Enantio-pyochelin: a novel siderophore produced by Pseudomonas fluorescens CHA0

RESUME Pour favoriser sa croissance en condition limitante de fer, le pathogène opportunistePseudomonas aeruginosa PAO1 sécrète un sidérophore nommé pyochéline. Celui-ci estproduit par un mécanisme de "thiotemplate", à partir de l'acide salicylique et de deuxmolécules de cystéine, et existe sous forme d'une paire de diastéréoisomèresinterconvertibles: pyochéline I (4'R, 2?R, 4?R) et pyochéline II (4'R, 2?S, 4?R). Deprécédentes études ont montré que la pyochéline induit l'expression de ses propres gènes debiosynthèse via le régulateur transcriptionnel PchR qui appartient à la famille AraC/XylS. Lapyochéline est donc non seulement un sidérophore mais également une molécule signale.Nous avons découvert que Pseudomonas fluorescens CHA0 sécrète une pyochélinestéréochimiquement distincte de celle produite par P. aeruginosa. Ce nouveau sidérophorefavorise la croissance de P. fluorescens en condition limitante en fer et induit l'expression deses propres gènes de biosynthèse. Cependant, cette molécule n'est pas reconnue commesidérophore ou molécule signale par P. aeruginosa. Réciproquement, la pyochéline estincapable de stimuler la croissance et la signalisation chez P. fluorescens. La structure dusiderophore de P. fluorescens CHA0 a été déterminée comme étant un antipode optique de lapyochéline et nommé énantio-pyochéline.La stéréospécificité de l'induction des gènes de biosynthèse de la pyochéline/énantiopyochélineest basée sur la stéréospécificité des protéines PchR de P. aeruginosa et P.fluorescens envers leur sidérophores-ligands respectifs. PchR est fonctionnel chez l'espècehétérologue, mais uniquement en présence de son propre ligand. Les récepteurs spécifiquesdes sidérophores pyochéline/enantio-pyochéline ne sont pas indispensables à la signalisationmais sont essentiels à l'incorporation du fer et à la croissance en carence de fer. Laconstruction de protéines hybrides et tronquées a révélé que le domaine N-terminal de PchRest l'élément déterminant pour la spécificité de la protéine vis-à-vis de son ligand. SUMMARY : The siderophore pyochelin is produced by the opportunistic pathogen Pseudomonas aeruginosa PAO1 and promotes growth under iron limitation. Pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine and exists as a pair of interconvertible diastereoisomers: pyochelin I (4'R, 2"R, 4"R) and pyochelin II (4'R, 2"S, 4"R). Pyochelin induces the expression of its biosynthesis and uptake genes via the transcriptional AraC/Xy1S family regulator PchR in a process termed pyochelin signaling. Pseudomonas fluorescens CHAO was found to make a stereochemically distinct pyochelin to P. aeruginosa. This siderophore promoted the growth of P. fluorescens under iron limitation and induced the expression of its biosynthesis genes but was not recognised as a siderophore or signaling molecule by P. aeruginosa. Reciprocally, pyochelin was unable to promote growth or signaling in P. fluorescens. The structure of the P. fluorescens CHAO siderophore was determined and found to be enantio-pyochelin, the optical antipode of pyochelin. Stereospecificity in induction of pyochelin/enantio-pyochelin biosynthesis genes was found to be due to stereospecificity of the homologous PchR proteins of P. aeruginosa and P. fluorescens towards their respective siderophore ligands. PchR was able to function in the heterologous species, but only if supplied with its native ligand. The pyochelin/enantiopyochelin receptors were not essential for signaling although both receptors are essential for iron uptake and growth under iron limitation. Construction of hybrid and truncated PchR proteins revealed that the N-terminal domain of PchR is responsible for siderophore recognition/stereospecificity.

Optimizacion de la produccion de biotensioactivos por Pseudomonas aeruginosa 44T1

En este trabajo se describen los resultados experimentales destinados a la optimización de la producción de biotensioactivos por Pseudomonas aeruginosa 44T1 en un medio mineral con glucosa como fuente de carbono. Se han ensayado diversos componentes del medio de cultivo y condiciones de incubación, siendo la relación C/N, la concentración de hierro así como la temperatura de incubación, los parámetros fundamentales que han incrementado los valores de CMC-1 como medida de la acumulación de tensioactivos.

Genome-wide search reveals a novel GacA-regulated small RNA in Pseudomonas species.

BACKGROUND: Small RNAs (sRNAs) are widespread among bacteria and have diverse regulatory roles. Most of these sRNAs have been discovered by a combination of computational and experimental methods. In Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium and opportunistic human pathogen, the GacS/GacA two-component system positively controls the transcription of two sRNAs (RsmY, RsmZ), which are crucial for the expression of genes involved in virulence. In the biocontrol bacterium Pseudomonas fluorescens CHA0, three GacA-controlled sRNAs (RsmX, RsmY, RsmZ) regulate the response to oxidative stress and the expression of extracellular products including biocontrol factors. RsmX, RsmY and RsmZ contain multiple unpaired GGA motifs and control the expression of target mRNAs at the translational level, by sequestration of translational repressor proteins of the RsmA family. RESULTS: A combined computational and experimental approach enabled us to identify 14 intergenic regions encoding sRNAs in P. aeruginosa. Eight of these regions encode newly identified sRNAs. The intergenic region 1698 was found to specify a novel GacA-controlled sRNA termed RgsA. GacA regulation appeared to be indirect. In P. fluorescens CHA0, an RgsA homolog was also expressed under positive GacA control. This 120-nt sRNA contained a single GGA motif and, unlike RsmX, RsmY and RsmZ, was unable to derepress translation of the hcnA gene (involved in the biosynthesis of the biocontrol factor hydrogen cyanide), but contributed to the bacterium's resistance to hydrogen peroxide. In both P. aeruginosa and P. fluorescens the stress sigma factor RpoS was essential for RgsA expression. CONCLUSION: The discovery of an additional sRNA expressed under GacA control in two Pseudomonas species highlights the complexity of this global regulatory system and suggests that the mode of action of GacA control may be more elaborate than previously suspected. Our results also confirm that several GGA motifs are required in an sRNA for sequestration of the RsmA protein.

Measurement of biologically available naphthalene in gas and aqueous phases by use of a Pseudomonas putida biosensor.

Genetically constructed microbial biosensors for measuring organic pollutants are mostly applied in aqueous samples. Unfortunately, the detection limit of most biosensors is insufficient to detect pollutants at low but environmentally relevant concentrations. However, organic pollutants with low levels of water solubility often have significant gas-water partitioning coefficients, which in principle makes it possible to measure such compounds in the gas rather than the aqueous phase. Here we describe the first use of a microbial biosensor for measuring organic pollutants directly in the gas phase. For this purpose, we reconstructed a bioluminescent Pseudomonas putida naphthalene biosensor strain to carry the NAH7 plasmid and a chromosomally inserted gene fusion between the sal promoter and the luxAB genes. Specific calibration studies were performed with suspended and filter-immobilized biosensor cells, in aqueous solution and in the gas phase. Gas phase measurements with filter-immobilized biosensor cells in closed flasks, with a naphthalene-contaminated aqueous phase, showed that the biosensor cells can measure naphthalene effectively. The biosensor cells on the filter responded with increasing light output proportional to the naphthalene concentration added to the water phase, even though only a small proportion of the naphthalene was present in the gas phase. In fact, the biosensor cells could concentrate a larger proportion of naphthalene through the gas phase than in the aqueous suspension, probably due to faster transport of naphthalene to the cells in the gas phase. This led to a 10-fold lower detectable aqueous naphthalene concentration (50 nM instead of 0.5 micro M). Thus, the use of bacterial biosensors for measuring organic pollutants in the gas phase is a valid method for increasing the sensitivity of these valuable biological devices.

Strategies of "Pseudomonas aeruginosa" to overcome copper limitation

Summary Copper is an important trace element and micronutrient for living organisms as it is the cofactor of several enzymes involved in diverse biological redox processes such as aerobic respiration, denitrification and photosynthesis. Despite its importance, copper may be poorly bioavailable in soils and aquatic environments, as well as in the human body, especially at physiological or alkaline pH. In this work, we have investigated the strategies that the versatile bacterium and opportunistic pathogen Pseudomonas aeruginosa has evolved to face and overcome copper limitation. The global response of the P. aeruginosa to copper limitation was assessed under aerobic conditions. Numerous iron uptake functions (including the siderophores pyoverdine and pyochelin) were down-regulated whereas expression of cioAB (encoding an alternative, copper-independent, cyanide-resistant ubiquinol oxidase) was up-regulated. Wild type P. aeruginosa was able to grow aerobically in a defined glucose medium depleted of copper by a copper chelator, whereas a cioAB mutant did not grow. Thus, P. aeruginosa relies on the CioAB enzyme to cope with severe copper deprivation. A quadruple cyo cco1 cco2 cox mutant, which was deleted for all known heme-copper terminal oxidases of P. aeruginosa, grew aerobically, albeit more slowly than did the wild type, indicating that the CioAB enzyme is capable of energy conservation. However, the expression of a cioA'-'lacZ fusion was less dependent on the copper status in the quadruple mutant than in the wild type, suggesting that copper availability might affect cioAB expression indirectly, via the function of the heme-copper oxidases. These results suggest that the CioAB enzyme can be used as a by-pass strategy to overcome severe copper limitation and perform aerobic respiration even if virtually no copper is available. The PA0114 gene, which encodes a protein of the SCOT/SenC family, was found to be important for copper acquisition and aerobic respiration in low copper conditions. A PA0114 (sent) mutant grew poorly in low copper media and had low terminal oxidase activity with TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine), but expressed the CioAB enzyme at elevated levels. Addition of copper reversed these phenotypes, suggesting that periplasmic copper capture by the SenC protein is another strategy that helps P. aeruginosa to adapt to copper deprivation. RESUME Le cuivre est un micronutriment important pour les organismes vivants. Il représente le cofacteur de plusieurs enzymes impliquées dans une multitude de processus biologiques tels que la respiration aérobie, la dénitrification et la photosynthèse. Malgré son importance, le cuivre peut être peu disponible dans les sols, les environnements aquatiques et le corps humain, spécialement à pH physiologique ou alcalin. Dans ce travail nous avons étudié les stratégies développées par la bactérie pathogène opportuniste Pseudomonas aeruginosa PAO1 afm de faire face et de surmonter le manque de cuivre. La réponse globale de P. aeruginosa à la carence de cuivre a été analysée dans des conditions aérobie. Les résultats obtenus ont montré que plusieurs gènes impliqués dans l'acquisition du fer, tels que les gènes codant pour les sidérophores (pyoverdine et pyochéline), étaient réprimés, tandis que l'expression de l'opéron cioAB, codant pour l'oxydase terminale insensible au cyanure (CIO), était augmentée. La souche sauvage P. aeruginosa est capable de croître dans un milieu où la concentration en cuivre est limitée, due à la présence d'un chélateur spéciftque de cuivre, tandis que le mutant cioAB ne croît pas dans ces conditions. Nous avons conclu que P. aeruginosa nécessite l'oxydase terminale CIO pour faire face à la carence en cuivre. Un quadruple mutant affecté dans toutes les oxydases dépendantes du cuivre (cyo ccol cco2 cox) et appartenant aux oxydases de type hème-cuivre, peut croître en aérobie, néanmoins plus lentement que la souche sauvage, ce qui montre que l'enzyme CIO est capable de conserver l'énergie. L'expression de la fusion rapportrice cioA'-'IacZ chez le quadruple mutant est moins dépendante de la disponibilité de cuivre que chez la souche sauvage. Ces résultats suggèrent que la disponibilité de cuivre influence l'expression de cioAB d'une façon indirecte, par le biais des oxydases terminales de type héme-cuivre. Il est donc possible qu'en cas de carence de cuivre, P. aeruginosa utilise l'enzyme CIO comme stratégie afin de surmonter ce manque et de réaliser la respiration aérobie. Nous avons démontré que le gène PA0114, codant pour une protéine appartenant à la famille SCO1/SenC, est important dans l'acquisition et dans la respiration aérobie dans des environnements où le cuivre est présent en faible concentration. En ces conditions, la croissance du mutant senC est faible; de plus, l'activité des oxydases terminales en présence du donneur d'électrons TMPD (N,N,N,N'-tetraméthyl-p-phénylenediamine) est basse. Toutefois, l'addition de cuivre au milieu de culture permet de restaurer le phénotype du type sauvage. Ces résultats montrent que la protéine SenC est capable d'acquérir le cuivre et représente donc une autre stratégie chez P. aeruginosa pour s'adapter à un manque de cuivre.

Characterization of PhlG, a hydrolase that specifically degrades the antifungal compound 2,4-diacetylphloroglucinol in the biocontrol agent Pseudomonas fluorescens CHA0.

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative DeltaphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a DeltaphlA DeltaphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a kcat of 33 s(-1) and a Km of 140 microM at 30 degrees C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.

Temperature-responsive sensing regulates biocontrol factor expression in Pseudomonas fluorescens CHA0.

In the plant-beneficial, root-colonizing strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively regulates the synthesis of biocontrol factors (mostly antifungal secondary metabolites) and contributes to oxidative stress response via the stress sigma factor RpoS. The backbone of this pathway consists of the GacS/GacA two-component system, which activates the expression of three small regulatory RNAs (RsmX, RsmY, RsmZ) and thereby counters translational repression exerted by the RsmA and RsmE proteins on target mRNAs encoding biocontrol factors. We found that the expression of typical biocontrol factors, that is, antibiotic compounds and hydrogen cyanide (involving the phlA and hcnA genes), was significantly lower at 35 degrees C than at 30 degrees C. The expression of the rpoS gene was affected in parallel. This temperature control depended on RetS, a sensor kinase acting as an antagonist of the GacS/GacA system. An additional sensor kinase, LadS, which activated the GacS/GacA system, apparently did not contribute to thermosensitivity. Mutations in gacS or gacA were epistatic to (that is, they overruled) mutations in retS or ladS for expression of the small RNAs RsmXYZ. These data are consistent with a model according to which RetS-GacS and LadS-GacS interactions shape the output of the Gac/Rsm pathway and the environmental temperature influences the RetS-GacS interaction in P. fluorescens CHA0.

Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.

Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.

A dual functional origin of transfer in the ICEclc genomic island of Pseudomonas knackmussii B13.

Genomic islands (GEIs) are large DNA segments, present in most bacterial genomes, that are most likely acquired via horizontal gene transfer. Here, we study the self-transfer system of the integrative and conjugative element ICEclc of Pseudomonas knackmussii B13, which stands model for a larger group of ICE/GEI with syntenic core gene organization. Functional screening revealed that unlike conjugative plasmids and other ICEs ICEclc carries two separate origins of transfer, with different sequence context but containing a similar repeat motif. Conjugation experiments with GFP-labelled ICEclc variants showed that both oriTs are used for transfer and with indistinguishable efficiencies, but that having two oriTs results in an estimated fourfold increase of ICEclc transfer rates in a population compared with having a single oriT. A gene for a relaxase essential for ICEclc transfer was also identified, but in vivo strand exchange assays suggested that the relaxase processes both oriTs in a different manner. This unique dual origin of transfer system might have provided an evolutionary advantage for distribution of ICE, a hypothesis that is supported by the fact that both oriT regions are conserved in several GEIs related to ICEclc.

Fast and simple epidemiological typing of Pseudomonas aeruginosa using the double-locus sequence typing (DLST) method.

Although the molecular typing of Pseudomonas aeruginosa is important to understand the local epidemiology of this opportunistic pathogen, it remains challenging. Our aim was to develop a simple typing method based on the sequencing of two highly variable loci. Single-strand sequencing of three highly variable loci (ms172, ms217, and oprD) was performed on a collection of 282 isolates recovered between 1994 and 2007 (from patients and the environment). As expected, the resolution of each locus alone [number of types (NT) = 35-64; index of discrimination (ID) = 0.816-0.964] was lower than the combination of two loci (NT = 78-97; ID = 0.966-0.971). As each pairwise combination of loci gave similar results, we selected the most robust combination with ms172 [reverse; R] and ms217 [R] to constitute the double-locus sequence typing (DLST) scheme for P. aeruginosa. This combination gave: (i) a complete genotype for 276/282 isolates (typability of 98%), (ii) 86 different types, and (iii) an ID of 0.968. Analysis of multiple isolates from the same patients or taps showed that DLST genotypes are generally stable over a period of several months. The high typability, discriminatory power, and ease of use of the proposed DLST scheme makes it a method of choice for local epidemiological analyses of P. aeruginosa. Moreover, the possibility to give unambiguous definition of types allowed to develop an Internet database ( http://www.dlst.org ) accessible by all.

PchR-box recognition by the AraC-type regulator PchR of Pseudomonas aeruginosa requires the siderophore pyochelin as an effector.

Under iron limitation, the opportunistic human pathogen Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted into the extracellular environment, pyochelin complexes ferric ions and delivers them, via the outer membrane receptor FptA, to the bacterial cytoplasm. Extracellular pyochelin also acts as a signalling molecule, inducing the expression of pyochelin biosynthesis and uptake genes by a mechanism involving the AraC-type regulator PchR. We have identified a 32 bp conserved sequence element (PchR-box) in promoter regions of pyochelin-controlled genes and we show that the PchR-box in the pchR-pchDCBA intergenic region is essential for the induction of the pyochelin biosynthetic operon pchDCBA and the repression of the divergently transcribed pchR gene. PchR was purified as a fusion with maltose-binding protein (MBP). Mobility shift assays demonstrated specific binding of MBP-PchR to the PchR-box in the presence, but not in the absence of pyochelin and iron. PchR-box mutations that interfered with pyochelin-dependent regulation in vivo, also affected pyochelin-dependent PchR-box recognition in vitro. We conclude that pyochelin, probably in its iron-loaded state, is the intracellular effector required for PchR-mediated regulation. The fact that extracellular pyochelin triggers this regulation suggests that the siderophore can enter the cytoplasm.

Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa.

The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic ornithine carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of proteins.

Characterization of a MexAB-OprM efflux system necessary for productive metabolism of Pseudomonas azelaica HBP1 on 2-hydroxybiphenyl.

Pseudomonas azelaica HBP1 is one of the few bacteria known to completely mineralize the biocide and toxic compound 2-hydroxybiphenyl (2-HBP), but the mechanisms of its tolerance to the toxicity are unknown. By transposon mutant analysis and screening for absence of growth on water saturating concentrations of 2-HBP (2.7 mM) we preferentially found insertions in three genes with high homology to the mexA, mexB, and oprM efflux system. Mutants could grow at 2-HBP concentrations below 100 μM but at lower growth rates than the wild-type. Exposure of the wild-type to increasing 2-HBP concentrations resulted in acute cell growth arrest and loss of membrane potential, to which the cells adapt after a few hours. By using ethidium bromide (EB) as proxy we could show that the mutants are unable to expel EB effectively. Inclusion of a 2-HBP reporter plasmid revealed that the wild-type combines efflux with metabolism at all 2-HBP concentrations, whereas the mutants cannot remove the compound and arrest metabolism at concentrations above 24 μM. The analysis thus showed the importance of the MexAB-OprM system for productive metabolism of 2-HBP.

Detection of plant-modulated alterations in antifungal gene expression in Pseudomonas fluorescens CHA0 on roots by flow cytometry.

The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.