Systematic discovery of pseudomonad genetic factors involved in sensitivity to tailocins

Tailocins are bactericidal protein complexes produced by a wide variety of bacteria that kill closely related strains and may play a role in microbial community structure. Thanks to their high specificity, tailocins have been proposed as precision antibacterial agents for therapeutic applications. Compared to tailed phages, with whom they share an evolutionary and morphological relationship, bacterially produced tailocins kill their host upon production but producing strains display resistance to self-intoxication. Though lipopolysaccharide (LPS) has been shown to act as a receptor for tailocins, the breadth of factors involved in tailocin sensitivity, and the mechanisms behind resistance to self-intoxication, remain unclear. Here, we employed genome-wide screens in four non-model pseudomonads to identify mutants with altered fitness in the presence of tailocins produced by closely related pseudomonads. Our mutant screens identified O-antigen composition and display as most important in defining sensitivity to our tailocins. In addition, the screens suggest LPS thinning as a mechanism by which resistant strains can become more sensitive to tailocins. We validate many of these novel findings, and extend these observations of tailocin sensitivity to 130 genome-sequenced pseudomonads. This work offers insights into tailocin–bacteria interactions, informing the potential use of tailocins in microbiome manipulation and antibacterial therapy.Subject terms: Functional genomics, Microbial ecology, Bacterial genetics, Soil microbiology     

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.   f luorescens i nsecticidal t oxin) that is related to the insect toxin Mcf ( M akes c aterpillars f loppy) 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.     

A direct quantitative agar-plate based assay for analysis of Pseudomonas protegens Pf-5 degradation of polyurethane films

A quantitative assay was developed for the direct measurement of polymer film degradation from bacterial colonies on agar plates. Small (1 mm diameter) colonies of Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens Pf-5) were used for this work. Interactions between the Pf-5 colonies and thin polyurethane (PU) coatings on ZnSe coupons were evaluated for degradation using infrared spectroscopy. Three different coatings were analyzed and were formed from 1) a colloidal, aqueous – based polyester PU (Impranil^® DLN); 2) an organic solvent – based polyester PU (Irogran); and 3) an organic solvent – based polyether PU (AS-P108). Over a 24 h time period at 30 °C, citrate exposed Pf-5 cultures rapidly degraded Impranil coatings, consistent with analogous zone clearing assays. However, the Irogran and AS-P108 PU's, which are not directly compatible with zone clearing assays, showed no measureable degradation by the coating assay under identical conditions. These results demonstrated the capability to evaluate any variety of polymer formulation as solid films under identical biological conditions. The results also show that rapid microbial degradation of colloidal polyurethanes such as Impranil are not necessarily representative of activity towards other PU materials.     

Phylogenetically closely related pseudomonads isolated from arthropods exhibit differential insect-killing abilities and genetic variations in insecticidal factors

Strains belonging to the Pseudomonas protegens and Pseudomonas chlororaphis species are able to control soilborne plant pathogens and to kill pest insects by producing virulence factors such as toxins, chitinases, antimicrobials or two-partner secretion systems. Most insecticidal Pseudomonas described so far were isolated from roots or soil. It is unknown whether these bacteria naturally occur in arthropods and how they interact with them. Therefore, we isolated P. protegens and P. chlororaphis from various healthy insects and myriapods, roots and soil collected in an agricultural field and a neighbouring grassland. The isolates were compared for insect killing, pathogen suppression and host colonization abilities. Our results indicate that neither the origin of isolation nor the phylogenetic position mirror the degree of insecticidal activity. Pseudomonas protegens strains appeared homogeneous regarding phylogeny, biocontrol and insecticidal capabilities, whereas P. chlororaphis strains were phylogenetically and phenotypically more heterogenous. A phenotypic and genomic analysis of five closely related P. chlororaphis isolates displaying varying levels of insecticidal activity revealed variations in genes encoding insecticidal factors that may account for the reduced insecticidal activity of certain isolates. Our findings point towards an adaption to insects within closely related pseudomonads and contribute to understand the ecology of insecticidal Pseudomonas.     

Forecasting of phenotypic and genetic outcomes of experimental evolution in Pseudomonas protegens

Experimental evolution with microbes is often highly repeatable under identical conditions, suggesting the possibility to predict short-term evolution. However, it is not clear to what degree evolutionary forecasts can be extended to related species in non-identical environments, which would allow testing of general predictive models and fundamental biological assumptions. To develop an extended model system for evolutionary forecasting, we used previous data and models of the genotype-to-phenotype map from the wrinkly spreader system in Pseudomonas fluorescens SBW25 to make predictions of evolutionary outcomes on different biological levels for Pseudomonas protegens Pf-5. In addition to sequence divergence (78% amino acid and 81% nucleotide identity) for the genes targeted by mutations, these species also differ in the inability of Pf-5 to make cellulose, which is the main structural basis for the adaptive phenotype in SBW25. The experimental conditions were changed compared to the SBW25 system to test if forecasts were extendable to a non-identical environment. Forty-three mutants with increased ability to colonize the air-liquid interface were isolated, and the majority had reduced motility and was partly dependent on the Pel exopolysaccharide as a structural component. Most (38/43) mutations are expected to disrupt negative regulation of the same three diguanylate cyclases as in SBW25, with a smaller number of mutations in promoter regions, including an uncharacterized polysaccharide synthase operon. A mathematical model developed for SBW25 predicted the order of the three main pathways and the genes targeted by mutations, but differences in fitness between mutants and mutational biases also appear to influence outcomes. Mutated regions in proteins could be predicted in most cases (16/22), but parallelism at the nucleotide level was low and mutational hot spot sites were not conserved. This study demonstrates the potential of short-term evolutionary forecasting in experimental populations and provides testable predictions for evolutionary outcomes in other Pseudomonas species.     

Characterization of methyl-accepting chemotaxis proteins (MCPs) for amino acids in plant-growth-promoting rhizobacterium Pseudomonas protegens CHA0 and enhancement of amino acid chemotaxis by MCP genes overexpression

ABSTRACT

Pseudomonas protegens CHA0, known as plant-growth-promoting rhizobacterium, showed positive chemotactic responses toward proteinaceous L-amino acids. Genomic analysis revealed that P. protegens CHA0 possesses four putative chemoreceptors for amino acids (designated CtaA, CtaB, CtaC, and CtaD, respectively). Pseudomonas aeruginosa PCT2, a mutant defective in chemotaxis to amino acids, harboring a plasmid containing each of ctaA, ctaB, ctaC, and ctaD showed chemotactic responses to 20, 4, 4, and 11 types of amino acids, respectively. To enhance chemotaxis toward amino acids, we introduced the plasmids containing ctaA, ctaB, ctaC, or ctaD into P. protegens CHA0. By overexpression of the genes, we succeeded in enhancing chemotaxis toward more than half of the tested ligands. However, unexpectedly, the P. protegens CHA0 transformants showed unchanged or decreased responses to some amino acids when compared to wild-type CHA0. We speculate that alternation of expression of a chemoreceptor may affect the abundance of other chemoreceptors.     

Two polyurethanases PueA and PueB are major extracellular lipases partly secreted by the mediation of their cognate ABC exporter AprDEF in Pseudomonas protegens Pf-5

Two polyurethanases PueA and PueB from Pseudomonas protegens Pf-5 have been reported to have hydrolytic activity against synthetic p-nitrophenyl palmitate of lipase substrate, and PueA may play a more effective role in this activity. However, it is still unknown whether PueA and PueB play similar parts in the lipase activity against natural acylglycerols and achieve the extracellular secretion via their cognate ABC exporter AprDEF. In this study, we investigated these questions through the construction of four markerless deletion mutants in Pf5139 (Δupp derivative of Pf-5), two heterologous co-expression strains and their three control strains in lipase-free Escherichia coli BL21(DE3), and detected their lipase activities by the tributyrin plate assay and the liquid culture assay. The results showed that PueA and PueB, classified as subfamily I.3 lipases, are major extracellular lipases involved in the uptake of oil in Pf-5, and PueA plays a leading role in extracellular lipase activity. In addition, the extracellular secretion of PueA and PueB can be partly mediated via AprDEF in Pf-5 and BL21(DE3). Finally, PueA and PueB are also able to achieve the extracellular secretion without the assistance of AprDEF in Pf-5 and BL21(DE3).     

Domain Shuffling in a Sensor Protein Contributed to the Evolution of Insect Pathogenicity in Plant-Beneficial Pseudomonas protegens

Pseudomonas protegens is a biocontrol rhizobacterium with a plant-beneficial and an insect pathogenic lifestyle, but it is not understood how the organism switches between the two states. Here, we focus on understanding the function and possible evolution of a molecular sensor that enables P. protegens to detect the insect environment and produce a potent insecticidal toxin specifically during insect infection but not on roots. By using quantitative single cell microscopy and mutant analysis, we provide evidence that the sensor histidine kinase FitF is a key regulator of insecticidal toxin production. Our experimental data and bioinformatic analyses indicate that FitF shares a sensing domain with DctB, a histidine kinase regulating carbon uptake in Proteobacteria. This suggested that FitF has acquired its specificity through domain shuffling from a common ancestor. We constructed a chimeric DctB-FitF protein and showed that it is indeed functional in regulating toxin expression in P. protegens. The shuffling event and subsequent adaptive modifications of the recruited sensor domain were critical for the microorganism to express its potent insect toxin in the observed host-specific manner. Inhibition of the FitF sensor during root colonization could explain the mechanism by which P. protegens differentiates between the plant and insect host. Our study establishes FitF of P. protegens as a prime model for molecular evolution of sensor proteins and bacterial pathogenicity.     

Complete Genome Sequence of the Biocontrol Strain Pseudomonas protegens Cab57 Discovered in Japan Reveals Strain-Specific Diversity of This Species

The biocontrol strain Pseudomonas sp. Cab57 was isolated from the rhizosphere of shepherd’s purse growing in a field in Hokkaido by screening the antibiotic producers. The whole genome sequence of this strain was obtained by paired-end and whole-genome shotgun sequencing, and the gaps between the contigs were closed using gap-spanning PCR products. The P. sp. Cab57 genome is organized into a single circular chromosome with 6,827,892 bp, 63.3% G+C content, and 6,186 predicted protein-coding sequences. Based on 16S rRNA gene analysis and whole genome analysis, strain Cab57 was identified as P. protegens. As reported in P. protegens CHA0 and Pf-5, four gene clusters (phl, prn, plt, and hcn) encoding the typical antibiotic metabolites and the reported genes associated with Gac/Rsm signal transduction pathway of these strains are fully conserved in the Cab57 genome. Actually strain Cab57 exhibited typical Gac/Rsm activities and antibiotic production, and these activities were enhanced by knocking out the retS gene (for a sensor kinase acting as an antagonist of GacS). Two large segments (79 and 115 kb) lacking in the Cab57 genome, as compared with the Pf-5 genome, accounted for the majority of the difference (247 kb) between these genomes. One of these segments was the complete rhizoxin analog biosynthesis gene cluster (ca. 79 kb) and another one was the 115-kb mobile genomic island. A whole genome comparison of those relative strains revealed that each strain has unique gene clusters involved in metabolism such as nitrite/nitrate assimilation, which was identified in the Cab57 genome. These findings suggest that P. protegens is a ubiquitous bacterium that controls its biocontrol traits while building up strain-specific genomic repertoires for the biosynthesis of secondary metabolites and niche adaptation.     

Pseudomonas fluorescens NZI7 repels grazing by C. elegans,a natural predator

The bacteriovorous nematode Caenorhabditis elegans has been used to investigate many aspects of animal biology, including interactions with pathogenic bacteria. However, studies examining C. elegans interactions with bacteria isolated from environments in which it is found naturally are relatively scarce. C. elegans is frequently associated with cultivation of the edible mushroom Agaricus bisporus, and has been reported to increase the severity of bacterial blotch of mushrooms, a disease caused by bacteria from the Pseudomonas fluorescens complex. We observed that pseudomonads isolated from mushroom farms showed differential resistance to nematode predation. Under nutrient poor conditions, in which most pseudomonads were consumed, the mushroom pathogenic isolate P. fluorescens NZI7 was able to repel C. elegans without causing nematode death. A draft genome sequence of NZI7 showed it to be related to the biocontrol strain P. protegens Pf-5. To identify the genetic basis of nematode repellence in NZI7, we developed a grid-based screen for mutants that lacked the ability to repel C. elegans. The mutants isolated in this screen included strains with insertions in the global regulator GacS and in a previously undescribed GacS-regulated gene cluster, ‘EDB'' (‘edible''). Our results suggest that the product of the EDB cluster is a poorly diffusible or cell-associated factor that acts together with other features of NZI7 to provide a novel mechanism to deter nematode grazing. As nematodes interact with NZI7 colonies before being repelled, the EDB factor may enable NZI7 to come into contact with and be disseminated by C. elegans without being subject to intensive predation.     

Gamma-glutamyl transpeptidase from two plant growth promoting rhizosphere fluorescent pseudomonads

Glutathione is the most abundant non-protein thiol compound present in many cells. Because this molecule is involved in many physiological processes, each cell maintains a critical level of glutathione. Gamma-glutamyl transpeptidase (GGT, E.C.2.3.2.2) is the key enzyme involved in the glutathione cycle. In the present study, GGT was isolated from two plant growth promoting rhizosphere isolates, Pseudomonas protegens strain Pf-5 and Pseudomonas fluorescens strain PfT-1. GGT in these strains is located in the periplasm and possessed good hydrolytic activity at pH 8.0. Strains Pf-5 and PfT-1 showed maximum enzyme activity when grown at 30–35 °C. The ggt gene from both the strains was cloned in pGEM-T cloning vector and sequenced. Subsequently, GGT expressed in Escherichia coli BL21(DE3) using the pET-28a(+) expression vector was purified and characterized. The enzymes are active in a wide range of pH and some divalent cations significantly enhanced the hydrolytic activity. These enzymes showed higher thermal stability as compared to those of other mesophilic strains, as they retained ~50 % of activity at 50 °C even after 12 h of incubation. The enzymes could also tolerate up to 3.0 M NaCl.     

Genomic-assisted characterisation of Pseudomonas sp. strain Pf4, a potential biocontrol agent in hydroponics

In an attempt to select potential biocontrol agents against Pythium spp. and Rhizoctonia spp. root pathogens for use in soilless systems, 12 promising bacteria were selected for further investigations. Sequence analysis of the 16S rRNA gene revealed that three strains belonged to the genus Enterobacter, whereas nine strains belonged to the genus Pseudomonas. In in vitro assays, one strain of Pseudomonas sp., Pf4, closely related to Pseudomonas protegens (formerly Pseudomonas fluorescens), showed noteworthy antagonistic activity against two strains of Pythium aphanidermatum and two strains of Rhizoctonia solani AG 1-IB, with average inhibition of mycelial growth >80%. Strain Pf4 was used for in vivo treatments on lamb’s lettuce against R. solani root rot in small-scale hydroponics. Pf4-treated and untreated plants were daily monitored for symptom development and after two weeks of infection, a significant protective effect of Pf4 against root rot was recorded. The survival and population density of Pf4 on roots were also checked, demonstrating a density above the threshold value of 10⁵?CFU?g^?1 of root required for disease suppression. Known loci for the synthesis of antifungal metabolites, detected using PCR, and draft-genome sequencing of Pf4 demonstrated that Pseudomonas sp. Pf4 has the potential to produce an arsenal of secondary metabolites (plt, phl, ofa and fit-rzx gene clusters) very similar to that of the well-known biocontrol P. protegens strain Pf-5.     

Production of Siderophores Increases Resistance to Fusaric Acid in Pseudomonas protegens Pf-5

Fusaric acid is produced by pathogenic fungi of the genus Fusarium, and is toxic to plants and rhizobacteria. Many fluorescent pseudomonads can prevent wilt diseases caused by these fungi. This study was undertaken to evaluate the effect of fusaric acid on P. protegens Pf-5 and elucidate the mechanisms that enable the bacterium to survive in the presence of the mycotoxin. The results confirm that fusaric acid negatively affects growth and motility of P. protegens. Moreover, a notable increase in secretion of the siderophore pyoverdine was observed when P. protegens was grown in the presence of fusaric acid. Concomitantly, levels of enzymes involved in the biosynthesis of pyoverdine and enantio-pyochelin, the second siderophore encoded by P. protegens, increased markedly. Moreover, while similar levels of resistance to fusaric acid were observed for P. protegens mutants unable to synthesize either pyoverdine or enanto-pyochelin and the wild type strain, a double mutant unable to synthesize both kinds of siderophores showed a dramatically reduced resistance to this compound. This reduced resistance was not observed when this mutant was grown under conditions of iron excess. Spectrophotometric titrations revealed that fusaric acid binds not only Fe²⁺ and Fe³⁺, but also Zn²⁺, Mn²⁺ and Cu²⁺, with high affinity. Our results demonstrate that iron sequestration accounts at least in part for the deleterious effect of the mycotoxin on P. protegens.     

Novel Lectin-Like Bacteriocins of Biocontrol Strain Pseudomonas fluorescens Pf-5

Bacteriocin LlpA, produced by Pseudomonas sp. strain BW11M1, is a peculiar antibacterial protein due to its homology to mannose-binding lectins mostly found in monocots (A. H. A. Parret, G. Schoofs, P. Proost, and R. De Mot, J. Bacteriol. 185:897-908, 2003). Biocontrol strain Pseudomonas fluorescens Pf-5 contains two llpA-like genes, named llpA1_Pf-5 and llpA2_Pf-5. Recombinant Escherichia coli cells expressing llpA1_Pf-5 or llpA2_Pf-5 acquired bacteriocin activity and secreted a 31-kDa protein cross-reacting with LlpA_BW11M1 antibodies. Antibacterial activity of the recombinant proteins was evidenced by gel overlay assays. Analysis of the antimicrobial spectrum indicated that LlpA1_Pf-5 and LlpA2_Pf-5 are able to inhibit P. fluorescens strains, as well as the related mushroom pathogen Pseudomonas tolaasii. LlpA-type bacteriocins are characterized by a domain structure consisting of tandem monocot mannose-binding lectin (MMBL) domains. Molecular phylogeny of these MMBL domains suggests that the individual MMBL domains within an LlpA protein have evolved separately toward a specific, as yet unknown, function or, alternatively, were acquired from different ancestral sources. Our observations are consistent with earlier observations, which hinted that MMBL-like bacteriocins represent a new family of antibacterial proteins, probably with a novel mode of action.     

Engineering Pseudomonas protegens Pf-5 to improve its antifungal activity and nitrogen fixation

In agricultural production, sustainability is currently one of the most significant concerns. The genetic modification of plant growth-promoting rhizobacteria may provide a novel way to use natural bacteria as microbial inoculants. In this study, the root-colonizing strain Pseudomonas protegens Pf-5 was genetically modified to act as a biocontrol agent and biofertilizer with biological nitrogen fixation activity. Genetic inactivation of retS enhanced the production of 2,4-diacetylphloroglucinol, which contributed for the enhanced antifungal activity. Then, the entire nitrogenase island with native promoter from Pseudomonas stutzeri DSM4166 was introduced into a retS mutant strain for expression. Root colonization patterns assessed via confocal laser scanning microscopy confirmed that GFP-tagged bacterial were mainly located on root surfaces and at the junctions between epidermal root cells. Moreover, under pathogen and N-limited double treatment conditions, the fresh weights of seedlings inoculated with the recombinant retS mutant-nif strain were increased compared with those of the control. In conclusion, this study has innovatively developed an eco-friendly alternative to the agrochemicals that will benefit global plant production significantly.     

Pyocin-mediated antagonistic interactions in Pseudomonas spp. isolated in James Ross Island,Antarctica

Interactions within bacterial communities are frequently mediated by the production of antimicrobial agents. Despite the increasing interest in research of new antimicrobials, studies describing antagonistic interactions among cold-adapted microorganisms are still rare. Our study assessed the antimicrobial interactions of 36 Antarctic Pseudomonas spp. and described the genetic background of these interactions in selected strains. The overall bacteriocinogeny was greater compared to mesophilic Pseudomonas non-aeruginosa species. R-type tailocins were detected on transmission electron micrographs in 16 strains (44.4%); phylogenetic analysis of the corresponding gene clusters revealed that the P. prosekii CCM 8878 tailocin was related to the Rp3 group, whereas the tailocin in Pseudomonas sp. CCM 8880 to the Rp4 group. Soluble antimicrobials were produced by eight strains (22.-2%); gene mining found pyocin L homologues in the genomes of P. prosekii CCM 8881 and CCM 8879 and pyocin S9-like homologues in P. prosekii CCM 8881 and Pseudomonas sp. CCM 8880. Analysis of secretomes confirmed the production of all S- and L-type pyocin genes. Our results suggest that bacteriocin-based inhibition plays an important role in interactions among Antarctic soil bacteria, and these native, cold-adapted microorganisms could be a promising source of new antimicrobials.