Metabolism of Phosphatidylglycerol, Phosphatidylethanolamine, and Cardiolipin of Bacillus stearothermophilus

The total phospholipid content of Bacillus stearothermophilus was constant during exponential growth, increased during the transition from the exponential to stationary phase of growth, and then slowly increased during the stationary phase. The first increase was a result of an increase in phosphatidylethanolamine; the second was a result of an increase in cardiolipin. Cessation of aeration of an exponentially growing culture or suspension in a nongrowth medium resulted in an immediate reduction in the rate of total phospholipid and phosphatidylethanolamine synthesis and a quantitative conversion of phosphatidylglycerol to cardiolipin. Cardiolipin appeared to be synthesized by the direct conversion of two molecules of phosphatidylglycerol to cardiolipin. After a 20-min pulse of (32)P, phosphatidylglycerol showed the most rapid loss of (32)P followed by cardiolipin, whereas phosphatidylethanolamine did not lose (32)P. The loss of (32)P from the total lipid pool, phosphatidylglycerol, and cardiolipin was biphasic, with rapid loss during the first two bacterial doublings followed by a greatly reduced rate of loss. The major loss of (32)P from the total phospholipid pool appeared to be by breakdown of cardiolipin. The loss of (32)P from the lipid pool was energy dependent (i.e., did not occur under anaerobic conditions or in the absence of an energy source) and was dependent on some factor other than the concentration of cardiolipin in the cells. The apparent conversion of phosphatidylglycerol to cardiolipin was independent of energy metabolism. Chloramphenicol reduced the rate of turnover of both phosphatidylglycerol and cardiolipin. The rate of lipid synthesis (all phospholipid components) was constant for about 10 min after the addition of chloramphenicol but diminished markedly after 20 min. Turnover of (32)P incorporated into phospholipid during a 30-min period prior to the addition of chloramphenicol was more rapid after the removal of chloramphenicol than that of (32)P incorporated during a 30-min period in the presence of chloramphenicol.     

Lipids of Salmonella typhimurium and Escherichia coli: structure and metabolism

The nature and quantity of the phospholipids of Salmonella typhimurium and Escherichia coli K-12 have been examined. The main classes of phospholipids, phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin have been completely characterized. Four minor compounds have been detected: phosphatidylserine, phosphatidic acid, and two partially characterized lipids. The phospholipid composition of the two organisms is quite similar, the only difference is the absence of one of the minor components and a decreased level of all components in E. coli. A study of the turnover of the phosphate in the phospholipids demonstrated no turnover in phosphatidylethanolamine, a slow turnover in phosphatidylglycerol, and a slow turnover in cardiolipin with, possibly, a transfer of phosphate from phosphatidylglycerol to cardiolipin. The amino acid phenylalanine is shown to become incorporated intact into lipidic compounds which have been partially characterized. Methods for the isolation and separation of lipids have been examined for their utility with these bacteria.     

The interaction of cardiolipin with rat liver carbamoyl phosphate synthetase I

A selective interaction of rat liver carbamoyl phosphate synthetase I with cardiolipin, and other anionic phospholipids, has been demonstrated. The enzymatic activity of the synthetase is inhibited by cardiolipin and, to a lesser extent, by phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine. This group of anionic phospholipids also induced a conformational change in the synthetase, yielding a species with increased exposure of the linkages between independently folded domains of the enzyme, as determined by limited proteolysis under nondenaturing conditions. The interaction of cardiolipin with carbamoyl phosphate synthetase I was a fairly slow process, with complex kinetics, and was apparently irreversible. The inclusion of Mg2+ or of MgATP in the incubation mixture prevented the cardiolipin effects. The zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine had negligible effects on the structure and activity of the synthetase. This interaction between cardiolipin and carbamoyl phosphate synthetase I potentially constitutes one of the mechanisms by which the synthetase forms its loose association with the inner mitochondrial membrane. Multiple mechanisms, including synthetase conformational changes, cardiolipin phase changes, and ATP/ADP binding site involvement, are possibly involved in the phospholipid/synthetase interaction and the resulting potential regulatory mechanism(s) for urea cycle activity.     

Altered phospholipid metabolism in a temperature-sensitive mutant of a thermophilic bacillus

The phospholipid metabolism of a temperature-sensitive mutant of a thermophilic bacillus was studied after the shift from a permissive (58°C) to a restrictive (65°C) growth temperature. During the short period of growth of the mutant at 65°C, the proportions of cardiolipin and its 3-acyl derivative (lyso-cardiolipin) increased, and the proportions of phosphatidylglycerol and phosphatidylethanolamine decreased on cell dry weight basis. In ³²P incorporation and turnover experiments, phosphatidylglycerol showed the most rapid uptake and loss of the label. Turnover of cardiolipin, limited to a short period, ceased 18 min after the shift, as did the turnover of phosphatidylethanolamine. In the absence of net phospholipid synthesis, there was a quantitative conversion of phosphatidylglycerol to cardiolipin and an increase in the proportion of lyso-cardiolipin. Chloramphenicol, added to the medium at the time of the shift, reduced the rate of phospholipid synthesis, prevented the increase in the proportions of cardiolipin and lyso-cardiolipin, and slowed the decrease in the proportions of the other two phospholipids. The results indicated a defect in the regulatory mechanism(s) of phospholipid metabolism in the mutant at the restrictive temperature.Nonstandard Abbreviations WT parental strain, thermophilic bacillus - TS-13 temperature-sensitive mutant of a thermophilic bacillus - CL cardiolipin - PG phosphatidylglycerol - PE phosphatidylethanolamine - l-CL lyso-cardiolipin     

Specific extraction of bacterial cardiolipin from sporulating Bacillus subtilis

A method for rapid purification of bacterial cardiolipin is presented. The cardiolipin level was first increased by suspending Bacillus subtilis cells in a buffer containing an uncoupling agent. At least 90% of the phosphatidylglycerol molecules were rapidly converted into cardiolipin. In sporulating strains, the accumulated cardiolipin appeared to be unextractable by conventional phospholipid extraction procedures. Sporulating bacteria were therefore treated first by a classical technique in order to eliminate lipids other than cardiolipin; a second extraction in a highly acidic medium then allowed us to quantitatively extract the remaining cardiolipin. Besides simplicity and rapidity, this method has the advantage of yielding cardiolipin in a nearly pure form from a relatively low number of bacteria.     

Phospholipids of the differentiating bacterium Caulobacter crescentus.

The phospholipid composition of the stalked and swarmer cell types of the differentiating, Gram-negative bacterium Caulobacter crescentus was determined. The phospholipid composition of the stalked cell type was 86.5% phosphatidylglycerol, 10.4% lysylphosphatidylglycerol, and 3.0% cardiolipin; that of the swarmer cell type was 84.1, 11.4, and 4.4% respectively. Phosphatidylethanolamine, which is a major phospholipid component of most Gram-negative bacteria, was totally absent.     

Modification of phospholipid composition in Pseudomonas putida A ATCC 12633 induced by contact with tetradecyltrimethylammonium

AIMS: The aim of this work was to establish if the response to tetradecyltrimethylammonium (TDTMA), a representative quaternary ammonium compound (QAC), involves changes in the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633. METHODS AND RESULTS: Pseudomonas putida was exposed to 50 mg l(-1) of TDTMA for 15 min, and PL composition was analysed. With respect to control values, phosphatidic acid and phosphatidylglycerol increased by 140% and 120%, respectively; cardiolipin decreased about 60%. In TDTMA-adapted bacteria, the most significant change was a 380% increase in phosphatidic acid. Accompanying this change was a 130% increase in phosphatidylglycerol and a 70% decrease in cardiolipin. The changes in adapted cells were reverted after two subcultures without biocide. CONCLUSIONS: Pseudomonas putida responded to TDTMA through quantitative changes in PLs with specific variations in the content of phosphatidic acid, phosphatidylglycerol and cardiolipin. These modifications indicated that these PLs are involved in cellular responses to QACs, utilizing phosphatidic acid principally to neutralize the high positive charge density given for the ammonium quaternary moiety from TDTMA. SIGNIFICANCE AND IMPACT OF THE STUDY: The changes in PL composition give a new insight about the response inflicted by Ps. putida when these bacteria are exposed to QACs.     

Membrane phospholipid composition of Caulobacter crescentus.

The phospholipid composition of Caulobacter crescentus CB13 and CB15 was determined. The acidic phospholipids, phosphatidylglycerol and cardiolipin, comprise approximately 87% of the total phospholipids. Neither phosphatidylethanolamine nor its precursor phosphatidylserine was detected. The outer and inner membranes of C. crescentus CB13 were separated, and phospholipid analysis revealed heterogeneity with respect to the relative amounts of phosphatidylglycerol and cardiolipin in the two membranes. As has been shown to be the case for other bacterial membranes, the concentration of cardiolipin increases and phosphatidylglycerol decreases as cell cultures enter stationary phase.     

Features of phospholipid composition of marine proteobacteria of Pseudoalteromonas species

The study of the phospholipid composition of 14 type strains of marine proteobacteria of the genus Pseudoalteromonas showed that phospholipids are the main polar lipid constituents of membranes in these proteobacteria. The phospholipid patterns of the strains studied were found to be similar and involved five phospholipids typical of gram-negative bacteria, namely, phosphatidylethanolamine, phosphatidylglycerol, bisphosphatidic acid, lysophosphatidylethanolamine, and phosphatidic acid. The major phospholipids were phosphatidylethanolamine and phosphatidylglycerol, which add up to 89-97% of total phospholipids; bisphosphatidic acid was dominant among minor phospholipids. The prevalence of phosphatidylethanolamine (62-77% of total phospholipids) and the absence of diphosphatidylglycerol are the characteristic features of most bacteria of this genus. As in Escherichia coli, the phospholipid composition of the marine proteobacteria depended on the presence of magnesium in the medium.     

Characterization of the cardiolipin synthetase activity of Escherichia coli envelopes

The synthesis of cardiolipin from phosphatidylglycerol catalyzed by isolated envelopes of Escherichia coli occurs without the utilization of endogenous CDP-diglyceride as a substrate. The synthesis of cardiolipin has been assayed distinct from the synthesis of bis-phosphatidic acid. Envelope fractions isolated from cultures exposed to treatments which increase the relative rate of cardiolipin synthesis in vivo were found not to have increased amounts of cardiolipin synthetase activity in vitro. We suggest that the relative increase of cardiolipin synthesis observed during these treatments stems from the lack of an energy requirement for the cardiolipin synthetase reaction and the presence of large amounts of cellular phosphatidylglycerol.     

Identification and characterization of human cardiolipin synthase

The mitochondrial phospholipid cardiolipin is synthesized from cytidinediphosphate-diacylglycerol and phosphatidylglycerol, a process catalyzed by the enzyme cardiolipin synthase. In this study, we identified a human candidate gene/cDNA for cardiolipin synthase, C20orf155. Expression of this candidate cDNA in the (cardiolipin synthase-deficient) crd1Delta yeast confirmed that it indeed encodes human cardiolipin synthase. Purified mitochondria of the crd1Delta expressing human cardiolipin synthase were used to characterize the enzyme. It has an alkaline pH optimum, requires divalent cations for activity and appears to have a different substrate preference for cytidinediphosphate-diacylglycerol species when compared to phosphatidylglycerol species. The possible implications for CL synthesis and remodeling are discussed.     

Phospholipids of marine proteobacteria of the genusPseudoalteromonas

The study of the phospholipid composition of 14 type strains of marine proteobacteria of the genusPseudoalteromonas showed that phospholipids are the main polar lipid constituents of membranes in these proteobacteria. The phospholipid patterns of the strains studied were found to be similar and involved five phospholipids typical of gram-negative bacteria, namely, phosphatidylethanolamine, phosphatidylglycerol, bisphosphatidic acid, lysophosphatidylethanolamine, and phosphatidic acid. The major phospholipids were phosphatidylethanolamine and phosphatidylglycerol, which add up to 89–97% of the total phospholipids; bisphosphatidic acid was dominant among minor phospholipids. The prevalence of phosphatidylethanolamine (62–77% of the total phospholipids) and the absence of diphosphatidylglycerol are the characteristic features of most bacteria of this genus. As inEscherichia coli, the phospholipid composition of the marine proteobacteria depended on the presence of magnesium in the medium.     

Activity of N-acetylmuramoyl-L-alanine amidase in phospholipidic environments

A purified preparation of N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), a murein hydrolase from Escherichia coli, was found to lose its activity during incubation in the presence of bacterial phospholipid suspensions. Whether it was co-dispersed with the phospholipids or added to sonicated phospholipid suspension, the enzyme was inhibited (or inactivated) from the first minutes of incubation at 37 degree C. As phosphatidylglycerol/cardiolipin ratio of the phospholipid suspension as increased (all other things being equal), a further decrease of amidase activity was observed. The highest losses of activity were found after co-dispersion of the enzyme and the substrate together with the phospholipids, the resulting suspension being formed of larger multilayered vesicles, as revealed by electron microscopy. In these conditions, the effect on enzyme activity was only partially accounted for by the proportion of the enzyme that was entrapped in the vesicles. The entrapment capacity of the enzyme (using a 35S-labelled enzyme preparation) and of the substrate (3H-labelled) by the multilamellar phospholipidic vesicles did not significantly change as a function of their relative content of phosphatidylglycerol and cardiolipin. The possible physiological meaning of the results is discussed is connection with our previous data and with other works related to membranous phospholipid distribution and to septum formation control in bacteria.     

Membrane phospholipid asymmetry in Bacillus amyloliquefaciens.

The phospholipid distribution in the membrane of Bacillus amyloliquefaciens was studied by using phospholipase C (B. cereus), phospholipase A2 (Crotalus), and the nonpenetrating chemical probe trinitrobenzenesulfonic acid. After treatment of intact protoplasts of B. amyloliquefaciens with either phospholipase, about 70% of total membrane phospholipid was hydrolyzed; specifically, about 90, 90, and 30% of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, respectively. Under these conditions, protoplasts remained intact and sealed. However, when protoplasts that were permeabilized by cold-shock treatment were incubated with either of the phospholipases, up to 80% of cardiolipin was hydrolyzed and phosphatidylglycerol and phosphatidylethanolamine were hydrolyzed virtually to completion. In intact cells, 92% of the phosphatidylethanolamine could be labeled with trinitrobenzenesulfonic acid under conditions in which the reagent did not penetrate the membrane to any significant extent. These results indicate that 70% of total phospholipid of this bacillus exists in the outer half of the bilayer. The distribution of phosphatidylethanolamine in this bilayer is highly asymmetric with it being located predominantly in the outer half. The results with phospholipases suggest that the distributions of cardiolipin and phosphatidylglycerol are also asymmetric but independent confirmation of this is required.     

The phospholipid composition ofBradyrhizobium spp

The phospholipid composition of two strains ofBradyrhizobium is reported. In contrast to previous studies [Bunn CR, Elkan GH (1970) Can J Microbiol 17:291–295; and Gerson T, Patel JJ (1975) Appl Microbiol 30:193–198], we determined that phosphatidylglycerol is a major phospholipid within this bacterial genus. Furthermore, neither phosphatidylserine nor phosphatidylinositol was detected within lipid extracts derived from these bacteria. In addition to phosphatidylglycerol, other major phospholipids ofBradyrhizobium were shown to include phosphatidylcholine, phosphatidylethanolamine, and cardiolipin. Possible explanations for the discrepancies between the present study and those of previous investigations are discussed.     

Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs.

The phospholipid composition and phospholipid fatty acid composition of purified Rickettsia prowazeki were determined. The lipid phosphorous content was 6.8 +/- 1.3 microgram/mg of total rickettsial protein. The major phospholipid was phosphatidylethanolamine (60 to 70%); phosphatidylglycerol constituted 20%, and phosphatidylcholine constituted 15%. Small amounts of phosphatidylserine and cardiolipin were detected. The principal fatty acids were 18:1, 16:1, and 16:0. The fatty acid composition of the phosphatidylcholine in the rickettsial extracts was very different than that of the other rickettsial phosphatides and very similar to that of normal yolk sac phosphatidylcholine. The specific of the phosphatidylcholine of rickettsiae grown in the presence of 32P was markedly lower than that of phosphatidylethanolamine and phosphatidylglycerol. It is suggested that the phosphatidylcholine in the rickettsial extract is yolk sac derived and either tightly absorbed or exchanged into the rickettsial membrane.     

Effect of age on the membrane lipid composition of Streptococcus sanguis

The cell membrane of Streptococcus sanguis contains three classes of lipid: neutral lipid, glycolipid and phospholipid. A striking difference in membrane lipid composition between cells in the exponential and in the stationary phases of growth was observed. During the exponential phase, approx. 37–45%, 14–19% and 37–45% of the lipids synthesized were found to be neutral lipid, glycolipid and phospholipid, respectively. The amount of lipid synthesized reached a maximum at the early stationary phase. The amount of phospholipid drastically declined thereafter and that of neutral lipid slightly declined. In contrast, the amount of glycolipid markedly increased and exceeded the amount of phospholipid. The phospholipid present during the exponential phase was found to be mainly phosphatidylglycerol (82–88%) and a small amount of cardiolipin (12–18%). At the stationary phase, the amount of phosphatidylglycerol greatly decreased and reached approx. 16% of that in the early stationary phase, while cardiolipin steadily increased and became the major phospholipid in the late stationary phase. The glycolipid was found to be composed of mainly mono- and diglucosyldiglycerides. At the end of the experiment (after 8 h incubation), the distribution of lipids was found to be: neutral lipid, 46%; glycolipid (monoglucosyldiglyceride, 28%; diglucosyldiglyceride, 13%) 41%; and phospholipid (phosphatidylglycerol, 3%, cardiolipin, 8%) 13%.     

An improved two dimensional thin-layer chromatography system for the separation of phosphatidylglycerol and its derivatives.

A two dimensional thin-layer chromatography system has been devised for the improved separations of phosphatidylglycerol and its derivaties, cardiolipin and bis/monoacylglyceryl)phosphate, from the other phospholipid components of tissue total lipid extracts. The system employs silica gel G plates prepared with 0.4 M boric acid. Linear recovery of added phosphatidylglycerol was found, and phosphatidylglycerol did not cochromatograph with N, N-dimethylphosphatidylethanolamine in this system. The phospholipid class composition of various rat tissues and a Morris 7777 hepatoma has been determined and compared with values from the literature.     

Viability of an Escherichia coli pgsA null mutant lacking detectable phosphatidylglycerol and cardiolipin

Phosphatidylglycerol, the most abundant acidic phospholipid in Escherichia coli, has been considered to play specific roles in various cellular processes and is believed to be essential for cell viability. It is functionally replaced in some cases by cardiolipin, another abundant acidic phospholipid derived from phosphatidylglycerol. However, we now show that a null pgsA mutant is viable, if the major outer membrane lipoprotein is deficient. The pgsA gene normally encodes phosphatidylglycerophosphate synthase that catalyzes the committed step in the biosynthesis of these acidic phospholipids. In the mutant, the activity of this enzyme and both phosphatidylglycerol and cardiolipin were not detected (less than 0.01% of total phospholipid, both below the detection limit), although phosphatidic acid, an acidic biosynthetic precursor, accumulated (4.0%). Nonetheless, the null mutant grew almost normally in rich media. In low-osmolarity media and minimal media, however, it could not grow. It did not grow at temperatures over 40 degrees C, explaining the previous inability to construct a null pgsA mutant (W. Xia and W. Dowhan, Proc. Natl. Acad. Sci. USA 92:783-787, 1995). Phosphatidylglycerol and cardiolipin are therefore nonessential for cell viability or basic life functions. This notion allows us to formulate a working model that defines the physiological functions of acidic phospholipids in E. coli and explains the suppressing effect of lipoprotein deficiency.     

Moraxella catarrhalis Expresses a Cardiolipin Synthase That Impacts Adherence to Human Epithelial Cells

The major phospholipid constituents of Moraxella catarrhalis membranes are phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin (CL). However, very little is known regarding the synthesis and function of these phospholipids in M. catarrhalis. In this study, we discovered that M. catarrhalis expresses a cardiolipin synthase (CLS), termed MclS, that is responsible for the synthesis of CL within the bacterium. The nucleotide sequence of mclS is highly conserved among M. catarrhalis isolates and is predicted to encode a protein with significant amino acid similarity to the recently characterized YmdC/ClsC protein of Escherichia coli. Isogenic mclS mutant strains were generated in M. catarrhalis isolates O35E, O12E, and McGHS1 and contained no observable levels of CL. Site-directed mutagenesis of a highly conserved HKD motif of MclS also resulted in a CL-deficient strain. Moraxella catarrhalis, which depends on adherence to epithelial cells for colonization of the human host, displays significantly reduced levels of adherence to HEp-2 and A549 cell lines in the mclS mutant strains compared to wild-type bacteria. The reduction in adherence appears to be attributed to the absence of CL. These findings mark the first instance in which a CLS has been related to a virulence-associated trait.