A second quorum-sensing system regulates cell surface properties but not phenazine antibiotic production in Pseudomonas aureofaciens

The root-associated biological control bacterium Pseudomonas aureofaciens 30-84 produces a range of exoproducts, including protease and phenazines. Phenazine antibiotic biosynthesis by phzXYFABCD is regulated in part by the PhzR-PhzI quorum-sensing system. Mutants defective in phzR or phzI produce very low levels of phenazines but wild-type levels of exoprotease. In the present study, a second genomic region of strain 30-84 was identified that, when present in trans, increased beta-galactosidase activity in a genomic phzB::lacZ reporter and partially restored phenazine production to a phzR mutant. Sequence analysis identified two adjacent genes, csaR and csaI, that encode members of the LuxR-LuxI family of regulatory proteins. No putative promoter region is present upstream of the csaI start codon and no lux box-like element was found in either the csaR promoter or the 30-bp intergenic region between csaR and csaI. Both the PhzR-PhzI and CsaR-CsaI systems are regulated by the GacS-GacA two-component regulatory system. In contrast to the multicopy effects of csaR and csaI in trans, a genomic csaR mutant (30-84R2) and a csaI mutant (30-84I2) did not exhibit altered phenazine production in vitro or in situ, indicating that the CsaR-CsaI system is not involved in phenazine regulation in strain 30-84. Both mutants also produced wild-type levels of protease. However, disruption of both csaI and phzI or both csaR and phzR eliminated both phenazine and protease production completely. Thus, the two quorum-sensing systems do not interact for phenazine regulation but do interact for protease regulation. Additionally, the CsaI N-acylhomoserine lactone (AHL) signal was not recognized by the phenazine AHL reporter 30-84I/Z but was recognized by the AHL reporters Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136(pCF240). Inactivation of csaR resulted in a smooth mucoid colony phenotype and formation of cell aggregates in broth, suggesting that CsaR is involved in regulating biosynthesis of cell surface components. Strain 30-84I/I2 exhibited mucoid colony and clumping phenotypes similar to those of 30-84R2. Both phenotypes were reversed by complementation with csaR-csaI or by the addition of the CsaI AHL signal. Both quorum-sensing systems play a role in colonization by strain 30-84. Whereas loss of PhzR resulted in a 6.6-fold decrease in colonization by strain 30-84 on wheat roots in natural soil, a phzR csaR double mutant resulted in a 47-fold decrease. These data suggest that gene(s) regulated by the CsaR-CsaI system also plays a role in the rhizosphere competence of P. aureofaciens 30-84.     

Negative Cross-Communication among Wheat Rhizosphere Bacteria: Effect on Antibiotic Production by the Biological Control Bacterium Pseudomonas aureofaciens 30-84

Phenazine antibiotic production in the biological control bacterium Pseudomonas aureofaciens 30-84 is regulated in part via the PhzR/PhzI N-acyl homoserine lactone (AHL) system. Previous work showed that a subpopulation of the wheat rhizosphere community positively affected phenazine gene expression in strain 30-84 via AHL signals (E. A. Pierson, D. W. Wood, J. A. Cannon, F. M. Blachere, and L. S. Pierson III, Mol. Plant-Microbe Interact. 11:1078-1084, 1998). In the present work, a second subpopulation, one that negatively affected phenazine gene expression, was identified from this rhizosphere community. Strain 30-84 grown in conditioned medium (CM) from several strains produced lower levels of phenazines (1.5- to 9.3-fold) than control when grown in CM from the strain 30-84I₁/I₂. Growth of the phzB::lacZ reporter strain 30-84Z in this CM resulted in decreased lacZ expression (4.3- to 9.2-fold) compared to growth of the control strain in CM, indicating that inhibition of phzB occurred at the level of gene expression. Preliminary chemical and biological characterizations suggested that these signals, unlike other identified negative signals, were not extractable in ethyl acetate. Introduction of extra copies of phzR and phzI, but not phzI alone, in trans into strain 30-84Z reduced the negative effect on phzB::lacZ expression. The presence of negative-signal-producing strains in a mixture with strain 30-84 reduced strain 30-84's ability to inhibit the take-all disease pathogen in vitro. Together, the results from the previous work on the positive-signal subpopulation and the present work on the negative-signal subpopulation suggest that cross-communication among members of the rhizosphere community and strain 30-84 may control secondary metabolite production and pathogen inhibition.     

Quorum-sensing regulation in soil pseudomonads

228 strains of soil and rhizosphere pseudomonads isolated in different geographic zones were screened, with the use of two tester systems, for the capacity to produce N-acyl-homoserine lactones (AHLs), which are autoinducers involved in quorum-sensing (QS) regulation. AHL production was found in 11.4% of the strains investigated. In five Pseudomonas chlororaphis strains shown to be active AHL producers and chosen for further study, PCR identified two QS systems that involved the phzI, phzR, csaI, and csaR genes; this finding suggests the conservative nature of these regulation systems in P. chloroaphis. Strain P. chlororaphis 449, chosen as a model object and studied in greater detail, produced three AHL species including N-butanoyl-homoserine lactone and N-hexanoyl-homoserine lactone. This strain produced three types of phenazine antibiotics, as well as siderophores and cyanide; it also exhibited antagonistic properties toward a wide spectrum of phytopathogenic fungi. The phzI and csaI genes, coding for synthases of AHLs of two types, were cloned and sequenced; mutants with knocked-out phzI and csaI genes were obtained. With the use of transposon mutagenesis and the gene substitution method, mutations were obtained in the global expression regulator genes gacS, coding for the GacA-GacS regulation system kinase, and rpoS, coding for the sigma S subunit of RNA polymerase. The effect of these mutations on the AHL synthesis and on the regulation of various metabolic processes in P. chlororaphis was studied.     

Quorum sensing systems of regulation,synthesis of phenazine antibiotics,and antifungal activity in rhizospheric bacterium <Emphasis Type="Italic">pseudomonas chlororaphis</Emphasis> 449

Strain Pseudomonas chlororaphis 449, an antagonist of a broad spectrum of phytopathogenic microorganisms isolated from the maize rhizosphere, was shown to produce three phenazine antibiotics: phenazine-1-carboxylic acid (PCA), 2-hydroxylphenazine-1-carboxylic acid (2-OH-PCA), and 2-hydroxylphenazine (2-OH-PHZ). Two Quorum Sensing (QS) systems of regulation were identified: Phz/R and CsaI/R. Genes phzI and csaI were cloned and sequenced. Cells of strain 449 synthesize at least three types of AHL: N-butanoyl-L-homoserine lactone (C₄-AHL), N-hexanoyl-L-homoserine lactone (C₆-AHL), and N-(3-oxo-hexanoyl)-L-homoserine lactone (30C₆-AHL). Transposon mutagenesis was used to generate mutants of strain 449 deficient in synthesis of phenazines, which carried inactivated phzA and phzB genes of the phenazine operon and gene phzO. Mutations phzA ^? and phzB ^? caused a drastic reduction in the antagonistic activity of bacteria toward phytopathogenic fungi. Both mutants lost the ability to protect cucumber and leguminous plants against phytopathogenic fungi Rhizoctonia solani and Sclerotinia sclerotiorum. These results suggest a significant role of phenazines in the antagonistic activity of P. chlororaphis 449.     

Negative cross-communication among wheat rhizosphere bacteria: effect on antibiotic production by the biological control bacterium Pseudomonas aureofaciens 30-84

Phenazine antibiotic production in the biological control bacterium Pseudomonas aureofaciens 30-84 is regulated in part via the PhzR/PhzI N-acyl homoserine lactone (AHL) system. Previous work showed that a subpopulation of the wheat rhizosphere community positively affected phenazine gene expression in strain 30-84 via AHL signals (E. A. Pierson, D. W. Wood, J. A. Cannon, F. M. Blachere, and L. S. Pierson III, Mol. Plant-Microbe Interact. 11:1078-1084, 1998). In the present work, a second subpopulation, one that negatively affected phenazine gene expression, was identified from this rhizosphere community. Strain 30-84 grown in conditioned medium (CM) from several strains produced lower levels of phenazines (1.5- to 9.3-fold) than control when grown in CM from the strain 30-84I(1)/I(2). Growth of the phzB::lacZ reporter strain 30-84Z in this CM resulted in decreased lacZ expression (4.3- to 9.2-fold) compared to growth of the control strain in CM, indicating that inhibition of phzB occurred at the level of gene expression. Preliminary chemical and biological characterizations suggested that these signals, unlike other identified negative signals, were not extractable in ethyl acetate. Introduction of extra copies of phzR and phzI, but not phzI alone, in trans into strain 30-84Z reduced the negative effect on phzB::lacZ expression. The presence of negative-signal-producing strains in a mixture with strain 30-84 reduced strain 30-84's ability to inhibit the take-all disease pathogen in vitro. Together, the results from the previous work on the positive-signal subpopulation and the present work on the negative-signal subpopulation suggest that cross-communication among members of the rhizosphere community and strain 30-84 may control secondary metabolite production and pathogen inhibition.     

N-(3-Hydroxyhexanoyl)-l-Homoserine Lactone Is the Biologically Relevant Quormone That Regulates the phz Operon of Pseudomonas chlororaphis Strain 30-84

Phenazine production by Pseudomonas fluorescens 2-79 and P. chlororaphis isolates 30-84 and PCL1391 is regulated by quorum sensing through the activator PhzR and acyl-homoserine lactones (acyl-HSLs) synthesized by PhzI. PhzI from P. fluorescens 2-79 produces five acyl-HSLs that include four 3-hydroxy species. Of these, N-(3-hydroxyhexanoyl)-HSL is the biologically relevant ligand for PhzR. The quorum-sensing systems of P. chlororaphis strains 30-84 and PCL1391 have been reported to produce and respond to N-(hexanoyl)-HSL. These differences were of interest since PhzI and PhzR of strain 2-79 share almost 90% sequence identity with orthologs from strains 30-84 and PCL1391. In this study, as assessed by thin-layer chromatography, the three strains produce almost identical complements of acyl-HSLs. The major species produced by P. chlororaphis 30-84 were identified by mass spectrometry as 3-OH-acyl-HSLs with chain lengths of 6, 8, and 10 carbons. Heterologous bacteria expressing cloned phzI from strain 30-84 produced the four 3-OH acyl-HSLs in amounts similar to those seen for the wild type. Strain 30-84, but not strain 2-79, also produced N-(butanoyl)-HSL. A second acyl-HSL synthase of strain 30-84, CsaI, is responsible for the synthesis of this short-chain signal. Strain 30-84 accumulated N-(3-OH-hexanoyl)-HSL to the highest levels, more than 100-fold greater than that of N-(hexanoyl)-HSL. In titration assays, PhzR_30-84 responded to both N-(3-OH-hexanoyl)- and N-(hexanoyl)-HSL with equal sensitivities. However, only the 3-OH-hexanoyl signal is produced by strain 30-84 at levels high enough to activate PhzR. We conclude that strains 2-79, 30-84, and PCL1391 use N-(3-OH-hexanoyl)-HSL to activate PhzR.     

Plants genetically modified to produce N-acylhomoserine lactones communicate with bacteria.

N-acylhomoserine lactones (AHLs) play a critical role in plant/microbe interactions. The AHL, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), induces exoenzymes that degrade the plant cell wall by the pathogenic bacterium Erwinia carotovora. Conversely, the antifungal activity of the biocontrol bacterium Pseudomonas aureofaciens 30-84 is due (at least in part) to phenazine antibiotics whose synthesis is regulated by N-hexanoylhomoserine lactone (HHL). Targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in plants of the cognate AHL signaling molecules (OHHL and HHL). The AHLs produced by the transgenic plants were sufficient to induce target gene expression in several recombinant bacterial AHL biosensors and to restore biocontrol activity to an HHL-deficient P. aureofaciens strain. In addition, pathogenicity was restored to an E. carotovora strain rendered avirulent as a consequence of a mutation in the OHHL synthase gene, carI. The ability to generate bacterial quorum-sensing signaling molecules in the plant offers novel opportunities for disease control and for manipulating plant/microbe interactions.     

phzO, a gene for biosynthesis of 2-hydroxylated phenazine compounds in Pseudomonas aureofaciens 30-84

Certain strains of root-colonizing fluorescent Pseudomonas spp. produce phenazines, a class of antifungal metabolites that can provide protection against various soilborne root pathogens. Despite the fact that the phenazine biosynthetic locus is highly conserved among fluorescent Pseudomonas spp., individual strains differ in the range of phenazine compounds they produce. This study focuses on the ability of Pseudomonas aureofaciens 30-84 to produce 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA) and 2-hydroxyphenazine from the common phenazine metabolite phenazine-1-carboxylic acid (PCA). P. aureofaciens 30-84 contains a novel gene located downstream from the core phenazine operon that encodes a 55-kDa aromatic monooxygenase responsible for the hydroxylation of PCA to produce 2-OH-PCA. Knowledge of the genes responsible for phenazine product specificity could ultimately reveal ways to manipulate organisms to produce multiple phenazines or novel phenazines not previously described.     

Cloning and heterologous expression of the phenazine biosynthetic locus from Pseudomonas aureofaciens 30-84.

Pseudomonas aureofaciens strain 30-84 suppresses take-all disease of wheat caused by Gaeumannomyces graminis var. tritici. Three antibiotics, phenazine-1-carboxylic acid, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine, were responsible for disease suppression. Tn5-induced mutants deficient in production of one or more of the antibiotics (Phz-) were significantly less suppressive than the parental strain. Cosmids pLSP259 and pLSP282 from a genomic library of strain 30-84 restored phenazine production and fungal inhibition to 10 different Phz- mutants. Sequences required for production of the phenazines were localized to a segment of approximately 2.8 kilobases that was present in both cosmids. Expression of this locus in Escherichia coli required the introduction of a functional promoter, was orientation-specific, and resulted in the production of all three phenazine antibiotics. These results strongly suggest that the cloned sequences encode a major portion of the phenazine biosynthetic pathway.     

ExpI and PhzI Are Descendants of the Long Lost Cognate Signal Synthase for SdiA

SdiA of E. coli and Salmonella is a LuxR homolog that detects N-acyl homoserine lactones (AHLs). Most LuxR homologs function together with a cognate AHL synthase (a LuxI homolog), but SdiA does not. Instead, SdiA detects AHLs produced by other bacterial species. In this report, we performed a phylogenetic analysis of SdiA. The results suggest that one branch of the Enterobacteriaceae obtained a rhlR/rhlI pair by horizontal transfer. The Erwinia and Pantoea branches still contain the complete pair where it is known as expR/expI and phzR/phzI, respectively. A deletion event removed the luxI homolog from the remainder of the group, leaving just the luxR homolog known as sdiA. Thus ExpR and PhzR are SdiA orthologs and ExpI and PhzI are descendants of the long lost cognate signal synthase of SdiA.     

Survival of GacS/GacA Mutants of the Biological Control Bacterium Pseudomonas aureofaciens 30-84 in the Wheat Rhizosphere

GacS/GacA comprises a two-component regulatory system that controls the expression of secondary metabolites required for the control of plant diseases in many pseudomonads. High mutation frequencies of gacS and gacA have been observed in liquid culture. We examined whether gacS/gacA mutants could competitively displace the wild-type populations on roots and thus pose a threat to the efficacy of biological control. The survival of a gac mutant alone and in competition with the wild type on roots was examined in the biological control strain Pseudomonas aureofaciens 30-84. In this bacterium, GacS/GacA controls the expression of phenazine antibiotics that are inhibitory to plant pathogenic fungi and enhance the competitive survival of the bacterium. Wheat seedlings were inoculated with strain 30-84, and bacteria were recovered from roots after 21 days in sterile or nonsterile soil to check for the presence of gacS or gacA mutants. Although no mutants were detected in the inoculum, gacS/gacA mutants were recovered from 29 out of 31 roots and comprised up to 36% of the total bacterial populations. Southern hybridization analysis of the recovered gacA mutants did not indicate a conserved mutational mechanism. Replacement series analysis on roots utilizing strain 30-84 and a gacA mutant (30-84.gacA) or a gacS mutant (30-84.A2) demonstrated that although the mutant population partially displaced the wild type in sterile soil, it did not do so in natural soil. In fact, in natural soil final rhizosphere populations of wild-type strain 30-84 starting from mixtures were at least 1.5 times larger than would be predicted from their inoculation ratio and generally were greater than or equal to the population of wild type alone despite lower inoculation rates. These results indicate that although gacS/gacA mutants survive in natural rhizosphere populations, they do not displace wild-type populations. Better survival of wild-type populations in mixtures with mutants suggests that mutants arising de novo or introduced within the inoculum may be beneficial for the survival of wild-type populations in the rhizosphere.     

GacS-dependent regulation of enzymic and antifungal activities and synthesis of <Emphasis Type="Italic">N</Emphasis>-acylhomoserine lactones in rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

Pseudomonas chlororaphis strain 449 isolated from the rhizosphere of maize suppresses numerous plant pathogens in vitro. The strain produces phenazine antibiotics and synthesizes at least three types of quorum sensing signaling molecules, N-acylhomoserine lactones. Here we have shown that the rhizospheric P. chlororaphis strains 449, well known strain 30–84 as well as two other P. chlororaphis strains exhibit polygalacturonase activity. Using mini-Tn5 transposon mutagenesis, four independent mutants of strain P. chlororaphis 449 with insertion of mini-Tn5 Km2 in gene gacS of two-component GacA-GacS system of global regulation were selected. All these mutant strains were deficient in production of extracellular proteinase(s), phenazines, N-acylhomoserine lactones synthesis, and did not inhibit the growth of G⁺ bacteria in comparison with the wild type strain. The P. chlororaphis 449-06 gacS ⁻ mutant studied in greater detail was deficient in polygalacturonase, pectin methylesterase activities, swarming motility and antifungal activity. It is the first time the involvement of GacA-GacS system in the regulation of enzymes of pectin metabolism, polygalacturonase and pectin methylesterase, was demonstrated in fluorescent pseudomonads.     

A Seven-Gene Locus for Synthesis of Phenazine-1-Carboxylic Acid by Pseudomonas fluorescens 2-79

Pseudomonas fluorescens 2-79 produces the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA), which is active against a variety of fungal root pathogens. In this study, seven genes designated phzABCDEFG that are sufficient for synthesis of PCA were localized within a 6.8-kb BglII-XbaI fragment from the phenazine biosynthesis locus of strain 2-79. Polypeptides corresponding to all phz genes were identified by analysis of recombinant plasmids in a T7 promoter/polymerase expression system. Products of the phzC, phzD, and phzE genes have similarities to enzymes of shikimic acid and chorismic acid metabolism and, together with PhzF, are absolutely necessary for PCA production. PhzG is similar to pyridoxamine-5′-phosphate oxidases and probably is a source of cofactor for the PCA-synthesizing enzyme(s). Products of the phzA and phzB genes are highly homologous to each other and may be involved in stabilization of a putative PCA-synthesizing multienzyme complex. Two new genes, phzX and phzY, that are homologous to phzA and phzB, respectively, were cloned and sequenced from P. aureofaciens 30-84, which produces PCA, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine. Based on functional analysis of the phz genes from strains 2-79 and 30-84, we postulate that different species of fluorescent pseudomonads have similar genetic systems that confer the ability to synthesize PCA.     

Mutants of Burkholderia cenocepacia with a change in synthesis of N-acyl-homoserine lactones—Signal molecules of quorum sensing regulation

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding Lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.     

A Quorum-Quenching Approach To Investigate the Conservation of Quorum-Sensing-Regulated Functions within the Burkholderia cepacia Complex

Taxonomic studies of the past few years have shown that the Burkholderia cepacia complex, a heterogeneous group of B. cepacia-like organisms, consists of at least nine species. B. cepacia complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time, B. cepacia complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All B. cepacia complex species investigated thus far use quorum-sensing (QS) systems that rely on N-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the Bacillus sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described B. cepacia complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested B. cepacia complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and Caenorhabditis elegans killing efficiency was regulated by AHL in the large majority of strains investigated.