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PubMed2002

Structural basis and specificity of acyl-homoserine lactone signal production in bacterial quorum sensing.

Synthesis and detection of acyl-homoserine lactones (AHLs) enables many gram-negative bacteria to engage in quorum sensing, an intercellular signaling mechanism that activates differentiation to virulent and biofilm lifestyles. The AHL synthases catalyze acylation of S-adenosyl-L-methionine by acyl-acyl carrier protein and lactonization of the methionine moiety to give AHLs. The crystal structure of the AHL synthase, EsaI, determined at 1.8 A resolution, reveals a remarkable structural similarity to the N-acetyltransferases and defines a common phosphopantetheine binding fold as the catalytic core. Critical residues responsible for catalysis and acyl chain specificity have been identified from a modeled substrate complex and verified through functional analysis in vivo. A mechanism for the N-acylation of S-adenosyl-L-methionine by 3-oxo-hexanoyl-acyl carrier protein is proposed.

4-Butyrolactone↗
PubMed1998

Self perception in bacteria: quorum sensing with acylated homoserine lactones.

A variety of Gram-negative bacteria produce membrane permeant, acylated homoserine lactone (HL) pheromones that act as cell density cues. Synthesis and response to these factors requires proteins homologous to the Luxl acylhomoserine lactone synthase and the LuxR transcription factor from Vibrio fischeri. Recent genetic and biochemical studies have begun to provide a mechanistic understanding of acyl HL dependent gene regulation. Examination of the role of acyl HLs in diverse bacteria positions LuxR-Luxl type systems within an increasingly broad regulatory context and suggests that, in some bacteria, they comprise a global regulatory circuit.

4-Butyrolactone↗
PubMed2006

Antibody interference with N-acyl homoserine lactone-mediated bacterial quorum sensing.

Many bacterial pathogens coordinate their virulence factor expression in a cell density-dependent manner. This population-dependent coordination of gene expression in bacteria has been termed "quorum sensing" (QS). N-Acyl homoserine lactones (AHLs) are used by over 70 Gram-negative bacterial species as autoinducers. Inhibition of QS signaling might represent a new target for antimicrobial therapy. Here we report the hapten design, synthesis, generation of monoclonal antibodies (mAbs) against AHLs, and the evaluation of these mAbs for their ability to blunt QS signaling and inhibit virulence factor expression in P. aeruginosa. The mAbs can be envisioned as a tool for future investigations into AHL-based QS, which may aid in gaining new insights into the pathogenesis of P. aeruginosa and may ultimately lead to the development of new strategies to combat bacterial diseases.

4-Butyrolactone↗
PubMed2004

Rational design and synthesis of new quorum-sensing inhibitors derived from acylated homoserine lactones and natural products from garlic.

Parallel solution-phase synthesis of sulfide AHL analogues (10a-s) by one-pot or a sequential approach is reported. The corresponding sulfoxides 13a-e and sulfones 14a-e were prepared to expand the diversity of the 19-member array of sulfides . Likewise, dithianes 12a-c were prepared with similarity both to sulfides 10a-s and to bioactive structures from garlic. Design and biological screening of all compounds presented in this work targeted inhibition of quorum-sensing comprising competitive inhibition of transcriptional regulators LuxR and LasR. The design was based on critical interactions within the binding-site and structural motifs in molecular components isolated from garlic, 7 and 8, shown to be quorum-sensing inhibitors but not antibiotics. A potent quorum-sensing inhibitor N-(heptylsulfanylacetyl)-l-homoserine lactone (10c) was identified. Together with data collected for the other analogues, the resulting structure-activity relationship led to a hypothesis in which competitive binding was assumed.

4-Butyrolactone↗
PubMed2003

Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes.

N-acylhomoserine lactones (AHLs) are used as signal molecules by many quorum-sensing Proteobacteria. Diverse plant and animal pathogens use AHLs to regulate infection and virulence functions. These signals are subject to biological inactivation by AHL-lactonases and AHL-acylases. Previously, little was known about the molecular details underlying the latter mechanism. An AHL signal-inactivating bacterium, identified as a Ralstonia sp., was isolated from a mixed-species biofilm. The signal inactivation encoding gene from this organism, which we call aiiD, was cloned and successfully expressed in Escherichia coli and inactivated three AHLs tested. The predicted 794-amino-acid polypeptide was most similar to the aculeacin A acylase (AAC) from Actinoplanes utahensis and also shared significant similarities with cephalosporin acylases and other N-terminal (Ntn) hydrolases. However, the most similar homologues of AiiD are deduced proteins of undemonstrated function from available Ralstonia, Deinococcus and Pseudomonas genomes. LC-MS analyses demonstrated that AiiD hydrolyses the AHL amide, releasing homoserine lactone and the corresponding fatty acid. Expression of AiiD in Pseudomonas aeruginosa PAO1 quenched quorum sensing by this bacterium, decreasing its ability to swarm, produce elastase and pyocyanin and to paralyze nematodes. Thus, AHL-acylases have fundamental implications and hold biotechnological promise in quenching quorum sensing.

4-Butyrolactone↗
PubMed2001

Production of N-acyl homoserine lactones by gram-negative bacteria isolated from contact lens wearers.

The purpose of this study was to investigate the production of N-acyl-homoserine lactone (AHL) signal molecules in ocular gram-negative bacteria. A total of 91 ocular strains isolated from contact lens adverse response patients and asymptomatic subjects were used in the study. These included Acinetobacter, Aeromonas hydrophila, Escherichia coli, Haemophilus influenzae, Klebsiella oxytoca, Pseudomonas aeruginosa, Serratia liquefaciens, Serratia marcescens, and Stenotrophomonas maltophilia. The biosensor strains Chromobacterium violaceum mutant CV026 and Agrobacterium tumefaciens A136 were used for detection of AHL signal molecules. The majority of A. hydrophila, P. aeruginosa, and S. liquefaciens strains produced more than one AHL molecule. Serratia marcescens strains were AHL positive only under detection of A136. The rest of the test species did not show any AHL production under the current detection system. These findings indicate that AHL-mediated quorum-sensing systems are present in some of the ocular bacteria, and the different signal molecules may be involved with the quorum-sensing pathway in the other bacterial species.

4-Butyrolactone↗
PubMed2000

hexA of Erwinia carotovora ssp. carotovora strain Ecc71 negatively regulates production of RpoS and rsmB RNA, a global regulator of extracellular proteins, plant virulence and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone.

The soft-rotting bacterium, Erwinia carotovora ssp. carotovora (E. c. carotovora), produces an array of extracellular enzymes (= exoenzymes), including pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel) and protease (Prt), as well as HarpinEcc, the elicitor of hypersensitive reaction (HR). The production of these exoenzymes and HarpinEcc responds to plant products and the quorum-sensing signal [N-(3-oxohexanoyl)-L-homoserine lactone; OHL] and is subject to both transcriptional and post-transcriptional regulation. hexA of E. c. carotovora strain Ecc71 (hereafter hexA71), like that of another E. c. carotovora strain, negatively controls the production of exoenzymes, OHL and virulence in E. c. carotovora strain Ecc71. In addition to exoenzymes, HexA71 negatively regulates the expression of hrpNEcc, the structural gene for HarpinEcc. Exoenzyme overproduction is abolished by OHL deficiency in a HexA- and Ohll- double mutant, indicating that HexA and OHL are components of a common regulatory pathway controlling exoenzyme production. HexA71 negatively affects RpoS, as the levels of this alternative sigma factor are higher in the HexA- mutant than in the HexA+ strain. However, a HexA- and RpoS double mutant produces higher levels of exoenzymes and transcripts of pel-1, peh-1 and celVgenes than the HexA- and RpoS+ parent. Thus, the elevated levels of RpoS protein in the HexA- mutant do not account for exoenzyme overproduction. The following evidence associates for the first time the phenotypic changes in the HexA mutant to overproduction of rsmB RNA, a global regulator of exoenzymes, HarpinEcc, OHL and secondary metabolites. Analyses of rsmB transcripts and expression of an rsmB-lacZoperon fusion in E. c. carotovora strain Ecc71 revealed that HexA71 negatively regulates transcription of rsmB. Multiple copies of hexA71+ DNA suppress various phenotypes, including exoenzyme production in E. c. carotovora strain Ecc71, and concomitantly inhibit the production of rsmB, pel-1, peh-1, celV and hrpNEcc transcripts. Multiple copies of rsmB+ DNA, on the other hand, stimulate exoenzyme production by relieving the negative effects of a chromosomal copy of hexA+. The occurrence of hexA homologues and the negative effect of the dosage of hexA71 DNA on rsmB transcripts were also detected in other E. c. carotovora strains as well as Erwinia carotovora atroseptica and Erwinia carotovora betavasculorum. Extrapolating from the findings with LrhA, the Escherichia coli homologue of HexA, and the presence of sprE homologues in E. carotovora subspecies, we propose that HexA71 controls several regulatory pathways in E. carotovora including rsmB transcription and the production of SprEEcc which, in turn, affects RpoS levels. A model is presented that integrates the findings presented here and our current knowledge of the major regulatory network that controls exoprotein production in soft-rotting Erwinia carotovora subspecies.

4-Butyrolactone↗
PubMed2005

Three-dimensional structure of the quorum-quenching N-acyl homoserine lactone hydrolase from Bacillus thuringiensis.

The three-dimensional structure of the N-acyl-l-homoserine lactone hydrolase (AHL lactonase) from Bacillus thuringiensis has been determined, by using single-wavelength anomalous dispersion (SAD) phasing, to 1.6-angstroms resolution. AHLs are produced by many Gram-negative bacteria as signaling molecules used in quorum-sensing pathways that indirectly sense cell density and regulate communal behavior. Because of their importance in pathogenicity, quorum-sensing pathways have been suggested as potential targets for the development of novel therapeutics. Quorum-sensing can be disrupted by enzymes evolved to degrade these lactones, such as AHL lactonases. These enzymes are members of the metallo-beta-lactamase superfamily and contain two zinc ions in their active sites. The zinc ions are coordinated to a number of ligands, including a single oxygen of a bridging carboxylate and a bridging water/hydroxide ion, thought to be the nucleophile that hydrolyzes the AHLs to ring-opened products, which can no longer act as quorum signals.

Bacillus thuringiensis↗
PubMed2001

Two enzymes of diacylglyceryl-O-4'-(N,N,N,-trimethyl)homoserine biosynthesis are encoded by btaA and btaB in the purple bacterium Rhodobacter sphaeroides.

Betaine lipids are ether-linked, nonphosphorous glycerolipids that resemble the more commonly known phosphatidylcholine in overall structure. Betaine lipids are abundant in many eukaryotes such as nonseed plants, algae, fungi, and amoeba. Some of these organisms are entirely devoid of phosphatidylcholine and, instead, contain a betaine lipid such as diacylglyceryl-O-4'-(N,N,N,-trimethyl)homoserine. Recently, this lipid also was discovered in the photosynthetic purple bacterium Rhodobacter sphaeroides where it seems to replace phosphatidylcholine under phosphate-limiting growth conditions. This discovery provided the opportunity to study the biosynthesis of betaine lipids in a bacterial model system. Mutants of R. sphaeroides deficient in the biosynthesis of the betaine lipid were isolated, and two genes essential for this process, btaA and btaB, were identified. It is proposed that btaA encodes an S-adenosylmethionine:diacylglycerol 3-amino-3-carboxypropyl transferase and btaB an S-adenosylmethionine-dependent N-methyltransferase. Both enzymatic activities can account for all reactions of betaine lipid head group biosynthesis. Because the equivalent reactions have been proposed for different eukaryotes, it seems likely that orthologs of btaA/btaB may be present in other betaine lipid-containing organisms.

Aminoacyltransferases↗
PubMed1999

Acyl homoserine-lactone quorum-sensing signal generation.

Acyl homoserine lactones (acyl-HSLs) are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression. These signals are synthesized by members of the LuxI family of proteins. To understand the mechanism of acyl-HSL synthesis we have purified the Pseudomonas aeruginosa RhlI protein and analyzed the kinetics of acyl-HSL synthesis by this enzyme. Purified RhlI catalyzes the synthesis of acyl-HSLs from acyl-acyl carrier proteins and S-adenosylmethionine. An analysis of the patterns of product inhibition indicated that RhlI catalyzes signal synthesis by a sequential, ordered reaction mechanism in which S-adenosylmethionine binds to RhlI as the initial step in the enzymatic mechanism. Because pathogenic bacteria such as P. aeruginosa use acyl-HSL signals to regulate virulence genes, an understanding of the mechanism of signal synthesis and identification of inhibitors of signal synthesis has implications for development of quorum sensing-targeted antivirulence molecules.

4-Butyrolactone↗
PubMed2006

Long- and short-chain plant-produced bacterial N-acyl-homoserine lactones become components of phyllosphere, rhizosphere, and soil.

Two N-acyl-homoserine lactone (acyl-HSL) synthase genes, lasI from Pseudomonas aeruginosa and yenI from Yersinia enterocolitica, were introduced into tobacco, individually and in combination. Liquid chromatograph-tandem mass spectrometry and thin-layer chromatography confirmed products of lasI and yenI activity in single and cotransformants. Cotransformants expressing plastid-localized LasI and YenI synthases produced the major acyl-HSLs for each synthase in all tissues tested. Total acyl-HSL signals accumulated in leaf tissue up to 3 pmol/mg of fresh weight, half as much in stem tissue, and approximately 10-fold less in root tissues. Acyl-HSLs were present in aqueous leaf washes from greenhouse-grown transgenic plants. Transgenic lines grown for 14 days under axenic conditions produced detectable levels of acyl-HSLs in root exudates. Ethyl acetate extractions of rhizosphere and nonrhizosphere soil from transgenically grown plants contained active acyl-HSLs, whereas plant-free soil or rhizosphere and nonrhizosphere soil from wild-type plants lacked detectable amounts of acyl-HSLs. This work shows that bioactive acyl-HSLs are exuded from leaves and roots and accumulate in the phytosphere of plants engineered to produce acyl-HSLs. These data further suggest that plants that are bioengineered to synthesize acyl-HSLs can foster beneficial plant-bacteria communications or deter deleterious interactions. Therefore, it is feasible to use bioengineered plants to supplement soils with specific acyl-HSLs to modulate bacterial phenotypes and plant-associated bacterial community structures.

4-Butyrolactone↗
PubMed1998

Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria.

Many gram-negative bacteria regulate expression of specialized gene sets in response to population density. This regulatory mechanism, called autoinduction or quorum-sensing, is based on the production by the bacteria of a small, diffusible signal molecule called the autoinducer. In the most well-studied systems the autoinducers are N-acylated derivatives of L-homoserine lactone (acyl-HSL). Signal specificity is conferred by the length, and the nature of the substitution at C-3, of the acyl side-chain. We evaluated four acyl-HSL bioreporters, based on tra of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudomonas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HSLs, and alkanoyl-HSLs with chain lengths ranging from C4 to C12. The traG::lacZ fusion reporter from the A. tumefaciens Ti plasmid was the single most sensitive and versatile detector of the four. Using this reporter, we screened 106 isolates representing seven genera of bacteria that associate with plants. Most of the Agrobacterium, Rhizobium, and Pantoea isolates and about half of the Erwinia and Pseudomonas isolates gave positive reactions. Only a few isolates of Xanthomonas produced a detectable signal. We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography. Among the pseudomonads and erwinias, most produced a single dominant activity chromatographing with the properties of N-(3-oxo-hexanoyl)-L-HSL. However, a few of the erwinias, and the P. fluorescens and Ralstonia solanacearum isolates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlike any of our standards. The few positive xanthomonas, and almost all of the agrobacteria, produced small amounts of a compound with the chromatographic properties of N-(3-oxo-octanoyl)-L-HSL. Members of the genus Rhizobium showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals. Several isolates produced extremely nonpolar compounds indicative of very long acyl side-chains. Production of these compounds suggests that quorum-sensing is common as a gene regulatory mechanism among gram-negative plant-associated bacteria.

4-Butyrolactone↗
PubMed2000

Plants secrete substances that mimic bacterial N-acyl homoserine lactone signal activities and affect population density-dependent behaviors in associated bacteria.

In gram-negative bacteria, many important changes in gene expression and behavior are regulated in a population density-dependent fashion by N-acyl homoserine lactone (AHL) signal molecules. Exudates from pea (Pisum sativum) seedlings were found to contain several separable activities that mimicked AHL signals in well-characterized bacterial reporter strains, stimulating AHL-regulated behaviors in some strains while inhibiting such behaviors in others. The chemical nature of the active mimic compounds is currently unknown, but all extracted differently into organic solvents than common bacterial AHLs. Various species of higher plants in addition to pea were found to secrete AHL mimic activities. The AHL signal-mimic compounds could prove to be important in determining the outcome of interactions between higher plants and a diversity of pathogenic, symbiotic, and saprophytic bacteria.

4-Butyrolactone↗
PubMed2004

The assimilation of gamma-butyrolactone in Agrobacterium tumefaciens C58 interferes with the accumulation of the N-acyl-homoserine lactone signal.

Agrobacterium tumefaciens C58 communicates using N-acyl-homoserine lactones (acyl-HSL) and contains two lactonase-encoding genes, attM and aiiB, the products of which are capable of inactivating the acyl-HSL signal. In A. tumefaciens A6, the expression of the attKLM operon is controlled by the transcriptional repressor encoded by an adjacent gene, attJ. An attJ::Tn5 mutant does not accumulate acyl-HSL because of the constitutive expression of the lactonase AttM, the activity of which inactivates acyl-HSL. In this work, the attKLM operon of A. tumefaciens C58 was shown to be involved in an assimilative pathway of gamma-butyrolactone (GBL), gamma-hydroxybutyrate (GHB), and succinate semialdehyde (SSA), in which AttM and AttL are key enzymes for GBL and GHB assimilation. The expression of the attKLM promoter was activated in the presence of GBL, GHB, and SSA. Under these conditions, A. tumefaciens C58 did not accumulate the acyl-HSL that it naturally synthesizes, and also became able to inactivate exogenous acyl-HSL signals. Therefore, in A. tumefaciens C58, the assimilative pathway of gamma-butyrolactone interferes with the acyl-HSL signaling.

4-Butyrolactone↗
PubMed2000

The Aspergillus nidulans cysA gene encodes a novel type of serine O-acetyltransferase which is homologous to homoserine O-acetyltransferases.

The Aspergillus nidulans cysA gene was cloned by functional complementation of the cysA1 mutation that impairs the synthesis of O:-acetylserine. The molecular nature of cysA1 and cysA103 alleles was characterized; a nucleotide substitution and a frame shift were found in the former and a deletion mutation in the latter. The CYSA protein is 525 amino acids long and is encoded by an uninterrupted open reading frame. Expression of the cysA gene appears not to be regulated by sulfur, carbon and nitrogen sources. Protein sequence analysis reveals extensive similarity to homoserine O:-acetyltransferases, particularly the bacterial ones, and no homology with known serine O:-acetyltransferases. The authors propose that the CYSA protein is analogous to serine O:-acetyltransferases, i.e. it catalyses the same reaction but has an independent evolutionary origin.

Acetyltransferases↗
PubMed1997

Expression of an Aspartate Kinase Homoserine Dehydrogenase Gene Is Subject to Specific Spatial and Temporal Regulation in Vegetative Tissues, Flowers, and Developing Seeds.

Although the regulation of amino acid synthesis has been studied extensively at the biochemical level, it is still not known how genes encoding amino acid biosynthesis enzymes are regulated during plant development. In the present report, we have used the [beta]-glucuronidase (GUS) reporter gene to study the regulation of expression of an Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase (AK/HSD) gene in transgenic tobacco plants. The polypeptide encoded by the AK/HSD gene comprises two linked key enzymes in the biosynthesis of aspartate-family amino acids. AK/HSD-GUS gene expression was highly stimulated in apical and lateral meristems, lateral buds, young leaves, trichomes, vascular and cortical tissues of growing stems, tapetum and other tissues of anthers, pollen grains, various parts of the developing gynoecium, developing seeds, and, in some transgenic plants, also in stem and leaf epidermal trichomes. AK/HSD-GUS gene expression gradually dimished upon maturation of leaves, stems, floral tissues, and embryos. GUS expression was relatively low in roots. During seed development, expression of the AK/HSD gene in the embryo was coordinated with the initiation and onset of storage protein synthesis, whereas in the endosperm it was coordinated with the onset of seed desiccation. Upon germination, AK/HSD-GUS gene expression in the hypocotyl and the cotyledons was significantly affected by light. The expression pattern of the A. thaliana AK/HSD-GUS reporter gene positively correlated with the levels of aspartate-family amino acids and was also very similar to the expression pattern of the endogenous tobacco AK/HSD mRNA as determined by in situ hybridization.

Journal Article↗
PubMed1980

Comparison of sensitive and desensitized forms of maize homoserine dehydrogenase.

The properties of homoserine dehydrogenase (EC 1.1.1.3) isolated from shoots of young etiolated seedlings of Zea mays L. var. earliking can be reversibly altered by dialysis against an appropriate buffer. Treatment with 500 millimolar potassium phosphate buffer (pH 7.5) in the absence of l-threonine results in diminished regulatory control such that the enzyme becomes less sensitive to feedback inhibition. The physical and regulatory properties of experimentally altered and unaltered enzymes are compared with those of enzyme isolated from shoots of older seedlings. Multiple forms of both sensitive and insensitive enzymes are identified, and a model which is consistent with the observed isozymes and the difference in regulatory properties of enzymes obtained from seedlings of different ages is proposed. The initially sensitive enzyme is postulated to undergo a conformational change followed by formation of insensitive multimeric aggregated forms. The experimental conditions which facilitate alteration of the enzyme are discussed in relation to conditions which could occur in vivo.

Journal Article↗
PubMed1981

Effects of plant age and extraction conditions on the properties of homoserine dehydrogenase isolated from maize seedlings.

Homoserine dehydrogenase (EC 1.1.1.3) was extracted from shoots of etiolated seedlings of Zea mays L. which had been grown for periods ranging from three to thirteen days. Both the amount of enzyme extracted and its regulatory properties, as measured by the sensitivity of the enzyme to inhibition by the feedback modulator, l-threonine, were found to be a function of seedling age and extraction conditions. Equivalent amounts of enzyme with similar properties could be isolated from young seedlings under a variety of conditions. Extraction media containing comparatively low concentrations of the buffer component and a high concentration of dithioerythritol were found to be required for optimum extraction of the enzyme from shoots of seedlings grown longer than four days and from leaves of light-grown plants. In the absence of dithioerythritol, diminished regulatory control was observed to be a direct function of seedling age. Evidence of rapid desensitization of the enzyme during extraction was obtained from experiments in which dithioerythritol was added to extracts prepared in the absence of a thiol compound. Therefore, previous observations of growth-dependent desensitization in a number of plants could be due to incomplete extraction or to changes in cellular factors which inactivate and/or alter the enzyme. Whether the enzyme itself becomes increasingly susceptible to alteration during seedling growth remains to be established.

Journal Article↗