Mechanistically compatible mixtures of bacterial antagonists improve biological control of fire blight of pear.

Mixtures of biological control agents can be superior to individual agents in suppressing plant disease, providing enhanced efficacy and reliability from field to field relative to single biocontrol strains. Nonetheless, the efficacy of combinations of Pseudomonas fluorescens A506, a commercial biological control agent for fire blight of pear, and Pantoea vagans strain C9-1 or Pantoea agglomerans strain Eh252 rarely exceeds that of individual strains. A506 suppresses growth of the pathogen on floral colonization and infection sites through preemptive exclusion. C9-1 and Eh252 produce peptide antibiotics that contribute to disease control. In culture, A506 produces an extracellular protease that degrades the peptide antibiotics of C9-1 and Eh252. We hypothesized that strain A506 diminishes the biological control activity of C9-1 and Eh252, thereby reducing the efficacy of biocontrol mixtures. This hypothesis was tested in five replicated field trials comparing biological control of fire blight using strain A506 and A506 aprX::Tn5, an extracellular protease-deficient mutant, as individuals and combined with C9-1 or Eh252. On average, mixtures containing A506 aprX::Tn5 were superior to those containing the wild-type strain, confirming that the extracellular protease of A506 diminished the biological control activity of C9-1 and Eh252 in situ. Mixtures of A506 aprX::Tn5 and C9-1 or Eh252 were superior to oxytetracycline or single biocontrol strains in suppressing fire blight of pear. These experiments demonstrate that certain biological control agents are mechanistically incompatible, in that one strain interferes with the mechanism by which a second strain suppresses plant disease. Mixtures composed of mechanistically compatible strains of biological control agents can suppress disease more effectively than individual biological control agents.

Control of fire blight by Pseudomonas fluorescens A506 and Pantoea vagans C9-1 applied as single strains and mixed inocula.

The biological control agents Pseudomonas fluorescens A506 and Pantoea vagans C9-1 were evaluated individually and in combination for the suppression of fire blight of pear or apple in 10 field trials inoculated with the pathogen Erwinia amylovora. The formulation of pathogen inoculum applied to blossoms influenced establishment of the pathogen and the efficacy of biological control. Pantoea vagans C9-1 suppressed fire blight in all five trials in which the pathogen was applied as lyophilized cells but in none of the trials in which the pathogen was applied as freshly harvested cells. In contrast, Pseudomonas fluorescens A506 reduced disease significantly in only one trial. A mixture of the two strains also suppressed fire blight, but the magnitude of disease suppression over all field trials (averaging 32%) was less than that attained by C9-1 alone (42%). The two biological control agents did not antagonize one another on blossom surfaces, and application of the mixture of A506 and C9-1 to blossoms resulted in a greater proportion of flowers having detectable populations of at least one bacterial antagonist than the application of individual strains. Therefore, the mixture of A506 and C9-1 provided less disease control than expected based upon the epiphytic population sizes of the antagonists on blossom surfaces. We speculate that the biocontrol mixture was less effective than anticipated due to incompatibility between the mechanisms by which A506 and C9-1 suppress disease.

Role of rpfF in virulence and exoenzyme production of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule of soybean.

Ten strains of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule of soybean, which were isolated from various soybean growing regions of Thailand, produced an extracellular diffusible factor (DSF) related to a well-characterized quorum sensing molecule produced by other Xanthomonas spp. Genomic DNA of the 10 strains of X. axonopodis pv. glycines contained rpfF, a gene encoding for the biosynthesis of the DSF in X. campestris pv. campestris. The rpfF gene from one strain of X. axonopodis pv. glycines was fully sequenced, and the 289 aa product is closely related to RpfF of other Xanthomonas spp. (95 to 98% identical). Three independently generated rpfF mutants of X. axonopodis pv. glycines strain No12-2 were defective in the production of a DSF, as expected if rpfF encodes for DSF biosynthesis in X. axonopodis pv. glycines. The rpfF mutants of X. axonopodis pv. glycines exhibited reduced virulence on soybean and produced less than wild-type levels of extracellular polysaccharide and the extracellular enzymes carboxylmethylcellulase, protease, endo-beta-1,4-mannanase, and pectate lyase. Transcripts for three genes that encode for the extracellular enzymes protease, endoglucanase, and pectate lyase were at lower abundance in an rpfF mutant than in the parental strain of X. axonopodis pv. glycines. These results indicate that X. axonopodis pv. glycines produces a diffusible signal related to the DSF of X. campestris pv. campestris, which contributes to virulence and exoenzyme production by this phytopathogenic bacterium.

Antibiosis Contributes to Biological Control of Fire Blight by Pantoea agglomerans Strain Eh252 in Orchards.

ABSTRACT Fire blight, caused by Erwinia amylovora, is the most serious bacterial disease of pear and apple trees. Biological control with strains of Pantoea agglomerans (syn. Erwinia herbicola) may provide an effective disease management strategy for fire blight. Most strains of P. agglomerans evaluated for suppression of fire blight produce compounds that inhibit the growth of E. amylovora in culture. The role of these inhibitory compounds in fire blight suppression in orchard environments has not been studied. In seven field trials in Oregon, we compared the population dynamics and disease suppression with P. agglomerans Eh252, a strain that produces a single antibiotic, with its near-isogenic antibiotic-deficient derivative, strain 10:12. Water or suspensions of Eh252 or 10:12 (1 x 10(8) CFU/ml) were applied at 30 and 70% bloom to pear or apple trees. Aqueous suspensions of freeze-dried cells of E. amylovora (3 x 10(5) CFU/ml) were applied at full bloom. Additional trees were treated with streptomycin or oxytetracycline at 30 and 70% bloom and in some experiments, 1 day after application of the pathogen. Population sizes of Eh252 or 10:12 on pear blossoms were estimated by spreading dilutions of blossom washes on culture media. Average population sizes of Eh252 and 10:12 on blossoms ranged from 10(5) to 10(7) CFU, and in five of six trials, the relative area under the population curve of Eh252 was not significantly different than that of its derivative 10:12. Both Eh252 and 10:12 reduced the growth of the pathogen on blossoms compared with inoculated water-treated controls. Eh252 significantly decreased the incidence of fire blight in six of seven field trials compared with the incidence on water-treated trees, and 10:12 similarly reduced the incidence of fire blight in four of seven trials. In three of seven field trials, trees treated with Eh252 had a significantly lower incidence of fire blight compared with trees treated 3 with 10:12. Overall,3 Eh252 reduced the incidence of fire blight by 55 +/- 8%, 10:12 by 30 +/- 6%, streptomycin by 75 +/- 4%, and oxytetracycline by 16 +/- 14%. The effectiveness of strain 10:12 compared with water treatment indicates that other mechanisms (e.g., competitive exclusion or habitat modification) also contribute to disease suppression by P. agglomerans. The increased suppression of fire blight by the parental strain Eh252 compared with the antibiotic-deficient mutant 10:12 indicates that antibiosis is an important mechanism of biological control of fire blight.

Role of iron in rhizobacteria-mediated induced systemic resistance of cucumber.

ABSTRACT Seed treatment with the rhizosphere bacterium Serratia marcescens strain 90-166 suppressed anthracnose of cucumber, caused by Colleto-trichum orbiculare, through induced systemic resistance (ISR). When the iron concentration of a planting mix was decreased by addition of an iron chelator, suppression of cucumber anthracnose by strain 90-166 was significantly improved. Strain 90-166 produced 465 +/- 70 mg/liter of catechol siderophore, as determined by the Rioux assay in deferrated King's medium B. The hypothesis that a catechol siderophore produced by strain 90-166 may be responsible for induction of systemic resistance by this strain was tested by evaluating disease suppression by a mini-Tn5-phoA mutant deficient in siderophore production. Sequence analysis of genomic DNA flanking the mini-Tn5-phoA insertion identified the target gene as entA, which encodes an enzyme in the catechol siderophore biosynthetic pathways of several bacteria. Severity of anthracnose of cucumbers treated with the entA mutant was not significantly different (P = 0.05) from the control, whereas plants treated with wild-type 90-166 had significantly less disease (P = 0.05) than the control. Total (internal and external) population sizes of 90-166 and the entA mutant on roots did not differ significantly (P = 0.05) at any sample time, whereas internal population sizes of the entA mutant were significantly lower (P = 0.05) than those of the wild-type strain at two sampling times. These data suggest that catechol siderophore biosynthesis genes in Serratia marcescens 90-166 are associated with ISR but that this role may be indirect via a reduction in internal root populations.

Lon protease influences antibiotic production and UV tolerance of Pseudomonas fluorescens Pf-5.

Pseudomonas fluorescens Pf-5 is a soil bacterium that suppresses plant pathogens due in part to its production of the antibiotic pyoluteorin. Previous characterization of Pf-5 revealed three global regulators, including the stationary-phase sigma factor sigma(S) and the two-component regulators GacA and GacS, that influence both antibiotic production and stress response. In this report, we describe the serine protease Lon as a fourth global regulator influencing these phenotypes in Pf-5. lon mutants overproduced pyoluteorin, transcribed pyoluteorin biosynthesis genes at enhanced levels, and were more sensitive to UV exposure than Pf-5. The lon gene was preceded by sequences that resembled promoters recognized by the heat shock sigma factor sigma(32) (sigma(H)) of Escherichia coli, and Lon accumulation by Pf-5 increased after heat shock. Therefore, sigma(H) represents the third sigma factor (with sigma(S) and sigma(70)) implicated in the regulation of antibiotic production by P. fluorescens. Lon protein levels were similar in stationary-phase and exponentially growing cultures of Pf-5 and were not positively affected by the global regulator sigma(S) or GacS. The association of antibiotic production and stress response has practical implications for the success of disease suppression in the soil environment, where biological control organisms such as Pf-5 are likely to encounter environmental stresses.

Utilization of heterologous siderophores enhances levels of iron available to Pseudomonas putida in the rhizosphere.

Pseudomonas spp. have the capacity to utilize siderophores produced by diverse species of bacteria and fungi, and the present study was initiated to determine if siderophores produced by rhizosphere microorganisms enhance the levels of iron available to a strain of Pseudomonas putida in this natural habitat. We used a previously described transcriptional fusion (pvd-inaZ) between an iron-regulated promoter (pvd) and the ice nucleation reporter gene (inaZ) to detect alterations in iron availability to P. putida. Ice nucleation activity (INA) expressed from the pvd-inaZ fusion by P. putida N1R or N1R Pvd(-), a derivative deficient in the production of a pyoverdine siderophore, was inversely related to the concentration of ferric citrate in a culture medium. In culture, INA expressed by N1R Pvd(-) (pvd-inaZ) was reduced in the presence of the ferric complex of pseudobactin-358, a pyoverdine siderophore produced by P. putida WCS358 that can be utilized as a source of iron by N1R Pvd(-). In the rhizosphere of cucumbers grown in sterilized soil, N1R Pvd(-) (pvd-inaZ) expressed INA, indicating that iron availability was sufficiently low in that habitat to allow transcription of the iron-regulated pvd promoter. Coinoculation with WCS358 or N1R significantly decreased INA expressed by N1R Pvd(-) (pvd-inaZ) in the rhizosphere, whereas coinoculation with a pyoverdine-deficient mutant of WCS358 did not reduce INA expressed by N1R Pvd(-) (pvd-inaZ). These results indicate that iron availability to N1R Pvd(-) (pvd-inaZ) in the rhizosphere was enhanced by the presence of another strain of P. putida that produces a pyoverdine that N1R Pvd(-) (pvd-inaZ) was able to utilize as a source of iron. In culture, strain N1R Pvd(-) also utilized ferric complexes of the siderophores enterobactin and aerobactin as sources of iron. In the rhizosphere of cucumbers grown in sterilized soil, INA expressed by N1R Pvd(-) (pvd-inaZ) was reduced in the presence of strains of Enterobacter cloacae that produced enterobactin, aerobactin, or both siderophores, but INA expressed by N1R Pvd(-) (pvd-inaZ) was not altered in the presence of a mutant of E. cloacae deficient in both enterobactin and aerobactin production. Therefore, the iron status of P. putida was altered by siderophores produced by an unrelated bacterium coinhabiting the rhizosphere. Finally, we demonstrated that INA expressed by N1R containing pvd-inaZ in the rhizosphere differed between plants grown in sterilized versus nonsterilized field soil. The results of this study demonstrate that (i) P. putida expresses genes for pyoverdine production and uptake in the rhizosphere, but the level of gene expression is influenced by other bacteria that coexist with P. putida in this habitat, and (ii) diverse groups of microorganisms can alter the availability of chemical resources in microbial habitats on root surfaces.

Characterization of the pyoluteorin biosynthetic gene cluster of Pseudomonas fluorescens Pf-5.

Ten genes (plt) required for the biosynthesis of pyoluteorin, an antifungal compound composed of a bichlorinated pyrrole linked to a resorcinol moiety, were identified within a 24-kb genomic region of Pseudomonas fluorescens Pf-5. The deduced amino acid sequences of eight plt genes were similar to the amino acid sequences of genes with known biosynthetic functions, including type I polyketide synthases (pltB, pltC), an acyl coenzyme A (acyl-CoA) dehydrogenase (pltE), an acyl-CoA synthetase (pltF), a thioesterase (pltG), and three halogenases (pltA, pltD, and pltM). Insertions of the transposon Tn5 or Tn3-nice or a kanamycin resistance gene in each of these genes abolished pyoluteorin production by Pf-5. The presumed functions of the eight plt products are consistent with biochemical transformations involved in pyoluteorin biosynthesis from proline and acetate precursors. Isotope labeling studies demonstrated that proline is the primary precursor to the dichloropyrrole moiety of pyoluteorin. The deduced amino acid sequence of the product of another plt gene, pltR, is similar to those of members of the LysR family of transcriptional activators. pltR and pltM are transcribed divergently from the pltLABCDEFG gene cluster, and a sequence with the characteristics of a LysR binding site was identified within the 486-bp intergenic region separating pltRM from pltLABCDEFG. Transcription of the pyoluteorin biosynthesis genes pltB, pltE, and pltF, assessed with transcriptional fusions to an ice nucleation reporter gene, was significantly greater in Pf-5 than in a pltR mutant of Pf-5. Therefore, PltR is proposed to be a transcriptional activator of linked pyoluteorin biosynthesis genes.

Epiphytic colonization of pear stigmas and hypanthia by bacteria during primary bloom.

ABSTRACT Pear blossoms were sampled during various stages of bloom in 1991 and 1992 from orchards at Cashmere, WA, and Corvallis and Medford, OR, for epiphytic populations of culturable bacteria. On stigmatic surfaces, bacteria were isolated from 2 to 32% of blossoms prior to petal expansion and from 47 to 94% of blossoms by petal fall. In general, a lower percentage of hypanthia than stigmas supported bacterial populations. Randomly selected bacteria isolated at population levels of >/=10(4) CFU/tissue were identified by fatty acid methyl ester analysis. Diverse genera of gram-negative and -positive bacteria were identified from the Medford and Cashmere field sites. Pseudomonas syringae and Pseudomonas viri-diflava were isolated from all sites and were the predominant species detected at Corvallis, where they were isolated from 28% of the blossoms sampled on a given date. Because most pear blossoms do not support detectable populations (>/=10(2) CFU/tissue) of culturable bacteria prior to petal expansion, we speculate that introduced biocontrol agents may become established with minimal competition from indigenous epiphytes at early bloom stages.

Microbial Antagonism at the Root Level Is Involved in the Suppression of Fusarium Wilt by the Combination of Nonpathogenic Fusarium oxysporum Fo47 and Pseudomonas putida WCS358.

ABSTRACT Two biological control agents, nonpathogenic Fusarium oxysporum Fo47 and Pseudomonas putida WCS358, were evaluated for suppression of Fusarium wilt of flax grown in nutrient solution and for suppression of the population density and metabolic activity of the causal organism F. oxysporum f. sp. lini strain Foln3GUS on root surfaces. Due to the presence of an introduced gusA reporter gene construct in Foln3GUS, the pathogen expressed beta-glucuronidase activity that was related to its carbon metabolism. At a Fo47 to Foln3GUS inoculum ratio of 100:1, both the population density of the pathogen and the beta-glucuronidase activity on and in flax roots were reduced by the nonpathogenic strain, and Fusarium wilt was suppressed. At a Fo47 to Foln3GUS inoculum ratio of 10:1, Fo47 decreased the severity of Fusarium wilt to a smaller extent and it also reduced beta-glucuronidase activity without reducing the density of Foln3GUS on flax roots. At a nonpathogenic to pathogenic Fusarium strains ratio of 10:1, the addition of P. putida WCS358 further suppressed Fusarium wilt and the density of the pathogen at the root level, whereas a mutant of WCS358 deficient in pseudobactin production had no significant effect. Iron availability to WCS358 on flax roots, assessed by ice-nucleation activity conferred from a transcriptional fusion (pvd-inaZ) of an ice-nucleation reporter gene to an iron-regulated promoter, was sufficiently low to allow pseudobactin production. P. putida WCS358 did not reduce the severity of Fusarium wilt of flax when inoculated without Fo47, and it did not improve disease suppression achieved by high inoculum doses of Fo47 (a Fo47 to Foln3GUS ratio of 100:1). Together, these data provide evidence that (i) suppression of Fusarium wilt of flax by Fo47 is related to reductions in the population density and metabolic activity of the pathogen on the root surface; (ii) WCS358 can enhance the biological control activity of Fo47, but this enhancement depends on the population of Fo47 relative to the pathogen; and (iii) pseudobactin contributes to suppression of Fusarium wilt by the combination of Fo47 and WCS358 on roots in which conditions are conducive to pseudobactin production by the bacterium.

The two-component regulators GacS and GacA influence accumulation of the stationary-phase sigma factor sigmaS and the stress response in Pseudomonas fluorescens Pf-5.

Three global regulators are known to control antibiotic production by Pseudomonas fluorescens. A two-component regulatory system comprised of the sensor kinase GacS (previously called ApdA or LemA) and GacA, a member of the FixJ family of response regulators, is required for antibiotic production. A mutation in rpoS, which encodes the stationary-phase sigma factor sigmaS, differentially affects antibiotic production and reduces the capacity of stationary-phase cells of P. fluorescens to survive exposure to oxidative stress. The gacA gene of P. fluorescens Pf-5 was isolated, and the influence of gacS and gacA on rpoS transcription, sigmaS levels, and oxidative stress response of Pf-5 was determined. We selected a gacA mutant of Pf-5 that contained a single nucleotide substitution within a predicted alpha-helical region, which is highly conserved among the FixJ family of response regulators. At the entrance to stationary phase, sigmaS content in gacS and gacA mutants of Pf-5 was less than 20% of the wild-type level. Transcription of rpoS, assessed with an rpoS-lacZ transcriptional fusion, was positively influenced by GacS and GacA, an effect that was most evident at the transition between exponential growth and stationary phase. Mutations in gacS and gacA compromised the capacity of stationary-phase cells of Pf-5 to survive exposure to oxidative stress. The results of this study provide evidence for the predominant roles of GacS and GacA in the regulatory cascade controlling stress response and antifungal metabolite production in P. fluorescens.

Establishment of Bacterial Antagonists of Erwinia amylovora on Pear and Apple Blossoms as Influenced by Inoculum Preparation.

The influence of inoculum preparation on the establishment of bacterial antagonists that suppress fire blight and Erwinia amylovora on blossoms was evaluated. Aqueous suspensions of Pseudomonas fluorescens A506, E. herbicola C9-1R, or E. amylovora 153N were prepared from cells harvested from the surface of an agar medium or from cells that were lyophilized after culture under similar conditions. Bacterial suspensions (1 x 10(8) CFU/ml) were sprayed on pear and apple trees at 50% bloom near midday. The incidence of recovery (proportion of blossoms containing detectable populations) and the population sizes of the bacteria on individual blossoms with detectable populations were followed over a period of several days. Fluorescent microspheres (1 mum in diameter) were added to sprays at a concentration of 1 x 10(7) microspheres per ml to mark blossoms that were open during application of bacteria. After dilution-plating, the stigmas and styles of each blossom were examined for the presence of microspheres with an epifluorescence microscope. In three of five trials, bacteria applied as suspensions of lyophilized cells were recovered from a greater proportion of blossoms than bacterial cells harvested directly from culture media. Every blossom harvested within 6 days after spraying had microspheres present on the surfaces of the styles and stigmas; thus, lack of establishment of detectable populations, rather than escape of blossoms from spray inoculation, accounted for the differences in proportion of blossoms colonized by the different preparations of bacteria. The use of lyophilized cells in field trials decreased variability in the establishment of bacteria on blossoms.

Availability of iron to Pseudomonas fluorescens in rhizosphere and bulk soil evaluated with an ice nucleation reporter gene.

The biological availability of iron in the rhizosphere was assessed by evaluating ice nucleation activity (INA) expressed in situ by Pseudomonas fluorescens Pf-5 containing a transcriptional fusion (pvd-inaZ) of an iron-regulated promoter to an ice nucleation reporter gene (inaZ). Pf-5 containing pvd-inaZ expresses INA that is inversely related to the iron availability of a growth medium (J. E. Loper and S. E. Lindow, Appl. Environ. Microbiol. 60:1934-1941, 1994). INA expressed by rhizosphere populations of Pf-5 containing pvd-inaZ was at a maximum within 12 to 24 h following inoculation of the bacterium onto bean roots and typically decreased gradually during the following 4 days. Iron availability in the soil, which was altered by the addition of chelators, influenced INA expressed by rhizosphere populations of Pf-5 containing pvd-inaZ. In soil adjusted to a pH of 7.0 or 8.0 by adding Ca(OH)2, rhizosphere populations of Pf-5 containing pvd-inaZ expressed greater INA, indicating lower iron availability, than they did in the nonamended soil at a pH of 5.4. Similarly, rhizosphere populations of Pf-5 containing pvd-inaZ expressed less INA in an agricultural soil of pH 5.4 than in other agricultural soils ranging in pH from 6.4 to 7.7. These results conform to the predictions of chemical models stating that pH is a major factor influencing iron availability in soil solutions. The results of this study indicate that P. fluorescens Pf-5 encountered an iron-limited environment immediately after it was inoculated onto bean roots planted in agricultural field soils. One to two days after the bacterium was inoculated onto root surfaces, however, iron became more available to rhizosphere populations of Pf-5. We speculate that iron acquisition systems of plants and other rhizosphere organisms may provide available sources of iron to established rhizosphere populations of P. fluorescens.

EcbI and EcbR: homologs of LuxI and LuxR affecting antibiotic and exoenzyme production by Erwinia carotovora subsp. betavasculorum.

Erwinia carotovora subsp. betavasculorum Ecb168 causes vascular necrosis and root rot of sugar beet and produces an antibiotic(s) that is antagonistic against other Erwinia spp. EcbI- mutants of Ecb168, each containing a single transposon insertion in the ecbI gene (for Erwinia carotovora subsp. betavasculorum inducer), do not produce detectable levels of extracellular protease or antibiotic(s), and express less pectate lyase activity and virulence than the wild-type strain. A plasmid containing the cloned ecbI gene complemented the EcbI- mutants for these phenotypes. Protease production by EcbI- mutants grown on agar surfaces was restored by neighboring cells of Escherichia coli containing ecbI. Production of a diffusible N-acylhomoserine lactone autoinducer by wild-type Ecb168 was detected with indicator strains of E. coli and Agrobacterium tumefaciens. EcbI- mutant strains did not produce an autoinducer detected by the indicator strains. Antibiotic production by EcbI- mutants was restored by cell-free culture supernatants of Ecb168 or E. coli containing a cloned ecbI gene. The predicted amino acid sequence of EcbI is similar to those of CarI, ExpI, and HsII, three LuxI homologs required for production of a diffusible N-acylhomoserine lactone autoinducer in Erwinia carotovora subsp. carotovora. A luxR homolog, termed ecbR (for Erwinia carotovora subsp. betavasculorum regulator), is convergently transcribed and overlaps with ecbI by 17 bp at their 3' ends. These results are consistent with the hypothesis that a quorum-sensing system related to the prototypic luxI-luxR gene pair controls antibiotic and exoenzyme production in Erwinia carotovora subsp. betavasculorum.

Identification and sequence analysis of the genes encoding a polyketide synthase required for pyoluteorin biosynthesis in Pseudomonas fluorescens Pf-5.

Pyoluteorin is a chlorinated antifungal metabolite of mixed polyketide/amino-acid origin produced by certain strains of Pseudomonas spp., including the soil bacterium Pseudomonas fluorescens Pf-5. Sequence analysis of a gene cluster required for pyoluteorin biosynthesis by Pf-5 (Kraus, J., Loper, J., 1995. Appl. Environ. Microbiol. 61, 849-854) has identified two genes whose deduced peptide sequences exhibit characteristics of both fungal and bacterial Type I polyketide synthases (PKSs). The pyoluteorin PKS does not contain a loading domain that is typically present in bacterial Type I PKSs. Furthermore, this PKS possesses an acyltransferase domain that does not contain the conserved residues surrounding the active-site motif typically found in domains of similar function. Based on the organization of the functional domains within the pyoluteorin PKS, we propose a biosynthetic pathway analogous to non-aromatic polyketide biosynthesis within the Actinomycete bacteria that is responsible for the formation of the resorcinol moiety of pyoluteorin.

The sigma factor sigma s affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5.

Pseudomonas fluorescens Pf-5, a rhizosphere-inhabiting bacterium that suppresses several soilborne pathogens of plants, produces the antibiotics pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol. A gene necessary for pyrrolnitrin production by Pf-5 was identified as rpoS, which encodes the stationary-phase sigma factor sigma s. Several pleiotropic effects of an rpoS mutation in Escherichia coli also were observed in an RpoS- mutant of Pf-5. These included sensitivities of stationary-phase cells to stresses imposed by hydrogen peroxide or high salt concentration. A plasmid containing the cloned wild-type rpoS gene restored pyrrolnitrin production and stress tolerance to the RpoS- mutant of Pf-5. The RpoS- mutant overproduced pyoluteorin and 2,4-diacetyl-phloroglucinol, two antibiotics that inhibit growth of the phytopathogenic fungus Pythium ultimum, and was superior to the wild type in suppression of seedling damping-off of cucumber caused by Pythium ultimum. When inoculated onto cucumber seed at high cell densities, the RpoS- mutant did not survive as well as the wild-type strain on surfaces of developing seedlings. Other stationary-phase-specific phenotypes of Pf-5, such as the production of cyanide and extracellular protease(s) were expressed by the RpoS- mutant, suggesting that sigma s is only one of the sigma factors required for the transcription of genes in stationary-phase cells of P. fluorescens. These results indicate that a sigma factor encoded by rpoS influences antibiotic production, biological control activity, and survival of P. fluorescens on plant surfaces.

A global regulator of secondary metabolite production in Pseudomonas fluorescens Pf-5.

Mutations in the apdA (for antibiotic production) gene of the plant root-colonizing bacterium Pseudomonas fluorescens Pf-5 pleiotropically abolish the production of an array of antibiotics, including pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol, as well as the production of tryptophan side chain oxidase, hydrogen cyanide, and an extracellular protease. The lack of production of secondary metabolites by ApdA- mutants was correlated with the loss of inhibition of the phytopathogenic fungus Rhizoctonia solani in culture. Sequencing of the apdA region identified an open reading frame of 2,751 bp. The predicted amino acid sequence of the apdA gene contains conserved domains of the histidine kinases that serve as sensor components of prokaryotic two-component regulatory systems. The apdA nucleotide and predicted amino acid sequences are strikingly similar to the sequences of lemA and repA, genes encoding putative sensor kinases that are required for the pathogenicity of Pseudomonas syringae pv. syringae and Pseudomonas viridiflava, respectively. Introduction of the cloned apdA+ gene restored the wild-type phenotype to both LemA- mutants of P. syringae and ApdA- mutants of Pf-5. The 101-kDa ApdA protein reacted with an anti-LemA antiserum, further demonstrating the similarity of ApdA to LemA. These results show that apdA encodes a putative sensor kinase component of a classical two-component regulatory system that is required for secondary-metabolite production by P. fluorescens Pf-5.

Characterization of a Genomic Region Required for Production of the Antibiotic Pyoluteorin by the Biological Control Agent Pseudomonas fluorescens Pf-5.

A 21-kb region required for the biosynthesis of the polyketide antibiotic pyoluteorin by the biological control agent Pseudomonas fluorescens Pf-5 was identified and cloned. Seven previously isolated mutants deficient in pyoluteorin production (Plt(sup-)) had Tn5 insertions spanning the 21-kb region. Sequences flanking Tn5 inserts were cloned from genomic DNA of three Plt(sup-) mutants and used as probes to identify wild-type alleles of the plt loci from a genomic library of Pf-5. Five cosmids containing overlapping regions of genomic DNA hybridized to one or more of the probes. One cosmid, pJEL1938, contained the entire 21-kb region and, when introduced into a Plt(sup-) mutant, partially restored pyoluteorin production. To study the expression of the genes required for pyoluteorin biosynthesis, the transposon Tn3-nice, which contains a promoterless ice nucleation gene (inaZ) and a type I neomycin phosphotransferase gene, was introduced into the genomic plt region of Pf-5. Carbon sources that influenced pyoluteorin production by Pf-5 had parallel effects on ice nucleation activity of Pf-5 containing a genomic plt::Tn3-nice fusion, indicating that inaZ was transcribed from a promoter of the plt region. Cells of Pf-5 containing a genomic plt::Tn3-nice fusion expressed ice nucleation activity on cotton and cucumber seeds planted in field soil. The expression of plt genes by Pf-5 in the cucumber spermosphere was delayed in comparison with expression in the cotton spermosphere. This study demonstrates that genes required for pyoluteorin production were expressed in situ by the biological control bacterium.

Derivation of Mutants of Erwinia carotovora subsp. betavasculorum Deficient in Export of Pectolytic Enzymes with Potential for Biological Control of Potato Soft Rot.

Erwinia carotovora subsp. betavasculorum Ecb168 produces an antibiotic(s) that suppresses growth of the related bacterium Erwinia carotovora subsp. carotovora in culture and in wounds of potato tubers. Strain Ecb168 also produces and secretes pectolytic enzymes and causes a vascular necrosis and root rot of sugar beet. Genes (out) involved in secretion of pectolytic enzymes by Ecb168 were localized to two HindIII fragments (8.5 and 10.5 kb) of Ecb168 genomic DNA by hybridization to the cloned out region of E. carotovora subsp. carotovora and by complementation of Out mutants of E. carotovora subsp. carotovora. Out mutants of Ecb168, which did not secrete pectate lyase into the culture medium, were obtained when deletions internal to either HindIII fragment were introduced into the genome of Ecb168 through marker exchange mutagenesis. Out mutants of Ecb168 were complemented to the Out phenotype by introduction of the corresponding cloned HindIII fragment. Out mutants of Ecb168 were less virulent than the Out parental strain on potato tubers. Strain Ecb168 and Out derivatives inhibited the growth of E. carotovora subsp. carotovora in culture, indicating that the uncharacterized antibiotic(s) responsible for antagonism was exported through an out-independent mechanism. Strain Ecb168 and Out derivatives reduced the establishment of large populations of E. carotovora subsp. carotovora in wounds of potato tubers and suppressed tuber soft rot caused by E. carotovora subsp. carotovora.

A biological sensor for iron available to bacteria in their habitats on plant surfaces.

A sensor responsive to iron was constructed by fusing a promoterless ice nucleation activity gene (inaZ) to an iron-regulated promoter of a genomic region involved in pyoverdine (fluorescent siderophore) (pvd) production in Pseudomonas syringae. Cells of Pseudomonas fluorescens and P. syringae that contained the pvd-inaZ fusion expressed iron-responsive ice nucleation activity in the bean rhizosphere and phyllosphere, respectively, and in culture. Addition of Fe(III) to leaves or soil reduced the apparent transcription of the pvd-inaZ reporter gene, as shown by a reduction in the number of ice nuclei produced, indicating that Fe(III) was primarily responsible for mediating transcription of the pvd-inaZ gene even in natural environments. A Pseudomonas sp. strain having an intact iceC gene, which conferred Fe-insensitive expression of ice nucleation activity, was included in all studies to account for small strain- or environment-dependent differences in the ability of bacterial cells to produce ice nuclei. Thus, a comparison of the ice nucleation activity conferred by pvd-inaZ with the activity conferred by iceC revealed the bioavailability of iron in culture or natural habitats. The relative ice nucleation activities expressed by strains containing iceC or pvd-inaZ indicated that, while not abundant, Fe(III) is not present at extremely low concentrations at all microsites colonized by bacteria on plant surfaces. Biological sensors that are constructed by fusing inaZ to chemically responsive promoters provide a novel way to characterize chemical constituents of microbial habitats.