crescentus was found to consist primarily of 16- and 18-carbon fatty acids, both saturated and monounsaturated, in agreement with the findings of Chow and Schmidt (J. Gen. Microbiol. 83:359-373, 1974). In addition, two minor but as yet unidentified fatty acids were detected. Chromatographic mobilities suggested that these fatty acids may be a cyclopropane and a branched-chain fatty acid. In addition, we demonstrated that the fatty acid composition of wild-type C. crescentus can be altered by growing the cells in medium supplemented with any one of a variety of unsaturated fatty acids. Linoleic acid, a diunsaturated fatty acid which is not synthesized by C. crescentus, was incorporated into phospholipids without apparent modification. In addition, we found that C. crescentus, like Escherichia coli, synthesizes vaccenic acid (18:1 delta 11,cis) rather than oleic acid (18:1 delta 9,cis). This result allowed us to deduce that the mechanism of fatty acid desaturation in C. crescentus is anaerobic, as it is in E. coli. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. Neither of these mutants resembled the E. coli unsaturated fatty acid auxotrophs, which have defined enzymatic lesions in fatty acid biosynthesis. Rather, the mutants appeared to have defects relating to the complex coordination of membrane biogenesis and cell cycle events in C. crescentus.     

RENEWAL OF FATTY ACIDS IN THE MEMBRANES OF VISUAL CELL OUTER SEGMENTS

The renewal of fatty acids in the visual cells and pigment epithelium of the frog retina was studied by autoradiographic analysis of animals injected with tritiated palmitic, stearic, or arachidonic acids. Most of the radioactive material could be extracted from the retina with chloroform-methanol, indicating that the fatty acids had been esterified in lipids. Analysis of the extracts, after injection of [³H]palmitic acid, revealed that the radioactivity was predominantly in phospholipid. Palmitic acid was initially concentrated in the pigment epithelium, particularly in oil droplets which are storage sites for vitamin A esterified with fatty acid. The cytoplasm, but not the nucleus of these cells, was also heavily labeled. Radioactive fatty acid was bound immediately to the visual cell outer segment membranes, including detached rod membranes which had been phagocytized by the pigment epithelium. This is believed to be due to fatty acid exchange in phospholipid molecules already situated in the membranes. Gradually, the concentration of radioactive material in the visual cell outer segment membranes increased, apparently as a result of the addition of new phospholipid molecules, possibly augmented by the transfer from the pigment epithelium of esterified vitamin A. Injected fatty acid became particularly concentrated in new membranes which are continually assembled at the base of rod outer segments. This localized concentration was short-lived, apparently due to the rapid renewal of fatty acid. The results support the conclusion that rods renew the lipids of their outer segments by membrane replacement, whereas both rods and cones renew the membrane lipids by molecular replacement, including fatty acid exchange and replacement of phospholipid molecules in existing membranes.     

The outer membrane of Proteus mirabilis. II. The extractable lipid fraction and electron-paramagnetic resonance analysis of the outer and cytoplasmic membranes.

1. The lipid fraction extracted from the outer and cytoplasmic membranes of Proteus mirabilis with chloroform/methanol consisted almost entirely of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. 2. The phospholipid content of the cytoplasmic membrane was more than twice that of the outer membrane (38% as against 18% of the total dry weight) and the proportions of the three phospholipids differed somewhat in the two membranes. Yet, the fatty acid composition of the extractable lipids was essentially the same in both membranes. 3. The freedom of motion of spin-labeled fatty acids in the outer membrane of P. mirabilis depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe is an associate lipid structure with the properties of a bilayer. Nevertheless, the mobility of the probe was more restricted in the outer membrane than in the cytoplasmic membrane, indicating a higher viscosity of the outer membrane. 4. Chloroform/methanol completely removed the phospholipids from the outer membrane, leaving the lipopolysaccharide moiety intact. The motion of spin-labeled fatty acids in the extracted membranes was, however, highly restricted, suggesting that, in the native outer membrane, the local environment of the probe is composed of phospholipids rather than lipopolysaccharide. Aqueous acetone extraction removed only 75-80% of the phospholipids of the outer membrane. Nevertheless, the mobility of the spin-labeled fatty acid remained highly restricted, suggesting the existence of two phospholipid environments in the outer membrane differing in the nature of their association with the lipopolysaccharide and protein moieties.     

The biosynthetic incorporation of short-chain linear saturated fatty acids by Acholeplasma laidlawii B may suppress cell growth by perturbing membrane lipid polar headgroup distribution

Acholeplasma laidlawii B cells made fatty acid auxotrophic by growth in the presence of the biotin-binding agent avidin grow increasingly poorly at 37 degrees C when supplemented with single exogenous linear saturated fatty acids of decreasing hydrocarbon chain length. Interestingly, this progressive decrease in growth yields with decreasing hydrocarbon chain length is not observed when cells are cultured in the presence of other classes of exogenous fatty acids. Moreover, normal growth is observed is other types of fatty acids with equivalent or shorter hydrocarbon chain lengths, indicating that poor growth in the presence of short-chain linear saturated fatty acids cannot be due to a decrease in membrane lipid bilayer thickness per se. To understand the molecular basis of such growth inhibition, we determined the growth yields, membrane lipid fatty acid and polar headgroups compositions, and phase state and fluidity of the membrane lipids in cells progressively biosynthetically enriched in tridecanoic acid (13:0) or dodecanoic acid (12:0). The growth of fatty acid auxotrophic A. laidlawii B cells grown in the presence of binary combinations of an exogenous fatty acid which supports normal growth on its own and 13:0 or 12:0 revealed that growth inhibition is not observed until 13:0 and 12:0 biosynthetic incorporation levels reach about 90 and 60 mol %, respectively, after which growth is markedly inhibited. Differential scanning calorimetric analyses of membranes from cells maximally enriched in 13:0 indicate that the lipid gel/liquid-crystalline phase transition temperature is unexpectedly high but that at the growth temperature of 37 degrees C, the membrane lipid bilayer is almost exclusively in the liquid-crystalline state but is certainly not excessively fluid. However, high levels of 13:0 incorporation produce a greatly elevated level of the high melting, reversed nonlamellar phase-preferring lipid component monoglucosyl diacylglycerol, and greatly reduced levels of all other membrane lipid components. This marked elevation of monoglucosyl diacylglycerol levels can be rationalized as a regulatory response which maintains the lamellar/nonlamellar phase-forming propensity of the total membrane lipid mixture relatively constant in the face of the biosynthetic incorporation of increasing quantities of short-chain saturated fatty acids, which favor the lamellar phase. However, this lipid biosynthetic response produces a marked decline in the levels of anionic phospholipid and phosphoglycolipid which are probably required to maintain the minimal negative surface charge density of the lipid bilayer, which we suggest is responsible for the observed growth inhibition. This work shows that the lipid biosynthetic regulatory mechanisms present in this organism may sometimes operate at cross purposes such that it is not possible to simultaneously optimize all of the biologically relevant physical properties of the membrane lipid bilayer.     

Preparation and chemical properties of the outer membrane of a moderately halophilic gram-negative bacterium.

Outer membranes, almost free from peptidoglycan components, were prepared from a moderately halophilic gram-negative bacterium grown in a medium containing 2 M NaCl. The outer membrane was easily released, leaving mureinoplasts, by mild desalting in a 20% sucrose solution containing 50 mM tris(hydroxymethyl)aminomethane-HCl buffer, pH 7.8. The membrane was recovered by treatment with DNase I and CsCl buoyant density centrifugation. Chemical analyses revealed that the outer membrane was mainly composed of 31% protein, about 20% extractable lipids (mainly phospholipids), and lipopolysaccharides. The proteins had about 18 mol % excess of acidic over basic amino acids. The phospholipids comprised phosphatidyl ethanolamine, phosphatidyl glycerol, cardiolipin, and an unidentified phospholipid containing glucose, which seemed mainly associated with the outer membrane. The content of lipopolysaccharides in the outer membrane was calculated arbitrarily as 30% from the heptose content. A unique feature of these lipopolysaccharides seemed to be higher lipid content than found in lipopolysaccharides of other gram-negative bacteria. The major fatty acids of bound lipids of the outer membrane resembled those of the lipopolysaccharides obtained from cell envelope preparation and contained high concentrations of 3-hydroxy lauric acid.     

Inhibition of Candida parapsilosis Fatty Acid Synthase (Fas2) Induces Mitochondrial Cell Death in Serum

We have recently observed that a fatty acid auxotrophic mutant (fatty acid synthase, Fas2Δ/Δ) of the emerging human pathogenic yeast Candida parapsilosis dies after incubation in various media including serum. In the present study we describe the mechanism for cell death induced by serum and glucose containing media. We show that Fas2Δ/Δ yeast cells are profoundly susceptible to glucose leading us to propose that yeast cells lacking fatty acids exhibit uncontrolled metabolism in response to glucose. We demonstrate that incubation of Fas2Δ/Δ yeast cells with serum leads to cell death, and this process can be prevented with inhibition of protein or DNA synthesis, indicating that newly synthesized cellular components are detrimental to the mutant cells. Furthermore, we have found that cell death is mediated by mitochondria. Suppression of electron transport enzymes using inhibitors such as cyanide or azide prevents ROS overproduction and Fas2Δ/Δ yeast cell death. Additionally, deletion of mitochondrial DNA, which encodes several subunits for enzymes of the electron transport chain, significantly reduces serum-induced Fas2Δ/Δ yeast cell death. Therefore, our results show that serum and glucose media induce Fas2Δ/Δ yeast cell death by triggering unbalanced metabolism, which is regulated by mitochondria. To our knowledge, this is the first study to critically define a link between cytosolic fatty acid synthesis and mitochondrial function in response to serum stress in C. parapsilosis.     

Fatty-acid biosynthesis in a branched-chain alpha-keto acid dehydrogenase mutant of Streptomyces avermitilis

Fatty-acid biosynthesis by a branched-chain alpha-keto acid dehydrogenase (bkd) mutant of Streptomyces avermitilis was analyzed. This mutant is unable to produce the appropriate precursors of branched-chain fatty acid (BCFA) biosynthesis, but unlike the comparable Bacillus subtilis mutant, was shown not to have an obligate growth requirement for these precursors. The bkd mutant produced only straight-chain fatty acids (SCFAs) with membrane fluidity provided entirely by unsaturated fatty acids (UFAs), the levels of which increased dramatically compared to the wild-type strain. The levels of UFAs increased in both the wild-type and bkd mutant strains as the growth temperature was lowered from 37 degrees C to 24 degrees C, suggesting that a regulatory mechanism exists to alter the proportion of UFAs in response either to a loss of BCFA biosynthesis, or a decreased growth temperature. No evidence of a regulatory mechanism for BCFAs was observed, as the types of these fatty acids, which contribute significantly to membrane fluidity, did not alter when the wild-type S. avermitilis was grown at different temperatures. The principal UFA produced by S. avermitilis was shown to be delta 9-hexadecenoate, the same fatty acid produced by Escherichia coli. This observation, and the inability of S. avermitilis to convert exogenous labeled palmitate to the corresponding UFA, was shown to be consistent with an anaerobic pathway for UFA biosynthesis. Incorporation studies with the S. avermitilis bkd mutant demonstrated that the fatty acid synthase has a remarkably broad substrate specificity and is able to process a wide range of exogenous branched chain carboxylic acids into unusual BCFAs.     

Effects of Oleate Starvation in a Fatty Acid Auxotroph of Escherichia coli K-12

The effects of oleate starvation on an oleate auxotroph of Escherichia coli K-12 were investigated. Following removal of oleate from the mutant growing in a minimal glycerol-peptone medium, the cells stopped making deoxyribonucleic acid, ribonucleic acid, protein, and phospholipids; they began to die exponentially and finally lysed. During oleate starvation in minimal medium minus peptone, inhibition of macromolecular syntheses and death occurred; however, lysis did not follow. When growth ceased, no further dying was observed. It is shown that none of the early effects (inhibition of macromolecular syntheses and death) can be due to leakiness of the cells, induction of a prophage or a colicin, or lack of energy sources. The cause of inhibition of macromolecular syntheses remained unknown. Since the rate of death was the same as the generation time under different conditions, it appears that death is due to the defective synthesis of some cellular structure (quite possibly, cytoplasmic membrane) during phospholipid deficiency. Lysis was found to require protein synthesis; electron microscopy revealed a peculiar type of "lysis from within"; i.e., the shape of the cells did not change but fragmentation of the inner layer of the cell envelope occurred. The murein was found to be unaltered. Most likely, lysis was a consequence of the cell's attempt to synthesize cytoplasmic membrane with altered phospholipid composition or during phospholipid deficiency. Several membrane functions (respiration, adenosine triphosphate formation, permeability) existing before oleate removal were not lost during starvation. Therefore, general damage to the membrane did not occur, and it could be that most, if not all, described effects were due to defective de novo membrane synthesis.     

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris

Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.     

Monounsaturated but not polyunsaturated fatty acids are required for growth of the deep-sea bacterium Photobacterium profundum SS9 at high pressure and low temperature

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by P. profundum SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.