Molecular cloning of the cls gene responsible for cardiolipin synthesis in Escherichia coli and phenotypic consequences of its amplification.

The cls gene responsible for cardiolipin synthesis in Escherichia coli K-12 was cloned in a 5-kilobase-pair DNA fragment inserted in a mini-F vector, pML31, and then subcloned into a 2.0-kilobase-pair fragment inserted in pBR322. The initial selection of the gene was accomplished in a cls pss-1 double mutant that had lesions in both cardiolipin and phosphatidylserine synthases and required either the cls or the pss gene product for normal growth at 42 degrees C in a broth medium, NBY, supplemented with 200 mM sucrose. The cloned gene was identified as the cls gene by the recovery and amplification of both cardiolipin and cardiolipin synthase in a cls mutant as well as by the integration of a pBR322 derivative into its genetic locus at 27 min on the chromosome of a polA1 mutant. The maxicell analysis indicated that a protein of molecular weight 46,000 is the gene product. The cls gene is thus most likely the structural gene coding for cardiolipin synthase. Hybrid plasmids of high copy numbers containing the cls gene were growth inhibitory to pss-I mutants under the above selective conditions, whereas they inhibited neither the growth of pss-I mutants at 30 degrees C nor that of pss+ strains at any temperature. Amplification of cardiolipin synthase activity was observed, but was not proportional to the probable gene dosage (the enzyme activity was at most 10 times that in wild-type cells), and cardiolipin synthesis in vivo was at the maximum 1.5 times that in wild-type strains, implying the presence in E. coli cells of a mechanism that avoids cardiolipin overproduction, which is possibly disadvantageous to proper membrane functions.     

Disruption of the Escherichia coli cls gene responsible for cardiolipin synthesis

The cls gene of Escherichia coli is responsible for the synthesis of a major membrane phospholipid, cardiolipin, and has been proposed to encode cardiolipin synthase. This gene cloned on a pBR322 derivative was disrupted by either insertion of or replacement with a kanamycin-resistant gene followed by exchange with the homologous chromosomal region. The proper genomic disruptions were confirmed by Southern blot hybridization and a transductional linkage analysis. Both types of disruptants had essentially the same properties; cardiolipin synthase activity was not detectable, but the strains grew well, although their growth rates and final culture densities were lower than those of the corresponding wild-type strains and strains with the classical cls-1 mutation. A disruptant harboring a plasmid that carried the intact cls gene grew normally. The results indicate that the cls gene and probably the cardiolipin synthase are dispensable for E. coli but may confer growth or survival advantages. Low but definite levels of cardiolipin were synthesized by all the disruptants. Cardiolipin content of the cls mutants depended on the dosage of the pss gene, and attempts to transfer a null allele of the cls gene into a pss-1 mutant were unsuccessful. We point out the possibilities of minor cardiolipin formation by phosphatidylserine synthase and of the essential nature of cardiolipin for the survival of E. coli cells.     

Alteration of phospholipid composition by combined defects in phosphatidylserine and cardiolipin synthases and physiological consequences in Escherichia coli.

Escherichia coli K-12 derivatives with a common genetic background carrying, either alone or in combination, the pss-1 allele coding for a temperature-sensitive phosphatidylserine synthase (A. Ohta and I. Shibuya, J. Bacteriol. 132:434-443, 1977) and cls- for a defective cardiolipin synthase (G. Pluschke et al., J. Biol. Chem. 253:5048-5055, 1978) were constructed. The phospholipid polar headgroup compositions of these strains were significantly different from each other depending on their genotypes and growth temperature, whereas other membrane characteristics such as the total phospholipid content, fatty acid composition, membrane protein profile, and lipopolysaccharide content were practically the same, suggesting that the phenotypes of these strains were the direct consequences of abnormalities in membrane phospholipid composition. The cls pss-1 double mutation caused an unusual accumulation of phosphatidylglycerol with an extremely low content of cardiolipin. The cls mutation alone was found to give a growth defect, and its introduction into a pss-1 mutant resulted in an enhanced temperature sensitivity of growth. Addition to a broth medium of a proper concentration of sucrose, NaCl, Mg2+, or Ca2+ allowed the growth of a pss-1 mutant at otherwise nonpermissive temperature, but a pss-1 cls double mutant required the combined addition of sucrose or NaCl and MgCl2 for full growth at 42 degrees C. The possible mechanisms for these physiological consequences of the mutations are discussed on a molecular basis. The remedial effects of culture supplements allowed the pss-1 mutants to grow at 42 degrees C resulting in enhanced abnormalities of membrane phospholipid composition.     

Correlation of 3,4-dihydroxybutyl 1-phosphonate resistance with a defect in cardiolipin synthesis in Escherichia coli.

Escherichia coli treated for 1 h with 100 microM rac-3,4-dihydroxybutyl 1-phosphonate (DBP), a glycerol-3-phosphate analog, die when sorted at 5 degrees C, whereas the viability of untreated cells is relatively unaffected. This observation formed the basis of a selection procedure that was used to isolate mutants that are partially resistant to DBP. One such mutant, strain 6204, is constitutive for DBP transport, exhibits a particularly high degree of cold resistance, has the same doubling time as the parent, and is similar to the parent strain in terms of incorporation of DBP into the lipid fraction. Glycerol-3-phosphate and phosphatidylglycerol phosphate synthetases obtained from strain 6204 and its parent were identical in terms of DBP recognition. The parent strain is killed when incubated in the presence of a combination of 70 microM rac-DBP and 0.25% deoxycholate, whereas strain 6204 continues to grow, albeit more slowly, in the presence of this combination. Strain 6204 can be distinguished from the parent strain on agar plates (low phosphate minimal medium with glucuronate as the sole carbon source) containing 15 microM rac-DBP. The insertion of Tn10 near the 6204 mutation has facilitated genetic manipulations. All phenotypic effects attributed to strain 6204 appear to be due to a single mutation. Genetic analysis indicates that Tn10, inserted near the gene responsible for DBP resistance, maps in the vicinity of 27 min. Three-factor crosses reveal a gene order of hemA-Dbpr-Tn10(zch)-trp. The only gene for phosphoglyceride metabolism known to map in this region is the gene associated with cardiolipin synthetase, cls. Genetic results suggest that the mutation responsible for DBP resistance maps in or very near cls. Analysis of the lipids isolated from untreated strain 6204 (and from each of the transductants prepared by P1 vir-mediated transfer of DBP resistance of wild-type strains) reveals that cardiolipin synthesis is defective. These results strongly suggest that the mutation responsible for DBP resistance has its primary effect on cardiolipin synthesis. To further test this hypothesis, strains with an authentic cls mutation were constructed and examined for resistance to DBP. These strains had growth properties that were identical with those of strain 6204. Wild-type strains and mutants defective in cardiolipin synthesis were treated with DBP and 20 mM magnesium or calcium chloride. Simultaneous treatment of either cell type with DBP and divalent cation not only failed to stimulate growth but, quite the contrary, had a marked synergistic growth inhibitory effect.     

Characterization of Staphylococcus aureus cardiolipin synthases 1 and 2 and their contribution to accumulation of cardiolipin in stationary phase and within phagocytes

In many bacteria, including Staphylococcus aureus, progression from the logarithmic to the stationary phase is accompanied by conversion of most of bacterial membrane phosphatidylglycerol (PG) to cardiolipin (CL). Phagocytosis of S. aureus by human neutrophils also induces the conversion of most bacterial PG to CL. The genome of all sequenced strains of S. aureus contains two open reading frames (ORFs) predicting proteins encoded with ～30% identity to the principal CL synthase (cls) of Escherichia coli. To test whether these ORFs (cls1 and cls2) encode cardiolipin synthases and contribute to CL accumulation in S. aureus, we expressed these proteins in a cls strain of E. coli and created isogenic single and double mutants in S. aureus. The expression of either Cls1 or Cls2 in CL-deficient E. coli resulted in CL accumulation in the stationary phase. S. aureus with deletion of both cls1 and cls2 showed no detectable CL accumulation in the stationary phase or after phagocytosis by neutrophils. CL accumulation in the stationary phase was due almost solely to Cls2, whereas both Cls1 and Cls2 contributed to CL accumulation following phagocytosis by neutrophils. Differences in the relative contributions of Cls1 and Cls2 to CL accumulation under different triggering conditions suggest differences in the role and regulation of these two enzymes.     

Amplification and substantial purification of cardiolipin synthase of Escherichia coli.

A simple, specific, and sensitive assay procedure for cardiolipin synthase of Escherichia coli has been developed. This measures the radioactivity of glycerol formed from phosphatidyl [2-3H]glycerol and is mainly based on the findings that 400 mM phosphate and 0.015% Triton X-100 markedly activate the enzyme. Cardiolipin synthase was amplified 760-fold upon induction with isopropyl beta-D-thiogalactoside in cells harboring a pBR322 derivative in which the cls gene encoding this enzyme was preceded by the tac promoter. Under these conditions, cardiolipin content increased, membrane potential decreased, spheroplasts became fragile, cells lost viability, and inducer-resistant mutants appeared at a high frequency. The amplification enabled the isolation of an enzyme preparation with a specific activity approximately 10,000-times higher than that of wild-type whole cell lysate. This purification was simply achieved by extraction of the crude membrane fraction with Triton X-100 and a single phosphocellulose column chromatography. This preparation, together with the crude envelope fraction, was used to characterize the basic properties of E. coli cardiolipin synthase, some of which were utilized in setting up the assay conditions.     

A second Escherichia coli protein with CL synthase activity

The Escherichia coli open reading frame f413, which has the potential to code for a polypeptide homologous to cardiolipin (CL) synthase, has been cloned. Its polypeptide product has a molecular mass of 48 kDa, is membrane-bound, and catalyzes CL formation but does not hydrolyze CL. A comparison of the sequences predicted for the polypeptides encoded by f413 and cls indicates that the N-terminal residues specified by cls may be unnecessary for CL synthase activity. Construction of a truncated cls gene and characterization of its polypeptide product have confirmed this conclusion.     

A Pseudomonas putida cardiolipin synthesis mutant exhibits increased sensitivity to drugs related to transport functionality

Biological membranes have evolved different mechanisms to modify their composition in response to chemical stimuli in a process called 'homeoviscous adaptation'. Among these mechanisms, modifications in the ratio of saturated/unsaturated fatty acids and in cis/trans fatty acid isomers, cyclopropanation and changes in the phospholipids head group composition have been observed. To further understand the role of phospholipid head groups in solvent stress adaptation, we knocked out the cls (cardiolipin synthase) gene in Pseudomonas putida DOT-T1E. As expected, cls mutant membranes contained less cardiolipin than those of the wild-type strain. Although no significant growth rate defect was observed in the cls mutant compared with the wild-type strain, mutant cells were significantly smaller than the wild-type cells. The cls mutant was more sensitive to toluene shocks and to several antibiotics than the parental strain, suggesting either that the RND efflux pumps involved in the extrusion of these drugs were not working efficiently or that membrane permeability was altered in the mutant. Membranes of the cls mutant strain seemed to be more rigid than those of the parental strain, as observed by measurements of fluorescence polarization using the DPH probe, which intercalates into the membranes. Ethidium bromide is pumped out in Pseudomonas putida by at least one RND efflux pump involved in antibiotic and solvent resistance, and the higher rate of accumulation of ethidium bromide inside mutant cells indicated that functioning of the efflux pumps was compromised as a consequence of the alteration in phospholipid head group composition.     

Cis/trans isomerisation of unsaturated fatty acids in a cardiolipin synthase knock-out mutant of Pseudomonas putida P8

The gene encoding cardiolipin synthase ( cls) from the phenol-degrading bacterium Pseudomonas putida P8, which rapidly adapts its membrane lipids to the presence of organic solvents by cis/trans isomerisation of unsaturated fatty acids, was isolated and completely sequenced. The functionality of the predicted gene product was proven by constructing a knock-out mutant that was significantly reduced in its growth rate both at elevated temperatures and in the presence of membrane-active solvents. Though the mutant showed a clear phenotype it was still able to synthesise trace amounts of cardiolipin. As an increase in cardiolipin (diphosphatidylglycerol) content is known to function as a long term membrane adaptation mechanism in pseudomonads, we tested whether the mutant compensates for the lack of the Cls by increased cis/trans isomerisation of unsaturated fatty acids. Increase in cis/trans isomerisation of unsaturated fatty acids was observed for the mutant at zero and low concentrations of 4-chlorophenol; however, cis/trans isomerisation is not able to fully compensate for the lack of cardiolipin production. Possibly, other long-term adaptation mechanisms are instrumental in compensating for the missing cardiolipin synthesis. As the cis/trans isomerase is activated similarly in the mutant and the wildtype, cis/trans isomerisation and cardiolipin production do not display mutual dependency.     

Genetic regulation of cardiolipin synthase in Escherichia coli.

Recombinant DNA and genetic techniques were used to construct Escherichia coli strains SOH92 [phi(cls-lacZ+)] and SOH93 [phi(cls-'lacZ)hyb]. beta-Galactosidase (116 kDa) synthesized by strain SOH92 was primarily present in the particulate fraction. Strain SOH92 produced about 20-fold more beta-galactosidase activity than strain SOH93. Expression of clsphilacZ in both SOH92 and SOH93 was influenced by the terminal electron acceptor (increasing in the order oxygen, nitrate, fumarate) when the cells were cultured in minimal medium with glycerol as the sole carbon source. As strains SOH92 and SOH93 progressed from early to late log growth phase under aerobic conditions in LB broth, clsphilacZ expression increased about 2.5-fold. Fusion strains containing a pss-1 allele had an increased cardiolipin (CL) level, but no corresponding increase in clsphilacZ expression was observed. A cls::Tn10dTet null mutation was introduced into SOH92 and SOH93. The strains produced less CL, but no corresponding changes in clsphilacZ expression were observed. A high copy number plasmid bearing the cls gene had no effect on clsphilacZ expression. Taken together, these results indicate that cls is not subject to autogenous regulation.     

Cardiolipin deficiency in Rhodobacter sphaeroides alters the lipid profile of membranes and of crystallized cytochrome oxidase, but structure and function are maintained

Many recent studies highlight the importance of lipids in membrane proteins, including in the formation of well-ordered crystals. To examine the effect of changes in one lipid, cardiolipin, on the lipid profile and the production, function, and crystallization of an intrinsic membrane protein, cytochrome c oxidase, we mutated the cardiolipin synthase (cls) gene of Rhodobacter sphaeroides, causing a >90% reduction in cardiolipin content in vivo and selective changes in the abundances of other lipids. Under these conditions, a fully native cytochrome c oxidase (CcO) was produced, as indicated by its activity, spectral properties, and crystal characteristics. Analysis by MALDI tandem mass spectrometry (MS/MS) revealed that the cardiolipin level in CcO crystals, as in the membranes, was greatly decreased. Lipid species present in the crystals were directly analyzed for the first time using MS/MS, documenting their identities and fatty acid chain composition. The fatty acid content of cardiolipin in R. sphaeroides CcO (predominantly 18:1) differs from that in mammalian CcO (18:2). In contrast to the cardiolipin dependence of mammalian CcO activity, major depletion of cardiolipin in R. sphaeroides did not impact any aspect of CcO structure or behavior, suggesting a greater tolerance of interchange of cardiolipin with other lipids in this bacterial system.     

Isolation,purification and characterization of Cardiolipin synthase from Mycobacterium phlei {PRIVATE}

It has been observed that mycobacterial species has high content of cardiolipin (CL) in their cell membranes more so pathogenic mycobacteria and in bacteria CL activates polymerases, gyrases by removing the bound ADP. Therefore, in the present study cardiolipin synthase (cls) which catalyses the formation of CL was isolated purified and characterized from the cell membrane of Mycobacterium phlei. The purified cls obtained from C-18 RP-HPLC column had a molecular weight of 58 kDa with an isoelectric point of 4.5. The enzyme activity (11.5+0.15 µM of CL phosphorous. ml-1 minute-1 for PG as substrate and 14+0.35µM of CL phosphorous. ml-1 minute-1 for CDP-DG as substrate) was optimal at pH 4.8 and showed K_M values of 55+0.05µM and 2.56+0.04µM for phosphatidyl glycerol and CDP-diacylglycerol, respectively, with an absolute requirement of Mg²⁺ and Mn²⁺ ions for its activity however, Ca²⁺ ions inhibited the activity of the cls. The partial amino acid sequence of cls showed significant homology with pgsA3 gene of M. tuberculosis and in this organism the CL biosynthesis is very high having three genes coding for PLs biosynthesis therefore, enzymes involved in CL biosynthesis may be an attractive drug target in the development of new antimycobacterial drugs.     

crinkled leaves 8--a mutation in the large subunit of ribonucleotide reductase--leads to defects in leaf development and chloroplast division in Arabidopsis thaliana

The crinkled leaves8 (cls8) mutant of Arabidopsis thaliana displays a developmental phenotype of abnormal leaf and flower morphology, reduced root growth and bleached leaf sections. Map-based cloning identified the mutation as being within the gene encoding the large subunit of ribonucleotide reductase (RNR1), the enzyme that catalyses the rate-limiting step in the production of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Levels of dTTP and dATP were significantly reduced in cls8. Two further mutant cls8 alleles and cls8::RNAi plants show similar or more severe phenotypes. The cls8-1 mutant has fewer copies of the chloroplast genome, and fewer, larger chloroplasts than wild-type plants. The ultrastructure of the chloroplast, however, appears normal in cls8-1 leaves. We present evidence that, under conditions of limited dNTP supply, the inhibition of chloroplast DNA replication may be the primary factor in inducing aberrant growth.     

Identity of the Escherichia coli cls and nov genes.

cls and nov mutants have similar increased sensitivities to novobiocin and reduced levels of cardiolipin, both of which can be corrected by plasmid-borne copies of either wild-type gene. A comparison of the DNA sequences of both genes further verifies their identity.     

Calcium-sensitive cls4 mutant of Saccharomyces cerevisiae with a defect in bud formation.

A calcium-sensitive cls4 mutant of Saccharomyces cerevisiae ceased dividing in the presence of 100 mM CaCl2, producing large, round, unbudded cells. Since its DNA replication and nuclear division still continued after interruption of normal budding, the cls4 mutant had a defect in bud formation in Ca2+-rich medium. Its calcium content and calcium uptake activity were the same as those of the wild-type strain, suggesting that the primary defect of the mutation was not in a Ca2+ transport system. Genetic analysis showed that the cls4 mutation did not complement the cdc24-1 mutation, which is known to be a temperature-sensitive mutation affecting bud formation and localized cell surface growth at a restrictive temperature. Moreover, cls4 was tightly linked to cdc24, and a yeast 3.4-kilobase-pair DNA fragment carrying both the CLS4 and CDC24 genes was obtained. These results suggest that the cls4 mutation is allelic to the cdc24 mutation. Thus, Ca2+ ion seems to control bud formation and bud-localized cell surface growth.     

Isolation and characterization of Ca2+-sensitive mutants of Saccharomyces cerevisiae

Thirty Ca2+-sensitive (cls: calcium sensitive) mutants of Saccharomyces cerevisiae were isolated by replica-plating. These mutants, which each had a single recessive chromosomal mutation, were divided into 18 complementation groups. Some cls mutants showed a phenotype of specific sensitivity to Ca2+, while others showed phenotypes of sensitivities to several divalent cations. From measurements of the calcium contents and initial rates of Ca2+ uptake of the cls mutants, 16 of the 18 cls complementation groups were classified into four types: type I mutants (cls5, cls6, cls13, cls14, cls15, cls16, cls17, and cls18) had both elevated calcium contents and increased uptake activities. A type II mutant (cls4) had a normal calcium content and normal uptake activity; type III mutants (cls1, cls2 and cls3) had elevated calcium contents but normal initial rates of Ca2+ uptake; type IV mutants (cls8, cls9, cls10 and cls11) had normal calcium contents but increased initial rates of Ca2+ uptake. Two of the mutants (cls7 and cls12) had intermediate biochemical properties. The primary defects of these four types of cls mutants were considered in terms of the Ca2+ transport system(s). Both type I and type III mutants, which had elevated calcium contents, simultaneously showed a trifluoperazine-sensitive phenotype, suggesting a close correlation of this phenotype with elevated calcium content. In addition, all type IV mutants were unable to utilize nonfermentable sugars. One CLS gene, CLS7, was located on the left arm of chromosome V.     

Phospholipid composition and phenotypic correction of an envC division mutant of Escherichia coli.

The cytoplasmic and outer membranes of a nonconditional chain-forming mutant, Escherichia coli PM61 envC, were separated by sucrose density gradient centrifugation. The phosphatidylglycerol/cardiolipin ratio in both membrane fractions was about one-third as high as in the parental strain P678. The increased level of cardiolipin in PM61 membranes is the result of an alteration of the polyglycerophosphatide cycle. It was found that the turnover rate of phosphatidylglycerol is more rapid in PM61 than in the parental strain but that its cardiolipin turnover is not significantly different. The envC mutation can be corrected phenotypically by increasing the osmolarity of the medium. In the presence of 0.6 M sucrose, the population of PM61 is composed of short rods, and the phosphatidylglycerol/cardiolipin ratio is shifted to that of the parent. The phosphatidylglycerol turns over more slowly, whereas the cardiolipin turns over more rapidly in both strains. Thus, the increase of external osmolarity acts on phospholipid metabolism as well as on an unknown step involved in the mechanism of cell division of the envC mutant.