Increase of ornithine amino lipid content in a sulfonolipid-deficient mutant of Cytophaga johnsonae

The gram-negative gliding bacterium Cytophaga johnsonae contains not only large quantities of unusual sulfonolipids but also, as we report here, a second class of unusual lipids. These lipids were detected and quantified by two-dimensional thin-layer chromatography of lipids from cells grown in the presence of [14C]acetate and shown by chemical studies to be alpha-N-(3-fatty acyloxy fatty acyl)ornithines. Like the sulfonolipids, these ornithine lipids were localized in the outer membrane (whereas phosphatidylethanolamine was the predominant lipid of the inner membrane). In a sulfonolipid-deficient mutant, the missing lipid was replaced, specifically, by an increased amount of ornithine lipid. Cells grown in liquid media contained predominantly ornithine lipids with nonhydroxylated residues in the O-fatty acyl position. In contrast, surface-grown cells contained a high proportion of ornithine lipids in which the O-fatty acyl group was 3-hydroxylated. The sulfonolipids and ornithine lipids are apparently coregulated in the sense that, regardless of perturbations caused by mutation or growth conditions, their total amounts remain constant at 40% of total cell lipid.     

Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae.

Cytophaga johnsonae displays many features that make it an excellent model of bacterial gliding motility. Unfortunately, genetic analyses of C. johnsonae, or any related gliding bacteria, were not possible because of a complete lack of selectable markers, cloning vectors, transposons, and convenient methods of gene transfer. As a first step toward a molecular analysis of gliding motility of C. johnsonae, we developed these genetic techniques and tools. Common broad-host-range plasmids and transposons did not function in C. johnsonae. We identified one Bacteroides transposon, Tn4351, that could be introduced into C. johnsonae on plasmid R751 by conjugation from Escherichia coli. Tn4351 inserted in the C. johnsonae genome and conferred erythromycin resistance. Tn-4351 insertions resulted in auxotrophic mutations and motility mutations. We constructed novel plasmids and cosmids for genetic analyses of C. johnsonae. These cloning vectors are derived from a small cryptic plasmid (pCP1) that we identified in the fish pathogen Cytophaga psychrophila D12. These plasmids contain the ermF (erythromycin resistance) gene from Tn4351 and a variety of features that facilitate propagation and selection in E. coli and conjugative transfer from E. coli to C. johnsonae.     

Temperate phages and bacteriocins of the gliding bacterium Cytophaga johnsonae

A collection of 30 independently isolated strains of Cytophaga johnsonae was screened for the presence of temperate bacteriophages. Two strains were found to harbour phages. The newly isolated phages differ in several respects from the 43 previously isolated phages for C. johnsonae. Both phages are polyhedral, approximately 60 nm in diameter, and have no apparent tail structure. They are chloroform sensitive, and plaque formation is inhibited by agar. Both are capable of establishing a stable association with host cells. Twenty-nine of the 30 strains produced diffusible substances that specifically inhibited the growth of other C. johnsonae strains or closely related species and that could not be propagated. These substances appear to be bacteriocins, some of which, like bacteriophages, are active only against motile cells, while other inhibit nonmotile as well as motile cells. One of each of these two types of bacteriocins was partially characterized and both were found to be proteinaceous in nature and bactericidal in effect.     

Structural characterization of the carbohydrate backbone of the lipopolysaccharide of Vibrio parahaemolyticus O-untypeable strain KX-V212 isolated from a patient

Vibrio parahaemolyticus strain KX-V212 of a novel serotype, which does not belong to any of the known 13 O-serotypes of this vibrio, was isolated from a patient. Its O-antigen harbors a unique strain-specific O-antigenic factor(s), in addition to that shared by the O-antigen of V. parahaemolyticus serotype O2. A carbohydrate backbone nonasaccharide was isolated from the lipopolysaccharide (LPS) of strain KX-V212 by dephosphorylation, reduction and deacylation and found to consist of one residue each of D-glucose, D-galactose, D-GlcN, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and 5-acetamido-7-(N-acetyl-D-alanyl)amino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (Non5Ac7Ala), and two residues each of D-GlcA and L-glycero-D-manno-heptose (LD-Hep). Analysis of the isolated and deacylated lipid A showed that this oligosaccharide was an artifact resulting from a loss of one GlcN residue from the lipid A backbone. Therefore, the carbohydrate backbone of the LPS is a decasaccharide having the structure shown below. The initial LPS contains also D-GalA and phosphoethanolamine at unknown positions. Both similarity and differences are observed between the LPS of V. parahaemolyticus serotype O2 and strain KX-V212. [carbohydrate structure: see text]     

Structure of a novel glucosamine-containing phosphoglycolipid from Deinococcus radiodurans

The structure of a major novel lipid from Deinococcus radiodurans has been determined to be 2'-O-(1,2-diacyl-sn-glycero-3-phospho)-3'-O-(alpha-N-acetylglucosaminyl) -N- glyceroyl alkylamine. The lipid was shown to contain a phosphatidic acid backbone by digestion with phospholipase A2 and by hydrolysis with hydrofluoric acid. Using a combination of chemical and NMR spectroscopic techniques, the structure of this lipid was elucidated and compared with that of a similar phosphoglycolipid reported earlier (Anderson, R., and Hansen, K. (1985) J. Biol. Chem. 260, 12219-12223) in which galactose was found in place of N-acetylglucosamine. The fatty acid compositions of the two lipids were similar.     

Phosphatidylglyceroylalkylamine, a novel phosphoglycolipid precursor in Deinococcus radiodurans.

We report here the structure of a previously uncharacterized phospholipid in the radiation-resistant bacterium Deinococcus radiodurans. This phospholipid, designated lipid 4, was shown by chemical analysis, HF hydrolysis, and nuclear magnetic resonance spectroscopy to be phosphatidylglyceroylalkylamine. Lipid 4 thus contains the unusual lipid constituents glyceric acid and alkylamines, which have previously been identified in two complex phosphoglycolipids from this organism. By [32P]phosphate pulse-chase labeling techniques, lipid 4 was shown to be the precursor of the complex phosphoglycolipids alpha-galactosyl- and alpha-N-acetylglucosaminylphosphatidylglyceroylalkylamine. While phosphatidylglyceroylalkylamine is rapidly biosynthesized from Pi, its subsequent glycosylation occurs much more slowly. Therefore, we conclude that the final glycosylation step is the rate-limiting event in the biosynthesis of the complex phosphoglycolipids alpha-galactosyl- and alpha-N-acetylglucosaminyl-phosphatidylglyceroylalkylamine.     

A novel lipid transfer protein from the dill Anethum graveolens L.: isolation,structure, heterologous expression,and functional characteristics

A novel lipid transfer protein, designated as Ag‐LTP, was isolated from aerial parts of the dill Anethum graveolens L. Structural, antimicrobial, and lipid binding properties of the protein were studied. Complete amino acid sequence of Ag‐LTP was determined. The protein has molecular mass of 9524.4 Da, consists of 93 amino acid residues including eight cysteines forming four disulfide bonds. The recombinant Ag‐LTP was overexpressed in Escherichia coli and purified. NMR investigation shows that the Ag‐LTP spatial structure contains four α ‐helices, forming the internal hydrophobic cavity, and a long C‐terminal tail. The measured volume of the Ag‐LTP hydrophobic cavity is equal to ~800 A³, which is much larger than those of other plant LTP1s. Ag‐LTP has weak antifungal activity and unpronounced lipid binding specificity but effectively binds plant hormone jasmonic acid. Our results afford further molecular insight into biological functions of LTP in plants. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

The major protein from lipid bodies of maize. Characterization and structure based on cDNA cloning

In plant seeds, the storage triacylglycerol is packed in discrete particles called lipid bodies which consist of a lipid core surrounded by a phospholipid monolayer with embedded proteins. We have cloned and sequenced a nearly full-length cDNA for the major protein (L3) associated with the lipid bodies of maize. The L3-cDNA clone was identified by hybrid-selected translation analysis and contains the complete 3' noncoding region and an open reading frame of 432 nucleotides. This open reading frame encodes a polypeptide with amino acid composition, hydrophobicity, and predicted protease digestion pattern which correlate well with those of the authentic L3 protein. Analyses of predicted secondary structure and local hydropathy of the deduced amino acid sequence suggest three structural domains in the protein. An internal domain of 72 contiguous hydrophobic or neutral amino acids is bounded at the amino-terminal side by a hydrophilic alpha-helix and on the carboxyl-terminal side by an amphipathic alpha-helix. The data suggest that L3 is uniquely suited to interact with both lipid and phospholipid moieties of the lipid body. A simple model for the topology of L3 on the lipid body is proposed. The unusual structure of the lipid body protein is discussed and compared to those of the two well-studied classes of lipid-associated proteins, apolipoproteins and intrinsic membrane proteins.     

The structure of the rough-type lipopolysaccharide from Shewanella oneidensis MR-1, containing 8-amino-8-deoxy-Kdo and an open-chain form of 2-acetamido-2-deoxy-D-galactose

The LPS from Shewanella oneidensis strain MR-1 was analysed by chemical methods and by NMR spectroscopy and mass spectrometry. The LPS contained no polysaccharide O-chain, and its carbohydrate backbone had the following structure: (1S)-GalNAco-(1-->4,6)-alpha-Gal-(1-->6)-alpha-Gal-(1-->3)-alpha-Gal-(1-P-3)-alpha-DDHep-(1-->5)-alpha-8-aminoKdo4R-(2-->6)-beta-GlcN4P-(1-->6)-alpha-GlcN1P, where R is P or EtNPP. There are several novel aspects to this LPS. It contains a novel linking unit between the core polysaccharide and lipid A moieties, namely 8-amino-3,8-dideoxy-D-manno-octulosonic acid (8-aminoKdo) and a residue of 2-acetamido-2-deoxy-D-galactose (N-acetylgalactosamine, GalNAco) in an open-chain form, linked as cyclic acetal to O-4 and O-6 of D-galactopyranose. The structure contains a phosphodiester linkage between the alpha-D-galactopyranose and D-glycero-D-manno-heptose (DDHep) residues.     

The structure of bacillomycin L, an antibiotic from Bacillus subtilis (author's transl)]

The structure of bacillomycin L, an antifungal agent isolated from the culture medium of a strain of Bacillus subtilis, has been investigated. The peptide moiety contains one mole each of D-aspartic acid, L-aspartic acid, L-glutamine, L-serine, D-serine, L-threonine, and D-tyrosine. The lipid moiety is a mixture of 3-amino-12-methyltridecanoic acid (46%), 3-amino-12-methyltetradecanoic acid (38%, 3-amino-14-methylpentadecanoic acid (11%), and two minor homologues. The peptide sequence and the cyclic structure were determined by structural analysis of the peptides obtained by mild acid hydrolysis. The molecular weight was determined by the thermoosmotic method; this showed that bacillomycin L has a monomeric structure which is given in Formula 1.     

The enzymic conversion of linoleic acid into 9-(nona-1′,3′-dienoxy)non-8-enoic acid, a novel unsaturated ether derivative isolated from homogenates of Solanum tuberosum tubers

1. A major component of the lipids in aqueous (pH7.5) homogenates of tuber tissue from Solanum tuberosum was isolated and characterized as 9-(nona-1',3'-dienoxy)non-8-enoic acid. 2. This novel unsaturated ether fatty acid derivative, which contains a butadienylvinyl ether function, has the structure: [Formula: see text] and is formed from linoleic acid by a sequence of enzymic reactions. 3. A precursor of the unsaturated ether derivative is 9-d-hydroperoxyoctadeca-10,12-dienoic acid, formed by the action of S. tuberosum lipoxygenase on linoleic acid. 4. An enzyme that converts the fatty acid hydroperoxide into the unsaturated ether derivative was isolated from S. tuberosum. The pH optimum of this enzyme is approx. 9, although the overall conversion of linoleic acid into the ether derivative is maximal at pH7.5. 5. An unusual feature of this pathway is the insertion of an oxygen atom into the alkyl chain of a fatty acid. 6. This novel mechanism may play a role in the breakdown of polyunsaturated fatty acids to volatile products in plants.     

Phosphonosphingoglycolipid,a novel sphingolipid from the viscera of Turbo cornutus

A novel lipid which contained long-chain base, fatty acid, galactose and N-methylaminoethylphosphonic acid in an equimolar was isolated from the viscera of Turbo cornutus.The methods used for the structural elucidation of this lipid were partial acid hydrolysis, alkaline hydrolysis, periodate oxidation and Smith degradation. The structure of breakdown products were mainly identified by combined gas chromatography and mass spectrometry.The structure of the novel lipid was determined to be 1-O-[6′-O-(N-methylaminoethylphosphonyl) galactopyranosyl] ceramide.Mass spectra of galactose-N-methylaminoethylphosphonate and glycerol-N-methylaminoethylphosphonate are given.     

Structure of human lysosomal membrane glycoprotein 1. Assignment of disulfide bonds and visualization of its domain arrangement

The amino acid sequence of one of the major lysosomal membrane glycoproteins, lysosome-associated membrane protein 1 (lamp-1), was deduced from its cDNA sequence (Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S. R. (1988) J. Biol. Chem. 263, 18920-18928). This amino acid sequence suggests that lamp-1 contains a hinge-like structure and could form disulfide bridges that are observed in the immunoglobulin superfamily. To test this possibility, we have determined the positions of the disulfide bridges by isolating and sequencing cystine-containing peptides which contain disulfide bridges. The results indicate that disulfide arrangement of lamp-1 is different from that of immunoglobulins. Each molecule contains, in total, four loops formed by disulfide bonds, and each loop contains 36-39 amino acid residues. However, none of the disulfide bonds connects two domains that are separated by a hinge-like structure. The results indicate that the hinge region has no ordered structure, and the relative positions of the two domains can be altered in space. Examination of the ultrastructure of lamp-1 by electron microscopy showed that the hinge-like structure actually functions as a hinge. These results indicate that the lamp-1 molecule represents a novel family of glycoproteins with unique structural properties.     

Isolation and Characterization of 2-Keto-3-Deoxyoctonate-Lipid A from a Heptose-Deficient Mutant of Escherichia coli

A heptose-deficient mutant of Escherichia coli has been isolated and from it a glycolipid, consisting of lipid A and 2-keto-3-deoxyoctonate (KDO), has been extracted with diisobutylketone-acetic acid-water. Based on beta-hydroxymyristic acid, the extractable glycolipid accounts for a major portion of the total lipid A in this mutant. A glycolipid, purified from the lipid extract by a combination of silicic acid and Sephadex LH-60 chromatography, contains glucosamine, phosphate, KDO, acetyl groups, and fatty acids in the following molar ratios: 1:2:2:1.7:5. These components account for over 80% of the lipid by weight. The fatty acid pattern of the glycolipid is typical of lipid A, the major component being beta-hydroxymyristic acid. The lipid also contains an amino sugar which appears to be 4-amino-4-deoxyarabinose. With the use of an ion-exchange paper chromatographic technique, gram-negative bacteria can be rapidly screened for the presence of this glycolipid. The mutant is believed to have a leaky defect in either biosynthesis of heptose or its incorporation into lipopolysaccharide. The lipopolysaccharide from the mutant contains only about a third as much heptose, glucose, and galactose as the parent CR34, a K-12 derivative. Chemical analysis and phage typing suggest that CR34 contains an incomplete core polysaccharide devoid of glucosamine.     

The structure of the core region of the lipopolysaccharide from Shewanella algae BrY, containing 8-amino-3,8-dideoxy-D-manno-oct-2-ulosonic acid

The structure of the carbohydrate backbone of the lipid A-core region of the LPS from Shewanella algae strain BrY was analysed. The LPS was N,O-deacylated to give three products, which were isolated and studied by chemical methods, NMR and mass spectrometry: [Carbohydrate structures: see text]. All monosaccharides except L-rhamnose had the D-configuration. This LPS presents a second example (after S. oneidensis) of the structure with a novel linking unit between the core and lipid A moieties, 8-amino-3,8-dideoxy-D-manno-oct-2-ulosonic acid (8-amino-Kdo).     

The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics

The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II-binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics.     

Chemical analysis of and degradation studies on the cell wall lipopolysaccharide of Rhodopseudomonas capsulata

A lipopolysaccharide (LPS) has been isolated from the gram-negative photosynthetic bacterium Rhodopseudomonas capsulata. Chemical analysis revealed the presence of d-glucose, d-galactose, l-rhamnose, 3-O-methyl-l-rhamnose (l-acofriose), d-glucosamine, 2-keto-3-deoxyoctonate, and neuraminic acid. The LPS does not contain l-glycero-d-mannoheptose, a typical component of the LPS of enteric bacteria. Fatty acid analysis showed that, apart from lauric acid, two hydroxy fatty acids (hydroxycaproic and hydroxymyristic acids) are the main components. By hydrolysis in weak acid, the LPS has been separated into a polysaccharide part (degraded polysaccharide) and a lipid part (lipid A). Presumably the lipid A contains a glucosamine backbone. Whereas the OH-groups of glucosamine are esterified with lauric and hydroxycaproic acids, hydroxymyristic acid is linked to the amino group of the sugar. By separation of the degraded polysaccharide by gel filtration, a fraction has been isolated which inhibited hemagglutination in a system containing antiserum, obtained by immunization of rabbits with whole cells, and isolated LPS. This fraction, which includes the determinant group, contains the sugars glucose, rhamnose, and acofriose. A second fraction obtained in this way was found to be serologically inactive and is composed of glucose, galactose, neuraminic acid, and phosphate.     

'Lipoteichoic acid' of Bifidobacterium bifidum subspecies pennsylvanicum DSM 20239. A lipoglycan with monoglycerophosphate side chains

The lipid macroamphiphile of Bifidobacterium bifidum subsp. pennsylvanicum DSM 20239 was extracted with phenol/water and purified by treatment with nucleases and hydrophobic interaction chromatography. From analytical data, the results of Smith degradation, hydrolysis with HF and methylation studies, the following structure is proposed: (formula; see text) where n and m are approximately 7-10 and 8-15, respectively. The monoglycerophosphate residues have the sn-glycero-1-phosphate configuration; 20-50% of them are substituted with L-alanine in ester linkage. The lipid anchor is most likely a galactosyldiacylglycerol, part of which carries a third fatty acid. This is the first example among gram-positive bacteria of a glycerophosphate-containing lipid macroamphiphile that carries the glycerophosphate residues as monomeric side chains on a lipoglycan. Further, it contains L-alanine in place of the D-alanine found in lipoteichoic acids.     

A Rhizobium leguminosarum AcpXL mutant produces lipopolysaccharide lacking 27-hydroxyoctacosanoic acid

The structure of the lipid A from Rhizobium etli and Rhizobium leguminosarum lipopolysaccharides (LPSs) lacks phosphate and contains a galacturonosyl residue at its 4' position, an acylated 2-aminogluconate in place of the proximal glucosamine, and a very long chain omega-1 hydroxy fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0). The 27OHC28:0 moiety is common in lipid A's among members of the Rhizobiaceae and also among a number of the facultative intracellular pathogens that form chronic infections, e.g., Brucella abortus, Bartonella henselae, and Legionella pneumophila. In this paper, a mutant of R. leguminosarum was created by placing a kanamycin resistance cassette within acpXL, the gene which encodes the acyl carrier protein for 27OHC28:0. The result was an LPS containing a tetraacylated lipid A lacking 27OHC28:0. A small amount of the mutant lipid A may contain an added palmitic acid residue. The mutant is sensitive to changes in osmolarity and an increase in acidity, growth conditions that likely occur in the nodule microenvironment. In spite of the probably hostile microenvironment of the nodule, the acpXL mutant is still able to form nitrogen-fixing root nodules even though the appearance and development of nodules are delayed. Therefore, it is possible that the acpXL mutant has a host-inducible mechanism which enables it to adapt to these physiological changes.