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.

Differential Contribution of Plant-Beneficial Functions from Pseudomonas kilonensis F113 to Root System Architecture Alterations in Arabidopsis thaliana and Zea mays.

Fluorescent pseudomonads are playing key roles in plant-bacteria symbiotic interactions due to the multiple plant-beneficial functions (PBFs) they are harboring. The relative contributions of PBFs to plant-stimulatory effects of the well-known plant growth-promoting rhizobacteria Pseudomonas kilonensis F113 (formerly P. fluorescens F113) were investigated using a genetic approach. To this end, several deletion mutants were constructed, simple mutants ΔphlD (impaired in the biosynthesis of 2,4-diacetylphloroglucinol [DAPG]), ΔacdS (deficient in 1-aminocyclopropane-1-carboxylate deaminase activity), Δgcd (glucose dehydrogenase deficient, impaired in phosphate solubilization), and ΔnirS (nitrite reductase deficient), and a quadruple mutant (deficient in the four PBFs mentioned above). Every PBF activity was quantified in the wild-type strain and the five deletion mutants. This approach revealed few functional interactions between PBFs in vitro. In particular, biosynthesis of glucose dehydrogenase severely reduced the production of DAPG. Contrariwise, the DAPG production impacted positively, but to a lesser extent, phosphate solubilization. Inoculation of the F113 wild-type strain on Arabidopsis thaliana Col-0 and maize seedlings modified the root architecture of both plants. Mutant strain inoculations revealed that the relative contribution of each PBF differed according to the measured plant traits and that F113 plant-stimulatory effects did not correspond to the sum of each PBF relative contribution. Indeed, two PBF genes (ΔacdS and ΔnirS) had a significant impact on root-system architecture from both model plants, in in vitro and in vivo conditions. The current work underscored that few F113 PBFs seem to interact between each other in the free-living bacterial cells, whereas they control in concert Arabidopsis thaliana and maize growth and development.

Evolutionary and ecological role of extracellular contractile injection systems: from threat to weapon.

Contractile injection systems (CISs) are phage tail-related structures that are encoded in many bacterial genomes. These devices encompass the cell-based type VI secretion systems (T6SSs) as well as extracellular CISs (eCISs). The eCISs comprise the R-tailocins produced by various bacterial species as well as related phage tail-like structures such as the antifeeding prophages (Afps) of Serratia entomophila, the Photorhabdus virulence cassettes (PVCs), and the metamorphosis-associated contractile structures (MACs) of Pseudoalteromonas luteoviolacea. These contractile structures are released into the extracellular environment upon suicidal lysis of the producer cell and play important roles in bacterial ecology and evolution. In this review, we specifically portray the eCISs with a focus on the R-tailocins, sketch the history of their discovery and provide insights into their evolution within the bacterial host, their structures and how they are assembled and released. We then highlight ecological and evolutionary roles of eCISs and conceptualize how they can influence and shape bacterial communities. Finally, we point to their potential for biotechnological applications in medicine and agriculture.

Relation of pest insect-killing and soilborne pathogen-inhibition abilities to species diversification in environmental Pseudomonas protegens.

Strains belonging to the Pseudomonas protegens phylogenomic subgroup have long been known for their beneficial association with plant roots, notably antagonising soilborne phytopathogens. Interestingly, they can also infect and kill pest insects, emphasising their interest as biocontrol agents. In the present study, we used all available Pseudomonas genomes to reassess the phylogeny of this subgroup. Clustering analysis revealed the presence of 12 distinct species, many of which were previously unknown. The differences between these species also extend to the phenotypic level. Most of the species were able to antagonise two soilborne phytopathogens, Fusarium graminearum and Pythium ultimum, and to kill the plant pest insect Pieris brassicae in feeding and systemic infection assays. However, four strains failed to do so, likely as a consequence of adaptation to particular niches. The absence of the insecticidal Fit toxin explained the non-pathogenic behaviour of the four strains towards Pieris brassicae. Further analyses of the Fit toxin genomic island evidence that the loss of this toxin is related to non-insecticidal niche specialisation. This work expands the knowledge on the growing Pseudomonas protegens subgroup and suggests that loss of phytopathogen inhibition and pest insect killing abilities in some of these bacteria may be linked to species diversification processes involving adaptation to particular niches. Our work sheds light on the important ecological consequences of gain and loss dynamics for functions involved in pathogenic host interactions of environmental bacteria.

Inhibition of broomrape germination by 2,4-diacetylphloroglucinol produced by environmental Pseudomonas.

Parasitic weeds such as broomrapes (Phelipanche ramosa and Orobanche cumana) cause severe damage to crops and their development must be controlled. Given that phloroglucinol compounds (PGCs) produced by environmental Pseudomonas could be toxic towards certain plants, we assessed the potential herbicidal effect of the bacterial model Pseudomonas ogarae F113, a PGCs-producing bacterium, on parasitic weed. By combining the use of a mutagenesis approach and of pure PGCs, we evaluated the in vitro effect of PGC-produced by P. ogarae F113 on broomrape germination and assessed the protective activity of a PGC-producing bacteria on oilseed rape (Brassica napus) against P. ramosa in non-sterile soils. We showed that the inhibition of the germination depends on the PGCs molecular structure and their concentrations as well as the broomrape species and pathovars. This inhibition caused by the PGCs is irreversible, causing a brown coloration of the broomrape seeds. The inoculation of PGCs-producing bacteria limited the broomrape infection of P. ramosa, without affecting the host growth. Moreover, elemental profiling analysis of oilseed rape revealed that neither F113 nor applied PGCs affected the nutrition capacity of the oilseed rape host. Our study expands the knowledge on plant-beneficial Pseudomonas as weed biocontrol agents and opens new avenues for the development of natural bioherbicides to enhance crop yield.

Molecular and evolutionary basis of O-antigenic polysaccharide-driven phage sensitivity in environmental pseudomonads.

IMPORTANCE: The application of plant-beneficial microorganisms to protect crop plants is a promising alternative to the usage of chemicals. However, biocontrol research often faces difficulties in implementing this approach due to the inconsistency of the bacterial inoculant to establish itself within the root microbiome. Beneficial bacterial inoculants can be decimated by the presence of their natural predators, notably bacteriophages (also called phages). Thus, it is important to gain knowledge regarding the mechanisms behind phage-bacteria interactions to overcome this challenge. Here, we evidence that the major long O-antigenic polysaccharide (O-PS, O-antigen) of the widely used model plant-beneficial bacterium Pseudomonas protegens CHA0 is the receptor of its natural predator, the phage ΦGP100. We examined the distribution of the gene cluster directing the synthesis of this O-PS and identified signatures of horizontal gene acquisitions. Altogether, our study highlights the importance of bacterial cell surface structure variation in the complex interplay between phages and their Pseudomonas hosts.

The conserved iol gene cluster in Pseudomonas is involved in rhizosphere competence.

The Pseudomonas genus has shown great potential as a sustainable solution to support agriculture through its plant-growth-promoting and biocontrol activities. However, their efficacy as bioinoculants is limited by unpredictable colonization in natural conditions. Our study identifies the iol locus, a gene cluster in Pseudomonas involved in inositol catabolism, as a feature enriched among superior root colonizers in natural soil. Further characterization revealed that the iol locus increases competitiveness, potentially caused by an observed induction of swimming motility and the production of fluorescent siderophore in response to inositol, a plant-derived compound. Public data analyses indicate that the iol locus is broadly conserved in the Pseudomonas genus and linked to diverse host-microbe interactions. Together, our findings suggest the iol locus as a potential target for developing more effective bioinoculants for sustainable agriculture.

Changes in structure and assembly of a species-rich soil natural community with contrasting nutrient availability upon establishment of a plant-beneficial Pseudomonas in the wheat rhizosphere.

BACKGROUND: Plant-beneficial bacterial inoculants are of great interest in agriculture as they have the potential to promote plant growth and health. However, the inoculation of the rhizosphere microbiome often results in a suboptimal or transient colonization, which is due to a variety of factors that influence the fate of the inoculant. To better understand the fate of plant-beneficial inoculants in complex rhizosphere microbiomes, composed by hundreds of genotypes and multifactorial selection mechanisms, controlled studies with high-complexity soil microbiomes are needed. RESULTS: We analysed early compositional changes in a taxa-rich natural soil bacterial community under both exponential nutrient-rich and stationary nutrient-limited growth conditions (i.e. growing and stable communities, respectively) following inoculation with the plant-beneficial bacterium Pseudomonas protegens in a bulk soil or a wheat rhizosphere environment. P. protegens successfully established under all conditions tested and was more abundant in the rhizosphere of the stable community. Nutrient availability was a major factor driving microbiome composition and structure as well as the underlying assembly processes. While access to nutrients resulted in communities assembled mainly by homogeneous selection, stochastic processes dominated under the nutrient-deprived conditions. We also observed an increased rhizosphere selection effect under nutrient-limited conditions, resulting in a higher number of amplicon sequence variants (ASVs) whose relative abundance was enriched. The inoculation with P. protegens produced discrete changes, some of which involved other Pseudomonas. Direct competition between Pseudomonas strains partially failed to replicate the observed differences in the microbiome and pointed to a more complex interaction network. CONCLUSIONS: The results of this study show that nutrient availability is a major driving force of microbiome composition, structure and diversity in both the bulk soil and the wheat rhizosphere and determines the assembly processes that govern early microbiome development. The successful establishment of the inoculant was facilitated by the wheat rhizosphere and produced discrete changes among other members of the microbiome. Direct competition between Pseudomonas strains only partially explained the microbiome changes, indicating that indirect interactions or spatial distribution in the rhizosphere or soil interface may be crucial for the survival of certain bacteria. Video Abstract.

From microbiome composition to functional engineering, one step at a time.

SUMMARYCommunities of microorganisms (microbiota) are present in all habitats on Earth and are relevant for agriculture, health, and climate. Deciphering the mechanisms that determine microbiota dynamics and functioning within the context of their respective environments or hosts (the microbiomes) is crucially important. However, the sheer taxonomic, metabolic, functional, and spatial complexity of most microbiomes poses substantial challenges to advancing our knowledge of these mechanisms. While nucleic acid sequencing technologies can chart microbiota composition with high precision, we mostly lack information about the functional roles and interactions of each strain present in a given microbiome. This limits our ability to predict microbiome function in natural habitats and, in the case of dysfunction or dysbiosis, to redirect microbiomes onto stable paths. Here, we will discuss a systematic approach (dubbed the N+1/N-1 concept) to enable step-by-step dissection of microbiome assembly and functioning, as well as intervention procedures to introduce or eliminate one particular microbial strain at a time. The N+1/N-1 concept is informed by natural invasion events and selects culturable, genetically accessible microbes with well-annotated genomes to chart their proliferation or decline within defined synthetic and/or complex natural microbiota. This approach enables harnessing classical microbiological and diversity approaches, as well as omics tools and mathematical modeling to decipher the mechanisms underlying N+1/N-1 microbiota outcomes. Application of this concept further provides stepping stones and benchmarks for microbiome structure and function analyses and more complex microbiome intervention strategies.

Pivotal role of O-antigenic polysaccharide display in the sensitivity against phage tail-like particles in environmental Pseudomonas kin competition.

Environmental pseudomonads colonize various niches including insect and plant environments. When invading these environments, bacteria are confronted with the resident microbiota. To oppose with closely related strains, they rely on narrow-spectrum weaponry such as tailocins, i.e., phage tail-like particles. Little is known about the receptors for these tailocins especially among phylogenetically closely related species. Here, we studied the interaction between an R-tailocin from Pseudomonas protegens CHA0 and a targeted kin, Pseudomonas protegens Pf-5. Using genome-wide transposon insertion sequencing, we identified that lipopolysaccharides are involved in the sensitivity of Pf-5 towards the tailocin of CHA0. By generating Pf-5 lipopolysaccharide mutants and exposing them to extracted tailocin, we specified the two O-antigenic polysaccharides (O-PS) targeted by the tailocin. We affirmed the role of these O-PS through competition assays in vitro as well as in insects. Further, we demonstrate that O-PS are double-edge swords that are responsible for the sensitivity of P. protegens towards tailocins and phages produced by their kin, but shield bacteria from the immune system of the insect. Our results shed light on the trade-off that bacteria are confronted with, where specific O-PS decorations can both be of benefit or disadvantage depending on the host environment and its bacterial inhabitants.

Live cell dynamics of production, explosive release and killing activity of phage tail-like weapons for Pseudomonas kin exclusion.

Interference competition among bacteria requires a highly specialized, narrow-spectrum weaponry when targeting closely-related competitors while sparing individuals from the same clonal population. Here we investigated mechanisms by which environmentally important Pseudomonas bacteria with plant-beneficial activity perform kin interference competition. We show that killing between phylogenetically closely-related strains involves contractile phage tail-like devices called R-tailocins that puncture target cell membranes. Using live-cell imaging, we evidence that R-tailocins are produced at the cell center, transported to the cell poles and ejected by explosive cell lysis. This enables their dispersal over several tens of micrometers to reach targeted cells. We visualize R-tailocin-mediated competition dynamics between closely-related Pseudomonas strains at the single-cell level, both in non-induced condition and upon artificial induction. We document the fatal impact of cellular self-sacrifice coupled to deployment of phage tail-like weaponry in the microenvironment of kin bacterial competitors, emphasizing the necessity for microscale assessment of microbial competitions.

Spatially Restricted Immune Responses Are Required for Maintaining Root Meristematic Activity upon Detection of Bacteria.

Plants restrict immune responses to vulnerable root parts. Spatially restricted responses are thought to be necessary to avoid constitutive responses to rhizosphere microbiota. To directly demonstrate the importance of spatially restricted responses, we expressed the plant flagellin receptor (FLS2) in different tissues, combined with fluorescent defense markers for immune readouts at cellular resolution. Our analysis distinguishes responses appearing cell autonomous from apparently non-cell-autonomous responses. It reveals lignification as a general immune response, contrasting suberization. Importantly, our analysis divides the root meristem into a central zone refractory to FLS2 expression and a cortex that is sensitized by FLS2 expression, causing meristem collapse upon stimulation. Meristematic epidermal expression generates super-competent lines that detect native bacterial flagellin and bypass the weak or absent response to commensals, providing a powerful tool for studying root immunity. Our manipulations and readouts demonstrate incompatibility of meristematic activity and defense and the importance of cell-resolved studies of plant immune responses.

Field Site-Specific Effects of an Azospirillum Seed Inoculant on Key Microbial Functional Groups in the Rhizosphere.

The beneficial effects of plant growth-promoting Rhizobacteria (PGPR) entail several interaction mechanisms with the plant or with other root-associated microorganisms. These microbial functions are carried out by multiple taxa within functional groups and contribute to rhizosphere functioning. It is likely that the inoculation of additional PGPR cells will modify the ecology of these functional groups. We also hypothesized that the inoculation effects on functional groups are site specific, similarly as the PGPR phytostimulation effects themselves. To test this, we assessed in the rhizosphere of field-grown maize the effect of seed inoculation with the phytostimulatory PGPR Azospirillum lipoferum CRT1 on the size and/or diversity of selected microbial functional groups important for plant growth, using quantitative polymerase chain reaction and/or Illumina MiSeq metabarcoding. The functional groups included bacteria able to fix nitrogen (a key nutrient for plant growth), producers of 1-aminocyclopropane-1-carboxylate (ACC) deaminase (which modulate ethylene metabolism in plant and stimulate root growth), and producers of 2,4-diacetylphloroglucinol (an auxinic signal enhancing root branching). To test the hypothesis that such ecological effects were site-specific, the functional groups were monitored at three different field sites, with four sampling times over two consecutive years. Despite poor inoculant survival, inoculation enhanced maize growth. It also increased the size of functional groups in the three field sites, at the maize six-leaf and flowering stages for diazotrophs and only at flowering stage for ACC deaminase and 2,4-diacetylphloroglucinol producers. Sequencing done in the second year revealed that inoculation modified the composition of diazotrophs (and of the total bacterial community) and to a lesser extent of ACC deaminase producers. This study revealed an ecological impact that was field specific (even though a few taxa were impacted in all fields) and of unexpected magnitude with the phytostimulatory Azospirillum inoculant, when considering microbial functional groups. Further methodological developments are needed to monitor additional functional groups important for soil functioning and plant growth under optimal or stress conditions.

Draft Genome Sequence of Pseudomonas sp. Strain LD120, Isolated from the Marine Alga Saccharina latissima.

We report the draft genome sequence of Pseudomonas sp. strain LD120, which was isolated from a brown macroalga in the Baltic Sea. The genome of this marine Pseudomonas protegens subgroup bacterium harbors biosynthetic gene clusters for toxic metabolites typically produced by members of this Pseudomonas subgroup, including 2,4-diacetylphloroglucinol, pyoluteorin, and rhizoxin analogs.

Transcriptome plasticity underlying plant root colonization and insect invasion by Pseudomonas protegens.

Pseudomonas protegens shows a high degree of lifestyle plasticity since it can establish both plant-beneficial and insect-pathogenic interactions. While P. protegens protects plants against soilborne pathogens, it can also invade insects when orally ingested leading to the death of susceptible pest insects. The mechanism whereby pseudomonads effectively switch between lifestyles, plant-beneficial or insecticidal, and the specific factors enabling plant or insect colonization are poorly understood. We generated a large-scale transcriptomics dataset of the model P. protegens strain CHA0 which includes data from the colonization of wheat roots, the gut of Plutella xylostella after oral uptake and the Galleria mellonella hemolymph after injection. We identified extensive plasticity in transcriptomic profiles depending on the environment and specific factors associated to different hosts or different stages of insect infection. Specifically, motor-activity and Reb toxin-related genes were highly expressed on wheat roots but showed low expression within insects, while certain antimicrobial compounds (pyoluteorin), exoenzymes (a chitinase and a polyphosphate kinase), and a transposase exhibited insect-specific expression. We further identified two-partner secretion systems as novel factors contributing to pest insect invasion. Finally, we use genus-wide comparative genomics to retrace the evolutionary origins of cross-kingdom colonization.

Secondary metabolites from plant-associated Pseudomonas are overproduced in biofilm.

Plant rhizosphere soil houses complex microbial communities in which microorganisms are often involved in intraspecies as well as interspecies and inter-kingdom signalling networks. Some members of these networks can improve plant health thanks to an important diversity of bioactive secondary metabolites. In this competitive environment, the ability to form biofilms may provide major advantages to microorganisms. With the aim of highlighting the impact of bacterial lifestyle on secondary metabolites production, we performed a metabolomic analysis on four fluorescent Pseudomonas strains cultivated in planktonic and biofilm colony conditions. The untargeted metabolomic analysis led to the detection of hundreds of secondary metabolites in culture extracts. Comparison between biofilm and planktonic conditions showed that bacterial lifestyle is a key factor influencing Pseudomonas metabolome. More than 50% of the detected metabolites were differentially produced according to planktonic or biofilm lifestyles, with the four Pseudomonas strains overproducing several secondary metabolites in biofilm conditions. In parallel, metabolomic analysis associated with genomic prediction and a molecular networking approach enabled us to evaluate the impact of bacterial lifestyle on chemically identified secondary metabolites, more precisely involved in microbial interactions and plant-growth promotion. Notably, this work highlights the major effect of biofilm lifestyle on acyl-homoserine lactone and phenazine production in P. chlororaphis strains.

T6SS contributes to gut microbiome invasion and killing of an herbivorous pest insect by plant-beneficial Pseudomonas protegens.

Pseudomonas protegens are multi-talented plant-colonizing bacteria that suppress plant pathogens and stimulate plant defenses. In addition, they are capable of invading and killing agriculturally important plant pest insects that makes them promising candidates for biocontrol applications. Here we assessed the role of type VI secretion system (T6SS) components of type strain CHA0 during interaction with larvae of the cabbage pest Pieris brassicae. We show that the T6SS core apparatus and two VgrG modules, encompassing the respective T6SS spikes (VgrG1a and VgrG1b) and associated effectors (RhsA and Ghh1), contribute significantly to insect pathogenicity of P. protegens in oral infection assays but not when bacteria are injected directly into the hemolymph. Monitoring of the colonization levels of P. protegens in the gut, hemolymph, and excrements of the insect larvae revealed that the invader relies on T6SS and VgrG1a module function to promote hemocoel invasion. A 16S metagenomic analysis demonstrated that T6SS-supported invasion by P. protegens induces significant changes in the insect gut microbiome affecting notably Enterobacteriaceae, a dominant group of the commensal gut bacteria. Our study supports the concept that pathogens deploy T6SS-based strategies to disrupt the commensal microbiota in order to promote host colonization and pathogenesis.

Updated Genome Sequence and Annotation for the Full Genome of Pseudomonas protegens CHA0.

Minor differences in the previously obtained genome of Pseudomonas protegens CHA0 were detected after resequencing the strain. Based on this, the genome size slightly increased. Additionally, we performed a manual annotation of genes involved in biocontrol and insect pathogenicity. This annotation version will be the basis for upcoming genome studies.

Genomic, phylogenetic and catabolic re-assessment of the Pseudomonas putida clade supports the delineation of Pseudomonas alloputida sp. nov., Pseudomonas inefficax sp. nov., Pseudomonas persica sp. nov., and Pseudomonas shirazica sp. nov.

Bacteria of the Pseudomonas putida group are studied for a large panel of properties ranging from plant growth promotion and bioremediation to pathogenicity. To date, most of the classification of individual pseudomonads from this group relies on 16S RNA gene analysis, which is insufficient for accurate taxonomic characterization within bacterial species complexes of the Pseudomonas putida group. Here, a collection of 20 of these bacteria, isolated from various soils, was assessed via multi-locus sequence analysis of rpoD, gyrB and rrs genes. The 20 strains clustered in 7 different clades of the P. putida group. One strain per cluster was sequenced and results were compared to complete genome sequences of type strains of the P. putida group. Phylogenetic analyses, average nucleotide identity data and digital DNA hybridizations, combined to phenotypic characteristics, resulted in the proposition and description of four new species i.e. Pseudomonas alloputida Kh7 T (= LMG 29756 T = CFBP 8484 T) sp. nov., Pseudomonas inefficax JV551A3 T (= DSM108619 T = CFBP 8493 T) sp. nov., Pseudomonas persica RUB6 T (= LMG 29757 T = CFBP 8486 T) sp. nov. and Pseudomonas shirazica VM14 T (= LMG 29953 T = CFBP 8487 T) sp. nov.

Genome Sequence of the Pseudomonas protegens Phage ΦGP100.

We report here the complete annotated genome sequence of ΦGP100, a lytic bacteriophage of the Podoviridae family. ΦGP100 was isolated from rhizosphere soil in Switzerland and infects specifically strains of Pseudomonas protegens that are known for their plant-beneficial activities. Phage ΦGP100 has a 50,547-bp genome with 76 predicted open reading frames.