A suppressor of fab1 challenges hypotheses on the role of thylakoid unsaturation in photosynthetic function

Leaf membrane lipids of the Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis 1 (fab1) mutant contain a 35% to 40% increase in the predominant saturated fatty acid 16:0, relative to wild type. This increase in membrane saturation is associated with loss of photosynthetic function and death of mutant plants at low temperatures. We have initiated a suppressor screen for mutations that allow survival of fab1 plants at 2 degrees C. Five suppressor mutants identified in this screen all rescued the collapse of photosynthetic function observed in fab1 plants. While fab1 plants died after 5 to 7 weeks at 2 degrees C, the suppressors remained viable after 16 weeks in the cold, as judged by their ability to resume growth following a return to 22 degrees C and to subsequently produce viable seed. Three of the suppressors had changes in leaf fatty acid composition when compared to fab1, indicating that one mechanism of suppression may involve compensating changes in thylakoid lipid composition. Surprisingly, the suppressor phenotype in one line, S31, was associated with a further substantial increase in lipid saturation. The overall leaf fatty acid composition of S31 plants contained 31% 16:0 compared with 23% in fab1 and 17% in wild type. Biochemical and genetic analysis showed that S31 plants contain a new allele of fatty acid desaturation 5 (fad5), fad5-2, and are therefore partially deficient in activity of the chloroplast 16:0 Delta7 desaturase. A double mutant produced by crossing fab1 to the original fad5-1 allele also remained alive at 2 degrees C, indicating that the fad5-2 mutation is the suppressor in the S31 (fab1 fad5-2) line. Based on the biophysical characteristics of saturated and unsaturated fatty acids, the increased 16:0 in fab1 fad5-2 plants would be expected to exacerbate, rather than ameliorate, low-temperature damage. We propose instead that a change in shape of the major thylakoid lipid, monogalactosyldiacylglycerol, mediated by the fad5-2 mutation, may compensate for changes in lipid structure resulting from the original fab1 mutation. Our identification of mutants that suppress the low-temperature phenotype of fab1 provides new tools to understand the relationship between thylakoid lipid structure and photosynthetic function.     

Photoinhibition in mutants of Arabidopsis deficient in thylakoid unsaturation

Thylakoid lipid composition in higher plants is characterized by a high level of fatty acid unsaturation. We have screened four mutants of Arabidopsis that have reduced levels of fatty acid unsaturation. Three of the mutant lines tested, fad5, fad6, and the fad3-2 fad7-2 fad8 triple mutant, were more susceptible to photoinhibition than wild-type Arabidopsis, whereas one mutant, fab1, was indistinguishable from wild type. The fad3-2 fad7-2 fad8 triple mutant, which contains no trienoic fatty acids in its thylakoid membranes, was most susceptible to photoinhibition. Detailed investigation of photoinhibition in the triple mutant revealed that the rate of photoinactivation of PSII was the same in wild-type and mutant plants. However, the recovery of photoinactivated PSII was slower in fad3-2 fad7-2 fad8, relative to wild type, at all temperatures below 27 degrees C. These results indicate that trienoic fatty acids of thylakoid membrane lipids are required for low-temperature recovery from photoinhibition in Arabidopsis.     

A Mutant of Arabidopsis Deficient in the Elongation of Palmitic Acid

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by an increased level of 16:0 and a concomitant decrease of 18-carbon fatty acids as a consequence of a single recessive nuclear mutation at the fab1 locus. Quantitative analysis of the fatty acid composition of individual lipids established that lipids synthesized by both the prokaryotic and eukaryotic pathways were affected by the mutation. Direct enzyme assays demonstrated that the mutant plants were deficient in the activity of 3-ketoacyl-acyl carrier protein synthase II; therefore, it is inferred that fab1 may encode this enzyme. Labeling experiments with [14C]acetate and lipase positional analysis indicated that the mutation results in a small shift in the partitioning of lipid synthesis between the prokaryotic and eukaryotic pathways. Synthesis of chloroplast lipids by the prokaryotic pathway was increased with a corresponding reduction in the eukaryotic pathway.     

A Mutant of Arabidopsis with Increased Levels of Stearic Acid

A mutation at the fab2 locus of Arabidopsis caused increased levels of stearate in leaves. The increase in leaf stearate in fab2 varied developmentally, and the largest increase occurred in young leaves, where stearate accounted for almost 20% of total leaf fatty acids. The fatty acid composition of leaf lipids isolated from the fab2 mutant showed increased stearate in all the major glycerolipids of both the chloroplast and extrachloroplast membranes. Although the stearate content was increased, the fab2 mutant still contained abundant amounts of 18:1, 18:2, and 18:3 fatty acids. These results are consistent with the expectations for a mutation partially affecting the action of the stromal stearoyl-acyl carrier protein desaturase. Positional analysis indicated that the extra 18:0 is excluded with high specificity from the sn-2 position of both chloroplast and extrachloroplast glycerolipids. Although stearate content was increased in all the major leaf membrane lipids, the amount of increase varied considerably among the different lipids, from a high of 25% of fatty acids in phosphatidylcholine to a low of 2.9% of fatty acids in monogalactosyldiacylglycerol.     

The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function

The peroxisome, sole site of beta-oxidation in Saccharomyces cerevisiae, is known to be required for optimal growth in the presence of fatty acid. Screening of the haploid yeast deletion collection identified approximately 130 genes, 23 encoding peroxisomal proteins, necessary for normal growth on oleic acid. Oleate slightly enhances growth of wild-type yeast and inhibits growth of all strains identified by the screen. Nonperoxisomal processes, among them chromatin modification by H2AZ, Pol II mediator function, and cell-wall-associated activities, also prevent oleate toxicity. The most oleate-inhibited strains lack Sap190, a putative adaptor for the PP2A-type protein phosphatase Sit4 (which is also required for normal growth on oleate) and Ilm1, a protein of unknown function. Palmitoleate, the other main unsaturated fatty acid of Saccharomyces, fails to inhibit growth of the sap190delta, sit4delta, and ilm1delta strains. Data that suggest that oleate inhibition of the growth of a peroxisomal mutant is due to an increase in plasma membrane porosity are presented. We propose that yeast deficient in peroxisomal and other functions are sensitive to oleate perhaps because of an inability to effectively control the fatty acid composition of membrane phospholipids.     

Role of the Bacillus subtilis fatty acid desaturase in membrane adaptation during cold shock

In our attempt to understand the cold shock response of Bacillus subtilis, we report on the role of the B. subtilis fatty acid desaturase (FA-D) Des during membrane adaptation to low temperatures and demonstrate its importance during cold shock. A des null mutant was constructed and analysed in comparison with its parental strain. Growth studies and large-scale comparative fatty acid (FA) analysis revealed a severe cold-sensitive phenotype of the des deletion mutant during the absence of isoleucine and showed that four unsaturated fatty acid (UFA) species differing in length, branching pattern and position of the double bond are synthesized in B. subtilis JH642 but not in the des null mutant. Apart from the lack of UFA synthesis, the FA-D deletion strain showed a dramatically altered saturated fatty acid (SFA) profile at the onset of the stationary growth phase in the presence of exogenous isoleucine sources. Expression of des integrated in trans at the amyE locus of the des deletion strain not only cured the cold-sensitive phenotype observed for the des mutant but allowed much better growth than in strain JH642 after a shift from 37 degrees C to 15 degrees C. These results show that, during cold shock adaptation, des expression can completely replace the isoleucine-dependent, long-term, FA branching adaptation mechanism. We conclude that the crucial aspect in cold adaptation of the cytoplasmic membrane is not its specific molecular composition but rather its physical status in terms of its fluidity.     

Alterations in normal fatty acid composition in a temperature-sensitive mutant of a thermophilic bacillus

A temperature-sensitive mutant of a thermophilic bacillus was isolated which was unable to maintain membrane integrity at high temperature. The mutant appeared to lose cytoplasmic contents, as indicated by a decrease in turbidity and cell refractivity, when shifted from a permissive (52° C) to a restrictive (65° C) temperature. Cell number remained fairly constant, however. At the approximate onset of the decline in turbidity, viability decreased and net synthesis of ribonucleic acid, deoxyribonucleic acid, and protein ceased. Both chloramphenicol and sucrose were effective in retarding the decline in turbidity at 65° C. An abnormal fatty acid composition at high temperature suggested that the lesion in the mutant involved lipid synthesis. The proportion of fatty acids with a high melting point (> 55° C) increased in the parent from 42% at 42° C to 69% at 65° C. Similar changes were not made by the mutant. An abnormal phospholipid composition was also observed in the mutant at 42° C and 52° C. However, at 58° C, the maximum growth temperature of the mutant, the proportion of major phospholipids (phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin) was similar to the parent strain. The mutant's apparent loss of membrane stability at high temperature and its inability to regulate fatty acid and phospholipid composition in a normal manner suggested that (i) the temperature-sensitivity of the mutant may be a result of a defect in normal lipid metabolism at high temperature and (ii) the normal changes in fatty acid composition observed at increased growth temperatures may be an essential feature of thermophily.A preliminary report of this work was presented at the 73rd Annual Meeting of the American Society for Microbiology, Miami Beach, Florida, May 6–11, 1973.     

Altered acyltransferase activity in Escherichia coli associated with mutations in acyl coenzyme A synthetase

Growth of a temperature-sensitive general fatty acid synthesis mutant of Escherichia coli K12 at its restrictive temperature in the presence of exogenous palmitate results in lysis of the bacterium. Under these conditions, palmitate is incorporated into membrane phospholipid to a high level. Mutants of bacteria restricting this incorporation (having a palmitate-resistant phenotype) have been isolated and one such mutant, strain L8-2/3, has been further characterized. This mutant has lowered acyl-CoA synthetase (fadD) activity (25-33% of normal) and consequently is defective in fatty acid uptake. This lowered uptake could explain the palmitate-resistant phenotype of strain L8-2/3. However, both in vivo (fatty acid composition and positional distribution data) and in vitro (acyltransferase activity measurements) experiments suggest that this mutant is also altered in its acyltransferase activities. The mutation(s) of strain L8-2/3 appears to allow increased (approximately 2-fold) incorporation of myristate (and possible unsaturated fatty acids) into position 2 of 1-acyl-sn-glycerol 3-phosphate but normal palmitate incorporation into the same position. The incorporation of palmitate, myristate, and oleate into position 1 of sn-glycerol 3-phosphate by strain L8-2/3 is also higher than that observed with the parent, strain L8-2. Replacing the partially defective fadD gene of strain L8-2/3 with a wild type allele conferred on this strain the palmitate sensitivity and the acyltransferase activity of the parent strain L8-2. This finding, taken together with other data, suggests that acyl-CoA synthetase interacts with the acyltransferase(s) in some manner to influence the fatty acid specificity of the acyltransferase.     

Composition,structure and phase transition in yeast fatty acid auxotroph membranes: Spin labels and freeze-fracture

• 1 Fatty acid desaturase mutant yeast cells have been enriched with 2 fatty acids having substantially different physical properties.
• 2 Differences in fatty acid composition are reflected in the ESR determined phase transitions and the freeze-fracture morphology of the tonoplast.
• 3 In these cells, fatty acid composition, rather than the position of the phase transition relative to growth temperature, appears to be the more important variable in determining membrane morphology.
• 4 The freezing process used in the freeze-fracture technique does not appear to cause a demixing of the lipid components of the membrane.

     

Lipid and protein composition and thermotropic lipid phase transitions in fatty acid-homogeneous membranes of Acholeplasma laidlawii B

The membrane composition and lipid physical properties have been systematically investigated as a function of fatty acid composition for a series of Acholeplasma laidlawii B membrane preparations made homogeneous in various fatty acids by growing cells on single fatty acids and avidin, a potent fatty acid synthetic inhibitor. The membrane protein molecular weight distribution is essentially constant as a function of fatty acid composition, but the lipid/protein ratio varies over a 2-fold range when different fatty acid growth supplements are used. The membrane lipid head-group composition varies somewhat under these conditions, particularly in the ratio of the two major neutral glycolipids. Differential thermal analytical investigations of the thermotropic phase transitions of various combinations of membrane components suggest that these compositional changes are unlikely to result in qualitative changes in the nature of lipid-protein or lipid-lipid interactions, although lesser changes of a quantitative nature probably do occur. The total lipids of membranes made homogeneous in their lipid fatty acyl chain composition exhibit sharper than normal gel-to-liquid-crystalline phase transitions of which midpoint temperatures correlate very well with the phase transition temperatures of synthetic hydrated phosphatidylcholines with like acyl chains. Our results indicate that using avidin and suitable fatty acids to grow A. laidlawii B, it is possible to manipulate the position and the sharpness of the membrane lipid phase transition widely and independently without causing major modifications in other aspects of the membrane composition. This fact makes the fatty acid-homogeneous A. laidlawii B membrane a very useful biological membrane preparation in which to study lipid physical properties and their functional consequences.     

A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

The fab1 mutant of Arabidopsis is partially deficient in activity of beta-ketoacyl-[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16:0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a cDNA from Brassica napus encoding a KAS II enzyme resulted in complementation of both mutant phenotypes. The dual complementation by expression of the single gene proves that low-temperature damage is a consequence of altered membrane unsaturation. The fab1 mutation is a single nucleotide change in Arabidopsis KAS2 that results in a Leu337Phe substitution. The Leu337 residue is conserved among plant and bacterial KAS proteins, and in the crystal structures of E. coli KAS I and KAS II, this leucine abuts a phenylalanine whose imidazole ring extends into the substrate binding cavity causing the fatty acid chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size-exclusion chromatography and was severely impaired in condensation activity. These results suggest that the molecular defect in fab1 plants is a structural instability of the KAS2 gene product induced by insufficient space for the imidazole ring of the mutant phenylalanine residue.     

Novel mutations affecting leaf stearate content and plant size in Arabidopsis

 The fab2-1 mutant of Arabidopsis is an extreme dwarf as a direct result of an increase in the levels of stearate (18 : 0) in membrane lipids. We isolated a series of lines in which second-site suppressor mutations partly alleviate the dwarf phenotype. In all four of the suppressor lines examined, restoration of more normal morphology is accompanied by decreases in leaf 18 : 0 content. Three of the isolated suppressors suppress the high stearate phenotype in both leaves and seeds. The effects of one of the suppressors, TW2-1, is limited to the leaves. A second allele at the fab2 locus, fab2-2, was also identified and plants homozygous for this allele where intermediate in both plant size and 18 : 0 content between wild-type Arabidopsis and fab2-1 mutants. The alleles at fab2 and the suppressor mutations provided a total of nine genotypes which were analyzed to demonstrate a clear-cut relationship between leaf 18 : 0 content (0.7–19.6% of total leaf fatty acids) and reductions in plant size (24–4 mm). These results illustrate the utility of suppressor analysis for addressing problems in biochemistry and plant biology. They also indicate that the genetic control of plant lipid composition is more complex than previously appreciated. Received: 24 January 1997 / Accepted: 14 February 1997     

Mutant of Escherichia coli Deficient in the Synthesis of cis-Vaccenic Acid

A temperature-sensitive unsaturated fatty acid (fabA) auxotroph of Escherichia coli was found also to be deficient in the elongation of palmitoleic acid to cis-vaccenic acid. Reversion and transductional analyses demonstrate that this second phenotype and the fabA mutation are independent in action and are not cotransduced. The deficiency in conversion of palmitoleic acid to cis-vaccenic acid was also demonstrated in vitro, and these results strongly suggest this phenotype is due to a deficiency in an elongation enzyme. We suggest that the phenotype may have been selected during growth because it can physiologically compensate for the fabA lesion. In fab(+) strains, the inability to synthesize cis-vaccenic acid is physiologically asymptomatic. Such strains grow normally at all temperatures tested and are not sodium sensitive. Although the parental strain has an increased amount of cis-vaccenic acid in cells grown at 15 C, the mutant does not. Since the mutant grows normally at 15 C, the data indicate that increased amounts of cis-vaccenic acid are not required for growth at 15 C.     

Cyanobacterial monogalactosyldiacylglycerol-synthesis pathway is involved in normal unsaturation of galactolipids and low-temperature adaptation of Synechocystis sp. PCC 6803

We characterized certain physiological functions of cyanobacterial monoglucosyldiacylglycerol using a Synechocystis sp. PCC 6803 mutant in which the gene for monoglucosyldiacylglycerol synthase had been disrupted and its function complemented by inclusion of an Arabidopsis monogalactosyldiacylglycerol synthase gene. By using this method, we prepared the first viable monoglucosyldiacylglycerol-deficient mutant of cyanobacterium and found that monoglucosyldiacylglycerol is not essential for its growth and photosynthesis under a set of “normal growth conditions” when monogalactosyldiacylglycerol is adequately supplied by the Arabidopsis monogalactosyldiacylglycerol synthase. The mutant had healthy thylakoid membranes and normal pigment content. The membrane lipid composition of the mutant was similar with that of WT except lack of monoglucosyldiacylglycerol and a slight increase in the level of phosphatidylglycerol at both normal and low temperatures. However, the ratio of unsaturated fatty acids in monogalactosyldiacylglycerol and digalactosyldiacylglycerol was reduced in the mutant compared with WT. Although the growth of the mutant was indistinguishable with that of WT at normal growth temperature, it was markedly retarded at low temperature compared with that of WT. Our data indicated the possibility that cyanobacterial monogalactosyldiacylglycerol-synthesis pathway might be required for the adequate unsaturation level of fatty acids in galactolipids and affect the low-temperature sensitivity.     

Genetic analysis and characterization of a Caulobacter crescentus mutant defective in membrane biogenesis.

A mutant of Caulobacter crescentus has been isolated which has an auxotrophic requirement for unsaturated fatty acids or biotin for growth on medium containing glucose as the carbon source. This mutant exhibits a pleiotropic phenotype which includes (i) the auxotrophic requirement, (ii) cell death in cultures attempting to grow on glucose in the absence of fatty acids or biotin, and (iii) a major change in the outer membrane protein composition before cell death. This genetic lesion did not appear to affect directly a fatty acid biosynthetic reaction because fatty acid and phospholipid syntheses were found to continue in the absence of supplement. Oleic acid repressed fatty acid biosynthesis and induced fatty acid degradation in the wild-type parent, AE5000 . The mutant strain, AE6000 , was altered in both of these regulatory functions. The AE6000 mutant also showed specific inhibition of the synthesis of outer membrane and flagellar proteins. Total phospholipid, DNA, RNA, and protein syntheses were unaffected. The multiple phenotypes of the AE6000 mutant were found to cosegregate and to map between hclA and lacA on the C. crescentus chromosome. The defect in this mutant appears to be associated with a regulatory function in membrane biogenesis and provides evidence for a direct coordination of membrane protein synthesis and lipid metabolism in C. crescentus.     

Hyperthermic sensitivity and growth stage in Escherichia coli

Hyperthermic sensitivities of Escherichia coli B/r and Bs-1 were determined for lag-, midlog-, and stationary-phase cells at 47, 48, and 49 degrees C. In both strains midlog-phase cells were strikingly more heat sensitive (100-fold greater killing after 4 h at 48 degrees C) than stationary-phase cells, with intermediate sensitivity for lag-phase cells. In contrast to the reported difference in the radiation sensitivity between these two strains, very little difference in heat sensitivity was seen. Patterns of fatty acid composition of both strains were very similar at each phase of growth. From midlog to stationary phase, 16:1 and 18:1 unsaturated fatty acids decrease from 16 and 30% to 0.5 and 3%, respectively, while the C17 and C19 cyclopropane fatty acids increase from 7 and 3% to 22 and 25%, respectively. Concomitant with these changes in fatty acid composition, substantially higher membrane microviscosity values were recorded for stationary-phase cells. Total membrane microviscosity was positively associated with the C17 and C19 cyclopropane fatty acid composition and with cell survival following hyperthermia. In contrast to hyperthermic sensitivity, radiation survival differences between B/r and Bs-1 are little affected by growth stage. We propose that these results are consistent with a critical influence of membrane lipids on cellular hyperthermic sensitivity and further that the target sites for radiation and hyperthermia are different in these cells.     

Copper toxicity towards Saccharomyces cerevisiae: dependence on plasma membrane fatty acid composition.

One major mechanism of copper toxicity towards microorganisms is disruption of plasma membrane integrity. In this study, the influence of plasma membrane fatty acid composition on the susceptibility of Saccharomyces cerevisiae to Cu2+ toxicity was investigated. Microbial fatty acid composition is highly variable, depending on both intrinsic and environmental factors. Manipulation was achieved in this study by growth in fatty acid-supplemented medium. Whereas cells grown under standard conditions contained only saturated and monounsaturated fatty acids, considerable incorporation of the diunsaturated fatty acid linoleate (18:2) (to more than 65% of the total fatty acids) was observed in both whole-cell homogenates and plasma membrane-enriched fractions from cells grown in linoleate-supplemented medium. Linoleate enrichment had no discernible effect on the growth of S. cerevisiae. However, linoleate-enriched cells were markedly more susceptible to copper-induced plasma membrane permeabilization. Thus, after addition of Cu(NO3)2, rates of cellular K+ release (loss of membrane integrity) were at least twofold higher from linoleate-supplemented cells than from unsupplemented cells; this difference increased with reductions in the Cu2+ concentration supplied. Levels of cellular Cu accumulation were also higher in linoleate-supplemented cells. These results were correlated with a very marked dependence of whole-cell Cu2+ toxicity on cellular fatty acid unsaturation. For example, within 10 min of exposure to 5 microM Cu2+, only 3% of linoleate-enriched cells remained viable (capable of colony formation). In contrast, 100% viability was maintained in cells previously grown in the absence of a fatty acid supplement. Cells displaying intermediate levels of linoleate incorporation showed intermediate Cu2+ sensitivity, while cells enriched with the triunsaturated fatty acid linolenate (18:3) were most sensitive to Cu2+. These results demonstrate for the first time that changes in cellular and plasma membrane fatty acid compositions can dramatically alter microbial sensitivity to copper.     

Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis

Using chemical mutagenesis, mutants of Hansenula polymorpha that were defective in fatty acid synthesis were selected based on their growth requirements on saturated fatty acid mixtures. One mutant (S7) was incapable of synthesizing polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. A genetic analysis demonstrated that the S7 strain had a double lesion affecting fatty acid synthesis and Δ¹²-desaturation. A segregant with a defect in PUFA synthesis (H69-2C) displayed normal growth characteristics in the temperature range of 20–42 °C through a modulation of the cellular fatty acid composition. Compared with the parental strain, this yeast mutant had increased sensitivity at low and high temperatures (15 and 48 °C, respectively) with an increased tolerance to oxidative stress. The responses to ethanol stress were similar for the parental and PUFA-defective strains. Myristic acid was also determined to play an essential role in the cell growth of H. polymorpha. These findings suggest that both the type of cellular fatty acids and the composition of fatty acids might be involved in the stress responsive mechanisms in this industrially important yeast.     

Cardiolipin Accumulation in the Inner and Outer Membranes of Escherichia coli Mutants Defective in Phosphatidylserine Synthetase

Mutants of Escherichia coli defective in phosphatidylserine synthetase (pss) make less phosphatidylethanolamine than normal cells, and they are temperature sensitive for growth. We have isolated a new mutant, designated RA2021, which is better than previously available strains in that the residual phosphatidylethanolamine level approaches 25% after 4 h at 42 degrees C. The total amount of phospholipid normalized to the density of the culture is about the same in RA2021 (pss-21) as in the isogenic wild-type RA2000 (pss(+)). Consequently, there is a net accumulation of polyglycerophosphatides in the mutant, particularly of cardiolipin. The addition of 10 to 20 mM MgCl(2) to a culture of RA2021 prolongs growth under nonpermissive conditions and prevents loss of cell viability, but it does not eliminate the temperature-sensitive phenotype. Divalent cations, like Mg(2+), do not correct the phospholipid composition of the mutant, but may act indirectly by balancing the negative charges of phosphatidylglycerol and cardiolipin. To determine the effects of the pss mutation on membrane composition, we have examined the subcellular distribution of the polyglycerophosphatides that accumulate in these strains. All of the excess anionic lipids of RA2021 are associated with the envelope fraction and are distributed equally between the inner and outer membranes. The protein compositions of the isolated membranes do not differ significantly in the mutant and wild type. The fatty acid composition of RA2021 is almost the same as wild type at 30 degrees C, but there is more palmitic and cyclopropane fatty acid at 42 degrees C. These results demonstrate that the modification of the polar lipid composition observed in pss mutants affects both membranes and that cardiolipin, which is not ordinarily present in large quantities, can accumulate in the outer membrane when it is overproduced by the cell. The altered polar headgroup composition of the outer membrane in pss mutants may account, in part, for their hypersensitivity to the aminoglycoside antibiotics.