gacA Gene Research Articles

Multiple pathways impact the swarming motility of Pseudomonas fluorescens Pf0-1.

Swarming motility in pseudomonads typically requires both a functional flagellum and the production/secretion of a biosurfactant. Published work has shown that the wild-type Pseudomonas fluorescens Pf0-1 is swarming deficient due to a point mutation in the gacA gene, which until recently was thought to inactivate rather than attenuate the Gac/Rsm pathway. As a result, little is known about the underlying mechanisms that regulate swarming motility by P. fluorescens Pf0-1. Here, we demonstrate that a ΔrsmA ΔrsmE ΔrsmI mutant, which phenotypically mimics Gac/Rsm pathway overstimulation, is proficient at swarming motility. RsmA and RsmE appear to play a key role in this regulation. Transposon mutagenesis of the ΔrsmA ΔrsmE ΔrsmI mutant identified multiple factors that impact swarming motility, including pathways involved in flagellar synthesis and biosurfactant production/secretion. We find that loss of genes linked to biosurfactant Gacamide A biosynthesis or secretion impacts swarming motility, as does loss of the alternative sigma factor FliA, which results in a defect in flagellar function. Collectively, these findings provide evidence that P. fluorescens Pf0-1 can swarm if the Gac/Rsm pathway is activated, highlight the regulatory complexity of swarming motility in this strain, and demonstrate that the cyclic lipopeptide Gacamide A is utilized as a biosurfactant for swarming motility.IMPORTANCESwarming motility is a coordinated process that allows communities of bacteria to collectively move across a surface. For P. fluorescens Pf0-1, this phenotype is notably absent in the parental strain, and to date, little is known about the regulation of swarming in this strain. Here, we identify RsmA and RsmE as key repressors of swarming motility via modulating the levels of biosurfactant production/secretion. Using transposon mutagenesis and subsequent genetic analyses, we further identify potential regulatory mechanisms of swarming motility and link Gacamide A biosynthesis and transport machinery to swarming motility.

Uncovering the GacS-mediated role in evolutionary progression through trajectory reconstruction in Pseudomonas aeruginosa.

The genetic diversities of subpopulations drive the evolution of pathogens and affect their ability to infect hosts and cause diseases. However, most studies to date have focused on the identification and characterization of adaptive mutations in single colonies, which do not accurately reflect the phenotypes of an entire population. Here, to identify the composition of variant subpopulations within a pathogen population, we developed a streamlined approach that combines high-throughput sequencing of the entire population cells with genotyping of single colonies. Using this method, we reconstructed a detailed quorum-sensing (QS) evolutionary trajectory in Pseudomonas aeruginosa. Our results revealed a new adaptive mutation in the gacS gene, which codes for a histidine kinase sensor of a two-component system (TCS), during QS evolution. This mutation reduced QS activity, allowing the variant to sweep throughout the whole population, while still being vulnerable to invasion by the emerging QS master regulator LasR-null mutants. By tracking the evolutionary trajectory, we found that mutations in gacS facilitated QS-rewiring in the LasR-null mutant. This rapid QS revertant caused by inactive GacS was found to be associated with the promotion of ribosome biogenesis and accompanied by a trade-off of reduced bacterial virulence on host cells. In conclusion, our findings highlight the crucial role of the global regulator GacS in modulating the progression of QS evolution and the virulence of the pathogen population.

GacA reduces virulence and increases competitiveness in planta in the tumorigenic olive pathogen Pseudomonas savastanoi pv. savastanoi.

GacS/GacA is a widely distributed two-component system playing an essential role as a key global regulator, although its characterization in phytopathogenic bacteria has been deeply biased, being intensively studied in pathogens of herbaceous plants but barely investigated in pathogens of woody hosts. P. savastanoi pv. savastanoi (Psv) is characterized by inducing tumours in the stem and branches of olive trees. In this work, the model strain Psv NCPPB 3335 and a mutant derivative with a complete deletion of gene gacA were subjected to RNA-Seq analyses in a minimum medium and a medium mimicking in planta conditions, accompanied by RT-qPCR analyses of selected genes and phenotypic assays. These experiments indicated that GacA participates in the regulation of at least 2152 genes in strain NCPPB 3335, representing 37.9 % of the annotated CDSs. GacA also controls the expression of diverse rsm genes, and modulates diverse phenotypes, including motility and resistance to oxidative stresses. As occurs with other P. syringae pathovars of herbaceous plants, GacA regulates the expression of the type III secretion system and cognate effectors. In addition, GacA also regulates the expression of WHOP genes, specifically encoded in P. syringe strains isolated from woody hosts, and genes for the biosynthesis of phytohormones. A gacA mutant of NCPPB 3335 showed increased virulence, producing large immature tumours with high bacterial populations, but showed a significantly reduced competitiveness in planta. Our results further extend the role of the global regulator GacA in the virulence and fitness of a P. syringae pathogen of woody hosts.

Characterisation of pellicle-forming ability in clinical carbapenem-resistant Acinetobacter baumannii.

Acinetobacter baumannii was reported to have resistance towards carbapenems and the ability to form an air-liquid biofilm (pellicle) which contributes to their virulence. The GacSA two-component system has been previously shown to play a role in pellicle formation. Therefore, this study aims to detect the presence of gacA and gacS genes in carbapenem-resistant Acinetobacter baumannii (CRAB) isolates recovered from patients in intensive care units and to investigate their pellicle forming ability. The gacS and gacA genes were screened in 96 clinical CRAB isolates using PCR assay. Pellicle formation assay was performed in Mueller Hinton medium and Luria Bertani medium using borosilicate glass tubes and polypropylene plastic tubes. The biomass of the pellicle was quantitated using the crystal violet staining assay. The selected isolates were further assessed for their motility using semi-solid agar and monitored in real-time using real-time cell analyser (RTCA). All 96 clinical CRAB isolates carried the gacS and gacA genes, however, only four isolates (AB21, AB34, AB69 and AB97) displayed the ability of pellicle-formation phenotypically. These four pellicle-forming isolates produced robust pellicles in Mueller Hinton medium with better performance in borosilicate glass tubes in which biomass with OD570 ranging from 1.984±0.383 to 2.272±0.376 was recorded. The decrease in cell index starting from 13 hours obtained from the impedance-based RTCA showed that pellicle-forming isolates had entered the growth stage of pellicle development. These four pellicle-forming clinical CRAB isolates could be potentially more virulent, therefore further investigation is warranted to provide insights into their pathogenic mechanisms.

The combination of hexanal and geraniol in sublethal concentrations synergistically inhibits quorum sensing in Pseudomonas fluorescens-In vitro and in silico approaches.

Hexanal and geraniol are essential oil components with anti-quorum sensing (QS) activity against Pseudomonas fluorescens. This study demonstrated that QS inhibition (QSI) efficacy of the hexanal and geraniol combination (HG) was significantly higher when compared to those of their mono-counterparts at the same concentration. Tests on P. fluorescens motility, biofilm formation, acyl-homoserine lactones' (AHLs) production, gene expression in vitro, and molecular docking in silico were conducted to evaluate the synergistic effect of hexanal and geraniol on QSI. HG mixture at 0.5 minimal inhibitory concentration (MIC) showed a strong synergistic inhibition of biofilm formation (51.8%), motility (60.13%), and extracellular protease activity (58.9%) of P. fluorescens. The synthesis of AHLs, e.g., C8 -HSL and C12 -HSL, was inhibited by hexanal, geraniol, and HG; both AHLs are responsible for regulating virulence factors in P. fluorescens. The expression of pcoI and gacA genes regulating AHL synthetase and sensor kinase was significantly down-regulated by HG (0.29 and 0.38-fold) at 0.5 MIC. Hexanal and HG showed significant inhibition of the expression of pcoR and gacS genes, which are responsible for AHL receptor protein and response regulation; however, geraniol failed to downregulate the two genes. Molecular docking in silico also supported these findings. Hexanal, which gets inserted into the minor groove of pcoI/pcoR DNA fragments, inhibits the expression of both the genes. Both hexanal (-31.487kcal/mol) and geraniol (-25.716kcal/mol) had a higher binding affinity with PcoI protein than the halogenated furanone C30 (-24.829kcal/mol), which is a known competitor of AHLs. Similarly, hexanal and geraniol strongly bind to the PcoR protein also. It was found that HG at 0.5 MIC could effectively inhibit QS by suppressing the expression of pcoR/gacS and gacA/gacS genes and therefore, could inhibit the motility and biofilm formation of P. fluorescens. The present study indicated that HG at sub-MIC as QS inhibitor could be further developed as a new preservative of agri-food products.

Modulation of Arabidopsis thaliana growth by volatile substances emitted by Pseudomonas and Serratia strains.

Many volatile compounds secreted by bacteria play an important role in the interactions of microorganisms, can inhibit the growth of phytopathogenic bacteria and fungi, can suppress or stimulate plant growth and serve as infochemicals presenting a new type of interspecies communication. In this work, we investigated the effect of total pools of volatile substances and individual volatile organic compounds (VOCs) synthesized by the rhizosphere bacteria Pseudomonas chlororaphis 449 and Serratia plymuthica IC1270, the soil-borne strain P. fluorescens B-4117 and the spoiled meat isolate S. proteamaculans 94 on Arabidopsis thaliana plants. We showed that total gas mixtures secreted by these strains during their growth on Luria-Bertani agar inhibited A. thaliana growth. Hydrogen cyanide synthesis was unnecessary for the growth suppression. A decrease in the inhibition level was observed for the strain P. chlororaphis 449 with a mutation in the gacS gene, while inactivation of the rpoS gene had no effect. Individual VOCs synthesized by these bacteria (1-indecene, ketones 2-nonanone, 2-heptanone, 2-undecanone, and dimethyl disulfide) inhibited the growth of plants or killed them. Older A. thaliana seedlings were more resistant to VOCs than younger seedlings. The results indicated that the ability of some volatiles emitted by the rhizosphere and soil bacteria to inhibit plant growth should be considered when assessing the potential of such bacteria for the biocontrol of plant diseases.

7-hydroxytropolone is the main metabolite responsible for the fungal antagonism of Pseudomonas donghuensis strain SVBP6.

Pseudomonas donghuensis strain SVBP6, an isolate from an agricultural plot in Argentina, displays a broad-spectrum and diffusible antifungal activity, which requires a functional gacS gene but could not be ascribed yet to known secondary metabolites typical of Pseudomonas biocontrol species. Here, we report that Tn5 mutagenesis allowed the identification of a gene cluster involved in both the fungal antagonism and the production of a soluble tropolonoid compound. The ethyl acetate extract from culture supernatant showed a dose-dependent inhibitory effect against the phytopathogenic fungus Macrophomina phaseolina. The main compound present in the organic extract was identified by spectroscopic and X-ray analyses as 7-hydroxytropolone (7HT). Its structure and tautomerism was confirmed by preparing the two key derivatives 2,3-dimethoxy- and 2,7-dimethoxy-tropone. 7HT, but not 2,3- or 2,7-dimethoxy-tropone, mimicked the fungal inhibitory activity of the ethyl acetate extract from culture supernatant. The activity of 7HT, as well as its production, was barely affected by the presence of up to 50 μM added iron (Fe+2 ). To summarize, P. donghuensis SVBP6 produces 7HT under the positive control of the Gac-Rsm cascade and is the main active metabolite responsible for the broad-spectrum inhibition of different phytopathogenic fungi.

An Extract Produced by Bacillus sp. BR3 Influences the Function of the GacS/GacA Two-Component System in Pseudomonas syringae pv. tomato DC3000.

The GacS/GacA two-component system is essential for virulence in many plant pathogenic bacteria, and thus represents a promising anti-virulence target. In the present study, we isolated and screened rhizobacteria that were capable of inhibiting the expression of the gacS gene in the phytopathogenic bacterium Pseudomonas syringae pv. tomato (Pto) DC3000. One candidate inhibitor bacterium, BR3 was obtained and identified as a Bacillus sp. strain based on 16s rRNA gene sequence analysis. Besides the gacS gene, the GacA-dependent small RNA genes rsmZ and rsmY were repressed transcriptionally when DC3000 was treated with an extract from strain BR3. Importantly, the extract also influenced bacterial motility, the expression of type three secretion system effector AvrPto, and the plant hypersensitive response triggered by strain DC3000. The results suggested that the extract from strain BR3 might offer an alternative method to control bacterial diseases in plants by targeting the GacS/GacA system.

Genomic insights into the broad antifungal activity, plant-probiotic properties, and their regulation, in Pseudomonas donghuensis strain SVBP6.

Plant-growth promotion has been linked to the Pseudomonas genus since the beginning of this research field. In this work, we mined the genome of an Argentinean isolate of the recently described species P. donghuensis. Strain SVBP6, isolated from bulk soil of an agricultural plot, showed a broad antifungal activity and several other plant-probiotic activities. As this species has been recently described, and it seems like some plant-growth promoting (PGP) traits do not belong to the classical pseudomonads toolbox, we decide to explore the SVBP6 genome via an bioinformatic approach. Genome inspection confirmed our previous in vitro results about genes involved in several probiotic activities. Other genetic traits possibly involved in survival of SVBP6 in highly competitive environments, such as rhizospheres, were found. Tn5 mutagenesis revealed that the antifungal activity against the soil pathogen Macrophomina phaseolina was dependent on a functional gacS gene, from the regulatory cascade Gac-Rsm, but it was not due to volatile compounds. Altogether, our genomic analyses and in vitro tests allowed the phylogenetic assignment and provided the first insights into probiotic properties of the first P. donghuensis isolate from the Americas.

Genomic characterisation of environmental Pseudomonas aeruginosa isolated from dental unit waterlines revealed the insertion sequence ISPa11 as a chaotropic element.

The bacterium Pseudomonas aeruginosa is well known to have a remarkable adaptive capacity allowing it to colonise many environments. A recent study on environmental isolates from dental unit waterlines (DUWLs) hinted at a genetic clustering into two groups. Isolates from one of these groups, named cluster III, were shown to have unusual phenotypes for environmental isolates, such as an increased biofilm production. To have a better ecological view, more specifically on isolates from cluster III, the complete genomes of 39 isolates including 16 from DUWLs were sequenced. In addition to an investigation of antibiotic resistance and secretion system gene content, a molecular phylogeny allowed confirmation of the split of the 16 environmental isolates in two groups and also sheds light on a correlation between the phylogenetic positions and the serotypes of the isolates. Isolates from cluster III harboured insertion sequences ISPa11 inserted into the O-specific antigen biosynthesis clusters and the gene lasR, encoding for a master regulator of the quorum sensing. Investigation of key regulators revealed another truncated gene, gacS. Alteration in lasR and gacS genes was consistent with phenotypic assays confirming their inactivation. These results bring new perspectives regarding the ecological adaptive potential of P. aeruginosa.

Rhizoxin analogs, orfamide A and chitinase production contribute to the toxicity of Pseudomonas protegens strain Pf-5 to Drosophila melanogaster.

Pseudomonas protegens strain Pf-5 is a soil bacterium that was first described for its capacity to suppress plant diseases and has since been shown to be lethal to certain insects. Among these is the common fruit fly Drosophila melanogaster, a well-established model organism for studies evaluating the molecular and cellular basis of the immune response to bacterial challenge. Pf-5 produces the insect toxin FitD, but a ΔfitD mutant of Pf-5 retained full toxicity against D. melanogaster in a noninvasive feeding assay, indicating that FitD is not a major determinant of Pf-5's oral toxicity against this insect. Pf-5 also produces a broad spectrum of exoenzymes and natural products with antibiotic activity, whereas a mutant with a deletion in the global regulatory gene gacA produces none of these exoproducts and also lacks toxicity to D. melanogaster. In this study, we made use of a panel of Pf-5 mutants having single or multiple mutations in the biosynthetic gene clusters for seven natural products and two exoenzymes that are produced by the bacterium under the control of gacA. Our results demonstrate that the production of rhizoxin analogs, orfamide A, and chitinase are required for full oral toxicity of Pf-5 against D. melanogaster, with rhizoxins being the primary determinant.

A gacS deletion in Pseudomonas aeruginosa cystic fibrosis isolate CHA shapes its virulence.

Pseudomonas aeruginosa, a human opportunistic pathogen, is capable of provoking acute and chronic infections that are associated with defined sets of virulence factors. During chronic infections, the bacterium accumulates mutations that silence some and activate other genes. Here we show that the cystic fibrosis isolate CHA exhibits a unique virulence phenotype featuring a mucoid morphology, an active Type III Secretion System (T3SS, hallmark of acute infections), and no Type VI Secretion System (H1-T6SS). This virulence profile is due to a 426 bp deletion in the 3′ end of the gacS gene encoding an essential regulatory protein. The absence of GacS disturbs the Gac/Rsm pathway leading to depletion of the small regulatory RNAs RsmY/RsmZ and, in consequence, to expression of T3SS, while switching off the expression of H1-T6SS and Pel polysaccharides. The CHA isolate also exhibits full ability to swim and twitch, due to active flagellum and Type IVa pili. Thus, unlike the classical scheme of balance between virulence factors, clinical strains may adapt to a local niche by expressing both alginate exopolysaccharide, a hallmark of membrane stress that protects from antibiotic action, host defences and phagocytosis, and efficient T3S machinery that is considered as an aggressive virulence factor.

The freshwater sponge Ephydatia fluviatilis harbours diverse Pseudomonas species (Gammaproteobacteria, Pseudomonadales) with broad-spectrum antimicrobial activity.

Bacteria are believed to play an important role in the fitness and biochemistry of sponges (Porifera). Pseudomonas species (Gammaproteobacteria, Pseudomonadales) are capable of colonizing a broad range of eukaryotic hosts, but knowledge of their diversity and function in freshwater invertebrates is rudimentary. We assessed the diversity, structure and antimicrobial activities of Pseudomonas spp. in the freshwater sponge Ephydatia fluviatilis. Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprints of the global regulator gene gacA revealed distinct structures between sponge-associated and free-living Pseudomonas communities, unveiling previously unsuspected diversity of these assemblages in freshwater. Community structures varied across E. fluviatilis specimens, yet specific gacA phylotypes could be detected by PCR-DGGE in almost all sponge individuals sampled over two consecutive years. By means of whole-genome fingerprinting, 39 distinct genotypes were found within 90 fluorescent Pseudomonas isolates retrieved from E. fluviatilis. High frequency of in vitro antibacterial (49%), antiprotozoan (35%) and anti-oomycetal (32%) activities was found among these isolates, contrasting less-pronounced basidiomycetal (17%) and ascomycetal (8%) antagonism. Culture extracts of highly predation-resistant isolates rapidly caused complete immobility or lysis of cells of the protozoan Colpoda steinii. Isolates tentatively identified as P. jessenii, P. protegens and P. oryzihabitans showed conspicuous inhibitory traits and correspondence with dominant sponge-associated phylotypes registered by cultivation-independent analysis. Our findings suggest that E. fluviatilis hosts both transient and persistent Pseudomonas symbionts displaying antimicrobial activities of potential ecological and biotechnological value.

A bacterial symbiont is converted from an inedible producer of beneficial molecules into food by a single mutation in the gacA gene.

Stable multipartite mutualistic associations require that all partners benefit. We show that a single mutational step is sufficient to turn a symbiotic bacterium from an inedible but host-beneficial secondary metabolite producer into a host food source. The bacteria's host is a "farmer" clone of the social amoeba Dictyostelium discoideum that carries and disperses bacteria during its spore stage. Associated with the farmer are two strains of Pseudomonas fluorescens, only one of which serves as a food source. The other strain produces diffusible small molecules: pyrrolnitrin, a known antifungal agent, and a chromene that potently enhances the farmer's spore production and depresses a nonfarmer's spore production. Genome sequence and phylogenetic analyses identify a derived point mutation in the food strain that generates a premature stop codon in a global activator (gacA), encoding the response regulator of a two-component regulatory system. Generation of a knockout mutant of this regulatory gene in the nonfood bacterial strain altered its secondary metabolite profile to match that of the food strain, and also, independently, converted it into a food source. These results suggest that a single mutation in an inedible ancestral strain that served a protective role converted it to a "domesticated" food source.

Biocontrol performance evaluation of spontaneous mutants of Pseudomonas fluorescens VUPf5 generated during proliferation

VUPf5 genetic stability as a superior strain (of the 900 bacterial strains that were isolated from the rhizosphere of different regions of Iran) for the control of take-all disease of wheat was studied. The genetic stability of the strain in terms of, whether or not, the mass and reproduction of bacteria in liquid medium was determined. VUPf5 produces some secondary metabolites such as extracellular proteases, phenazine antibiotic, siderophore, hydrogen cyanide and volatile metabolites. The regulatory GacA/GacS system has involved as an important system in the production of secondary metabolites in the biocontrol fluorescent Pseudomonas spp. During proliferation, different colonies of bacteria were found in terms of morphology, such as colour (white instead of orange) and shape (approximately flat and larger) of the colony; and media pH that in the mutants was higher, and the production of some metabolites, such as protease, HCN and phenazine was surveyed. Also, mutants in the genetic comparison with the wild type were different. VUPf5 and different colonies were detected in gacA and gacS genes. This paper appears in colonies with low efficiency in the production of secondary metabolites is related to mutation in genes gacS and gacA.

Characterization of thegacA-dependent surface and coral mucus colonization by an opportunistic coral pathogenSerratia marcescensPDL100

Opportunistic pathogens rely on global regulatory systems to assess the environment and to control virulence and metabolism to overcome host defenses and outcompete host-associated microbiota. In Gammaproteobacteria, GacS/GacA is one such regulatory system. GacA orthologs direct the expression of the csr (rsm) small regulatory RNAs, which through their interaction with the RNA-binding protein CsrA (RsmA), control genes with functions in carbon metabolism, motility, biofilm formation, and virulence. The csrB gene was controlled by gacA in Serratia marcescens PDL100. A disruption of the S.marcescens gacA gene resulted in an increased fitness of the mutant on mucus of the host coral Acropora palmata and its high molecular weight fraction, whereas the mutant was as competitive as the wild type on the low molecular weight fraction of the mucus. Swarming motility and biofilm formation were reduced in the gacA mutant. This indicates a critical role for gacA in the efficient utilization of specific components of coral mucus and establishment within the surface mucopolysaccharide layer. While significantly affecting early colonization behaviors (coral mucus utilization, swarming motility, and biofilm formation), gacA was not required for virulence of S.marcescens PDL100 in either a model polyp Aiptasia pallida or in brine shrimp Artemia nauplii.

Isolation of Surfactant-Resistant Pseudomonads from the Estuarine Surface Microlayer

Bioremediation efforts often rely on the application of surfactants to enhance hydrocarbon bioavailability. However, synthetic surfactants can sometimes be toxic to degrading microorganisms, thus reducing the clearance rate of the pollutant. Therefore, surfactant-resistant bacteria can be an important tool for bioremediation efforts of hydrophobic pollutants, circumventing the toxicity of synthetic surfactants that often delay microbial bioremediation of these contaminants. In this study, we screened a natural surfactant-rich compartment, the estuarine surface microlayer (SML), for cultivable surfactant-resistant bacteria using selective cultures of sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB). Resistance to surfactants was evaluated by colony counts in solid media amended with critical micelle concentrations (CMC) of either surfactants, in comparison with non-amended controls. Selective cultures for surfactant-resistant bacteria were prepared in mineral medium also containing CMC concentrations of either CTAB or SDS. The surfactantresistant isolates obtained were tested by PCR for the Pseudomonas genus marker gacA gene and for the naphthalene-dioxygenase-encoding gene ndo. Isolates were also screened for biosurfactant production by the atomized oil assay. A high proportion of culturable bacterioneuston was tolerant to CMC concentrations of SDS or CTAB. The gacA-targeted PCR revealed that 64% of the isolates were Pseudomonads. Biosurfactant production in solid medium was detected in 9.4% of tested isolates, all affiliated with genus Pseudomonas. This study shows that the SML is a potential source of surfactant-resistant and biosurfactant-producing bacteria in which Pseudomonads emerge as a relevant group.

GacA directly regulates expression of several virulence genes in Pseudomonas syringae pv. tabaci 11528

A two-component system comprising GacS and GacA affects a large number of traits in many Gram-negative bacteria. However, the signals to which GacS responds, the regulation mechanism for GacA expression, and the genes GacA controls are not yet clear. In this study, several phenotypic tests and tobacco-leaf pathogenicity assays were conducted using a gacA deletion mutant strain (BL473) of Pseudomonas syringae pv. tabaci 11528. To determine the regulation mechanism for gacA gene expression and to identify GacA-regulated genes, we conducted quantitative RT-PCR and electrophoretic mobility shift assay (EMSA) experiments. The results indicated that virulence traits related to the pathogenesis of P. syringae pv. tabaci 11528 are regulated coordinately by GacA and iron availability. They also revealed that several systems coordinately regulate gacA gene expression in response to iron concentration and bacterial cell density and that GacA and iron together control the expression of several virulence genes. EMSA results provided genetic and molecular evidence for direct control of virulence genes by GacA.

The role of antibiosis and induced systemic resistance, mediated by strains of Pseudomonas chlororaphis, Bacillus cereus and B. amyloliquefaciens, in controlling blackleg disease of canola

Antibiotic-producing Pseudomonas chlororaphis strains DF190 and PA23, Bacillus cereus strain DFE4 and Bacillus amyloliquefaciens strain DFE16 were tested for elicitation of induced systemic resistance (ISR) and direct antibiosis in control of blackleg in canola caused by the fungal pathogen Leptosphaeria maculans. Inoculation of bacteria 24 h and 48 h prior to the pathogen was crucial for disease control. In systemic induction studies, the bacteria and culture extracts had lower but significant suppression of the blackleg lesion. When inoculated at the same wound site as the pathogen pycnidiospores, the bacterial culture extracts had significantly higher reduction of blackleg lesion development. However, localized plant defense-related enzyme activity at the site of inoculation was not induced by all the bacteria. Direct antifungal activity at the infection site seems to be the dominant mechanism mediating control of L. maculans. A Tn5-gacS mutant of strain PA23 exhibited a complete loss of antifungal and biocontrol activity, which was restored upon addition of the gacS gene in trans. Interestingly, a phenazine-minus derivative of PA23 that produces elevated levels of pyrrolnitrin exhibited the same or higher blackleg disease suppression compared to the wild type. These findings suggest that direct antifungal activity, possibly mediated by pyrrolnitrin, and some low level of induced systemic resistance is involved in P. chlororaphis biocontrol of blackleg.

Lethality and Developmental Delay in Drosophila melanogaster Larvae after Ingestion of Selected Pseudomonas fluorescens Strains

BackgroundThe fruit fly, Drosophila melanogaster, is a well-established model organism for probing the molecular and cellular basis of physiological and immune system responses of adults or late stage larvae to bacterial challenge. However, very little is known about the consequences of bacterial infections that occur in earlier stages of development. We have infected mid-second instar larvae with strains of Pseudomonas fluorescens to determine how infection alters the ability of larvae to survive and complete development.Methodology/Principal FindingsWe mimicked natural routes of infection using a non-invasive feeding procedure to study the toxicity of the three sequenced P. fluorescens strains (Pf0-1, SBW25, and Pf-5) to Drosophila melanogaster. Larvae fed with the three strains of P. fluorescens showed distinct differences in developmental trajectory and survival. Treatment with SBW25 caused a subset of insects to die concomitant with a systemic melanization reaction at larval, pupal or adult stages. Larvae fed with Pf-5 died in a dose-dependent manner with adult survivors showing eye and wing morphological defects. In addition, larvae in the Pf-5 treatment groups showed a dose-dependent delay in the onset of metamorphosis relative to control-, Pf0-1-, and SBW25-treated larvae. A functional gacA gene is required for the toxic properties of wild-type Pf-5 bacteria.Conclusions/SignificanceThese experiments are the first to demonstrate that ingestion of P. fluorescens bacteria by D. melanogaster larvae causes both lethal and non-lethal phenotypes, including delay in the onset of metamorphosis and morphological defects in surviving adult flies, which can be decoupled.