Involvement of Escherichia coli K-12 recombination functions in elimination of multicopy plasmid R6K by DNA-damaging agents

Abstract A neutral lipomannan has been isolated from the membranes of Micrococcus agilis . In contrast to the lipomannans from 2 strains of Micrococcus luteus , which contained succinic acid ranging from 5.1%–8.0%, the M. agilis lipomannan had no detectable succinyl residues and exhibited neutral behaviour on Concanavalin A-agarose rocket electrophoresis. As with the M. luteus lipomannans, mannose was the only sugar detectable (as alditol acetate) by GLC analysis in the purified M. agilis lipomannan. Fatty acids accounted for 2% of the M. agilis lipomannan and were predominantly C₁₅ branched-chain acids, with higher amounts of C₁₆ iso and C₁₇ anteiso than that found in the M. luteus polymers. Neither conditions of growth of the organism nor the method of membrane preparation appeared to be responsible for the absence of succinyl residues. This appears to be the first report of a neutral membrane amphiphile.     

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus.

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected. We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80 degrees C for 1 min, and purified by ultrafiltration and ethanol precipitation. The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem, Biophys. Res. Commun. 63, 875--800). A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB3H4. The polysaccharide was only slightly degraded (2%) by jack bean alpha-mannosidase and could precipitate 15 times its own weight of concanavalin A. The acidic polymers was also detected in the cell "periplasm" and was secreted from cells grown in defined media during the period of decelerating growth.     

An inositol containing lipomannan from Propionibacterium freudenreichii

Abstract A lipoglycan has been extracted from cells of Propionibacterium freudenreichii by the standard procedures used to isolate lipoteichoic acids from Gram-positive bacteria. The polymer was purified by chromatography and shown to contain mannose, inositol, glycerol, fatty acids and phosphate. The presence of the components of phosphatidylinositol suggests the lipoglycan may be a mannan anchored to the membrane by a covalently linked phosphatidylinositol although alternative structures cannot be excluded.     

Teichoic acids and lipids associated with the membrane of a Bacillus licheniformis mutant and the membrane lipids of the parental strain.

Bacillus licheniformis 6346 MH-1 and a phosphoglucomutase-deficient poorly lytic mutant, B. licheniformis 6346 MH-5, both contain cardiolipin, phosphatidyl ethanolamine, and phosphatidyl glycerol but are devoid of phosphoglycolipids. Gentiobiosyl diglyceride is present in the parent organism but glycolipids are absent from the mutant. Lipoteichoic acid was extracted from the whole cells of MH-5 with hot aqueous phenol and contained fatty acids, glucosamine, and 1,3-polyglycerol phosphate. The fatty acids were predominantly of the branched-chain type and were esterified to hydroxyl groups of a terminal glycerol residue. The polyglycerol phosphate chains contained, on average, 32 to 40 glycerol residues, some of which were substituted at the secondary hydroxyl group with alpha-N-acylglucosaminyl residues. Phenol extraction of the supernatant fluid that remained when walls were removed from preparations of disrupted cells of MH-5 yielded membrane teichoic acid, which consisted of substituted polyglycerol phosphate but was devoid of fatty acids.     

Use of Antibody to Membrane Adenosine Triphosphatase in the Study of Bacterial Relationships

An antiserum to Ca(2+)-activated adenosine triphosphatase from membranes of Micrococcus lysodeikticus cross-reacted in agar gels with membrane adenosine triphosphatases from other pigmented micrococci and related species. Species of Micrococcus and Sarcina showed different levels of inhibition of adenosine triphosphatase activities in heterologous reactions with antiserum. Inter- and intraspecific relationships based on the inhibition reaction were compared with an independent parameter, namely the quantitative and qualitative composition of the bacterial membrane phospholipids and fatty acids. The guanine plus cytosine contents in the deoxyribonucleic acid of the species studied correlated well with the serological cross-reactivity of adenosine triphosphatases from their membranes. The types of cross-bridges found in the peptidoglycans of these cocci were also compared with the other properties. The results suggest that an antiserum specific for a major membrane protein may be a reliable and most useful adjunct in studying bacterial serotaxonomy.     

Distribution of enzymes involved in mannan synthesis in plasma membranes and mesosomal vesicles of Micrococcus lysodeikticus.

The distribution of membrane-bound enzymes involved in mannan biosynthesis in plasma and mesosomal membranes of Micrococcus lysodeikticus has been investigated. Isolated mesosomal vesicles, unlike plasma membrane preparations, cannot catalyze the transfer of [14C]mannose from GDP-[14C]mannose into mannan. This appears to result from the inability of this membrane system to synthesize the carrier lipid [14C]mannosyl-1-phosphorylundecaprenol. In contrast, this is the major mannolipid synthesized from GDP-[14C]mannose by isolated plasma membranes. The possibility that substrate inaccessibility could account for the failure to detect the enzyme in isolated mesosomal vesicles appears unlikely from the lack of activity following disruption of the vesicles with ultrasound or with surface active agents. Both membrane preparations possessed the ability to catalyse the transfer of [14C]mannose from purified [14C]mannosyl-1-phosphorylundecaprenol into mannan. Furthermore, free mannan and mannan located on both unlabeled mesosomal and unlabeled plasma membranes could act as acceptors of [14C]mannosyl units from 14C-labeled carrier lipid located in prelabeled plasma membranes. The possibility that the juxtaposition of mesosomal vesicles and enveloping plasma membrane (i.e. the mesosomal sacculus) in vivo allows mannan, located on mesosomal vesicles, to accept mannosyl units from carrier lipid located in the sacculus membrane is discussed.     

Inhibition of cardiolipin synthesis by end-products and other complex lipids in membrane preparations of Micrococcus lysodeikticus.

Using membrane preparations of Micrococcus lysodeikticus, the end-products of cardiolipin synthesis, cardiolipin and glycerol, were shown to inhibit cardiolipin synthetase at several concentrations. Other phospholipids tested for inhibitory effects, phosphatidyl-ethanolamine, phosphatidylinositol, and phosphatidic acid were also shown to inhibit cardiolipin synthesis. Phosphatidic acid was considerably more inhibitory than cardiolipin, phosphatidylethanolamine was similar to cardiolipin, and phosphatidylinositol less inhibitory at the same concentrations. A non-phosphate-containing glycolipid was also inhibitory. In contrast, glycerophosphate had no effect on cardiolipin synthesis.     

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected.We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80°C for 1 min, and purified by ultrafiltration and ethanol precipitation.The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem. Biophys. Res. Commun. 63, 875–880).A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB³H₄. The polysaccharide was only slightly degraded (2%) by jack bean α-mannosidase and could precipitate 15 times its own weight of concanavalin A.The acidic polymer was also detected in the cell “periplasm” and was secreted from cells grown in defined media during the period of decelerating growth.     

Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannan and lipomannan of Mycobacterium tuberculosis

The recent availability (Hunter, S.W., Gaylord, H., and Brennan, P.J. (1986) J. Biol. Chem. 261, 12345-12351) of the well known arabinomannan of Mycobacterium leprae and Mycobacterium tuberculosis as the pure native lipoarabinomannan has resulted in its implication in key aspects of the immunopathogenesis of leprosy and tuberculosis. We had indicated that the lipid moiety of lipoarabinomannan is probably based on a diacylglycerol unit in that glycerol and the two fatty acids, hexadecanoate and 10-methyloctadecanoate, were identified. In addition, lipoarabinomannan was also shown to contain myo-inositol 1-phosphate. Evidence is now presented, based on selective radiolabeling and analysis of various cleavage fragments, that the inositol phosphate exists as both an alkalilable phosphodiester and as part of a phosphatidylinositol "membrane anchor." The mannan of M. tuberculosis was also isolated as the native lipomannan. It also apparently contains a phosphatidylinositol unit but is devoid of the alkali-labile inositol phosphate residues. These lipopolysaccharides are apparently multiglycosylated versions of the well known myocobacterial mannosyl phosphatidylinositols and are prokaryotic versions of the growing list of phosphatidylinositol-anchored macromolecules. Immunogold labeling demonstrates that lipoarabinomannan is a true antigenic capsular or extracellular product of M. tuberculosis. The presence of a phosphatidylinositol residue on lipoarabinomannan may explain its interaction with macrophage membranes and role in mycobacterial pathogenesis.     

Lipid composition of the electron transport membrane of Haemophilus parainfluenzae

The principal lipids associated with the electron transport membrane of Haemophilus parainfluenzae are phosphatidylethanolamine (78%), phosphatidylmonomethylethanolamine (0.4%), phosphatidylglycerol (18%), phosphatidylcholine (0.4%), phosphatidylserine (0.4%), phosphatidic acid (0.2%), and cardiolipin (3.0%). Phospholipids account for 98.4% of the extractible fatty acids. There are no glycolipids, plasmalogens, alkyl ethers, or lipo amino acid esters in the membrane lipids. Glycerol phosphate esters derived from the phospholipids by mild alkaline methanolysis were identified by their staining reactions, mobility on paper and ion-exchange column chromatography, and by the molar glycerol to phosphate ratios. Eleven diacyl phospholipids can be separated by two-dimensional thin-layer chromatography. Each lipid served as a substrate for phospholipase D, and had a fatty acid to phosphate ratio of 2:1. Each separated diacyl phospholipid was deacylated and the glycerol phosphate ester was identified by paper chromatography in four solvent systems. Of the 11 separated phospholipids, 3 were phosphatidylethanolamines, 2 were phosphatidylserines, and 2 were phosphatidylglycerols. Phosphatidylcholine, cardiolipin, and phosphatidic acid were found at a single location. Phosphatidylmonomethylethanolamine was found with the major phosphatidylethanolamine. Three distinct classes of phospholipids are separable according to their relative fatty acid compositions. (i) The trace lipids consist of two phosphatidylethanolamines, two phosphatidylserines, phosphatidylcholine, phosphatidic acid, and a phosphatidylglycerol. Each lipid represents less than 0.3% of the total lipid phosphate. These lipids are characterized by high proportions of the short (C(10) to C(14)) and long (C(19) to C(22)) fatty acids with practically no palmitoleic acid. (ii) The major phospholipids (93% of the lipid phosphate) are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylglycerol. These lipids contain a low proportion of the short (C(19)) fatty acids. Palmitic and palmitoleic acids represent over 80% of the total fatty acids. (iii) The fatty acid composition of the cardiolipin is intermediate between the other two classes. Both palmitoleic and the longer fatty acids represent a significant proportion of the total fatty acid.     

The linkage of sugar phosphate polymer to peptidoglycan in walls of Micrococcus sp. 2102.

1. Protein-free walls of Micrococcus sp. 2102 contain peptidoglycan, poly-(N-acetylglucosamine 1-phosphate) and small amounts of glycerol phosphate. 2. After destruction of the poly-(N-acetylglucosamine 1-phosphate) with periodate, the glycerol phosphate remains attached to the wall, but can be removed by controlled alkaline hydrolysis. The homogeneous product comprises a chain of three glycerol phosphates and an additional phosphate residue. 3. The poly-(N-acetylglucosamine 1-phosphate) is attached through its terminal phosphate to one end of the tri(glycerol phosphate). 4. The other end of the glycerol phosphate trimer is attached through its terminal phosphate to the 3-or 4-position of an N-acetylglucosamine. It is concluded that the sequence of residues in the sugar 1-phosphate polymer-peptidoglycan complex is: (N-acetylglucosamine 1-phosphate)24-(glycerol phosphate)3-N-acetylglucosamine 1-phosphate-muramic acid (in peptidoglycan). Thus in this organism the phosphorylated wall polymer is attached to the peptidoglycan of the wall through a linkage unit comprising a chain of three glycerol phosphate residues and an N-acetylglucosamine 1-phosphate, similar to or identical with the linkage unit in Staphylococcus aureus H.     

Structure of a Micrococcus lysodeikticus cell wall fragment containing phosphorylated sugars.

A polysaccharide-peptidoglycan complex containing different phosphorylated sugars from Micrococcus lysodeikticus cell wall has been isolated and purified. The peptidoglycan contained muramic acid 6-phosphate and N-acetylglucosamine 6-phosphate as phosphorylated sugars in addition to other sugar residues. Mild acid hydrolysis of the peptidoglycan and subsequent reduction of the released polysaccharide showed therein the presence of glucose and N-acetyl-glucosamine in the linkage of the external polysaccharide residues to the peptidoglycan through phosphodiester linkage. These data suggest the presence of polysaccharide chains linked to a peptidoglycan core through two phosphorylated sugars via two different terminal carbohydrate residues of the external polysaccharide chains in a same polymer.     

Visualizing complexes of phospholipids with Streptomyces phospholipase D by automated docking

The automated docking program AutoDock was used to dock nine phosphatidic acids (PAs), six phosphatidylcholines, five phosphatidylethanolamines, four phosphatidylglycerols, one phosphatidylinositol and two phosphatidylserines, which have two identical saturated fatty acid residues with an even numbers of carbon atoms, onto the active site of Streptomyces sp. PMF phospholipase D (PLD). Two PAs with one double bond on the fatty acid chain linked to the C2 of the glycerol residue were also docked. In general, binding energies become progressively more negative as fatty acid residues become longer. When these residues are of sufficient length, one is coiled against a hydrophobic cliff in a well that also holds the glycerol and phosphate residues and the head group, while the other generally is bound by a hydrophobic surface outside the well. Phosphatidylcholines have the only head group that is firmly bound by the active site, giving a possible structural explanation for the low selectivity of Streptomyces PLD for other phospholipid substrates.     

In vitro synthesis of the unit that links teichoic acid to peptidoglycan.

The role of cytidine diphosphate (CDP)-glycerol in gram-positive bacteria whose walls lack poly(glycerol phosphate) was investigated. Membrane preparations from Staphylococcus aureus H, Bacillus subtilis W23, and Micrococcus sp. 2102 catalyzed the incorporation of glycerol phosphate residues from radioactive CDP-glycerol into a water-soluble polymer. In toluenized cells of Micrococcus sp. 2102, some of this product became linked to the wall. In each case, maximum incorporation of glycerol phosphate residues required the presence of the nucleotide precursors of wall teichoic acid and of uridine diphosphate-N-acetylglucosamine. In membrane preparations capable of synthesizing peptidoglycan, vancomycin caused a decrease in the incorporation of isotope from CDP-glycerol into polymer. Synthesis of the poly (glycerol phosphate) unit thus depended at an early stage on the concomitant synthesis of wall teichoic acid and later on the synthesis of peptidoglycan. It is concluded that CDP-glycerol is the biosynthetic precursor of the tri(glycerol phosphate) linkage unit between teichoic acid and peptidoglycan that has recently been characterized in S. aureus H.     

Study of membrane proteins from Microccus lysodeikticus using immunochemical methods]

Using immunoelectrophoresis, the antigenicity of various protein fractions of the Micrococcus lysodeikticus membranes was evaluated. It was shown that both the peripheral and integral membrane proteins possess the antigenic determinants. The antiserum exhausted by the M. lysodeikticus mebranes loses its ability to interact with intergral proteins, which are not solubilized by Triton X-100. It was thus assumed that the integral proteins are exposed on the membrane surface constantly or periodically and that there exist no proteins which are completely and permanently incorporated into the lipid bilayer. The respiratory chain of the M. lysodeikticus membrane is inhibited by membrane immunoglobulins by 50%. This is probably due to the presence in the membrane antiserum of antibodies specific to the respiratory chain enzymes. Evidence for this assumption can be derived from the fact that partially purified cytochrome b556 forms a precipitation zone with the membrane antiserum and that the activity of membrane NADH-dehydrogenase is inhibited by a monoserum against NADH-dehydrogenase.     

Quantitative determination of phospholipids using the dyes Victoria blue R and B

Different phospholipids, except the choline-containing phospholipids phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, formed complexes with the dye Victoria blue R, which selectively partitioned into the chloroform phase of chloroform/ethylene glycol/glycerol biphasic solvent system, and were quantitatively estimated at 590 nm. Considerable amounts of water, alcohols, nonlipid phosphates, neutral lipids, free fatty acids, and some detergents did not interfere with the formation of phospholipid-dye complexes. This special advantage of the method described allowed combined phospholipid extraction and estimation procedures in one test tube. Because of its high sensitivity (about 24.00 OD units/mumol of phosphatidic acid and about 10.25 OD units/mumol of other phospholipids), specificity, and simplicity, the proposed phospholipid assay appears to be very useful for rapid analyses of lipid extracts as well as TLC spots or suspensions of biological materials, as demonstrated for membranes and cells of Micrococcus lysodeikticus. The applicability of the dye Victoria blue B to the quantitative determination of phospholipids, except phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, at 605 nm using chloroform/ethylene glycol/glycerol/water and pentane (hexane)/ethyl acetate/isopropanol/water biphasic solvent systems with similar sensitivities and of sodium dodecyl sulfate in the pentane-containing system with high sensitivity (22.96 OD units/mumol) is also shown. The adaptation of this phospholipid assay to the determination of phospholipases C and D and to the differential quantitation of choline-containing phospholipids using additional phospholipid estimation techniques is discussed.     

Importance of the O6 position of guanine residues in the binding of DNA methylase to DNA

Methylation of Micrococcus lysodeikticus DNA by purified DNA methylase isolated from L1210 leukaemia cells is potently and specifically inhibited by both hetero and homoribo and deoxyribopolynucleotides containing guanine residues. The inhibitory effect is unaffected by chain length, but is abolished when the O6 residue of guanine is substituted as in poly[d(O6MeG)]20. Potent inhibition is also shown by polyinosinic and polyxanthylic acids, but not by polyadenylic acid or by heteropolymers containing adenine and thymine. These results suggest that the 6-position of the purine nucleus is important in binding of the DNA methylase to a particular region of the DNA duplex and that the hydrogen bonding properties of this group are important in enzyme recognition.     

Alternative lipid remodelling pathways for glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins of Saccharomyces cerevisiae exist with two types of lipid moiety--diacylglycerol or ceramide--both of which contain 26:0 fatty acids. To understand at which stage of biosynthesis these long-chain fatty acids become incorporated into diacylglycerol anchors, we compared the phosphatidylinositol moieties isolated from myo-[2-(3)H]inositol-labelled protein anchors and from GPI intermediates. There is no evidence for the presence of long-chain fatty acids in any intermediate of GPI biosynthesis. However, GPI-anchored proteins contain either the phosphatidylinositol moiety characteristic of the precursor lipids or a version with a long-chain fatty acid in the sn-2 position of glycerol. The introduction of long-chain fatty acids into sn-2 occurs in the endoplasmic reticulum (ER) and is independent of the sn-2-specific acyltransferase SLC1. Analysis of ceramide anchors revealed the presence of two types of ceramide, one added in the ER and another more polar molecule which is found only on proteins which have reached the mid Golgi. In summary, the lipid of GPI-anchored proteins can be exchanged by at least three different remodelling pathways: (i) remodelling from diacylglycerol to ceramide in the ER as proposed previously; (ii) remodelling from diacylglycerol to a more hydrophobic diacylglycerol with a long-chain fatty acid in sn-2 in the ER; and (iii) remodelling to a more polar ceramide in the Golgi.     

Membrane-reversible H+-ATPase from Micrococcus lysodeikticus.

Treatment of phosphorylating fragments of bacterial membrane from Micrococcus lysodeikticus with trypsin leads to increase ATPase activity. As a result of this treatment, the membrane fragments acquire the ability to transform the ATP energy into transmembrane difference in potential. Dithiothreitol has a similar effect to that of trypsin on the membrane fragments from M. lysodeikticus. Dicyclohexylcarbodimide inhibits ATPase of the membrane fragments of M. lysodeikticus, and also the ATPase-reaction-coupled generation of membrane potential. It has been suggested that the increased ATPase activity of membranes from M. lysodeikticus during treatment with trypsin and dithiothreitol is connected with the effect of these agents on the protein inhibitor of ATPase.     

Occurrence of 9- and 13-keto-octadecadienoic acid in biological membranes oxygenated by the reticulocyte lipoxygenase

Membranes of intact rabbit reticulocytes and rat liver mitochondrial membranes oxygenated by the pure reticulocyte lipoxygenase contain 13-keto-9Z,11E-octadecadienoic acid and 9-keto-10E,12Z-octadecadienoic acid. In mitochondrial membranes not treated with lipoxygenase and in rabbit erythrocyte membranes these products were not detected. The chemical structure of the compounds has been identified by cochromatography with authentic standards on various types of HPLC columns, by uv and ir spectroscopy and GC/MS. In the membranes of rabbit reticulocytes up to 2% of the linoleate residues are present as its 9- and 13-keto derivatives. Most of the keto compounds (up to 90%) are esterified in the membrane ester lipids, only about 10% were found in the free fatty acid fraction. It is proposed that the keto dienoic fatty acids are formed via decomposition of hydroperoxy polyenoic fatty acids originating from the oxygenation of the membrane lipids by the reticulocyte lipoxygenase.