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PubMed2006

Mouse and human cell activation by N-dodecanoyl-DL-homoserine lactone, a Chromobacterium violaceum autoinducer.

Chromobacterium violaceum produces autoinducers, including homoserine lactones (HSLs), for genetic regulation. Among the seven HSLs derived from C. violaceum we evaluated, only C(12)-HSL stimulated the production of inflammatory cytokines in mammalian monocytic cell lines through the activation of the NF-kappaB signaling pathway besides their quorum-sensing role, like 3-oxo-C(12)-HSL from Pseudomonas aeruginosa.

4-Butyrolactone↗
PubMed2003

Modification of in vivo and in vitro T- and B-cell-mediated immune responses by the Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone.

N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL), a quorum-sensing molecule of Pseudomonas aeruginosa, plays an important role in the pathogenesis of the organism through its control of virulence factor expression. Several reports have suggested that OdDHL can also directly modulate host immune responses. However, the nature of the modulation is controversial, with different reports suggesting promotion of either humoral (Th2-mediated) or inflammatory (Th1-mediated) responses. This report describes a series of studies which demonstrate for the first time that in vivo administration of OdDHL can modulate the course of an antibody response, with an increase in ovalbumin (OVA)-specific immunogloblulin G1 (IgG1) but not IgG2a in OdDHL-treated OVA-immunized BALB/c mice compared to levels for controls. In vitro stimulation of lymphocytes from both Th1-biased C57Bl/6 and T-cell receptor transgenic mice and Th2-biased BALB/c mice in the presence of OdDHL demonstrated that OdDHL inhibits in vitro cytokine production in response to both mitogen and antigen, with gamma interferon (IFN-gamma) tending to be more inhibited than interleukin-4 (IL-4). In vitro mitogen or antigen restimulation of cells from mice treated with OdDHL in vivo shows effects on cytokine production which depend on the underlying immune bias of the mouse strain used, with a relative increase of IFN-gamma in Th1-biased C57Bl/6 mice and a relative increase of IL-4 in Th2-biased BALB/c mice. Thus, the mode of action of OdDHL on T-cell cytokine production is likely to be a relatively nonspecific one which accentuates an underlying immune response bias rather than one which specifically targets either Th1 or Th2 responses.

4-Butyrolactone↗
PubMed1971

Growth rate of Escherichia coli at elevated temperatures: reversible inhibition of homoserine trans-succinylase.

The preceding paper (10) showed that the growth of Escherichia coli is slowed, without killing, at 40 to 45 C, and that in the several strains tested the cause is a decrease in the activity of homoserine trans-succinylase. These temperatures are now shown to inhibit the enzyme directly, in crude extracts and after partial purification. The effect is rapid and is immediately reversible, unlike the progressive and slowly reversible changes of conventional denaturation.

Acyltransferases↗
PubMed1993

Role of serine 352 in the allosteric response of Serratia marcescens aspartokinase I-homoserine dehydrogenase I analyzed by using site-directed mutagenesis.

Aspartokinase I and homoserine dehydrogenase I (AKI-HDI) from Serratia marcescens Sr41 are encoded by the thrA gene as a single polypeptide chain. Previously, a single amino acid substitution of Ser-352 with Phe was shown to produce an AKI-HDI enzyme that is not subject to threonine-mediated feedback inhibition. To determine the role of Ser-352 in the allosteric response, the thrA gene was modified by using site-directed mutagenesis so that Ser-352 of the wild-type AKI-HDI was replaced by Ala, Arg, Asn, Gln, Glu, His, Leu, Met, Pro, Thr, Trp, Tyr, or Val. The Thr-352 and Pro-352 replacements rendered AKIs sensitive to threonine. The Tyr-352 and Asn-352 substitutions led to activation, rather than inhibition, of AKI by threonine. The other replacements conferred threonine insensitivity on AKI. The threonine sensitivity of HDI was also changed by the amino acid substitutions at Ser-352. The HDI carried by the Tyr-352 mutant AKI-HDI was activated by threonine. Single amino acid replacements at Ser-352 by Ala, Asn, Gln, His, Phe, Pro, Thr, or Tyr were introduced into truncated AKI-HDIs containing the AKI and the central regions. The AKI activity of the truncated AKI-HDI containing the first 468 amino acid residues was sensitive to threonine, and introduction of the amino acid replacements did not alter the threonine sensitivity of the AKI. Another truncated AKI-HDI containing the first 462 amino acid residues possessed threonine-resistant AKI, whereas the substitutions of Ser-352 with Ala and Pro rendered AKI sensitive to threonine. The replacement of GIn-351 with Phe activated AK1 of the truncated AKI-HDI in the presence of L-threonine. These findings suggest that Ser-352 of the central region of AKI-HDI is possibly a key residue involved with the allosteric regulation of both AKI and HDI activities.

Allosteric Regulation↗
PubMed1997

A homoserine lactone autoinducer regulates virulence of an insect-pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae).

N-beta-Hydroxybutanoyl homoserine lactone (HBHL), the autoinducer of the luminescent system of Vibrio harveyi, has been identified as the first small compound to restore virulence to avirulent mutants of Xenorhabdus nematophilus. HBHL stimulated the level of lipase activity excreted by avirulent X. nematophilus and lowered the phenoloxidase activity in the hemolymph of insects infected with X. nematophilus, parameters that are both associated with insect pathogenesis. Moreover, mortality of the insects infected with avirulent X. nematophilus was restored upon injection with HBHL. Chloroform extraction of medium conditioned with wild-type but not avirulent X. nematophilus led to the isolation of a compound with the same chromatographic mobility as HBHL as well as the ability to stimulate the luminescence of a dim autoinducer-dependent mutant of V. harveyi. Transfer of the V. harveyi lux operon into avirulent and wild-type X. nematophilus generated dim and bright luminescent strains, respectively, which responded to HBHL and an agonist and antagonist in a manner analogous to their effects on the luminescence of dim autoinducer-deficient and bright wild-type strains of V. harveyi, indicating that similar HBHL-dependent regulatory systems exist in these two bacterial species.

Animals↗
PubMed1998

Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens.

The TraR and TraI proteins of Agrobacterium tumefaciens mediate cell-density-dependent expression of the Ti plasmid tra regulon. TraI synthesizes the autoinducer pheromone N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL), while TraR is an 3-oxo-C8-HSL-responsive transcriptional activator. We have compared the abilities of 3-oxo-C8-HSL and 32 related compounds to activate expression of a TraR-regulated promoter. In a strain that expresses wild-type levels of TraR, only 3-oxo-C8-HSL was strongly stimulatory, four compounds were detectably active only at high concentrations, and the remaining 28 compounds were inactive. Furthermore, many of these compounds were potent antagonists. In contrast, almost all of these compounds were stimulatory in a congenic strain that overexpresses TraR and no compound was a potent antagonist. We propose a model in which autoinducers enhance the affinity of TraR either for other TraR monomers or for DNA binding sites and that overexpression of TraR potentiates this interaction by mass action. Wild-type A. tumefaciens released a rather broad spectrum of autoinducers, including several that antagonize induction of a wild-type strain. However, under all conditions tested, 3-oxo-C8-HSL was more abundant than any other analog, indicating that other released autoinducers do not interfere with tra gene induction. We conclude that (i) in wild-type strains, only 3-oxo-C8-HSL significantly stimulates tra gene expression, while many autoinducer analogs are potent antagonists; (ii) TraR overexpression increases agonistic activity of autoinducer analogs, allowing sensitive biodetection of many autoinducers; and (iii) autoinducer stimulatory activity is potentiated by TraR overproduction, suggesting that autoinducers may shift an equilibrium between TraR monomers and dimers or oligomers. When autoinducer specificities of other quorum-sensing proteins are tested, care should be taken not to overexpress those proteins.

Agrobacterium tumefaciens↗
PubMed1999

Swarming by Pseudomonas syringae B728a requires gacS (lemA) and gacA but not the acyl-homoserine lactone biosynthetic gene ahlI.

Pseudomonas syringae pv. syringae B728a, a causal agent of bacterial brown spot on snap beans, swarms with a characteristic dendritic pattern on semisolid (0.4%) agar plates. Filamentation of swarming cells of B728a was not observed. Mutations in either the gacS (formerly lemA) or gacA gene of B728a eliminate the ability of this P. syringae isolate to swarm without obvious effects on bacterial motility. Three field isolates showed a similar dependence on gacS for swarming. Since gacS and gacA mutants are known to be deficient in N-acyl-L-homoserine lactone (acyl-HSL) production, a mutant was constructed by disruption of the ahlI gene of B728a. This mutant did not make any acyl-HSL detectable by the Agrobacterium traG::lacZ reporter system, yet was unaffected in its ability to swarm. Other phenotypes of gacS and gacA mutations were similarly unaffected in the ahlI mutant.

4-Butyrolactone↗
PubMed1997

Sequence analysis of functional regions of homoserine dehydrogenase genes from L-lysine and L-threonine-producing mutants of Brevibacterium lactofermentum.

The homoserine dehydrogenase (HD) genes from Brevibacterium lactofermentum lysine- and threonine-producing mutants were cloned, using the polymerase chain reaction, and sequenced. We found the amino acid substitutions, Val104Ile in the lysine-producing mutants in which HD may cause leaky mutation and Ser393Phe in the threonine-producing mutant with feedback-insensitive HD.

Amino Acid Sequence↗
PubMed2001

IL-8 production in human lung fibroblasts and epithelial cells activated by the Pseudomonas autoinducer N-3-oxododecanoyl homoserine lactone is transcriptionally regulated by NF-kappa B and activator protein-2.

The destructive pulmonary inflammation associated with Pseudomonas aeruginosa colonization is caused, in part, by the production of the chemokine IL-8, which recruits neutrophils into the lung. The Pseudomonas autoinducer, N-3-oxododecanoyl homoserine lactone (3-O-C12-HSL), is a small lipid-soluble molecule that is essential in the regulation of many P. aeruginosa virulence factors, but little is known about how it affects eukaryotic cells. In this report we demonstrate that 3-O-C12-HSL is a potent stimulator of both IL-8 mRNA and protein from human fibroblasts and epithelial cells in vitro. The IL-8 produced from these 3-O-C12-HSL-stimulated cells was found to be functionally active by inducing the chemotaxis of neutrophils. To determine a mechanism for this IL-8 induction, deletion constructs of the IL-8 promoter were examined. It was found that the DNA region between nucleotides -1481 and -546 and the transcription factor NF-kappaB were essential for the maximal induction of IL-8 by 3-O-C12-HSL. This was confirmed by EMSAs, where 3-O-C12-HSL induced a shift with both AP-2 and NF-kappaB consensus DNA. The activation of NF-kappaB and subsequent production of IL-8 were found to be regulated by a mitogen-activated protein kinase pathway. These findings support the concept that the severe lung damage that accompanies P. aeruginosa infections is caused by an exuberant neutrophil response stimulated by 3-O-C12-HSL-induced IL-8. Understanding the mechanisms of 3-O-C12-HSL activation of lung structural cells may provide a means to help control lung damage during infections with P. aeruginosa.

4-Butyrolactone↗
PubMed2002

[Synthesis of signaling N-acyl-homoserine-lactones participating in quorum sensing in rhizosphere and soil bacteria Pseudomonas and Xanthomonas].

Signaling molecules assigned to N-acyl-homoserine-lactones (AHL) serve as autoinducers for the genes controlling the quorum sensing regulatory system. In many gram-negative bacteria, AHL are the key factors responsible for density-dependent regulation of exoenzyme and secondary metabolite production; they also participate in interaction between bacteria and higher organisms. The soil and rhisosphere bacteria Pseudomonas and Xanthomonas from different geographical zones of Russia and the former USSR were analyzed for the presence of the AHL producers. Screening was conducted by using a test system based on the mutant strain Chromobacterium violaceum, which was unable to synthesize AHL but produced a pigment violacein in the presence of exogenous AHL. The AHL-like compounds proved to be formed by 9.7% of the studied bacteria. Various Pseudomonas species differed in the capacity to synthesize this compounds. In at least a half of the isolated P. aureofaciens and P. aeruginosa, an intense AHL production was observed, whereas the AHL-producers were far less frequent among the P. fluorescens, P. chlororaphis, P. lemonnieri, P. geniculata, and P. putida. None of the 41 Xanthomonas maltophilia strains examined synthesized AHL.

Homoserine↗
PubMed1978

Aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12. Concentration-dependent dissociation to dimers in the presence of L-threonine.

The concentration-dependent association-dissociation equilibrium of the bifunctional enzyme aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12 has been investigated at pH 7.6 in the presence of 10 mM L-threonine and 0.1 M KCl by equilibrium gel permeation monitored by a single-photon counting spectrophotometer. The results obtained are consistent with the existence of a dimer-tetramer equilibrium with the association constant of 2.6 X 10(7) M-1 (deltaG0 = -9.9 kcal/mol of dimer). The limiting partition cross-sections estimated by a three-parameter least squares minimization procedure indicate that the molecular radii of the dimer and tetramer are 53.8 A and 70 A, respectively. Both the dimeric and tetrameric forms of the enzyme possess dehydrogenase activity. Treatment of the enzyme with the chaotropic salts, potassium thiocyanate or potassium trichloroacetate, generates a monomeric form that is devoid of dehydrogenase activity. The catalytically inactive monomeric form of the enzyme has a molecular radius between 43 and 45.5 A and a molecular weight of approximately 80,000 as determined by small zone gel chromatography and sedimentation equilibrium studies.

Alcohol Oxidoreductases↗
PubMedDate not supplied

[The effect of amino acids in the asparagine family on the aspartate kinase and homoserine dehydrogenase of ethionine-resistant mutants of Pseudomonas putida].

The activity of aspartate kinase and homoserin dehydrogenase from ethionine resistant mutants Pseudomonas putida 25 and 6 have been studied as affected by amino acids from the family of asparagine. They are characterized by a capacity to the surplus synthesis of methionine. It is shown that mutants have negative regulation of the level of activity of the studied enzymes. It is supposed that the mutations (or mutation) could take place which affected properties of enzymes, which participated directly in the biosynthesis of methionine, in the analogue resistant clones 25 and 6.

Asparagine↗
PubMed1983

Characterization of proteolysis fragments of aspartokinase I: homoserine dehydrogenase I. Fluorescence and circular dichroism studies.

Proteolysis of native aspartokinase-homoserine dehydrogenase by chymotrypsin, subtilisin, clostripain, and V8 protease yields active dehydrogenase fragments. Fluorescence and near-UV circular dichroism measurements demonstrate that the bulk of the spectroscopic signal observed in the native protein originates in the residual fragments. Kinetic studies and far-UV CD spectra further distribute the fragments into two groups. Even though the remaining dehydrogenase activity is no longer inhibited by L-threonine, ultrafiltration binding studies and far-UV CD spectra clearly demonstrate that one of the two sets of inhibitor-binding sites is still intact. Computer analysis of the far-UV CD data of the native protein and the isolated fragments in the presence and absence of L-threonine has been used to resolve contributions from helix, beta, turn, and aperiodic components. This analysis indicates that the binding of the inhibitor induces decreases in helix content and generation of aperiodic structure within the molecule. The changes observed are similar in the native molecule and the fragments.

Aspartokinase Homoserine Dehydrogenase↗
PubMed1995

Purification and characterization of homoserine dehydrogenase from spinach leaves.

Homoserine dehydrogenase (HSDH) has been purified to homogeneity from spinach leaves using ammonium sulphate fractionation followed by ion exchange chromatography, gel filtration and FPLC techniques. The purified enzyme has a relative molecular mass of 220,000 and subunit molecular mass of 55,000 and probably occurs as a tetramer. The enzyme was found to be sensitive to threonine and also exhibited aspartate kinase (AK) activity, which was also sensitive to threonine suggesting that it is a bifunctional protein. The enzyme protein also gave a positive cross reaction with antibodies raised against purified AK isoenzymes. Both HSDH and AK activities were stimulated by calcium and calmodulin.

Aspartate Kinase↗
PubMed2006

Restraining Erwinia virulence by expression of N-acyl homoserine lactonase gene pro3A-aiiA in Bacillus thuringiensis subsp leesis.

To widen the biological control function of a genetically modified Bacillus thuringiensis subsp leesis strain BMB-005, an acyl homoserine lactonase (AHL lactonase) gene aiiA transcribed by the promoter of insecticidal crystal protein coding gene cry3A, was transformed into strain BMB-005. The amount of AHL lactonase protein produced by transformant BMB821A was 2.4-fold more than that produced by BMB-005. AHL-degradation assay showed that transformant BMB821A could degrade more AHLs molecules than the original strain BMB-005. The result of Erwinia carotovora pathogenicity test showed that the parental strain BMB-005 had no restraint of Erwinia infection, but the transformants exhibited strong restraint of E. carotovora infection on potato slices and cactus stems. Insecticidal bioassay against lepidopteran Spodoptera exigua showed that both strain BMB-005 and transformant BMB821A were toxic to S. exigua. The toxicity of transformant BMB821A (LC(50) was 3.8) was a little attenuated comparing with the toxicity of the original strain BMB-005 (LC(50) was 2.9). The B. thuringiensis strain BMB-005 has high toxicity against Helicoverpa armigera, Plutella xylostella, and S. exigua. This work provided new strategy for developing genetically engineered multi-functional B. thuringiensis strain that possesses insecticidal activity together with restraint of bacterial pathogenicity.

Animals↗
PubMed2003

Analysis of N-acyl-L-homoserine lactones produced by Burkholderia cepacia with partial filling micellar electrokinetic chromatography--electrospray ionization-ion trap mass spectrometry.

A method for the analysis of N-acyl-L-homoserine lactones (AHLs) with micellar electrokinetic chromatography coupled to electrospray ionization-ion trap mass spectrometry, combining the flexibility of capillary electrophoresis with the unmatched structural information provided by mass spectrometry is presented. Different surfactants were evaluated, with sodium dodecyl sulfate (SDS) yielding the best results considering sensitivity and flexibility. We examined the interaction of AHLs with the SDS micelles at different analysis conditions and applied the optimized method to the analysis of a real bacterial sample. Two AHLs from Burkholderia cepacia colonizing the rhizosphere of traditional Indian rice cultivars could be unambiguously determined in an ethyl acetate extract with high resolution flexibility.

4-Butyrolactone↗
PubMed2004

Identification of homoserine lactone derivatives using the methionine functionalized solid phase synthesis by gas chromatography/mass spectrometry.

Combinatorial homoserine lactone mixtures and individual products were obtained from the methionine-functionalized resin in solid-phase synthesis. The four-step process consisting of a coupling step of an N-Fmoc-L-methionine, deprotection of N-Fmoc group, N-coupling with a carboxylic acid, and cleavage reaction through a polymer supported strategy is described. Gas chromatography-mass selective detector (GC-MSD) techniques provide the most powerful methods for identifying both the combinatorial mixtures and individual products.

4-Butyrolactone↗