Production of Indole-3-Acetic Acid in the Plant-Beneficial Strain Pseudomonas chlororaphis O6 Is Negatively Regulated by the Global Sensor Kinase GacS

Certain plant growth–promoting bacteria, such as Pseudomonas fluorescens 89B61 and Bacillus pumilus SE34, secreted high levels of indole-3-acetic acid (IAA) in tryptophan-amended medium in stationary phase as determined by chromogenic analysis and high-performance liquid chromatography. Two other growth-promoting strains, P. chlororaphis O6 and Serratia marcescens 90-166, did not produce these high levels of IAA. However, when the gacS mutant of P. chlororaphis O6 was grown in tryptophan-supplemented medium, IAA was detected in culture filtrates. IAA production by the gacS mutant in P. chlororaphis O6 was repressed in the tryptophan medium by complementation with the wild-type gacS gene. Thus, the global regulatory Gac system in P. chlororaphis O6 acts as a negative regulator of IAA production from trypophan.     

Lon protease negatively affects GacA protein stability and expression of the Gac/Rsm signal transduction pathway in Pseudomonas protegens

In Pseudomonas protegens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway controls secondary metabolism and suppression of fungal root pathogens via the expression of regulatory small RNAs (sRNAs). Because of its high cost, this pathway needs to be protected from overexpression and to be turned off in response to environmental stress such as the lack of nutrients. However, little is known about its underlying molecular mechanisms. In this study, we demonstrated that Lon protease, a member of the ATP‐dependent protease family, negatively regulated the Gac/Rsm cascade. In a lon mutant, the steady‐state levels and the stability of the GacA protein were significantly elevated at the end of exponential growth. As a consequence, the expression of the sRNAs RsmY and RsmZ and that of dependent physiological functions such as antibiotic production were significantly enhanced. Biocontrol of Pythium ultimum on cucumber roots required fewer lon mutant cells than wild‐type cells. In starved cells, the loss of Lon function prolonged the half‐life of the GacA protein. Thus, Lon protease is an important negative regulator of the Gac/Rsm signal transduction pathway in P. protegens.     

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.     

Thiamine-Auxotrophic Mutants of Pseudomonas fluorescens CHA0 Are Defective in Cell-Cell Signaling and Biocontrol Factor Expression

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 × 10⁻⁸ M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.     

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.     

The role of volatile and non-volatile antibiotics produced by Pseudomonas chlororaphis strain PA23 in its root colonization and control of Sclerotinia sclerotiorum

Pseudomonas chlororaphis strain PA23 has demonstrated excellent biocontrol in the canola phyllosphere. This bacterium produces the non-volatile antibiotics phenazine and pyrrolnitrin as well as the volatile antibiotics nonanal, benzothiazole and 2-ethyl-1-hexanol. In vitro experiments were conducted to study the effects of different mutations on the production of these three organic volatile antibiotics by PA23. In planta experiments in the greenhouse investigated the role of the non-volatile antibiotics on root colonization and biocontrol ability of PA23 against Sclerotinia sclerotiorum on sunflower. Analysis of phenazine- and pyrrolnitrin-deficient Tn mutants of PA23 revealed no differences in production of the three volatile antibiotics. On all sampling dates, PA23 applied alone or in combination with the mutants showed significantly higher (P = 0.05) root bacterial number and Sclerotinia wilt suppression (P = 0.05). Decline of the bacterial population seemed to be inversely proportional to/or negatively correlated with the number of antibiotics produced by PA23 but the relative importance of phenazine or pyrrolnitrin on root colonization and/or wilt suppression was not clear. In several cases, the strains producing at least one antibiotic maintained relatively higher bacterial numbers than non-producing strains. However, by 6 weeks after sowing, there was a rapid and significant (P = 0.05) increase in the proportion of introduced bacteria capable of producing at least one antibiotic over the total bacterial population. Furthermore, combining certain mutants with PA23 reduced the root colonization and biocontrol ability of PA23. Strain PA23-314 (gacS mutant) showed competitive colonization in comparison to the other mutants for most sampling dates.     

Characterisation of Pseudomonas chlororaphis subsp. aurantiaca strain Pa40 with the ability to control wheat sharp eyespot disease

This study details the isolation and characterisation of Pseudomonas chlororaphis subsp. aurantiaca strain Pa40, and is the first to examine P. chlororaphis for use in suppression of wheat sharp eyespot on wheat. Pa40 was isolated during an investigation aimed to identify biocontrol agents for Rhizoctonia cerealis. Over 500 bacterial strains were isolated from the rhizosphere of infected wheat and screened for in vitro antibiosis towards R. cerealis and ability to provide biocontrol in planta. Twenty‐six isolates showed highly antagonistic activity towards R. cerealis, in which Pseudomonas spp. and Bacillus spp. were predominant members of the antagonistic community. Strain Pa40 exhibited clear and consistent suppression of wheat sharp eyespot disease in a greenhouse study and suppression was comparable to that of chemical treatment with validamycin A. Pa40 was identified as P. chlororaphis subsp. aurantiaca by the Biolog identification system combined with 16S rDNA, atpD, carA and recA sequence analysis and biochemical and physiological characteristics. To determine broad‐spectrum applicability and the specific mechanisms involved in Pa40's pathogen suppression this strain was tested for antibiosis towards various phytopathogens and assayed for many biocontrol activities and plant‐beneficial traits. Strain Pa40 inhibited the growth of 10 of 13 phytopathogenic fungal strains and six of eight phytopathogenic bacteria tested. This original work characterises HCN, protease and siderophore production in P. chlororaphis. Each of these characteristics likely contributed to Pa40's biocontrol capabilities as well as stimulation of the hypersensitive response in tobacco and the presence of genes involved in the biosynthesis of phenazine, 2‐hydroxylated phenazine and pyrrolnitrin.     

Production of the antifungal compounds phenazine and pyrrolnitrin from Pseudomonas chlororaphis O6 is differentially regulated by glucose

Aims: To determine whether glucose in growth medium affects secondary metabolite production and biocontrol efficacy of Pseudomonas chlororaphis O6. Methods and Results: The secondary metabolites pyrrolnitrin and phenazines antagonize phytopathogenic fungi. The expression of the prnA gene encoding tryptophan halogenase, the first step in pyrrolnitrin biosynthesis, required the stationary‐phase sigma factor, RpoS. Mutations in rpoS and prnA in Ps. chlororaphis O6 eliminated antifungal activity against Rhizoctonia solani and Fusarium graminearum. Pyrrolnitrin production was reduced by glucose in growth media, whereas phenazine levels were increased. The efficacy of Ps. chlororaphis O6 in the biocontrol of tomato late blight was reduced by addition of glucose to the growth medium. Conclusions: Regulation by glucose of pyrrolnitrin production influenced the efficacy of the biocontrol of tomato leaf blight. Significance and Impact of the Study: The nutritional regulation of secondary metabolite production from a soil pseudomonad may account, at least in part, for the variability of biocontrol under field conditions.     

A mathematical model of the Gac/Rsm quorum sensing network in Pseudomonas fluorescens

I present a deterministic model of the dynamics of signal transduction and gene expression in the Gac/Rsm network of the soil-dwelling bacterium Pseudomonas fluorescens. The network is involved in quorum sensing and governs antifungal production in this important biocontrol agent. A central role is played by small untranslated RNAs, which sequester regulatory mRNA-binding proteins. The model provides a reasonable match to the available data, which consists primarily of time series from reporter gene fusions. I use the model to investigate the information-processing properties of the Gac/Rsm network, in part by comparing it to a simplified model capable of quorum sensing. The results suggest that the complexity and redundancy of the Gac/Rsm network have evolved to meet the conflicting requirements of high sensitivity to environmental conditions and a conservative, robust response to variability in parameter values. Similar systems exist in a wide variety of bacteria, where they control a diverse set of population-dependent behaviors. This makes them important subjects for mathematical models that can help link empirical understanding of network structure to theoretical insights into how these networks have evolved to function under natural conditions.     

Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHA0

In Pseudomonas fluorescens strain CHA0, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere. Two Tn5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P. syringae pv. syringae as well as that of P. fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0. This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease. Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA. Independent point and deletion mutations arose at different sites in the gacA gene. In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased. In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions.     

Insecticidal features displayed by the beneficial rhizobacterium Pseudomonas chlororaphis PCL1606

The biocontrol rhizobacterium Pseudomonas chlororaphis is one of the bacterial species of the P. fluorescens group where insecticide fit genes have been found. Fit toxin, supported with other antimicrobial compounds, gives the bacterial the ability to repel and to fight against eukaryotic organisms, such as nematodes and insect larvae, thus protecting the plant host and itself. Pseudomonas chlororaphis PCL1606 is an antagonistic rhizobacterium isolated from avocado roots and show efficient biocontrol against fungal soil-borne disease. The main antimicrobial compound produced by P. chlororaphis PCL606 is 2-hexyl-5-propyl resorcinol (HPR), which plays a crucial role in effective biocontrol against fungal pathogens. Further analysis of the P. chlororaphis PCL1606 genome showed the presence of hydrogen cyanide (HCN), pyrrolnitrin (PRN), and homologous fit genes. To test the insecticidal activity and to determine the bases for such activity, single and double mutants on the biosynthetic genes of these four compounds were tested in a Galleria mellonella larval model using inoculation by injection. The results revealed that Fit toxin and HPR in combination are involved in the insecticide phenotype of P. chlororaphis PCL1606, and additional compounds such as HCN and PRN could be considered supporting compounds.

     

肉桂酸衍生物对铜绿假单胞菌PAO1 III型分泌系统的影响

【目的】铜绿假单胞菌是引起医院获得性感染最常见的条件致病菌,而III型分泌系统(Type III secretion system,TTSS)是其致病的主要因子之一。本文从合成的21个肉桂酸衍生物中筛选影响TTSS效应子(Effector)产生的化合物,并初步研究其作用机制。【方法】将TTSS效应子合成基因exoS的转录报告质粒pAT-exoS转入菌株PAO1中,获得PAO1(pAT-exoS)。待筛选的化合物与PAO1(pAT-exoS)菌株共培养6 h后,检测exoS基因的表达,从中筛选影响exoS基因表达的化合物。【结果】筛选结果表明：21个化合物中,3个化合物抑制exoS基因表达,2个化合物则促进exoS基因表达。此外,化合物TS128、TS143和TS160对菌株生长有明显的抑制作用。Western blot实验进一步证实筛选得到的化合物TS108、TS128和TS165可抑制ExoS的产生;化合物TS139和TS143则促进ExoS的产生。为进一步研究抑制剂的作用机理,过量表达TTSS主要的调控因子exsA基因可部分消除抑制剂TS108和TS165的抑制效果;而rsmZ rsmY双基因突变体PAO6421中添加抑制剂TS108和TS165并不能显著抑制exoS基因的表达,同样,抑制剂TS108和TS165也不影响受Gac/Rsm信号传导系统调控的群体感应信号分子的产生。【结论】抑制剂TS108和TS165的作用机制可能主要是影响esxA基因,从而影响exoS基因表达及蛋白产量。     

Inactivation of gacS Does Not Affect the Competitiveness of Pseudomonas chlororaphis in the Arabidopsis thaliana Rhizosphere

Quorum-sensing-controlled processes are considered to be important for the competitiveness of microorganisms in the rhizosphere. They affect cell-cell communication, biofilm formation, and antibiotic production, and the GacS-GacA two-component system plays a role as a key regulator. In spite of the importance of this system for the regulation of various processes, strains with a Gac⁻ phenotype are readily recovered from natural habitats. To analyze the influence of quorum sensing and the influence of the production of the antibiotic phenazine-1-carboxamide on rhizosphere colonization by Pseudomonas chlororaphis, a gnotobiotic system based on Arabidopsis thaliana seedlings in soil was investigated. Transposon insertion mutants of P. chlororaphis isolate SPR044 carrying insertions in different genes required for the production of N-acyl-homoserine lactones and phenazine-1-carboxamide were generated. Analysis of solitary rhizosphere colonization revealed that after prolonged growth, the population of the wild type was significantly larger than that of the homoserine lactone-negative gacS mutant and that of a phenazine-1-carboxamide-overproducing strain. In cocultivation experiments, however, the population size of the gacS mutant was similar to that of the wild type after extended growth in the rhizosphere. A detailed analysis of growth kinetics was performed to explain this phenomenon. After cells grown to the stationary phase were transferred to fresh medium, the gacS mutant had a reduced lag phase, and production of the stationary-phase-specific sigma factor RpoS was strongly reduced. This may provide a relative competitive advantage in cocultures with other bacteria, because it permits faster reinitiation of growth after a change to nutrient-rich conditions. In addition, delayed entry into the stationary phase may allow more efficient nutrient utilization. Thus, GacS-GacA-regulated processes are not absolutely required for efficient rhizosphere colonization in populations containing the wild type and Gac⁻ mutants.