Marcella D. Henkels

Comparative Genomics of Plant-Associated Pseudomonas spp.: Insights into Diversity and Inheritance of Traits Involved in Multitrophic Interactions

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45–52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire.

Evaluation of streptomycin, oxytetracycline, and copper resistance of Erwinia amylovora isolated from pear orchards in Washington State

One hundred and thirty-eight pathogenic strains of E. amylovora were isolated from fire blight cankers of pear trees from 44 orchards in the major pear-growing regions of Washington State. Ninety-eight strains, isolated from 38 of the orchards sampled, were resistant to streptomycin (1 mg/ml). Streptomycin-resistant strains of E. amylovora were ubiquitous in all of the major pear-growing regions of Washington. None of the strains tested were resistant to oxytetracycline (25 μg/ml) or CuSO 4 (0.16 mM). Nevertheless, spontaneous mutants with tolerance to 0.16 mM CuSO 4 were observed at a frequency of 10 −6 -10 −7 mutant colonies per wild type colony in most strains

Molecular analysis of a novel gene cluster encoding an insect toxin in plant‐associated strains of Pseudomonas fluorescens

Pseudomonas fluorescens CHA0 and the related strain Pf-5 are well-characterized representatives of rhizosphere bacteria that have the capacity to protect crop plants from fungal root diseases, mainly by releasing a variety of exoproducts that are toxic to plant pathogenic fungi. Here, we report that the two plant-beneficial pseudomonads also exhibit potent insecticidal activity. Anti-insect activity is linked to a novel genomic locus encoding a large protein toxin termed Fit (for P. fluorescensinsecticidal toxin) that is related to the insect toxin Mcf (Makes caterpillars floppy) of the entomopathogen Photorhabdus luminescens, a mutualist of insect-invading nematodes. When injected into the haemocoel, even low doses of P. fluorescens CHA0 or Pf-5 killed larvae of the tobacco hornworm Manduca sexta and the greater wax moth Galleria mellonella. In contrast, mutants of CHA0 or Pf-5 with deletions in the Fit toxin gene were significantly less virulent to the larvae. When expressed from an inducible promoter in a non-toxic Escherichia coli host, the Fit toxin gene was sufficient to render the bacterium toxic to both insect hosts. Our findings establish the Fit gene products of P. fluorescens CHA0 and Pf-5 as potent insect toxins that define previously unappreciated anti-insect properties of these plant-colonizing bacteria.

Isolation and Identification of Rhizoxin Analogs from Pseudomonas fluorescens Pf-5 by Using a Genomic Mining Strategy

ABSTRACT The products synthesized from a hybrid polyketide synthase/nonribosomal peptide synthetase gene cluster in the genome of Pseudomonas fluorescens Pf-5 were identified using a genomics-guided strategy involving insertional mutagenesis and subsequent metabolite profiling. Five analogs of rhizoxin, a 16-member macrolide with antifungal, phytotoxic, and antitumor activities, were produced by Pf-5, but not by a mutant with an insertion in the gene cluster. The five rhizoxin analogs, one of which had not been described previously, were differentially toxic to two agriculturally important plant pathogens, Botrytis cinerea and Phytophthora ramorum. The rhizoxin analogs also caused swelling of rice roots, a symptom characteristic of rhizoxin itself, but were less toxic to pea and cucumber roots. Of the rhizoxin analogs produced by Pf-5, the predominant compound, WF-1360 F, and the newly described compound 22Z-WF-1360 F were most toxic against the two plant pathogens and three plant species. These rhizoxin analogs were tested against a panel of human cancer lines, and they exhibited potent but nonselective cytotoxicity. This study highlights the value of the genomic sequence of the soil bacterium P. fluorescens Pf-5 in providing leads for the discovery of novel metabolites with significant biological properties.

Lethality and developmental delay in Drosophila melanogaster larvae after ingestion of selected Pseudomonas fluorescens strains.

Background The 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 Findings We 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/Significance These 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.

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.

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-5s 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.

Pseudomonas protegens Pf-5 Causes Discoloration and Pitting of Mushroom Caps Due to the Production of Antifungal Metabolites

Bacteria in the diverse Pseudomonas fluorescens group include rhizosphere inhabitants known for their antifungal metabolite production and biological control of plant disease, such as Pseudomonas protegens Pf-5, and mushroom pathogens, such as Pseudomonas tolaasii. Here, we report that strain Pf-5 causes brown, sunken lesions on peeled caps of the button mushroom (Agaricus bisporus) that resemble brown blotch symptoms caused by P. tolaasii. Strain Pf-5 produces six known antifungal metabolites under the control of the GacS/GacA signal transduction system. A gacA mutant produces none of these metabolites and did not cause lesions on mushroom caps. Mutants deficient in the biosynthesis of the antifungal metabolites 2,4-diacetylphloroglucinol and pyoluteorin caused less-severe symptoms than wild-type Pf-5 on peeled mushroom caps, whereas mutants deficient in the production of lipopeptide orfamide A caused similar symptoms to wild-type Pf-5. Purified pyoluteorin and 2,4-diacetylphloroglucinol mimicked the symptoms caused by Pf-5. Both compounds were isolated from mushroom tissue inoculated with Pf-5, providing direct evidence for their in situ production by the bacterium. Although the lipopeptide tolaasin is responsible for brown blotch of mushroom caused by P. tolaasii, P. protegens Pf-5 caused brown blotch-like symptoms on peeled mushroom caps through a lipopeptide-independent mechanism involving the production of 2,4-diacetylphloroglucinol and pyoluteorin.

Characterization of Toxin Complex Gene Clusters and Insect Toxicity of Bacteria Representing Four Subgroups of Pseudomonas fluorescens

Ten strains representing four lineages of the Pseudomonas fluorescens group (P. chlororaphis, P. corrugata, P. koreensis, and P. fluorescens subgroups) were evaluated for toxicity to the tobacco hornworm Manduca sexta and the common fruit fly Drosophila melanogaster. The three strains within the P. chlororaphis subgroup exhibited both oral and injectable toxicity to the lepidopteran M. sexta. All three strains have the gene cluster encoding the FitD insect toxin and a ΔfitD mutant of P. protegens strain Pf-5 exhibited diminished oral toxicity compared to the wildtype strain. Only one of the three strains, P. protegens Pf-5, exhibited substantial levels of oral toxicity against the dipteran D. melanogaster. Three strains in the P. fluorescens subgroup, which lack fitD, consistently showed significant levels of injectable toxicity against M. sexta. In contrast, the oral toxicity of these strains against D. melanogaster was variable between experiments, with only one strain, Pseudomonas sp. BG33R, causing significant levels of mortality in repeated experiments. Toxin complex (Tc) gene clusters, which encode insecticidal properties in Photorhabdus luminescens, were identified in the genomes of seven of the ten strains evaluated in this study. Within those seven genomes, six types of Tc gene clusters were identified, distinguished by gene content, organization and genomic location, but no correlation was observed between the presence of Tc genes and insect toxicity of the evaluated strains. Our results demonstrate that members of the P. fluorescens group have the capacity to kill insects by both FitD-dependent and independent mechanisms.

An Interspecies Signaling System Mediated by Fusaric Acid Has Parallel Effects on Antifungal Metabolite Production by Pseudomonas protegens Strain Pf-5 and Antibiosis of Fusarium spp.

ABSTRACT Pseudomonas protegens strain Pf-5 is a rhizosphere bacterium that suppresses soilborne plant diseases and produces at least seven different secondary metabolites with antifungal properties. We derived mutants of Pf-5 with single and multiple mutations in biosynthesis genes for seven antifungal metabolites: 2,4-diacetylphoroglucinol (DAPG), pyrrolnitrin, pyoluteorin, hydrogen cyanide, rhizoxin, orfamide A, and toxoflavin. These mutants were tested for inhibition of the pathogens Fusarium verticillioides and Fusarium oxysporum f. sp. pisi. Rhizoxin, pyrrolnitrin, and DAPG were found to be primarily responsible for fungal antagonism by Pf-5. Previously, other workers showed that the mycotoxin fusaric acid, which is produced by many Fusarium species, including F. verticillioides, inhibited the production of DAPG by Pseudomonas spp. In this study, amendment of culture media with fusaric acid decreased DAPG production, increased pyoluteorin production, and had no consistent influence on pyrrolnitrin or orfamide A production by Pf-5. Fusaric acid also altered the transcription of biosynthetic genes, indicating that the mycotoxin influenced antibiotic production by Pf-5 at the transcriptional level. Addition of fusaric acid to the culture medium reduced antibiosis of F. verticillioides by Pf-5 and derivative strains that produce DAPG but had no effect on antibiosis by Pf-5 derivatives that suppressed F. verticillioides due to pyrrolnitrin or rhizoxin production. Our results demonstrated the importance of three compounds, rhizoxin, pyrrolnitrin, and DAPG, in suppression of Fusarium spp. by Pf-5 and confirmed that an interspecies signaling system mediated by fusaric acid had parallel effects on antifungal metabolite production and antibiosis by the bacterial biological control organism.