MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli

MsbA is an essential ABC transporter in Escherichia coli required for exporting newly synthesized lipids from the inner to the outer membrane. It remains uncertain whether or not MsbA catalyzes trans-bilayer lipid movement (i.e. flip-flop) within the inner membrane. We now show that newly synthesized lipid A accumulates on the cytoplasmic side of the inner membrane after shifting an E. coli msbA missense mutant to the non-permissive temperature. This conclusion is based on the selective inhibition of periplasmic, but not cytoplasmic, covalent modifications of lipid A that occur in polymysin-resistant strains of E. coli. The accessibility of newly synthesized phosphatidylethanolamine to membrane impermeable reagents, like 2,4,6-trinitrobenzene sulfonic acid, is also reduced severalfold. Our data showed that MsbA facilitates the rapid translocation of some lipids from the cytoplasmic to the periplasmic side of the inner membrane in living cells

Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC: Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants

The lpcC gene of Rhizobium leguminosarum and the lpsB gene of Sinorhizobium meliloti encode protein orthologs that are 58% identical over their entire lengths of about 350 amino acid residues. LpcC and LpsB are required for symbiosis with pea and Medicago plants, respectively. S. meliloti lpsB complements a mutant of R. leguminosarum defective in IpcC, but the converse does not occur. LpcC encodes a highly selective mannosyl transferase that utilizes GDP-mannose to glycosylate the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide precursor Kdo2-lipid IVA. We now demonstrate that LpsB can also efficiently mannosylate the same acceptor substrate as does LpcC. Unexpectedly, however, the sugar nucleotide selectivity of LpsB is greatly relaxed compared with that of LpcC. Membranes of the wild-type S. meliloti strain 2011 catalyze the glycosylation of Kdo2-[4′-32P]lipid IVA at comparable rates using a diverse set of sugar nucleotides, including GDP-mannose, ADP-mannose, UDP-glucose, and ADP-glucose. This complex pattern of glycosylation is due entirely to LpsB, since membranes of the S. meliloti lpsB mutant 6963 do not glycosylate Kdo2-[4′-32P]lipid IVA in the presence of any of these sugar nucleotides. Expression of lpsB in E. coli using a T7lac promoter-driven construct results in the appearance of similar multiple glycosyl transferase activities seen in S. meliloti 2011 membranes. Constructs expressing lpcC display only mannosyl transferase activity. We conclude that LpsB, despite its high degree of similarity to LpcC is a much more versatile glycosyltransferase, probably accounting for the inability of lpcC to complement S. meliloti lpsB mutants. Our findings have important implications for the regulation of core glycosylation in S. meliloti and other bacteria containing LpcC orthologs.Facultad de Ciencias Exacta

Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC: Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants

The lpcC gene of Rhizobium leguminosarum and the lpsB gene of Sinorhizobium meliloti encode protein orthologs that are 58% identical over their entire lengths of about 350 amino acid residues. LpcC and LpsB are required for symbiosis with pea and Medicago plants, respectively. S. meliloti lpsB complements a mutant of R. leguminosarum defective in IpcC, but the converse does not occur. LpcC encodes a highly selective mannosyl transferase that utilizes GDP-mannose to glycosylate the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide precursor Kdo2-lipid IVA. We now demonstrate that LpsB can also efficiently mannosylate the same acceptor substrate as does LpcC. Unexpectedly, however, the sugar nucleotide selectivity of LpsB is greatly relaxed compared with that of LpcC. Membranes of the wild-type S. meliloti strain 2011 catalyze the glycosylation of Kdo2-[4′-32P]lipid IVA at comparable rates using a diverse set of sugar nucleotides, including GDP-mannose, ADP-mannose, UDP-glucose, and ADP-glucose. This complex pattern of glycosylation is due entirely to LpsB, since membranes of the S. meliloti lpsB mutant 6963 do not glycosylate Kdo2-[4′-32P]lipid IVA in the presence of any of these sugar nucleotides. Expression of lpsB in E. coli using a T7lac promoter-driven construct results in the appearance of similar multiple glycosyl transferase activities seen in S. meliloti 2011 membranes. Constructs expressing lpcC display only mannosyl transferase activity. We conclude that LpsB, despite its high degree of similarity to LpcC is a much more versatile glycosyltransferase, probably accounting for the inability of lpcC to complement S. meliloti lpsB mutants. Our findings have important implications for the regulation of core glycosylation in S. meliloti and other bacteria containing LpcC orthologs.Facultad de Ciencias Exacta

Identification and quantification of dolichol and dolichoic acid in neuromelanin from substantia nigra of the human brain.

Neuromelanin (NM) isolated from the substantia nigra of the human brain is found to contain a series of dolichoic acids (dol-CA) containing 14–20 isoprene units. This is the first observation of dol-CA in a natural system. Using internally spiked nor-dolichol and nor-dolichoic acid standards, the concentrations of dolichol (dol) and dol-CA present in NM were determined. Remarkably, dol was only four times as abundant as dol-CA in NM. The distribution of dol-CA chains lengths in NM also differed from that of dol, suggesting that the enzyme(s) responsible for the conversion of dol to dol-CA prefer a dolichol substrate containing 19 isoprene units

Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC: Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants

The lpcC gene of Rhizobium leguminosarum and the lpsB gene of Sinorhizobium meliloti encode protein orthologs that are 58% identical over their entire lengths of about 350 amino acid residues. LpcC and LpsB are required for symbiosis with pea and Medicago plants, respectively. S. meliloti lpsB complements a mutant of R. leguminosarum defective in IpcC, but the converse does not occur. LpcC encodes a highly selective mannosyl transferase that utilizes GDP-mannose to glycosylate the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide precursor Kdo2-lipid IVA. We now demonstrate that LpsB can also efficiently mannosylate the same acceptor substrate as does LpcC. Unexpectedly, however, the sugar nucleotide selectivity of LpsB is greatly relaxed compared with that of LpcC. Membranes of the wild-type S. meliloti strain 2011 catalyze the glycosylation of Kdo2-[4′-32P]lipid IVA at comparable rates using a diverse set of sugar nucleotides, including GDP-mannose, ADP-mannose, UDP-glucose, and ADP-glucose. This complex pattern of glycosylation is due entirely to LpsB, since membranes of the S. meliloti lpsB mutant 6963 do not glycosylate Kdo2-[4′-32P]lipid IVA in the presence of any of these sugar nucleotides. Expression of lpsB in E. coli using a T7lac promoter-driven construct results in the appearance of similar multiple glycosyl transferase activities seen in S. meliloti 2011 membranes. Constructs expressing lpcC display only mannosyl transferase activity. We conclude that LpsB, despite its high degree of similarity to LpcC is a much more versatile glycosyltransferase, probably accounting for the inability of lpcC to complement S. meliloti lpsB mutants. Our findings have important implications for the regulation of core glycosylation in S. meliloti and other bacteria containing LpcC orthologs.Facultad de Ciencias Exacta

LMSD: LIPID MAPS structure database

The LIPID MAPS Structure Database (LMSD) is a relational database encompassing structures and annotations of biologically relevant lipids. Structures of lipids in the database come from four sources: (i) LIPID MAPS Consortium's core laboratories and partners; (ii) lipids identified by LIPID MAPS experiments; (iii) computationally generated structures for appropriate lipid classes; (iv) biologically relevant lipids manually curated from LIPID BANK, LIPIDAT and other public sources. All the lipid structures in LMSD are drawn in a consistent fashion. In addition to a classification-based retrieval of lipids, users can search LMSD using either text-based or structure-based search options. The text-based search implementation supports data retrieval by any combination of these data fields: LIPID MAPS ID, systematic or common name, mass, formula, category, main class, and subclass data fields. The structure-based search, in conjunction with optional data fields, provides the capability to perform a substructure search or exact match for the structure drawn by the user. Search results, in addition to structure and annotations, also include relevant links to external databases. The LMSD is publicly available a

Transit timing variation and activity in the WASP-10 planetary system

Transit timing analysis may be an effective method of discovering additional bodies in extrasolar systems which harbour transiting exoplanets. The deviations from the Keplerian motion, caused by mutual gravitational interactions between planets, are expected to generate transit timing variations of transiting exoplanets. In 2009 we collected 9 light curves of 8 transits of the exoplanet WASP-10b. Combining these data with published ones, we found that transit timing cannot be explained by a constant period but by a periodic variation. Simplified three-body models which reproduce the observed variations of timing residuals were identified by numerical simulations. We found that the configuration with an additional planet of mass of $\sim$0.1 $M_{\rm{J}}$ and orbital period of $\sim$5.23 d, located close to the outer 5:3 mean motion resonance, is the most likely scenario. If the second planet is a transiter, the estimated flux drop will be $\sim$0.3 per cent and can be observable with a ground-based telescope. Moreover, we present evidence that the spots on the stellar surface and rotation of the star affect the radial velocity curve giving rise to spurious eccentricity of the orbit of the first planet. We argue that the orbit of WASP-10b is essentially circular. Using the gyrochronology method, the host star was found to be $270 \pm 80$ Myr old. This young age can explain the large radius reported for WASP-10b.Comment: MNRAS accepte

NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis

Modification of specific Gram-negative bacterial cell envelope components, such as capsule, O-antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F. tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram-negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria

Evolution of an endofungal Lifestyle: Deductions from the Burkholderia rhizoxinica Genome

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia rhizoxinica </it>is an intracellular symbiont of the phytopathogenic zygomycete <it>Rhizopus microsporus</it>, the causative agent of rice seedling blight. The endosymbiont produces the antimitotic macrolide rhizoxin for its host. It is vertically transmitted within vegetative spores and is essential for spore formation of the fungus. To shed light on the evolution and genetic potential of this model organism, we analysed the whole genome of <it>B. rhizoxinica </it>HKI 0454 - a type strain of endofungal <it>Burkholderia </it>species.</p> <p>Results</p> <p>The genome consists of a structurally conserved chromosome and two plasmids. Compared to free-living <it>Burkholderia </it>species, the genome is smaller in size and harbors less transcriptional regulator genes. Instead, we observed accumulation of transposons over the genome. Prediction of primary metabolic pathways and transporters suggests that endosymbionts consume host metabolites like citrate, but might deliver some amino acids and cofactors to the host. The rhizoxin biosynthesis gene cluster shows evolutionary traces of horizontal gene transfer. Furthermore, we analysed gene clusters coding for nonribosomal peptide synthetases (NRPS). Notably, <it>B. rhizoxinica </it>lacks common genes which are dedicated to quorum sensing systems, but is equipped with a large number of virulence-related factors and putative type III effectors.</p> <p>Conclusions</p> <p><it>B. rhizoxinica </it>is the first endofungal bacterium, whose genome has been sequenced. Here, we present models of evolution, metabolism and tools for host-symbiont interaction of the endofungal bacterium deduced from whole genome analyses. Genome size and structure suggest that <it>B. rhizoxinica </it>is in an early phase of adaptation to the intracellular lifestyle (genome in transition). By analysis of tranporters and metabolic pathways we predict how metabolites might be exchanged between the symbiont and its host. Gene clusters for biosynthesis of secondary metabolites represent novel targets for genomic mining of cryptic natural products. <it>In silico </it>analyses of virulence-associated genes, secreted proteins and effectors might inspire future studies on molecular mechanisms underlying bacterial-fungal interaction.</p