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PubMed2005

Inter-kingdom signaling: deciphering the language of acyl homoserine lactones.

Bacteria use small secreted chemicals or peptides as auto-inducers to coordinately regulate gene expression within a population in a process called quorum sensing. Quorum sensing controls several important functions in different bacterial species, including the production of virulence factors and biofilm formation in Pseudomonas aeruginosa and bioluminescence in Vibrio fischeri. Many gram-negative bacterial species use acyl homoserine lactones as auto-inducers that function as ligands for transcriptional regulatory proteins. Several recent reports indicate that bacterial acyl homoserine lactones can also affect gene expression in host cells. Direct signaling also appears to function in the opposite direction as some eukaryotic cell types produce mimics that interact with quorum sensing systems in bacteria. Here, we will describe the evidence to support the existence of bi-directional inter-kingdom signaling via acyl homoserine lactones and eukaryotic mimics and discuss the potential molecular mechanisms that mediate these responses. The functional consequences of inter-kingdom signaling will be discussed in relation to both pathogenic and non-pathogenic bacterial-host interactions.

4-Butyrolactone↗
PubMed2003

Influence of food preservation parameters and associated microbiota on production rate, profile and stability of acylated homoserine lactones from food-derived Enterobacteriaceae.

Quorum-dependent regulation is mediated by N-acyl-L-homoserine lactones (AHLs) in several Gram-negative bacteria. The production of AHLs has typically been studied using pure bacteria cultures grown in nutrient-rich media at optimal temperature. AHLs are produced in several chill-stored foods by Gram-negative bacteria participating in spoilage. As part of our investigation of the role of AHLs in food quality, we studied the AHL production in two Enterobacteriaceae isolated from cold-smoked salmon under growth conditions typical of those found in cold-smoked salmon. We tested the influence of carbon source (glucose, sucrose, xylose, arabinose, mannose, mannitol and sorbitol), temperature (5 and 25 degrees C), salt concentration (0-7%), pH (6, 7 and 8) and co-existing lactic acid bacteria microflora on the AHL profile and production rate from Serratia proteamaculans strain B5a and Enterobacter agglomerans strain B6a. The two strains produced the same types of AHLs under all conditions tested. The specific AHL concentrations (moles/liter/OD(450)) changed slightly for both strains at the various conditions. S. proteamaculans strain B5a produced approximately 150 nM/OD(450) N-3-oxo-hexanoyl homoserine lactone (OHHL) and E. agglomerans strain B6a produced two major signals, OHHL and N-3-oxo-octanoyl homoserine lactone (OOHL) in a 1:9 ratio with a total concentration of approximately 3000 nM/OD(450). The AHL signal molecules became unstable with increasing pH (>7.5). In cold-smoked salmon, pH is approximately 6 and therefore only a low degree of pH-induced turnover is expected to occur in this product. Overall, our study demonstrates that food-derived Enterobacteriaceae produce AHLs of the same type and in the same magnitude when grown under food-relevant conditions as when grown in laboratory media at high temperature. Also, the AHLs produced in foods will be relatively stable and their regulatory impact lasting during storage.

4-Butyrolactone↗
PubMed2005

Acyl-homoserine lactones from Erwinia psidii R. IBSBF 435T, a guava phytopathogen (Psidium guajava L.).

The phytopathogen Erwinia psidii R. IBSBF 435T causes rot in branches, flowers, and fruits of guava (Psidium guajava L.), being responsible for crop losses, and has no effective control. It was demonstrated that this strain produces two compounds [S-(-)-N-hexanoyl and N-heptanoyl-homoserine lactone], both belonging to the class of quorum-sensing signaling substances. A protocol using gas chromatography-flame ionization detection with chiral stationary phase is described for the absolute configuration determination of a natural acyl-homoserine lactone. Biological assays with specific reporter and synthesis of identified substances are also described. This is the first report on the N-heptanoyl-homoserine lactone occurrence in the Erwinia genus.

4-Butyrolactone↗
PubMed2003

The thrH gene product of Pseudomonas aeruginosa is a dual activity enzyme with a novel phosphoserine:homoserine phosphotransferase activity.

The thrH gene product of Pseudomonas aeruginosa has been shown to complement both homoserine kinase (thrB gene product) and phosphoserine phosphatase (serB gene product) activities in vivo. Sequence comparison has revealed that ThrH is related to phosphoserine phosphatases (PSP, EC 3.1.3.3) and belongs to the l-2-haloacid dehalogenase-like protein superfamily. We have solved the crystal structures of ThrH in the apoform and in complex with a bound product phosphate. The structure confirms an overall fold similar to that of PSP. Most of the catalytic residues of PSP are also conserved in ThrH, suggesting that similar catalytic mechanisms are used by both enzymes. Spectrophotometry-based in vitro assays show that ThrH is indeed a phosphoserine phosphatase with a K(m) of 0.207 mm and k(cat) of 13.4 min(-1), comparable with those of other PSPs. More interestingly, using high pressure liquid chromatography-based assays, we have demonstrated that ThrH is able to further transfer the phosphoryl group to homoserine using phosphoserine as the phosphoryl group donor, indicating that ThrH has a novel phosphoserine:homoserine phosphotransferase activity.

Amino Acid Sequence↗
PubMed2002

Beta-ketoacyl acyl carrier protein reductase (FabG) activity of the fatty acid biosynthetic pathway is a determining factor of 3-oxo-homoserine lactone acyl chain lengths.

The two acyl-homoserine lactones (AHLs) N-(butyryl)-L-homoserine lactone and N-[3-oxododecanoyl]-L-homoserine lactone (3-oxo-C(12)-HSL) are required for quorum sensing in Pseudomonas aeruginosa. These AHLs derive their invariant lactone rings from S-adenosylmethionine and their variable acyl chains from the cellular acyl-acyl carrier protein (ACP) pool. This reaction is catalysed by specific AHL synthases, which exhibit acyl chain specificity. Culture supernatants of P. aeruginosa contain multiple 3-oxo-AHLs that differ in their acyl chain lengths but their physiological role, if any, remains unknown. An in vitro fatty acid-3-oxo-AHL synthesis system was established utilizing purified P. aeruginosa Fab proteins, ACP and the LasI 3-oxo-AHL synthase. In the presence of excess protein, substrates and cofactors, this system produced almost exclusively 3-oxo-C(12)-HSL. When the beta-ketoacyl-ACP reductase (FabG) catalysed step was made rate-limiting by switching from the preferred NADPH cofactor to NADH, increased levels of short chain 3-oxo-AHLs were produced, presumably because shorter-chain ketoacyl-ACPs accumulated and thus became LasI substrates. Consistent with these in vitro observations, a fabG(Ts) mutant produced increased amounts of 3-oxo-AHLs in vivo. Thus, in vitro and in vivo evidence indicated that modulation of FabG activity of the fatty acid biosynthetic pathway may determine the acyl chain lengths of these 3-oxo-AHLs and that the LasI 3-oxo-AHL synthase is sufficient for their synthesis.

4-Butyrolactone↗
PubMed1977

Changes in Enzyme Regulation during Growth of Maize: III. Intracellular Localization of Homoserine Dehydrogenase in Chloroplasts.

Extracts of leaf tissue of Zea mays L. seedlings were fractionated on nonlinear sucrose gradients to separate subcellular organelles. Homoserine dehydrogenase (EC 1.1.1.3) was identified in those fractions containing intact chloroplasts, as judged by the presence of chlorophyll and triosephosphate isomerase activity. Neither enzyme activity was detected in fractions containing ruptured chloroplasts, mitochondria, or microbodies. Quantitative measurements of enzyme activity and chlorophyll, and electron microscopic analysis of plastid preparations support the conclusion that maize mesophyll chloroplasts contain a significant fraction of the total cellular content of homoserine dehydrogenase.A survey of representative kinetic, regulatory, and physical properties did not reveal any significant differences between enzyme released from isolated, undamaged chloroplasts and that obtained from soluble cellular fractions.Examination of enzyme prepared from chloroplasts of different age seedlings indicated that the sensitivity of homoserine dehydrogenase to inhibition by the feedback modifier l-threonine was progressively diminished during growth of the plants. This systematic change in regulatory properties of the enzyme occurred to the same extent for the enzymes obtained from chloroplasts and soluble fractions.

Journal Article↗
PubMed1990

Differential Regulation of Maize Homoserine Dehydrogenase under Physiological Conditions.

Homoserine dehydrogenase is associated with the multibranched pathway of amino acid biosynthesis originating with aspartic acid. Like most of the related pathway enzymes, this enzyme is localized in chloroplasts. The activity and regulatory properties of the threonine-sensitive isozyme of homoserine dehydrogenase isolated from Zea mays var earliking were examined under variable conditions that could exist within chloroplasts. Catalytic activity is not significantly altered within the range of pHs that occur within these organelles, but inhibition of the enzyme by the pathway product, l-threonine, is markedly diminished at the alkaline pHs characteristic of illuminated chloroplasts. Inhibition by threonine is also subject to modulation by physiological levels of NADPH. Under conditions considered to represent the environment within unilluminated chloroplasts, the enzyme is severely inhibited by micromolar concentrations of threonine, but significant enzyme activity is retained under conditions that are likely to occur during illumination, even in the presence of millimolar levels of threonine. These results indicate that homoserine dehydrogenase may be subject to environmentally mediated regulation in vivo. Other observations support this concept and suggest that the intrinsic catalytic and regulatory properties of key enzymes could facilitate a direct link between light-dependent carbon and nitrogen assimilation and amino acid biosynthesis in chloroplasts of higher plants.

Journal Article↗
PubMed2006

N-acyl-L-homoserine lactone signal interception by Escherichia coli.

N-acyl-L-homoserine lactone (AHL) mediated quorum sensing is a widespread communication system in gram-negative bacteria which regulates a wide range of target genes in a cell density-dependent manner. Although Escherichia coli is not capable of synthesizing AHL molecules because it lacks an AHL synthase encoding gene, it does produce a predicted AHL receptor of the LuxR family, named SdiA. In this work, we used a promoter trap library to screen for E. coli MG1655 promoters whose expression was affected by synthetic N-hexanoyl-L-homoserine lactone (C6-HSL), and we identified six upregulated and nine downregulated promoters, which also responded to synthetic 3-oxo-N-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). The AHL responsiveness of these promoters was eliminated by knock-out of sdiA, and was temperature dependent, since the identified promoters showed a response at 30 degrees C but not, or only very weakly at 37 degrees C. In addition, in line with the observed induction of gadA encoding a glutamate decarboxylase, we could demonstrate an increased acid tolerance of E. coli upon exposure to C6-HSL. In conclusion, our work shows that E. coli has the capacity to alter its pattern of gene expression and its phenotypical properties in response to AHLs by means of the AHL responsive transcriptional regulator SdiA.

4-Butyrolactone↗
PubMed2003

Utilization of acyl-homoserine lactone quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1.

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42 degrees C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other gamma-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.

4-Butyrolactone↗
PubMed2006

Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1.

The relevance of the acyl homoserine lactone (acyl-HSL) quorum signals N-3-oxododecanoyl-homoserine lactone (3OC12HSL) and N-butanoyl-homoserine lactone to the biology and virulence of Pseudomonas aeruginosa is well investigated. Previously, P. aeruginosa was shown to degrade long-chain, but not short-chain, acyl-HSLs as sole carbon and energy sources (J. J. Huang, J.-I. Han, L.-H. Zhang, and J. R. Leadbetter, Appl. Environ. Microbiol. 69:5941-5949, 2003). A gene encoding an enzyme with acyl-HSL acylase activity, pvdQ (PA2385), was identified, but it was not required for acyl-HSL utilization. This indicated that P. aeruginosa encodes another acyl-HSL acylase, which we identify here. A comparison of total cell proteins of cultures grown with long-acyl acyl-HSLs versus other substrates implicated the involvement of a homolog of PvdQ, the product of gene PA1032, for which we propose the name QuiP. Transposon mutants of quiP were defective for growth when P. aeruginosa was cultured in medium containing decanoyl-HSL as a sole carbon and energy source. Complementation with a functional copy of quiP rescued this growth defect. When P. aeruginosa was grown in buffered lysogeny broth, constitutive expression of QuiP in P. aeruginosa led to decreased accumulations of the quorum signal 3OC12HSL, relative to the wild type. Heterologous expression of QuiP was sufficient to confer long-chain acyl-HSL acylase activity upon Escherichia coli. Examination of gene expression patterns during acyl-HSL-dependent growth of P. aeruginosa further supported the involvement of quiP in signal decay and revealed other genes also possibly involved. It is not yet known under which "natural" conditions quiP is expressed or how P. aeruginosa balances the expression of its quorum-sensing systems with the expression of its acyl-HSL acylase activities.

4-Butyrolactone↗
PubMed2003

Detection of N-(3-oxohexanoyl)-L-homoserine lactone in mice infected with Yersinia enterocolitica serotype O8.

Yersinia enterocolitica synthesizes N-acyl-L-homoserine lactone (AHL) signal molecules via the LuxR-LuxI homologues YenR-YenI. In this study we checked two prototypes of mouse-virulent Y. enterocolitica serotype O8 strains WA-314 and 8081 for AHL production in vitro and in vivo (mouse infection model). We used thin-layer chromatography in combination with the Escherichia coli AHL biosensor to identify the AHL species produced. We detected only OHHL [N-(3-oxohexanoyl)-L-homoserine lactone] and not HHL (N-hexanoyl-L-homoserine lactone) produced by Y. enterocolitica O8 in culture supernatant or infected mouse tissue. This is the first report demonstrating AHL production by yersiniae during infection.

4-Butyrolactone↗
PubMed2006

Quorum-sensing signal synthesis by the Yersinia pestis acyl-homoserine lactone synthase YspI.

The acyl-homoserine lactone molecular species (AHLs) produced by the Yersinia pestis AHL synthase YspI were identified by biochemical and physical/chemical techniques. Bioassays of extracts from culture supernatants of the recombinant YspI and wild-type Yersinia pestis showed similar profiles of AHLs. Analysis by liquid chromatography-mass spectrometry revealed that the predominant AHLs were N-3-oxooctanoyl-L-homoserine lactone and N-3-oxo-hexanoyl-L-homoserine lactone.

4-Butyrolactone↗
PubMed2004

Degradation of acyl-homoserine lactone molecules by Acinetobacter sp. strain C1010.

A bacterium C1010, isolated from the rhizospheres of cucumbers in fields in Korea, degraded the microbial quorum-sensing molecules, hexanoyl homoserine lactone (HHSL), and octadecanoyl homoserine lactone (OHSL). Morphological characteristics and 16S rRNA sequence analysis identified C1010 as Acinetobacter sp. strain C1010. This strain was able to degrade the acyl-homoserine lactones (AHLs) produced by the biocontrol bacterium, Pseudomonas chlororaphis O6, and a phytopathogenic bacterium, Burkholderia glumae. Co-cultivation studies showed that the inactivation of AHLs by C1010 inhibited production of phenazines by P. chlororaphis O6. In virulence tests, the C1010 strain attenuated soft rot symptom caused by Erwinia carotovora ssp. carotovora. We suggest Acinetobacter sp. strain C1010 could be a useful bacterium to manipulate biological functions that are regulated by AHLs in various Gram-negative bacteria.

4-Butyrolactone↗
PubMed1982

Isolation and characterization of an ether-linked homoserine lipid from the thylakoid membrane of Chlamydomonas reinhardtii 137+.

A polar diacylglycerolipid isolated from the phototrophic green alga Chlamydomonas reinhardtii 137+ (wild-type) displays chromatographic and chemical identity with the either-linked homoserine lipid 1(3),2-diacylglyceryl-(3)-O-4'(N,N,N-trimethyl)homoserine (DGTS) of the golden alga Ochromonas danica. Subcellularly, DGTS is a constituent of a fraction of thylakoid membranes purified from Chlamydomonas whole-cell homogenates. The proportion of DGTS in the photosynthetic lamellae is approximately 40% of the total found in the alga. Cellular and thylakoid-membrane DGTS both have an unsaturated:saturated ratio of about 1.8 and contain predominantly hexadecanoic and octadecanoic fatty acids. Quantitatively, the fatty acid complement of thylakoid DGTS is distinct from the cellular DGTS fatty acid profile. The results provide the first demonstrations that DGTS is a bona fide membrane lipid and, specifically, that ether-linked homoserine lipid is a component of the thylakoid membrane of a phototrophic green-plant cell.

Cell Membrane↗
PubMed1975

Regulation of aspartokinase and homoserine dehydrogenase in acetic acid bacteria.

The regulation of aspartokinase and homoserine dehydrogenase has been studied in three Acetobacter and two Gluconobacter species. Both enzymes were regulated by feedback inhibition. Aspartokinase was inhibited by L-threonine and concertedly inhibited by L-threonine plus L-lysine. The homoserine dehydrogenase was NADP-specific and was inhibited by L-threonine. Separation of the two enzymes by ammonium sulphate fractionation was possible in Acetobacter peroxydans, A. rancens and Gluconobacter melanogenus but not in A. liquefaciens or G. oxydans.

Acetobacter↗
PubMed1992

Preferred order random kinetic mechanism for homoserine dehydrogenase of Escherichia coli (Thr-sensitive) aspartokinase/homoserine dehydrogenase-I: equilibrium isotope exchange kinetics.

Isotope exchange kinetics at chemical equilibrium have been used to investigate the kinetic mechanism of homoserine dehydrogenase (EC 1.1.1.3) of the (Thr-sensitive) aspartokinase/homoserine dehydrogenase-I multifunctional enzyme from E. coli. For the reaction (L-ASA + NADPH + H+ = L-Hse + NADP+), at pH 9.0, 37 degrees C, Keq = 100 (+/- 20). Under these conditions, the rate for exchange of [14C]-L-homoserine (Hse) in equilibrium L-aspartate-beta-semialdehyde (ASA) is nearly twice that for the [3H]-NADP+ in equilibrium NADPH exchange. This indicates that covalent interconversion between reactants and products bound in the active site cannot be rate-limiting. Upon variation of the concentrations of all four substrates in constant ratio at equilibrium (to minimize dead-end complex formation), the Hse in equilibrium ASA exchange increased smoothly toward a maximum. In contrast, the NADP+ in equilibrium NADPH exchange rate increased to a maximum value at partial saturation, then decreased to approximately half the maximum rate. These data are consistent with a preferred-order random kinetic mechanism in which the dominant pathway involves association of NADPH prior to L-ASA and dissociation of L-Hse prior to NADP+.

Aspartic Acid↗
PubMed2004

Probing the active site of homoserine trans-succinylase.

Homoserine trans-succinylase is the first enzyme in methionine biosynthesis of Escherichia coli and catalyzes the activation of homoserine via a succinylation reaction. The in vivo activity of this enzyme is subject to tight regulation by several mechanisms, including repression and activation of gene expression, feedback inhibition, temperature regulation and proteolysis. This complex regulation reflects the key role of this enzyme in bacterial metabolism. Here, we demonstrate--using proteomics and high-resolution mass spectrometry--that succinyl is covalently bound to one of the two adjacent lysine residues at positions 45 and 46. Replacing these lysine residues by alanine abolished the enzymatic activity. These findings position the lysine residues, one of which is conserved, at the active site.

Acyltransferases↗
PubMed2003

Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant.

An aspartate kinase-homoserine dehydrogenase (AK-HSDH) cDNA of Arabidopsis thaliana has been cloned by functional complementation of a Saccharomyces cerevisiae strain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast aspartate kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously cloned A. thaliana AK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolated akthr2 cDNA in a HSDH-less hom6 yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficient hom3 yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm that akthr2 encodes a threonine-sensitive bifunctional enzyme. Transgenic Arabidopsis thaliana plants (containing a construct with the promoter region of akthr2 in front of the gus reporter gene) were generated to compare the expression pattern of the akthr2 gene with the pattern of akthr1 earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of the akthr2 gene in the stem, the gynoecium and during seed formation, while akthr1 is less expressed in roots.

Amino Acid Sequence↗