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Conditioned medium from Listeria innocua stimulates emergence from a resting state: not a response to E. coli quorum sensing autoinducer AI-2.

The lag phase of the bacterial growth curve is an important determinant in speeding the detection of pathogens. It is affected by many factors including the prevailing growth environment and inoculum size, as well as specific signal molecules. The elucidation of growth-regulating signal molecules is further facilitated by culturing cells in defined growth media. In this study, a defined medium capable of supporting growth of Listeria innocua at similar levels as obtained using a complex brain heart infusion (BHI) media was developed. Further, the effects of conditioned medium (CM) on population lag time of L. innocua was investigated using a rapid parallel approach (with an automated microtiter plate reader). Importantly, the lag phase was shortened by up to approximately 50% by the addition of CM from L. innocua cultures obtained late in the exponential phase. Finally, while L. innocua were found to secrete bacterial signaling autoinducer, AI-2, tests using Escherichia coli based CM having a 90-fold difference in AI-2 level suggested that the observed decrease in lag phase was not due to E. coli-derived AI-2 and was instead due to elements specific to L. innocua. These findings indicate secreted signal molecules may be found in CM that speed detection of L. innocua.

Cell Division↗

Synthetic analogues of the bacterial signal (quorum sensing) molecule N-(3-oxododecanoyl)-L-homoserine lactone as immune modulators.

Comparative immune modulatory activity for a range of synthetic analogues of a Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-l-homoserine lactone (3O, C(12)-HSL), is described. Twenty-four single or combination systematic alterations of the structural components of 3O, C(12)-HSL were introduced as described. Given the already defined immunological profile of the parent compound, 3O, C(12)-HSL, these compounds were assayed for their ability to inhibit murine and human leucocyte proliferation and TNF-alpha secretion by lipopolysaccharide (LPS) stimulated human leucocytes in order to provide an initial structure-activity profile. From IC(50) values obtained with a murine splenocyte proliferation assay, it is apparent that acylated l-homoserine lactones with an 11-13 C side chain containing either a 3-oxo or a 3-hydroxy group are optimal structures for immune suppressive activity. These derivatives of 3O, C(12)-HSL with monounsaturation and/or a terminal nonpolar substituent on the side chain were also potent immune suppressive agents. However, structures lacking the homoserine lactone ring, structures lacking the l-configuration at the chiral center, and those with polar substituents were essentially devoid of activity. The ability of compounds selected from the optimal activity range to modulate mitogen-driven human peripheral blood mononuclear cell proliferation and LPS-induced TNF-alpha secretion indicates the suitability of these compounds for further investigation in relation to their molecular mechanisms of action in TNF-alpha driven immunological diseases, particularly autoimmune diseases such as psoriasis, rheumatoid arthritis, and type 1 (autoimmune) diabetes.

Adjuvants, Immunologic↗

Biosurfactant production by Serratia marcescens SS-1 and its isogenic strain SMdeltaR defective in SpnR, a quorum-sensing LuxR family protein.

Serratia marcescens SS-1 and its SpnR-defective isogenic mutant, SMdeltaR, produced an extracellular surfactant able to decrease surface tension of water from 72 to 37 dyne cm(-1) (SMdeltaR strain) and to 45 dyne cm(-1) (SS-1 strain). The biosurfactant also emulsified kerosene and diesel with a maximum emulsion index of 77% (diesel and kerosene) for the SMdeltaR strain, and 72% (kerosene) and 40% (diesel) for the SS-1 strain. Deletion of spnR gene appeared to enhance biosurfactant production. Model simulations suggest that biosurfactant production by the two strains was growth-associated. The SMdeltaR strain had a yield coefficient of 22-32% g dry cell(-1), which is 32-50% higher than that of the SS-1 strain.

Emulsions↗

Structure of a bacterial quorum-sensing transcription factor complexed with pheromone and DNA.

Many proteobacteria are able to monitor their population densities through the release of pheromones known as N-acylhomoserine lactones. At high population densities, these pheromones elicit diverse responses that include bioluminescence, biofilm formation, production of antimicrobials, DNA exchange, pathogenesis and symbiosis. Many of these regulatory systems require a pheromone-dependent transcription factor similar to the LuxR protein of Vibrio fischeri. Here we present the structure of a LuxR-type protein. TraR of Agrobacterium tumefaciens was solved at 1.66 A as a complex with the pheromone N-3-oxooctanoyl-L-homoserine lactone (OOHL) and its TraR DNA-binding site. The amino-terminal domain of TraR is an alpha/beta/alpha sandwich that binds OOHL, whereas the carboxy-terminal domain contains a helix turn helix DNA-binding motif. The TraR dimer displays a two-fold symmetry axis in each domain; however, these two axes of symmetry are at an approximately 90 degree angle, resulting in a pronounced overall asymmetry of the complex. The pheromone lies fully embedded within the protein with virtually no solvent contact, and makes numerous hydrophobic contacts with the protein as well as four hydrogen bonds: three direct and one water-mediated.

Amino Acid Sequence↗

Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR.

The transcription factor LuxR activates gene expression in response to binding the signaling molecule 3-oxo-hexanoyl-homoserine lactone (3OC6HSL), an acyl-HSL with a carbonyl substituent at the third carbon of the acyl chain. We previously described a LuxR variant, LuxR-G2E, that activates gene expression by binding a broader range of acyl-HSLs, including straight-chain acyl-HSLs to which LuxR does not respond. Here, we use a dual positive-negative selection system to identify a variant of LuxR-G2E that retains the response to straight-chain acyl-HSLs, but no longer responds to 3OC6HSL. A single mutation, R67M, reduces LuxR-G2E's response to acyl-HSLs having a carbonyl substituent at the third carbon of the acyl chain. This specificity-enhancing mutation would not have been identified by positive selection alone. The dual selection system provides a rapid and reliable method for identifying LuxR variants that have or lack the desired response to a given set of acyl-HSL signals. LuxR variants with altered signaling specificities might become useful components for constructing artificial cell-cell communication systems that program population level behaviors.

Cell Communication↗

Structure of the Bacillus subtilis quorum-sensing peptide pheromone ComX.

The ComX pheromone is an extracellular signaling molecule that stimulates natural competence in response to crowding in the gram-positive bacterium Bacillus subtilis. The pheromone is formed by isoprenylation of an inactive precursor peptide, but its precise structure is not known. Here we report the structure of the ComX pheromone, showing that addition of a geranyl group to a tryptophan residue results in the formation of an unusual ring structure.

Amino Acid Sequence↗

Octopine-type Ti plasmids code for a mannopine-inducible dominant-negative allele of traR, the quorum-sensing activator that regulates Ti plasmid conjugal transfer.

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by two hierarchical signalling systems. Transfer is dependent on a subset of opines produced by the plant tumours induced by the bacterium. Induction also requires an acyl-homoserine lactone signal, called AAI, that is produced by the bacteria themselves. AAI is the co-inducer for TraR, the transcriptional activator required for expression of the tra regulon. Octopine induces conjugation of the octopine-mannityl opine-type Ti plasmids by regulating the expression of traR via OccR, the octopine-dependent activator of the opine regulon. We have discovered a second traR-like gene, trlR, on the octopine-mannityl opine-type Ti plasmids pTi15955 and pTiR10. This gene is located in an operon coding for a mannopine transport system and is expressed as part of the mannityl opine regulon. Sequence analysis indicated that trlR is a frameshift allele of traR, and the resulting protein lacks the carboxy-terminal domain thought to constitute the DNA-binding region of TraR. Expression of trlR inhibited octopine-induced conjugation of pTi15955 and pTiR10 by suppressing the TraR-mediated transcription of the tra and trb operons. Although TrlR had no effect on the expression of traR, TraR activated the expression of trlR. Southern hybridizations indicated that several other Ti and opine-catabolic plasmids contain more than one copy of genes homologous to traR. We propose that trlR is a dominant negative allele of traR and that TrlR inhibits conjugation by forming inactive heteromultimers with TraR.

Agrobacterium tumefaciens↗

Gelatinase biosynthesis-activating pheromone: a peptide lactone that mediates a quorum sensing in Enterococcus faecalis.

Biosynthesis of gelatinase, a virulence factor of Enterococcus faecalis, was found to be regulated in a cell density-dependent fashion in which its production is active in late log to early stationary phase. Addition of early stationary phase culture filtrate to medium shifted the onset of gelatinase production to that of mid-log phase, suggesting that E. faecalis secretes a gelatinase biosynthesis-activating pheromone (GBAP). GBAP was isolated from culture supernatant of E. faecalis OG1S-P. Structural analysis suggested GBAP to be an 11-residue cyclic peptide containing a lactone structure, in which the alpha-carboxyl group of the C-terminal amino acid is linked to a hydroxyl group of the serine of the third residue. A synthetic peptide possessing the deduced structure showed GBAP activity at nanomolar concentrations as did natural GBAP. Database searches revealed that GBAP corresponds to a C-terminal part of a 242-residue FsrB protein. Northern analysis showed that GBAP slowly induces the transcription of two operons, fsrB-fsrC encoding FsrB and a putative histidine kinase FsrC and gelE-sprE encoding gelatinase GelE and serine protease SprE. Strains with an insertion mutation in either fsrC or a putative response regulator gene fsrA failed to respond to GBAP, suggesting that the GBAP signal is transduced by a two-component regulatory system.

Amino Acid Sequence↗

The LuxR family protein SpnR functions as a negative regulator of N-acylhomoserine lactone-dependent quorum sensing in Serratia marcescens.

Serratia marcescens SS-1 produces at least four N-acylhomoserine lactones (AHLs) which were identified using high-resolution mass spectrometry and chemical synthesis, as N-(3-oxohexanoyl) homo-serine lactone (3-oxo-C6-HSL), N-hexanoyl- (C6-HSL), N-heptanoyl (C7-HSL) and N-octanoyl- (C8-HSL) homoserine lactone. These AHLs are synthesized via the LuxI homologue SpnI, and regulate via the LuxR homologue SpnR, the production of the red pigment, prodigiosin, the nuclease, NucA, and a biosurfactant which facilitates surface translocation. spnR overexpression and spnR gene deletion show that SpnR, in contrast to most LuxR homologues, acts as a negative regulator. spnI overexpression, the provision of exogenous AHLs and spnI gene deletion suggest that SpnR is de-repressed by 3-oxo-C6-HSL. In addition, long chain AHLs antagonize the biosurfactant-mediated surface translocation of S. marcescens SS-1. Upstream of spnI there is a gene which we have termed spnT. spnI and spnT form an operon and although database searches failed to reveal any spnT homologues, overexpression of this novel gene negatively affected both sliding motility and prodigiosin production.

4-Butyrolactone↗

Autoinducer binding by the quorum-sensing regulator TraR increases affinity for target promoters in vitro and decreases TraR turnover rates in whole cells.

TraR is an Agrobacterium transcriptional regulator whose activity requires the pheromone N-3-oxooctanoyl-L-homoserine lactone. TraR was purified as a complex with the pheromone and contained one pheromone molecule per protein monomer. TraR-pheromone complexes bound to a single DNA site and activated two promoters that flank this site. Promoter expression was elevated 30-fold by using a supercoiled template. Pheromone binding increased the affinity of TraR for this binding site. Pheromone also increased TraR abundance in vivo by causing a 20-fold decrease in TraR turnover rates.

Bacterial Proteins↗

The cin quorum sensing locus of Rhizobium etli CNPAF512 affects growth and symbiotic nitrogen fixation.

Rhizobium etli CNPAF512 produces an autoinducer that inhibits growth of Rhizobium leguminosarum bv. viciae 248 and activates the Agrobacterium tumefaciens tra reporter system. Production of this compound in R. etli is dependent on two genes, named cinR and cinI, postulated to code for a transcriptional regulator and an autoinducer synthase, respectively. NMR analysis of the purified molecule indicates that the R. etli autoinducer produced by CinI is a saturated long chain 3-hydroxy-acyl-homoserine lactone, abbreviated as 3OH-(slc)-HSL. Using cin-gusA fusions, expression of cinI and cinR was shown to be growth phase-dependent. Deletion analysis of the cinI promoter region indicates that a regulatory element negatively controls cinI expression. Mutational analysis revealed that expression of the cinI gene is positively regulated by the CinR/3OH-(slc)-HSL complex. Besides 3OH-(slc)-HSL, R. etli produces at least six other autoinducer molecules, for which the structures have not yet been revealed, and of which the synthesis requires the previously identified raiI and raiR genes. At least three different autoinducers, including a compound co-migrating with 3OH-(slc)-HSL, are produced in R. etli bacteroids isolated from bean nodules. This is further substantiated by the observation that cinI and cinR are both expressed under symbiotic conditions. Acetylene reduction activity of nodules induced by the cin mutants was reduced with 60-70% compared with wild-type nodules, indicating that the R. etli 3OH-(slc)-HSL is involved in the symbiotic process. This was further confirmed by transmission electron microscopy of nodules induced by the wild type and the cinI mutant. Symbiosomes carrying cinI mutant bacteroids did not fully differentiate compared with wild-type symbiosomes. Finally, it was observed that the cinR gene and raiR control growth of R. etli.

4-Butyrolactone↗

Expression and characterization of the peptidase domain of Streptococcus pneumoniae ComA, a bifunctional ATP-binding cassette transporter involved in quorum sensing pathway.

ComA, a member of the bacteriocin ATP-binding cassette transporters, is postulated to be responsible for both the processing of the propeptide ComC and secretion of the mature competence-stimulating peptide, which regulates the competence and subsequent genetic transformation in Streptococcus pneumoniae. A recombinant N-terminal peptidase domain of ComA, designated PEP, was expressed as a soluble protein in Escherichia coli, purified to homogeneity, and characterized. Gel-filtration analysis revealed that PEP functions as a monomer. The purified PEP exhibited an efficient proteolytic activity for the substrate ComC, which was cleaved after the double glycine site. The stability of PEP was examined by circular dichroism analyses. A convenient method for analyzing the proteolytic activity of PEP was developed, and the kinetic parameters for ComC were determined (k(cat) = 1.5 +/- 0.083 min(-1) and Km = 62 +/- 9.0 microM). Replacements of Cys17 of PEP with Ser or Ala and His96 with Ala resulted in complete loss of activity, indicating that both Cys17 and His96 are essential for the catalysis. Together with information from a protease data base, the N-terminal domain of ComA was concluded to belong to the same clan as the papain-like cysteine proteases. Mutant substrates, in which each of the double glycines was replaced with Ala, were cleaved very poorly by PEP. The mechanism of this strict substrate specificity is discussed on the basis of the sequence alignment with other cysteine proteases.

Adenosine Triphosphate↗

Sub-inhibitory concentrations of ceftazidime and tobramycin reduce the quorum sensing signals of Pseudomonas aeruginosa.

AIM: Concentrations of antimicrobials below minimum inhibitory concentration (subMIC) may reduce the production by Pseudomonas aeruginosa of virulence factors such as elastase. We sought to determine whether the reduction in elastase production may be mediated by a reduction in acyl-homoserine lactones. METHODS: Pseudomonas aeruginosa in broth was exposed to three conditions for ceftazidime and tobramycin: control, 6% MIC and 25% MIC. Elastase was assayed using elastin congo red. N-(3-Oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl-homoserine lactone (C4-HSL) were assayed using biosensor Escherichia coli. RESULTS: Elastase was unchanged with ceftazidime. Elastase was reduced by 16% at 6% MIC tobramycin and reduced by 70% at 25% MIC tobramycin (P<0.0001). As a percentage of control, C12-HSL was mean 69.4% (SEM 7.3%) at 6% MIC tobramycin, and 31.7% (3.3%) at 25% MIC tobramycin (P=0.0001). C12-HSL was 78.9% (5.3%) at 6% MIC ceftazidime and was 29.7% (1.8%) at 25% MIC ceftazidime (P=0.0001). Both ceftazidime and tobramycin were associated with reduced C4-HSL at 6% MIC and 25% MIC (P<0.03). CONCLUSIONS: SubMIC tobramycin but not ceftazidime reduced elastase production by P. aeruginosa. In contrast, subMIC concentrations of both antimicrobials reduced C12-HSL and C4-HSL. It is unlikely that reduced HSL is the sole explanation for the reduction in elastase.

Anti-Bacterial Agents↗

Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora: the role of expR(Ecc).

The production of the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the extracellular cell wall-degrading enzymes, is partly controlled by the diffusible signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). OHHL is synthesized by the product of the expI/carI gene. Linked to expI we found a gene encoding a putative transcriptional regulator of the LuxR-family. This gene, expR(Ecc), is transcribed convergently to the expI gene and the two open reading frames are partially overlapping. The ExpR(Ecc) protein showed extensive amino acid sequence similarity to the repressor EsaR from Pantoea stewartii subsp. stewartii (formerly Erwinia stewartii subsp. stewartii) and to the ExpR(Ech) protein of Erwinia chrysanthemi. Inactivation of the E. carotovora subsp. carotovora expR(Ecc) gene caused no decrease in virulence or production of virulence determinants in vitro. In contrast, there was a slight increase in the maceration capacity of the mutant strain. The effects of ExpR(Ecc) were probably mediated by changes in OHHL levels. Inactivation of expR(Ecc) resulted in increased OHHL levels during early logarithmic growth. In addition, overexpression of expR(Ecc) caused a clear decrease in the production of virulence determinants and part of this effect was likely to be caused by OHHL binding to ExpR(Ecc). ExpR(Ecc) did not appear to exhibit transcriptional regulation of expI, but the effect on OHHL was apparently due to other mechanisms.

Amino Acid Sequence↗