Effect of 3,4-dihydroxybutyl-1-phosphonate on cardiolipin synthesis in B. subtilis

Endogenous phosphatidylglycerol is rapidly transformed into cardiolipin when B. subtilis 168 cells were incubated in a buffer without an energy source. Upon addition of 3,4-dihydroxybutyl-1-phosphonate (DHBP), a synthetic glycerol 3-phosphate analogue, this synthesis was completely blocked after a short lag; if the cells were grown in the presence of the analogue, there was no lag. When membrane fractions were incubated with exogenous [32P]phosphatidylglycerol, free DHBP and glycerol 3-phosphate had no effect on [32P]cardiolipin synthesis, but phosphatidyl-DHBP and phosphatidylglycerolphosphate were potent inhibitors. These results are consistent with our hypothesis that phosphatidylglycerolphosphate, the phosphatidylglycerol precursor, might also be a physical inhibitor of cardiolipin synthesis.     

Biosynthesis of murein lipoprotein in Escherichia coli: effects of 3,4-dihydroxybutyl-1-phosphonate.

The effects of 3,4-dihydroxybutyl-1-phosphonate, a four-carbon analog of sn-glycerol 3-phosphate, on the biosynthesis of the glyceryl moiety in murein lipoprotein of Escherichia coli were studied. The compound at a concentration of 55 microM strong inhibits in the incorporation of [2-3H]glycerol radioactivity into lipoprotein by virtue of its inhibition of the synthesis of phosphatidylglycerol. On the other hand, the incorporation of prelabeled [2-3H]glycerol radioactivity into lipoprotein was only partially inhbited by 3,4-dihydroxybutyl-1-phosphonate even at a much higher concentration (1 mM). These data were consistent with the postulated pathway for the biosynthesis of the lipid moiety in lipoportein: cysteine-lipoprotein + phosphatidylglycerol leads to glycerylcystein-lipoprotein + phosphatidic acid.     

Function of phospholipids in Escherichia coli. Characterization of a mutant deficient in cardiolipin synthesis.

Screening of a collection of temperature-sensitive mutants of Escherichia coli for defects in phospholipid metabolism led to the isolation of a mutant deficient in cardiolipin synthesis. The defective gene, named cls, is closely linked to the trp marker and maps at about Minute 27 on the E. coli chromosome. After transfer of cls to a defined genetic background by transduction, the mutant has the following properties as compared to an isogenic wild type. Exponentially growing cells show a reduction in cardiolipin content by a factor of at least 15 (less than 0.2 mol % of the total phospholipids). A crude membrane fraction derived from the mutant is unable to synthesize cardiolipin from phosphatidylglycerol in vitro. The mutant has no distinctive phenotype regarding its growth properties, membrane-associated respiratory functions, or the ability to insert bacteriophage M13 coat protein into the cell envelope. The cls mutation confers a 5-times reduction in the turnover of the phosphate moiety of phosphatidylglycerol.     

Composition of cardiolipin molecular species in Escherichia coli.

The composition of the molecular species of acidic phospholipids in Escherichia coli B during the late exponential growth phase at 37 degrees C was determined. Two phosphatidyl groups of cardiolipin, the 3-(3-sn-phosphatidyl) and 1-(3-sn-phosphatidyl) moieties of cardiolipin, were isolated by limited hydrolysis with phospholipase C. No significant difference in the composition of the molecular species was found between the 3-(3-sn-phosphatidyl) and 1-(3-sn-phosphatidyl) moieties. On the other hand, the composition of the molecular species of phosphatidylglycerol was different from that of cardiolipin. Phosphatidylglycerol contained more of the 1-palmitoyl 2-cis-9,10-methylenehexadecanoyl and 1-palmitoyl 2-cis-11,12-methyleneoctadecanoyl species than did cardiolipin. The difference in the composition of the molecular species between cardiolipin and phosphatidylglycerol may depend on the difference in the turnover rates of both phospholipids.     

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.     

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.     

Further studies on the cardiolipin phosphodiesterase of Escherichia coli.

The cardiolipin phosphodiesterase of Escherichia coli was further characterized. This enzyme has a pH optimum of 7.0 and is Mg2+ dependent. Mn2+ and Co2+ could replace Mg2+ but other divalent cations were inhibitory or without effect. The enzyme is not periplasmic and does not appear to be associated with membrane fractions prepared by different methods. It is recovered as a soluble protein in the cytosol fraction but could not be readily purified because of its instability. With cell-free systems, a requirement for ATP or ADP could be shown under certain defined conditions. Other nucleotides were less effective or ineffective in stimulating the phosphodiesterase. The cells displayed the highest activity during the middle to late exponential stage but no marked requirement for ATP was apparent when the phosphodiesterase was obtained from such freshly grown cells. If, however, cells were starved for several hours in saline medium, the cardiolipin phosphodiesterase level fell and a requirement for added ATP could be shown. The cardiolipin phosphodiesterase is an enzyme distinct from cardiolipin synthase. The assay conditions are quite different from each of these enzymes as are their subcellular distributions.     

A pleiotropic defect of membrane synthesis in a thermosensitive mutant tsC42 of Escherichia coli.

Synthesis of membrane proteins in a thermosensitive mutant of Escherichia coli K12, tsC42, that has a defect in a mechanism of cell cycle-dependent duplication of membrane enzymes was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The cells were labeled differentially with [14C]- and [3H]arginine and the membrane proteins synthesized at nonpermissive and permissive temperatures were compared. The results showed that at the nonpermissive temperature, the syntheses of cytoplasmic membrane proteins and outer membrane proteins were reduced more than 70% and 50%, respectively. No significant accumulation of precursor molecules of membrane proteins at the nonpermissive temperature was detected in pulse-chase experiments. It is therefore assumed that the mutant has a defect in a gene that regulates the biosynthesis of many membrane proteins.     

Escherichia coli minicell membranes are enriched in cardiolipin

The phospholipid composition of Escherichia coli minicells has been studied as a model for the cell division site. Minicells appeared to be enriched in cardiolipin at the expense of phosphatidylglycerol. Mass spectrometry showed no differences between the gross acyl chain compositions of minicells and wild-type cells.     

Complementation of the xeroderma pigmentosum DNA repair synthesis defect with Escherichia coli UvrABC proteins in a cell-free system.

A newly developed cell-free system was used to study DNA repair synthesis carried out by extracts from human cell lines in vitro. Extracts from a normal human lymphoid cell line and from cell lines established from individuals with hereditary dysplastic nevus syndrome perform damage-dependent repair synthesis in plasmid DNA treated with cis- or trans-diamminedichloro-platinum(II) or irradiated with ultraviolet light. Cell extracts of xeroderma pigmentosum origin (complementation groups A, C, D, and G) are deficient in DNA repair synthesis. When damaged plasmid DNA was pretreated with purified Escherichia coli UvrABC proteins, xeroderma pigmentosum cell extracts were able to carry out DNA repair synthesis. The ability of E. coli UvrABC proteins to complement xeroderma pigmentosum cell extracts indicates that the extracts are deficient in incision, but can carry out later steps of repair. Thus the in vitro system provides results that are in agreement with the incision defect found from studies of xeroderma pigmentosum cells.     

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.     

Transport of 3,4-dihydroxybutyl-1-phosphonate, an analogue of sn-glycerol 3-phosphate.

3,4-Dihydroxybutyl-1-phosphonate (DHBP), an analogue of glycerol 3-phosphate, is actively transported by the sn-glycerol 3-phosphate transport system of Escherichia coli strain 8. The Km for the transport of DHBP is 200 microM.     

Vinylglycolate resistance in Escherichia coli.

Escherichia coli K-12 vinylglycolate-resistant mutants have been isolated and characterized. Two of the mutants, JSH 150 and JSH 151, have been determined to be double mutants, lacking both membrane-bound L-and D-lactate dehydrogenases. The lactate transport system is intact in all strains; both radioactive lactate and vinylglycolate are actively taken up. Likewise, the phosphoenolypyruvate-dependent phosphotransferase system for hexose uptake is active. Vinylglycolate, previously shown to inhibit the phosphoenolpyruvate-dependent phosphotransferase system, has very little effect in the double mutants. The extent of vinylglycolate inhibition in other mutants seems directly related to the activity of the lactate dehydrogenases. This indicates that vinylglycolate is oxidized to 2-keto-3-butenoate before inactivating the phosphoenolpyruvate-dependent phosphotransferase system. These results were found in whole cells and confirmed in isolated membrane vesicles.     

Beta-alanine synthesis in Escherichia coli.

The enzyme, aspartate 1-decarboxylase (L-aspartate 1-carboxy-lyase; EC 4.1.1.15), that catalyzes the reaction aspartate leads to beta-alanine + CO2 was found in extracts of Escherichia coli. panD mutants of E. coli are defective in beta-alanine biosynthesis and lack aspartate 1-decarboxylase. Therefore, the enzyme functions in the biosynthesis of the beta-alanine moiety of pantothenate. The genetic lesion in these mutants is closely linked to the other pantothenate (pan) loci of E. coli K-12.     

Characterization of the defect in the Escherichia coli mutT1 mutator gene.

With a probe constructed from the wild-type gene, a DNA fragment containing the entire mutT1 mutator gene was isolated and cloned into pUC18. Nucleotide sequence analysis revealed that the mutator defect was most likely due to an IS1 insertion into the wild-type gene.     

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.     

Tiamulin resistance mutations in Escherichia coli.

Forty "two-step" and 13 "three-step" tiamulin-resistant mutants of Escherichia coli PR11 were isolated and tested for alteration of ribosomal proteins. Mutants with altered ribosomal proteins S10, S19, L3, and L4 were detected. The S19, L3, and L4 mutants were studied in detail. The L3 and L4 mutations did not segregate from the resistance character in transductional crosses and therefore seem to be responsible for the resistance. Extracts of these mutants also exhibited an increased in vitro resistance to tiamulin in the polyuridylic acid and phage R17 RNA-dependent polypeptide synthesis systems, and it was demonstrated that this was a property of the 50S subunit. In the case of the S19 mutant, genetic analysis showed segregation between resistance and the S19 alteration and therefore indicated that mutation of a protein other than S19 was responsible for the resistance phenotype. The isolated ribosomes of the S19, L3, and L4 mutants bound radioactive tiamulin with a considerably reduced strength when compared with those of wild-type cells. The association constants were lower by factors ranging from approximately 20 to 200. When heated in the presence of ammonium chloride, these ribosomes partially regained their avidity for tiamulin.