LuxR Family Research Articles

Using surface enhanced Raman scattering to analyze the interactions of protein receptors with bacterial quorum sensing modulators.

Many members of the LuxR family of quorum sensing (QS) transcriptional activators, including LasR of Pseudomonas aeruginosa, are believed to require appropriate acyl-homoserine lactone (acyl-HSL) ligands to fold into an active conformation. The failure to purify ligand-free LuxR homologues in nonaggregated form at the high concentrations required for their structural characterization has limited the understanding of the mechanisms by which QS receptors are activated. Surface-enhanced Raman scattering (SERS) is a vibrational spectroscopy technique that can be applied to study proteins at extremely low concentrations in their active state. The high sensitivity of SERS has allowed us to detect molecular interactions between the ligand-binding domain of LasR (LasRLBD) as a soluble apoprotein and modulators of P. aeruginosa QS. We found that QS activators and inhibitors produce differential SERS fingerprints in LasRLBD, and in combination with molecular docking analysis provide insight into the relevant interaction mechanism. This study reveals signal-specific structural changes in LasR upon ligand binding, thereby confirming the applicability of SERS to analyze ligand-induced conformational changes in proteins.

Structural and Mechanistic Roles of Novel Chemical Ligands on the SdiA Quorum-Sensing Transcription Regulator

ABSTRACTBacteria engage in chemical signaling, termed quorum sensing (QS), to mediate intercellular communication, mimicking multicellular organisms. The LuxR family of QS transcription factors regulates gene expression, coordinating population behavior by sensing endogenous acyl homoserine lactones (AHLs). However, some bacteria (such as Escherichia coli) do not produce AHLs. These LuxR orphans sense exogenous AHLs but also regulate transcription in the absence of AHLs. Importantly, this AHL-independent regulatory mechanism is still largely unknown. Here we present several structures of one such orphan LuxR-type protein, SdiA, from enterohemorrhagic E. coli (EHEC), in the presence and absence of AHL. SdiA is actually not in an apo state without AHL but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life and serves as an energy source, signaling molecule, and substrate for membrane biogenesis. While exogenous AHL renders to SdiA higher stability and DNA binding affinity, OCL may function as a chemical chaperone placeholder that stabilizes SdiA, allowing for basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides crucial mechanistic insights into the ligand regulation of AHL-dependent and -independent function of LuxR-type proteins. Importantly, in addition to its contribution to basic science, this work has implications for public health, inasmuch as the SdiA signaling system aids the deadly human pathogen EHEC to adapt to a commensal lifestyle in the gastrointestinal (GI) tract of cattle, its main reservoir. These studies open exciting and novel avenues to control shedding of this human pathogen in the environment.

Identification of AstG1, A LAL family regulator that positively controls ansatrienins production in Streptomyces sp. XZQH13.

Ansamycins is a group of type I polyketides characterized by the unique starter unit 3-amino-5-hydroxybenzoic acid. This family of secondary metabolites shows diverse biological activities, well-known members of which include rifamycin, geldanamycin, and maytansine. Previously, we isolated an AHBA synthase gene-positive strain Streptomyces sp. XZQH13 containing a "silent" ansamycin biosynthetic gene cluster ast. The constitutive expression of the Large-ATP-binding regulators of the LuxR family regulator gene astG1 located within the cluster triggered the expression of the biosynthetic genes. Reverse transcription-PCR experiments showed that the expression of the key biosynthetic genes, astB4, astD1, and astF1, was induced in the astG1 overexpression mutant compared to the wild type. This led to the isolation of two known ansatrienins, hydroxymycotrienin A (1) and thiazinotrienomycin G (2), which were identified by analysis of the mass spectral and NMR spectral data, from the mutant. These observations suggest that astG1 is probably a pathway-specific positive regulator for the biosynthesis of ansatrienin.

Transcriptomic analysis of effects of triclosan on Mycobacterium bovis BCG

Tuberculosis is a leading cause of death worldwide and infects thousands of Americans annually. Triclosan (TCS) is used as an oxidative antibacterial in household products such as disinfectants, soaps, detergents, toothpaste. This study presents for the first time, the transcriptomic changes in M. bovis BCG after treatment with 0.1 mM TCS for 10 and 60 min. The results after 10 min exposure to TCS show up-regulations of three groups of functional classes genes in M. bovis BCG. First group are hyc group (hycD, hysP, hysQ, and hysE), which is related to energy production and conversion. Second group is related to oxidation stress of many metabolites, especially, accA2, accD2, fadE12, and fadE13. Third group is associated with ppe family which is distinctive to mycobacteria (ppe3, ppe18, and ppe61). After 60 min exposure the following genes were drastically down-regulated ppe61, hycD hycQ, and bcg0232, which express luxR family two component transcriptional regulators.

Improvement of Wuyiencin biosynthesis in Streptomyces wuyiensis CK-15 by identification of a key regulator, WysR.

Wuyiencin is produced by Streptomyces ahygroscopicus var. wuyiensis CK-15 and is widely used as an antifungal agent in agriculture. Analysis of wuyiencin biosynthetic gene clusters reveals wysR, a member of the LAL-family of transcriptional regulatory genes. WysR consists of an Nterminal PAS domain and a LuxR family C-terminal helix-turn-helix motif. However, the roles of wysR in wuyiencin biosynthesis are largely unknown. In this study, we showed that inactivation of wysR resulted in the complete loss of wuyiencin production, which could be restored by complementation with a single copy of wysR. Furthermore, we successfully increased wuyiencin production to a significantly higher level by overexpression of wysR in S. wuyiensis CK-15. Quantitative real-time RT-PCR analysis showed that WysR regulates wuyiencin biosynthesis by modulating other putative regulatory genes. Thus, WysR was identified as an activator of wuyiencin biosynthesis, and overexpression of wysR gene proved to be an effective strategy for improving wuyiencin production.

A conserved PapB family member, TosR, regulates expression of the uropathogenic Escherichia coli RTX nonfimbrial adhesin TosA while conserved LuxR family members TosE and TosF suppress motility.

A heterogeneous subset of extraintestinal pathogenic Escherichia coli (ExPEC) strains, referred to as uropathogenic E. coli (UPEC), causes most uncomplicated urinary tract infections. However, no core set of virulence factors exists among UPEC strains. Instead, the focus of the analysis of urovirulence has shifted to studying broad classes of virulence factors and the interactions between them. For example, the RTX nonfimbrial adhesin TosA mediates adherence to host cells derived from the upper urinary tract. The associated tos operon is well expressed in vivo but poorly expressed in vitro and encodes TosCBD, a predicted type 1 secretion system. TosR and TosEF are PapB and LuxR family transcription factors, respectively; however, no role has been assigned to these potential regulators. Thus, the focus of this study was to determine how TosR and TosEF regulate tosA and affect the reciprocal expression of adhesins and flagella. Among a collection of sequenced UPEC strains, 32% (101/317) were found to encode TosA, and nearly all strains (91% [92/101]) simultaneously carried the putative regulatory genes. Deletion of tosR alleviates tosA repression. The tos promoter was localized upstream of tosR using transcriptional fusions of putative promoter regions with lacZ. TosR binds to this region, affecting a gel shift. A 100-bp fragment 220 to 319 bp upstream of tosR inhibits binding, suggesting localization of the TosR binding site. TosEF, on the other hand, downmodulate motility when overexpressed by preventing the expression of fliC, encoding flagellin. Deletion of tosEF increased motility. Thus, we present an additional example of the reciprocal control of adherence and motility.

Structural Insights into a Novel Interkingdom Signaling Circuit by Cartography of the Ligand-Binding Sites of the Homologous Quorum Sensing LuxR-Family

Recent studies have identified a novel interkingdom signaling circuit, via plant signaling molecules, and a bacterial sub-family of LuxR proteins, bridging eukaryotes and prokaryotes. Indeed pivotal plant-bacteria interactions are regulated by the so called Plant Associated Bacteria (PAB) LuxR solo regulators that, although closely related to the quorum sensing (QS) LuxR family, do not bind or respond to canonical quorum sensing N-acyl homoserine lactones (AHLs), but only to specific host plant signal molecules. The large body of structural data available for several members of the QS LuxR family complexed with different classes of ligands (AHLs and other compounds), has been exploited to dissect the cartography of their regulatory domains through structure-based multiple sequence alignments, structural superimposition and a comparative analysis of the contact residues involved in ligand binding. In the absence of experimentally determined structures of members of the PAB LuxR solos subfamily, an homology model of its prototype OryR is presented, aiming to elucidate the architecture of its ligand-binding site. The obtained model, in combination with the cartography of the regulatory domains of the homologous QS LuxRs, provides novel insights into the 3D structure of its ligand-binding site and unveils the probable molecular determinants responsible for differences in selectivity towards specific host plant signal molecules, rather than to canonical QS compounds.

A regulatory gene (ECO‐orf4) required for ECO‐0501 biosynthesis in Amycolatopsis orientalis

ECO-0501 is a novel linear polyene antibiotic, which was discovered from Amycolatopsis orientalis. Recent study of ECO-0501 biosynthesis pathway revealed the presence of regulatory gene: ECO-orf4. The A. orientalis ECO-orf4 gene from the ECO-0501 biosynthesis cluster was analyzed, and its deduced protein (ECO-orf4) was found to have amino acid sequence homology with large ATP-binding regulators of the LuxR (LAL) family regulators. Database comparison revealed two hypothetical domains, a LuxR-type helix-turn-helix (HTH) DNA binding motif near the C-terminal and an N-terminal nucleotide triphosphate (NTP) binding motif included. Deletion of the corresponding gene (ECO-orf4) resulted in complete loss of ECO-0501 production. Complementation by one copy of intact ECO-orf4 restored the polyene biosynthesis demonstrating that ECO-orf4 is required for ECO-0501 biosynthesis. The results of overexpression ECO-orf4 on ECO-0501 production indicated that it is a positive regulatory gene. Gene expression analysis by reverse transcription PCR of the ECO-0501 gene cluster showed that the transcription of ECO-orf4 correlates with that of genes involved in polyketide biosynthesis. These results demonstrated that ECO-orf4 is a pathway-specific positive regulatory gene that is essential for ECO-0501 biosynthesis.

The LuxR family regulator Rv0195 modulates Mycobacterium tuberculosis dormancy and virulence

Tuberculosis is a leading global killer that has not been effectively controlled to date. The ability of the causative agent, Mycobacterium tuberculosis, to become dormant is one of the major reasons for extended chemotherapeutic regimens and wide epidemicity. The underlying mechanisms of M. tuberculosis dormancy are not fully understood. In the present work, a LuxR family transcription factor gene, Rv0195, was deleted in the virulent M. tuberculosis strain H37Rv. Rv0195 deletion did not affect bacterial growth and long-term survival under aerobiosis but decreased cell survival and the ability to rapidly recover from dormancy in an in vitro anaerobiosis model. The deletion also reduced intracellular survivability under hypoxic and reductive stress triggered by vitamin C. Microarray hybridization analysis showed that Rv0195 affected the expression of more than 180 genes under anaerobiosis, and these genes did not overlap with the known anaerobiosis-up-regulated DosR regulon genes. Furthermore, the Rv0195 deletion diminished bacterial virulence in human macrophage-like cells and resulted in reduced bacterial survival and pathogenicity in a C57BL/6 mouse infection model. These findings offer a novel insight into the mechanisms by which M. tuberculosis adapts to and recovers from dormancy and demonstrate that the dormancy regulator Rv0195 contributes to bacterial virulence.

A transposon-based strategy to identify the regulatory gene network responsible for landomycin E biosynthesis

We report here a transposon-based strategy to generate Streptomyces globisporus 1912 mutants with improved landomycin E production. The modified minitransposon with strong, outward-oriented promoters for the overexpression of downstream-situated genes has been applied for mutant library generation. Approximately 2500 mutants of S.globisporus 1912 were analyzed for landomycin E production, leading to the identification of several overproducers. Subcloning and sequencing of the sites of integration showed that some of the inactivated genes encode proteins with a similarity to known bacterial regulators such as TetR and LuxR families. One of the regulators (GntR type) has shown the strongest influence on the landomycin E production. Its ortholog (encoded by sco3269) in Streptomyces coelicolor was characterized in greater detail and showed similar effects on actinorhodin production and morphological differentiation.

MasABK Proteins Interact with Proteins of the Type IV Pilin System to Affect Social Motility of Myxococcus xanthus

Gliding motility is critical for normal development of spore-filled fruiting bodies in the soil bacterium Myxococcus xanthus. Mutations in mgl block motility and development but one mgl allele can be suppressed by a mutation in masK, the last gene in an operon adjacent to the mgl operon. Deletion of the entire 5.5 kb masABK operon crippled gliding and fruiting body development and decreased sporulation. Expression of pilAGHI, which encodes type IV pili (TFP) components essential for social (S) gliding, several cryptic pil genes, and a LuxR family protein were reduced significantly in the Δmas mutant while expression of the myxalamide operon was increased significantly. Localization and two-hybrid analysis suggest that the three Mas proteins form a membrane complex. MasA-PhoA fusions confirmed that MasA is an integral cytoplasmic membrane protein with a ≈100 amino acid periplasmic domain. Results from yeast two-hybrid assays showed that MasA interacts with the lipoprotein MasB and MasK, a protein kinase and that MasB and MasK interact with one another. Additionally, yeast two-hybrid analysis revealed a physical interaction between two gene products of the mas operon, MasA and MasB, and PilA. Deletion of mas may be accompanied by compensatory mutations since complementation of the Δmas social gliding and developmental defects required addition of both pilA and masABK.

Intercellular and intracellular signalling systems that globally control the expression of virulence genes in plant pathogenic bacteria

Plant pathogenic bacteria utilize complex signalling systems to control the expression of virulence genes at the cellular level and within populations. Quorum sensing (QS), an important intercellular communication mechanism, is mediated by different types of small molecules, including N-acyl homoserine lactones (AHLs), fatty acids and small proteins. AHL-mediated signalling systems dependent on the LuxI and LuxR family proteins play critical roles in the virulence of a wide range of Gram-negative plant pathogenic bacteria belonging to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Xanthomonas spp. and Xylella fastidiosa, members of the Gammaproteobacteria, however, possess QS systems that are mediated by fatty acid-type diffusible signal factors (DSFs). Recent studies have demonstrated that Ax21, a 194-amino-acid protein in Xanthomonas oryzae pv. oryzae, plays dual functions in activating a rice innate immune pathway through binding to the rice XA21 pattern recognition receptor and in regulating bacterial virulence and biofilm formation as a QS signal molecule. In xanthomonads, DSF-mediated QS systems are connected with the signalling pathways mediated by cyclic diguanosine monophosphate (c-di-GMP), which functions as a second messenger for the control of virulence gene expression in these bacterial pathogens.

Bacterial quorum sensing, cooperativity, and anticipation of stationary-phase stress

Acyl-homoserine lactone-mediated quorum sensing (QS) regulates diverse activities in many species of Proteobacteria. QS-controlled genes commonly code for production of secreted or excreted public goods. The acyl-homoserine lactones are synthesized by members of the LuxI signal synthase family and are detected by cognate members of the LuxR family of transcriptional regulators. QS affords a means of population density-dependent gene regulation. Control of public goods via QS provides a fitness benefit. Another potential role for QS is to anticipate overcrowding. As population density increases and stationary phase approaches, QS might induce functions important for existence in stationary phase. Here we provide evidence that in three related species of the genus Burkholderia QS allows individuals to anticipate and survive stationary-phase stress. Survival requires QS-dependent activation of cellular enzymes required for production of excreted oxalate, which serves to counteract ammonia-mediated alkaline toxicity during stationary phase. Our findings provide an example of QS serving as a means to anticipate stationary phase or life at the carrying capacity of a population by activating the expression of cytoplasmic enzymes, altering cellular metabolism, and producing a shared resource or public good, oxalate.

Are There Acyl-Homoserine Lactones within Mammalian Intestines?

Many Proteobacteria are capable of quorum sensing using N-acyl-homoserine lactone (acyl-HSL) signaling molecules that are synthesized by LuxI or LuxM homologs and detected by transcription factors of the LuxR family. Most quorum-sensing species have at least one LuxR and one LuxI homolog. However, members of the Escherichia, Salmonella, Klebsiella, and Enterobacter genera possess only a single LuxR homolog, SdiA, and no acyl-HSL synthase. The most obvious hypothesis is that these organisms are eavesdropping on acyl-HSL production within the complex microbial communities of the mammalian intestinal tract. However, there is currently no evidence of acyl-HSLs being produced within normal intestinal communities. A few intestinal pathogens, including Yersinia enterocolitica, do produce acyl-HSLs, and Salmonella can detect them during infection. Therefore, a more refined hypothesis is that SdiA orthologs are used for eavesdropping on other quorum-sensing pathogens in the host. However, the lack of acyl-HSL signaling among the normal intestinal residents is a surprising finding given the complexity of intestinal communities. In this review, we examine the evidence for and against the possibility of acyl-HSL signaling molecules in the mammalian intestine and discuss the possibility that related signaling molecules might be present and awaiting discovery.

FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis

BackgroundFK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses.ResultsThree putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively.ConclusionsOur results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis NRRL 18488. We have shown that regulatory mechanisms can differ substantially from other, even apparently closely similar FK506-producing strains, reported in literature. Finally, we have demonstrated the potential of these genetically modified strains of S. tsukubaensis for improving the yield of fermentative processes for production of FK506.

Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach

A great deal of research has been done to understand bacterial cell-to-cell signaling systems, but there is still a large gap in our current knowledge because the majority of microorganisms in natural environments do not have cultivated representatives. Metagenomics is one approach to identify novel quorum sensing (QS) systems from uncultured bacteria in environmental samples. In this study, fosmid metagenomic libraries were constructed from a forest soil and an activated sludge from a coke plant, and the target genes were detected using a green fluorescent protein (GFP)-based Escherichia coli biosensor strain whose fluorescence was screened by spectrophotometry. DNA sequence analysis revealed two pairs of new LuxI family N-acyl-L-homoserine lactone (AHL) synthases and LuxR family transcriptional regulators (clones N16 and N52, designated AubI/AubR and AusI/AusR, respectively). AubI and AusI each produced an identical AHL, N-dodecanoyl-L-homoserine lactone (C(12)-HSL), as determined by nuclear magnetic resonance (NMR) and mass spectrometry. Phylogenetic analysis based on amino acid sequences suggested that AusI/AusR was from an uncultured member of the Betaproteobacteria and AubI/AubR was very deeply branched from previously described LuxI/LuxR homologues in isolates of the Proteobacteria. The phylogenetic position of AubI/AubR indicates that they represent a QS system not acquired recently from the Proteobacteria by horizontal gene transfer but share a more ancient ancestry. We demonstrated that metagenomic screening is useful to provide further insight into the phylogenetic diversity of bacterial QS systems by describing two new LuxI/LuxR-type QS systems from uncultured bacteria.

The two-component system PrlS/PrlR of Brucella melitensis is required for persistence in mice and appears to respond to ionic strength

Bacterial adaptation to environmental conditions is essential to ensure maximal fitness in the face of several stresses. In this context, two-component systems (TCSs) represent a predominant signal transduction mechanism, allowing an appropriate response to be mounted when a stimulus is sensed. As facultative intracellular pathogens, Brucella spp. face various environmental conditions, and an adequate response is required for a successful infection process. Recently, bioinformatic analysis of Brucella genomes predicted a set of 15 bona fide TCS pairs, among which some have been previously investigated. In this report, we characterized a new TCS locus called prlS/R, for probable proline sensor-regulator. It encodes a hybrid histidine kinase (PrlS) with an unusual Na(+)/solute symporter N-terminal domain and a transcriptional regulator (belonging to the LuxR family) (PrlR). In vitro, Brucella spp. with a functional PrlR/S system form bacterial aggregates, which seems to be an adaptive response to a hypersaline environment, while a prlS/R mutant does not. We identified ionic strength as a possible signal sensed by this TCS. Finally, this work correlates the absence of a functional PrlR/S system with the lack of hypersaline-induced aggregation in particular marine Brucella spp.

Hierarchical Control on Polyene Macrolide Biosynthesis: PimR Modulates Pimaricin Production via the PAS-LuxR Transcriptional Activator PimM

Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimR. This regulator combines an N-terminal domain corresponding to the Streptomyces antibiotic regulatory protein (SARP) family of transcriptional activators with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family. The PimR SARP domain (PimRSARP) was expressed in Escherichia coli as a glutathione S-transferase (GST)–fused protein. Electrophoretic mobility shift assays showed that GST-PimRSARP binds a single target, the intergenic region between the regulatory genes pimR and pimMs in the pimaricin cluster. The PimRSARP-binding site was investigated by DNaseI protection studies, revealing that it contains three heptameric direct repeats adjusting to the consensus 5′-CGGCAAG-3′. Transcription start points of pimM and pimR promoters were identified by 5′-RACE, revealing that unlike other SARPs, PimRSARP does not interact with the -35 region of its target promoter. Quantitative transcriptional analysis of these regulatory genes on mutants on each of them has allowed the identification of the pimM promoter as the transcriptional target for PimR. Furthermore, the constitutive expression of pimM restored pimaricin production in a pimaricin-deficient strain carrying a deletion mutant of pimR. These results reveal that PimR exerts its positive effect on pimaricin production by controlling pimM expression level, a regulator whose gene product activates transcription from eight different promoters of pimaricin structural genes directly.

Molecular modeling and active site analysis of SdiA homolog, a putative quorum sensor for Salmonella typhimurium pathogenecity reveals specific binding patterns of AHL transcriptional regulators

Salmonella typhimurium is a Gram-negative bacterium responsible for human diseases including gastroenteritis and typhoid fever and its quorum sensing system is currently being intensively researched. Molecular modeling and binding site analysis of SdiA homolog, a putative quorum sensor of the LuxR family and responsible for S. typhimurium pathogenecity revealed a high structural homology of their active site with three other LuxR family proteins LasR from Pseudomonas aeruginosa, TraR from Agrobacterium tumifaciens and CviR from Chromobacterium violaceum. The results show that all the LuxR family proteins harbor three conserved amino acids Tryptophan (W67) and Aspartic acid (D80) for formation of hydrogen bridges and Tyrosine (Y71) for the hydrophobic interactions (corresponding to their position in S. typhimurium SdiA) with acyl homoserine lactones (AHL)-dependent transcriptional regulators. However, in addition to the above conserved residues, Arginine (R60) also plays an important role in S. typhimurium SdiA binding with its AHL auto inducers and the complex is found to be stronger because of the interactions between nitrogen atoms of Arginine with the carbonyl oxygen in the lactone ring of AHL. The specific binding patterns would be helpful in guiding both enzymatic studies as well as design of specific inhibitors to overcome S. typhimurium pathogenecity.

Genetic and Functional Characterization of Cyclic Lipopeptide White-Line-Inducing Principle (WLIP) Production by Rice Rhizosphere Isolate Pseudomonas putida RW10S2

The secondary metabolite mediating the GacS-dependent growth-inhibitory effect exerted by the rice rhizosphere isolate Pseudomonas putida RW10S2 on phytopathogenic Xanthomonas species was identified as white-line-inducing principle (WLIP), a member of the viscosin group of cyclic lipononadepsipeptides. WLIP producers are commonly referred to by the taxonomically invalid name "Pseudomonas reactans," based on their capacity to reveal the presence of a nearby colony of Pseudomonas tolaasii by inducing the formation of a visible precipitate ("white line") in agar medium between both colonies. This phenomenon is attributed to the interaction of WLIP with a cyclic lipopeptide of a distinct structural group, the fungitoxic tolaasin, and has found application as a diagnostic tool to identify tolaasin-producing bacteria pathogenic to mushrooms. The genes encoding the WLIP nonribosomal peptide synthetases WlpA, WlpB, and WlpC were identified in two separate genomic clusters (wlpR-wlpA and wlpBC) with an operon organization similar to that of the viscosin, massetolide, and entolysin biosynthetic systems. Expression of wlpR is dependent on gacS, and the encoded regulator of the LuxR family (WlpR) activates transcription of the biosynthetic genes and the linked export genes, which is not controlled by the RW10S2 quorum-sensing system PmrR/PmrI. In addition to linking the known phenotypes of white line production and hemolytic activity of a WLIP producer with WLIP biosynthesis, additional properties of ecological relevance conferred by WLIP production were identified, namely, antagonism against Xanthomonas and involvement in swarming and biofilm formation.