-
The Journal of Biological Chemistry Oct 2009Although the unsaturated fatty acid (UFA) synthetic pathway of Escherichia coli is the prototype of such pathways, several unresolved issues have accumulated over the...
Although the unsaturated fatty acid (UFA) synthetic pathway of Escherichia coli is the prototype of such pathways, several unresolved issues have accumulated over the years. The key players are the fabA and fabB genes. Earlier studies of fabA transcription showed that the gene was transcribed from two promoters, with one being positively regulated by the FadR protein. The other weaker promoter (which could not be mapped with the technology then available) was considered constitutive because its function was independent of FadR. However, the FabR negative regulator was recently shown to represses fabA transcription. We report that the weak promoter overlaps the FadR-dependent promoter and is regulated by FabR. This promoter is strictly conserved in all E. coli and Salmonella enterica genomes sequenced to date and is thought to provide insurance against inappropriate regulation of fabA transcription by exogenous saturated fatty acids. Also, the fabAup promoter, a mutant promoter previously isolated by selection for increased FabA activity, was shown to be a promoter created de novo by a four-base deletion within the gene located immediately upstream of fabA. Demonstration of the key UFA synthetic reaction catalyzed by FabB has been elusive, although it was known to catalyze an elongation reaction. Strains lacking FabB are UFA auxotrophs indicating that the enzyme catalyzes an essential step in UFA synthesis. Using thioesterases specific for hydrolysis of short chain acyl-ACPs, the intermediates of the UFA synthetic pathway have been followed in vivo for the first time. These experiments showed that a fabB mutant strain accumulated less cis-5-dodecenoic acid than the parental wild-type strain. These data indicate that the key reaction in UFA synthesis catalyzed by FabB is elongation of the cis-3-decenoyl-ACP produced by FabA.
Topics: 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; Bacterial Proteins; Escherichia coli K12; Escherichia coli Proteins; Fatty Acid Synthase, Type II; Fatty Acids, Monounsaturated; Hydro-Lyases; Promoter Regions, Genetic; Repressor Proteins; Salmonella enterica; Transcription, Genetic
PubMed: 19679654
DOI: 10.1074/jbc.M109.023440 -
Applied and Environmental Microbiology Aug 2007Stenotrophomonas maltophilia WR-C possesses an rpf/diffusible signal factor (DSF) cell-cell communication system. It produces cis-Delta2-11-methyl-dodecenoic acid, a...
Stenotrophomonas maltophilia WR-C possesses an rpf/diffusible signal factor (DSF) cell-cell communication system. It produces cis-Delta2-11-methyl-dodecenoic acid, a DSF, and seven structural derivatives, which require rpfF and rpfB for synthesis. Acquisition of iron from the environment is important for bacterial growth as well as the expression of virulence genes. We identified a gene homologous to fecA, which encodes a ferric citrate receptor that transports exogenous siderophore ferric citrate from the environment into the bacterial periplasm. Western blot analysis with anti-FecA-His(6) antibody showed that the FecA homologue was induced in the iron-depleted medium supplemented with a low concentration of ferric citrate. Deletion of rpfF or rpfB resulted in reduced FecA expression compared to the wild type. Synthetic DSF restored FecA expression by the DeltarpfF mutant to the wild-type level. Reverse transcription-PCR showed that the fecA transcript was decreased in the DeltarpfF mutant compared to the wild type. These data suggest that DSF affected the level of fecA mRNA. Transposon inactivation of crp, which encodes cyclic AMP (cAMP) receptor protein (CRP) resulted in reduced FecA expression and rpfF transcript level. Putative CRP binding sites were located upstream of the rpfF promoter, indicating that the effect of CRP on FecA is through the rpf/DSF pathway and by directly controlling rpfF. We propose that CRP may serve as a checkpoint for iron uptake, protease activity, and hemolysis in response to environmental changes such as changes in concentrations of glucose, cAMP, iron, or DSF.
Topics: Bacterial Proteins; Base Sequence; Biological Transport; Cyclic AMP Receptor Protein; DNA Transposable Elements; Escherichia coli Proteins; Ferric Compounds; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mutagenesis, Insertional; Receptors, Cell Surface; Sequence Analysis, DNA; Signal Transduction; Stenotrophomonas maltophilia
PubMed: 17574998
DOI: 10.1128/AEM.00366-07 -
European Journal of Biochemistry Apr 1989Lipid A from Rhodobacter capsulatus 37b4 consists of a D-glucosaminyl-(beta 1-6)-D-glucosamine disaccharide backbone, carrying diphosphorylethanolamine at C-1 of the...
Lipid A from Rhodobacter capsulatus 37b4 consists of a D-glucosaminyl-(beta 1-6)-D-glucosamine disaccharide backbone, carrying diphosphorylethanolamine at C-1 of the reducing glucosamine and phosphorylethanolamine at C-4' of the nonreducing glucosamine. 1,4'-Bisphosphorylated lipid A, lacking the polar head groups, was also encountered and contributed to the observed microheterogeneity in the phosphate substitution. The amino functions of both glucosamines are substituted almost entirely by the rare 3-oxotetradecanoic acid, which is a characteristic constituent of lipid A in the genus Rhodobacter. 3-Hydroxydecanoic acid is ester-bound at C-3 and C-3' of the glucosamine disaccharide and the one at the nonreducing glucosamine (C-3') is partially substituted by dodecenoic acid to form an ester-bound diester. In free lipid A, hydroxy groups at C-4 and C-6' of the glucosamine disaccharide are unsubstituted. C-6' being the putative attachment point of the lipopolysaccharide core. The nontoxic Rhodobacter capsulatus lipid A shows extensive serological cross-reaction with the toxic Salmonella lipid A. Structural similarities in the hydrophilic part of both types of lipid A, dissimilarities in the hydrophobic part and their impacts on serologic properties are discussed.
Topics: Binding Sites; Fatty Acids; Hydroxylation; Immune Sera; Lipid A; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Structure; Phosphates; Rhodopseudomonas; Structure-Activity Relationship
PubMed: 2714269
DOI: 10.1111/j.1432-1033.1989.tb14677.x -
Free Radical Biology & Medicine Feb 2012Many of the pathological effects of lipid peroxidation are mediated by aldehydes generated through fragmentation of lipid peroxides. Among these aldehydes, the...
Fragmentation of a linoleate-derived γ-hydroperoxy-α,β-unsaturated epoxide to γ-hydroxy- and γ-oxo-alkenals involves a unique pseudo-symmetrical diepoxycarbinyl radical.
Many of the pathological effects of lipid peroxidation are mediated by aldehydes generated through fragmentation of lipid peroxides. Among these aldehydes, the γ-hydroxy- and γ-oxo-α,β-alkenals, e.g., 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), are especially prone to modifying proteins and DNA through covalent adduction. In addition the "mirror image" γ-hydroxy- and γ-oxo-α,β-alkenal phospholipids can serve as high-affinity ligands for biological receptors triggering pathology. Therefore, the mechanisms by which these aldehydes are generated in vivo are under intense scrutiny. We now report observations supporting the intermediacy of a unique pseudo-symmetrical diepoxycarbinyl radical that accounts for the coproduction of HNE, ONE, and their mirror image analogues 9-hydroxy-12-oxo-10(E)-dodecenoic acid and 9-keto-12-oxo-10-dodecenoic acid upon fragmentation of 13-hydroperoxy-cis-9,10-epoxyoctadeca-11-enoic acid.
Topics: Aldehydes; Epoxy Compounds; Free Radicals; Hydrolysis; Iron; Linoleic Acids; Lipid Peroxidation; Oxidation-Reduction; Tandem Mass Spectrometry
PubMed: 22155057
DOI: 10.1016/j.freeradbiomed.2011.11.013 -
Journal of Bacteriology Oct 2013In the present study, we report the identification of a putative enoyl-coenzyme A (CoA) hydratase/isomerase that is required for synthesis of the biofilm dispersion...
In the present study, we report the identification of a putative enoyl-coenzyme A (CoA) hydratase/isomerase that is required for synthesis of the biofilm dispersion autoinducer cis-2-decenoic acid in the human pathogen Pseudomonas aeruginosa. The protein is encoded by PA14_54640 (PA0745), named dspI for dispersion inducer. The gene sequence for this protein shows significant homology to RpfF in Xanthomonas campestris. Inactivation of dspI was shown to abolish biofilm dispersion autoinduction in continuous cultures of P. aeruginosa and resulted in biofilms that were significantly greater in thickness and biomass than those of the parental wild-type strain. Dispersion was shown to be inducible in dspI mutants by the exogenous addition of synthetic cis-2-decenoic acid or by complementation of ΔdspI in trans under the control of an arabinose-inducible promoter. Mutation of dspI was also shown to abolish cis-2-decenoic acid production, as revealed by gas chromatography-mass spectrometry (GC-MS) analysis of cell-free spent culture medium. The transcript abundance of dspI correlated with cell density, as determined by quantitative reverse transcriptase (RT) PCR. This regulation is consistent with the characterization of cis-2-decenoic acid as a cell-to-cell communication molecule that regulates biofilm dispersion in a cell density-dependent manner.
Topics: Bacterial Proteins; Biofilms; Enoyl-CoA Hydratase; Fatty Acids, Monounsaturated; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Pseudomonas aeruginosa
PubMed: 23935049
DOI: 10.1128/JB.00707-13 -
Journal of Clinical Microbiology Jan 1979The acid metabolites and the cellular fatty acids of three strains of Neisseria meningitidis grown in a chemically defined liquid medium were determined with...
The acid metabolites and the cellular fatty acids of three strains of Neisseria meningitidis grown in a chemically defined liquid medium were determined with computerized frequency-pulsed electron capture gas-liquid chromatography. Five acids not previously reported were subsequently identified: isobutyric, octanoic, decenoic (C10:1), dodecenoic (C12:1), and tetradecenoic (C14:1). These acids were produced during active metabolism and were not detected as cellular constituents. The frequency-pulsed electron capture gas-liquid chromatography methods which we used provide a rapid, reliable, sensitive means of detecting both these and other metabolic and cellular acids in spent culture medium.
Topics: Chromatography, Gas; Culture Media; Fatty Acids; Mass Spectrometry; Neisseria meningitidis
PubMed: 107187
DOI: 10.1128/jcm.9.1.97-102.1979 -
Molecular Microbiology Jan 2006The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) recruits a diffusible signal factor (DSF), which has recently been structurally characterized as...
Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions.
The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) recruits a diffusible signal factor (DSF), which has recently been structurally characterized as cis-11-methyl-2-dodecenoic acid, as a cell-cell communication signal to synchronize virulence gene expression and biofilm dispersal. In this study, we showed that despite the existance of phenotype variations in different Xcc isolates, the DSF-mediated functions were in general conserved. To investigate the genomic profiles of DSF regulation, we designed and conducted oligomicroarray analysis by comparison of the gene expression patterns of wild-type strain XC1 and its DSF-deficient mutant XC1dF, as well as those of XC1dF in the presence or absence of DSF signals. The analyses led to identification of 165 genes, whose expression was significantly influenced by DSF signals. These genes encode proteins and enzymes belonging to at least 12 functional groups. In addition to those previously known DSF-dependent activities such as production of extracellular enzymes and extracellular polysaccharides, microarray analyses also revealed new functions mediated by DSF, such as flagellum synthesis, resistance to toxins and oxidative stress, and aerobic respiration. Phenotype analyses confirmed that DSF signalling contributed to resistance to toxin acriflavin and hydrogen peroxide, and to the survival of bacterial cells at different temperatures. We conclude that DSF cell-cell signalling is not only essential for co-ordinating the expression of virulence genes but also plays a vital role in keeping up the general competence of the pathogen in ecosystems.
Topics: Base Sequence; Cell Communication; DNA Primers; Genome, Bacterial; Mutation; Regulon; Xanthomonas campestris
PubMed: 16390454
DOI: 10.1111/j.1365-2958.2005.04961.x -
Molecular Plant-microbe Interactions :... Mar 2014The rpfF gene from Xylella fastidiosa, encoding the synthase for diffusible signal factor (DSF), was expressed in 'Freedom' grape to reduce the pathogen's growth and...
The rpfF gene from Xylella fastidiosa, encoding the synthase for diffusible signal factor (DSF), was expressed in 'Freedom' grape to reduce the pathogen's growth and mobility within the plant. Symptoms in such plants were restricted to near the point of inoculation and incidence of disease was two- to fivefold lower than in the parental line. Both the longitudinal and lateral movement of X. fastidiosa in the xylem was also much lower. DSF was detected in both leaves and xylem sap of RpfF-expressing plants using biological sensors, and both 2-Z-tetradecenoic acid, previously identified as a component of X. fastidiosa DSF, and cis-11-methyl-2-dodecenoic acid were detected in xylem sap using electrospray ionization mass spectrometry. A higher proportion of X. fastidiosa cells adhered to xylem vessels of the RpfF-expressing line than parental 'Freedom' plants, reflecting a higher adhesiveness of the pathogen in the presence of DSF. Disease incidence in RpfF-expressing plants in field trials in which plants were either mechanically inoculated with X. fastidiosa or subjected to natural inoculation by sharpshooter vectors was two- to fourfold lower in than that of the parental line. The number of symptomatic leaves on infected shoots was reduced proportionally more than the incidence of infection, reflecting a decreased ability of X. fastidiosa to move within DSF-producing plants.
Topics: Animals; Bacterial Proteins; Cell Adhesion; Disease Susceptibility; Fatty Acids, Monounsaturated; Gene Expression; Gene Expression Regulation, Bacterial; Green Fluorescent Proteins; Host-Pathogen Interactions; Insect Vectors; Mutation; Plant Diseases; Plant Roots; Plant Shoots; Plants, Genetically Modified; Spectrometry, Mass, Electrospray Ionization; Virulence; Vitis; Xylella; Xylem
PubMed: 24499029
DOI: 10.1094/MPMI-07-13-0197-FI -
Applied and Environmental Microbiology Oct 2015In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within...
In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within the ecological niche. We analyzed the potential interspecies signaling effect of the Burkholderia diffusible signal factor (BDSF) on Francisella novicida, a model organism for Francisella tularensis, and demonstrated that BDSF both inhibits the formation and causes the dispersion of Francisella biofilm. Specificity was demonstrated for the cis versus the trans form of BDSF. Using transcriptome sequencing, quantitative reverse transcription-PCR, and activity assays, we found that BDSF altered the expression of many F. novicida genes, including genes involved in biofilm formation, such as chitinases. Using a chitinase inhibitor, the antibiofilm activity of BDSF was also shown to be chitinase dependent. In addition, BDSF caused an increase in RelA expression and increased levels of (p)ppGpp, leading to decreased biofilm production. These results support our observation that exposure of F. novicida to BDSF causes biofilm dispersal. Furthermore, BDSF upregulated the genes involved in iron acquisition (figABCD), increasing siderophore production. Thus, this study provides evidence for a potential role and mechanism of diffusible signal factor (DSF) signaling in the genus Francisella and suggests the possibility of interspecies signaling between Francisella and other bacteria. Overall, this study suggests that in response to the interspecies DSF signal, F. novicida can alter its gene expression and regulate its biofilm formation.
Topics: Biofilms; Burkholderia; Fatty Acids, Monounsaturated; Francisella tularensis; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Microbial Interactions; Molecular Sequence Data; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Siderophores
PubMed: 26231649
DOI: 10.1128/AEM.02165-15 -
FEBS Letters Aug 2003Guava (Psidium guajava) hydroperoxide lyase (HPL) preparations were incubated with [1-(14)C](9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid for 1 min at 0...
Guava (Psidium guajava) hydroperoxide lyase (HPL) preparations were incubated with [1-(14)C](9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid for 1 min at 0 degrees C, followed by rapid extraction/trimethylsilylation. Analysis of the trimethylsilylated products by gas chromatography-mass spectrometry and radio-high-performance liquid chromatography revealed a single predominant (14)C-labelled compound, identified by its (1)H-nuclear magnetic resonance, ultraviolet and mass spectra as the trimethylsilyl ether/ester of (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid. Longer time incubations afford smaller yield of this enol due to its partial tautomerization into (9Z)-12-oxo-9-dodecenoic acid. The data obtained demonstrate that formation of (9Z)-12-oxo-9-dodecenoic acid in the HPL reaction is preceded by unstable enol oxylipin, and further suggest that hemiacetals are the true products of HPL catalysis.
Topics: Aldehyde-Lyases; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Fatty Alcohols; Gas Chromatography-Mass Spectrometry; Lauric Acids; Linolenic Acids; Lipid Peroxides; Plant Proteins; Psidium
PubMed: 12914919
DOI: 10.1016/s0014-5793(03)00758-0