Interplay between rhizospheric Pseudomonas chlororaphis strains lays the basis for beneficial bacterial consortia.

Pseudomonas chlororaphis (Pc) representatives are found as part of the rhizosphere-associated microbiome, and different rhizospheric Pc strains frequently perform beneficial activities for the plant. In this study we described the interactions between the rhizospheric Pc strains PCL1601, PCL1606 and PCL1607 with a focus on their effects on root performance. Differences among the three rhizospheric Pc strains selected were first observed in phylogenetic studies and confirmed by genome analysis, which showed variation in the presence of genes related to antifungal compounds or siderophore production, among others. Observation of the interactions among these strains under lab conditions revealed that PCL1606 has a better adaptation to environments rich in nutrients, and forms biofilms. Interaction experiments on plant roots confirmed the role of the different phenotypes in their lifestyle. The PCL1606 strain was the best adapted to the habitat of avocado roots, and PCL1607 was the least, and disappeared from the plant root scenario after a few days of interaction. These results confirm that 2 out 3 rhizospheric Pc strains were fully compatible (PCL1601 and PCL1606), efficiently colonizing avocado roots and showing biocontrol activity against the fungal pathogen Rosellinia necatrix. The third strain (PCL1607) has colonizing abilities when it is alone on the root but displayed difficulties under the competition scenario, and did not cause deleterious effects on the other Pc competitors when they were present. These results suggest that strains PCL1601 and PCL1606 are very well adapted to the avocado root environment and could constitute a basis for constructing a more complex beneficial microbial synthetic community associated with avocado plant roots.

A Large Tn7-like Transposon Confers Hyper-Resistance to Copper in Pseudomonas syringae pv. syringae.

Copper resistance mechanisms provide an important adaptive advantage to plant pathogenic bacteria under exposure to copper treatments. Copper resistance determinants have been described in Pseudomonas syringae pv. syringae (Pss) strains isolated from mango intimately associated with 62 kb plasmids belonging to the pPT23A family (PFP). It has been previously described that the indiscriminate use of copper-based compounds promotes the selection of copper resistant bacterial strains and constitutes a selective pressure in the evolution of copper resistance determinants. Hence, we have explored in this study the copper resistance evolution and the distribution of specific genetic determinants in two different Pss mango populations isolated from the same geographical regions, mainly from southern Spain with an average of 20 years of difference. The total content of plasmids, in particular the 62 kb plasmids, and the number of copper resistant Pss strains were maintained at similar levels over the time. Interestingly, the phylogenetic analysis indicated the presence of a phylogenetic subgroup (PSG) in the Pss mango phylotype, mostly composed of the recent Pss population analyzed in this study that was strongly associated with a hyper-resistant phenotype to copper. Genome sequencing of two selected Pss strains from this PSG revealed the presence of a large Tn7-like transposon of chromosomal location, which harbored putative copper and arsenic resistance genes (COARS Tn7-like). Transformation of the copper sensitive Pss UMAF0158 strain with some putative copper resistance genes and RT-qPCR experiments brought into light the role of COARS Tn7-like transposon in the hyper-resistant phenotype to copper in Pss.IMPORTANCECopper compounds have traditionally been used as standard bactericides in agriculture in the past few decades. However, the extensive use of copper has fostered the evolution of bacterial copper resistance mechanisms. Pseudomonas syringae is a plant pathogenic bacterium used worldwide as a model to study plant-pathogen interactions. The adaption of P. syringae to plant surface environment is the most important step prior to an infection. In this scenario, copper resistance mechanisms could play a key role in improving its epiphytic survival. In this work, a novel Tn7-like transposon of chromosomal location was detected in P. syringae pv. syringae strains isolated from mango. This transposon conferred the highest resistance to copper sulfate described to date for this bacterial phytopathogen. Understanding in depth the copper resistance mechanisms and their evolution are important steps to the agricultural industry to get a better improvement of disease management strategies.

Draft Genome Sequences of Three Pseudomonas fluorescens Strains Isolated from Marine Sponges Harvested off the West Coast of Ireland.

Three Pseudomonas sp. strains isolated from marine sponges have shown potential quorum sensing inhibition (QSI) activity. We sequenced the draft genomes of the three strains with the goal of determining which genes or gene cluster(s) could be potentially involved in the QSI activity. Average nucleotide identity (ANI) and phylogenetic analysis classified the three strains as belonging to the Pseudomonas fluorescens species.

Quorum Sensing Signaling Alters Virulence Potential and Population Dynamics in Complex Microbiome-Host Interactomes.

Despite the discovery of the first N-acyl homoserine lactone (AHL) based quorum sensing (QS) in the marine environment, relatively little is known about the abundance, nature and diversity of AHL QS systems in this diverse ecosystem. Establishing the prevalence and diversity of AHL QS systems and how they may influence population dynamics within the marine ecosystem, may give a greater insight into the evolution of AHLs as signaling molecules in this important and largely unexplored niche. Microbiome profiling of Stelletta normani and BD1268 sponge samples identified several potential QS active genera. Subsequent biosensor-based screening of a library of 650 marine sponge bacterial isolates identified 10 isolates that could activate at least one of three AHL biosensor strains. Each was further validated and profiled by Ultra-High Performance Liquid Chromatography Mass Spectrometry, with AHLs being detected in 8 out of 10 isolate extracts. Co-culture of QS active isolates with S. normani marine sponge samples led to the isolation of genera such as Pseudomonas and Paenibacillus, both of which were low abundance in the S. normani microbiome. Surprisingly however, addition of AHLs to isolates harvested following co-culture did not measurably affect either growth or biofilm of these strains. Addition of supernatants from QS active strains did however impact significantly on biofilm formation of the marine Bacillus sp. CH8a sporeforming strain suggesting a role for QS systems in moderating the microbe-microbe interaction in marine sponges. Genome sequencing and phylogenetic analysis of a QS positive Psychrobacter isolate identified several QS associated systems, although no classical QS synthase gene was identified. The stark contrast between the biodiverse sponge microbiome and the relatively limited diversity that was observed on standard culture media, even in the presence of QS active compounds, serves to underscore the extent of diversity that remains to be brought into culture.

Pseudomonas syringae pv. syringae Associated With Mango Trees, a Particular Pathogen Within the "Hodgepodge" of the Pseudomonas syringae Complex.

The Pseudomonas syringae complex comprises different genetic groups that include strains from both agricultural and environmental habitats. This complex group has been used for decades as a "hodgepodge," including many taxonomically related species. More than 60 pathovars of P. syringae have been described based on distinct host ranges and disease symptoms they cause. These pathovars cause disease relying on an array of virulence mechanisms. However, P. syringae pv. syringae (Pss) is the most polyphagous bacterium in the P. syringae complex, based on its wide host range, that primarily affects woody and herbaceous host plants. In early 1990s, bacterial apical necrosis (BAN) of mango trees, a critical disease elicited by Pss in Southern Spain was described for the first time. Pss exhibits important epiphytic traits and virulence factors, which may promote its survival and pathogenicity in mango trees and in other plant hosts. Over more than two decades, Pss strains isolated from mango trees have been comprehensively investigated to elucidate the mechanisms that governs their epiphytic and pathogenic lifestyles. In particular, the vast majority of Pss strains isolated from mango trees produce an antimetabolite toxin, called mangotoxin, whose leading role in virulence has been clearly demonstrated. Moreover, phenotypic, genetic and phylogenetic approaches support that Pss strains producers of BAN symptoms on mango trees all belong to a single phylotype within phylogroup 2, are adapted to the mango host, and produce mangotoxin. Remarkably, a genome sequencing project of the Pss model strain UMAF0158 revealed the presence of other factors that may play major roles in its different lifestyles, such as the presence of two different type III secretion systems, two type VI secretion systems and an operon for cellulose biosynthesis. The role of cellulose in increasing mango leaf colonization and biofilm formation, and impairing virulence of Pss, suggests that cellulose may play a pivotal role with regards to the balance of its different lifestyles. In addition, 62-kb plasmids belonging to the pPT23A-family of plasmids (PFPs) have been strongly associated with Pss strains that inhabit mango trees. Further, complete sequence and comparative genomic analyses revealed major roles of PFPs in detoxification of copper compounds and ultraviolet radiation resistance, both improving the epiphytic lifestyle of Pss on mango surfaces. Hence, in this review we summarize the research that has been conducted on Pss by our research group to elucidate the molecular mechanisms that underpin the epiphytic and pathogenic lifestyle on mango trees. Finally, future directions in this particular plant-pathogen story are discussed.

Genome Sequence of Paracoccus sp. JM45, a Bacterial Strain Isolated from a Marine Sponge with a Dual Quorum Sensing Inhibition Activity.

The draft genome sequence of Paracoccus sp. strain JM45, isolated from a marine sponge harvested off the west coast of Ireland, is reported here. Quorum sensing and quorum sensing inhibition activities have been reported recently for this bacterium, and genomic analysis supports its potential use for novel therapeutic development.

Disruption of N-acyl-homoserine lactone-specific signalling and virulence in clinical pathogens by marine sponge bacteria.

In recent years, the marine environment has been the subject of increasing attention from biotechnological and pharmaceutical industries. A combination of unique physicochemical properties and spatial niche-specific substrates, in wide-ranging and extreme habitats, underscores the potential of the marine environment to deliver on functionally novel bioactivities. One such area of ongoing research is the discovery of compounds that interfere with the cell-cell signalling process called quorum sensing (QS). Described as the next generation of antimicrobials, these compounds can target virulence and persistence of clinically relevant pathogens, independent of any growth-limiting effects. Marine sponges are a rich source of microbial diversity, with dynamic populations in a symbiotic relationship. In this study, we have harnessed the QS inhibition (QSI) potential of marine sponge microbiota and through culture-based discovery have uncovered small molecule signal mimics that neutralize virulence phenotypes in clinical pathogens. This study describes for the first time a marine sponge Psychrobacter sp. isolate B98C22 that blocks QS signalling, while also reporting dual QS/QSI activity in the Pseudoalteromonas sp. J10 and ParacoccusJM45. Isolation of novel QSI activities has significant potential for future therapeutic development, of particular relevance in the light of the pending perfect storm of antibiotic resistance meeting antibiotic drug discovery decline.

Pantoea agglomerans as a New Etiological Agent of a Bacterial Necrotic Disease of Mango Trees.

Bacterial apical necrosis of mango trees, a disease elicited by Pseudomonas syringae pv. syringae, is a primary limiting factor of mango crop production in the Mediterranean region. In this study, a collection of bacterial isolates associated with necrotic symptoms in mango trees similar to those produced by bacterial apical necrosis disease were isolated over five consecutive years in orchards from the Canary Islands. The bacterial isolates were characterized and identified as Pantoea agglomerans. Pathogenicity tests conducted on onion bulbs and mango plants confirmed that P. agglomerans strains isolated from mango trees are a new etiological agent of a bacterial necrotic disease in the Canary Islands. Pathogenicity plasmids of the pPATH family have been previously reported in P. agglomerans. The majority of putatively pathogenic (n = 23) and pathogenic (n = 4) P. agglomerans strains isolated from mango trees harbored four plasmids, one of which was close in size to the 135-kb pPATH pathogenicity plasmid. The analysis of the presence of two major genes in pPATH plasmids (repA and hrpJ) was undertaken in P. agglomerans strains isolated from mango trees. The hrpJ gene was detected in the 140-kb plasmid of pathogenic P. agglomerans strains isolated from mango trees but it showed differences in nucleotide sequences compared with other pathogenic strains. In contrast, the repA gene was not detected in any of the putatively pathogenic and pathogenic P. agglomerans strains isolated from mango trees. Finally, genetic characterization and phylogenetic analysis using the hrpJ gene and the housekeeping genes gyrB and rpoB showed that almost all P. agglomerans strains that were putatively pathogenic and pathogenic on mango trees clustered together, forming a differentiated phylogroup with respect to the other pathogenic P. agglomerans strains described from other hosts.

Coumarin: a novel player in microbial quorum sensing and biofilm formation inhibition.

Antibiotic resistance is a growing threat worldwide, causing serious problems in the treatment of microbial infections. The discovery and development of new drugs is urgently needed to overcome this problem which has greatly undermined the clinical effectiveness of conventional antibiotics. An intricate cell-cell communication system termed quorum sensing (QS) and the coordinated multicellular behaviour of biofilm formation have both been identified as promising targets for the treatment and clinical management of microbial infections. QS systems allow bacteria to adapt rapidly to harsh conditions, and are known to promote the formation of antibiotic tolerant biofilm communities. It is well known that biofilm is a recalcitrant mode of growth and it also increases bacterial resistance to conventional antibiotics. The pharmacological properties of coumarins have been well described, and these have included several that possess antimicrobial properties. More recently, reports have highlighted the potential role of coumarins as alternative therapeutic strategies based on their ability to block the QS signalling systems and to inhibit the formation of biofilms in clinically relevant pathogens. In addition to human infections, coumarins have also been found to be effective in controlling plant pathogens, infections in aquaculture, food spoilage and in reducing biofouling caused by eukaryotic organisms. Thus, the coumarin class of small molecule natural product are emerging as a promising strategy to combat bacterial infections in the new era of antimicrobial resistance.

Complete sequence and comparative genomic analysis of eight native Pseudomonas syringae plasmids belonging to the pPT23A family.

BACKGROUND: The pPT23A family of plasmids appears to be indigenous to the plant pathogen Pseudomonas syringae and these plasmids are widely distributed and widely transferred among pathovars of P. syringae and related species. pPT23A-family plasmids (PFPs) are sources of accessory genes for their hosts that can include genes important for virulence and epiphytic colonization of plant leaf surfaces. The occurrence of repeated sequences including duplicated insertion sequences on PFPs has made obtaining closed plasmid genome sequences difficult. Therefore, our objective was to obtain complete genome sequences from PFPs from divergent P. syringae pathovars and also from strains of P. syringae pv. syringae isolated from different hosts. RESULTS: The eight plasmids sequenced ranged in length from 61.6 to 73.8 kb and encoded from 65 to 83 annotated orfs. Virulence genes including type III secretion system effectors were encoded on two plasmids, and one of these, pPt0893-29 from P. syringae pv. tabaci, encoded a wide variety of putative virulence determinants. The PFPs from P. syringae pv. syringae mostly encoded genes of importance to ecological fitness including the rulAB determinant conferring tolerance to ultraviolet radiation. Heavy metal resistance genes encoding resistance to copper and arsenic were also present in a few plasmids. The discovery of part of the chromosomal genomic island GI6 from P. syringae pv. syringae B728a in two PFPs from two P. syringae pv. syringae hosts is further evidence of past intergenetic transfers between plasmid and chromosomal DNA. Phylogenetic analyses also revealed new subgroups of the pPT23A plasmid family and confirmed that plasmid phylogeny is incongruent with P. syringae pathovar or host of isolation. In addition, conserved genes among seven sequenced plasmids within the same phylogenetic group were limited to plasmid-specific functions including maintenance and transfer functions. CONCLUSIONS: Our sequence analysis further revealed that PFPs from P. syringae encode suites of accessory genes that are selected at species (universal distribution), pathovar (interpathovar distribution), and population levels (intrapathovar distribution). The conservation of type IV secretion systems encoding conjugation functions also presumably contributes to the distribution of these plasmids within P. syringae populations.

Mining Microbial Signals for Enhanced Biodiscovery of Secondary Metabolites.

The advent of metagenomics based biodiscovery has provided researchers with previously unforeseen access to the rich tapestry of natural bioactivity that exists in the biosphere. Unhindered by the "culturable bottleneck" that has severely limited the translation of the genetic potential that undoubtedly exists in nature, metagenomics nonetheless requires ongoing technological developments to maximize its efficacy and applicability to the discovery of new chemical entities.Here we describe methodologies for the detection and isolation of quorum sensing (QS) signal molecules from metagenomics libraries. QS signals have already shown considerable potential for the activation and "awakening" of biosynthetic gene clusters, bridging the existing divide between the natural product repertoire and the natural biosynthetic biodiversity hinted at by nature's blueprint. The QS pipeline from high-throughput robotics to functional screening and hit isolation is detailed, highlighting the multidisciplinary nature of progressive biodiscovery programs.

Integrated (Meta) Genomic and Synthetic Biology Approaches to Develop New Biocatalysts.

In recent years, the marine environment has been the subject of increasing attention from biotechnological and pharmaceutical industries as a valuable and promising source of novel bioactive compounds. Marine biodiscovery programmes have begun to reveal the extent of novel compounds encoded within the enormous bacterial richness and diversity of the marine ecosystem. A combination of unique physicochemical properties and spatial niche-specific substrates, in wide-ranging and extreme habitats, underscores the potential of the marine environment to deliver on functionally novel biocatalytic activities. With the growing need for green alternatives to industrial processes, and the unique transformations which nature is capable of performing, marine biocatalysts have the potential to markedly improve current industrial pipelines. Furthermore, biocatalysts are known to possess chiral selectivity and specificity, a key focus of pharmaceutical drug design. In this review, we discuss how the explosion in genomics based sequence analysis, allied with parallel developments in synthetic and molecular biology, have the potential to fast-track the discovery and subsequent improvement of a new generation of marine biocatalysts.

Emerging concepts promising new horizons for marine biodiscovery and synthetic biology.

The vast oceans of the world, which comprise a huge variety of unique ecosystems, are emerging as a rich and relatively untapped source of novel bioactive compounds with invaluable biotechnological and pharmaceutical potential. Evidence accumulated over the last decade has revealed that the diversity of marine microorganisms is enormous with many thousands of bacterial species detected that were previously unknown. Associated with this diversity is the production of diverse repertoires of bioactive compounds ranging from peptides and enzymes to more complex secondary metabolites that have significant bioactivity and thus the potential to be exploited for innovative biotechnology. Here we review the discovery and functional potential of marine bioactive peptides such as lantibiotics, nanoantibiotics and peptidomimetics, which have received particular attention in recent years in light of their broad spectrum of bioactivity. The significance of marine peptides in cell-to-cell communication and how this may be exploited in the discovery of novel bioactivity is also explored. Finally, with the recent advances in bioinformatics and synthetic biology, it is becoming clear that the integration of these disciplines with genetic and biochemical characterization of the novel marine peptides, offers the most potential in the development of the next generation of societal solutions.

Deciphering the role of coumarin as a novel quorum sensing inhibitor suppressing virulence phenotypes in bacterial pathogens.

The rapid unchecked rise in antibiotic resistance over the last few decades has led to an increased focus on the need for alternative therapeutic strategies for the treatment and clinical management of microbial infections. In particular, small molecules that can suppress microbial virulence systems independent of any impact on growth are receiving increased attention. Quorum sensing (QS) is a cell-to-cell signalling communication system that controls the virulence behaviour of a broad spectrum of bacterial pathogens. QS systems have been proposed as an effective target, particularly as they control biofilm formation in pathogens, a key driver of antibiotic ineffectiveness. In this study, we identified coumarin, a natural plant phenolic compound, as a novel QS inhibitor, with potent anti-virulence activity in a broad spectrum of pathogens. Using a range of biosensor systems, coumarin was active against short, medium and long chain N-acyl-homoserine lactones, independent of any effect on growth. To determine if this suppression was linked to anti-virulence activity, key virulence systems were studied in the nosocomial pathogen Pseudomonas aeruginosa. Consistent with suppression of QS, coumarin inhibited biofilm, the production of phenazines and swarming motility in this organism potentially linked to reduced expression of the rhlI and pqsA quorum sensing genes. Furthermore, coumarin significantly inhibited biofilm formation and protease activity in other bacterial pathogens and inhibited bioluminescence in Aliivibrio fischeri. In light of these findings, coumarin would appear to have potential as a novel quorum sensing inhibitor with a broad spectrum of action.

Mangotoxin production of Pseudomonas syringae pv. syringae is regulated by MgoA.

BACKGROUND: The antimetabolite mangotoxin is a key factor in virulence of Pseudomonas syringae pv. syringae strains which cause apical necrosis of mango trees. Previous studies showed that mangotoxin biosynthesis is governed by the mbo operon. Random mutagenesis led to the identification of two other gene clusters that affect mangotoxin biosynthesis. These are the gacS/gacA genes and mgo operon which harbors the four genes mgoBCAD. RESULTS: The current study shows that disruption of the nonribosomal peptide synthetase (NRPS) gene mgoA resulted in loss of mangotoxin production and reduced virulence on tomato leaves. Transcriptional analyses by qPCR and promoter reporter fusions revealed that mbo expression is regulated by both gacS/gacA and mgo genes. Also, expression of the mgo operon was shown to be regulated by gacS/gacA. Heterologous expression under the native promoter of the mbo operon resulted in mangotoxin production in non-producing P. syringae strains, but not in other Pseudomonas species. Also introduction of the mbo and mgo operons in nonproducing P. protegens Pf-5 did not confer mangotoxin production but did enhance transcription of the mbo promoter. CONCLUSIONS: From the data obtained in this study, we conclude that both mbo and mgo operons are under the control of the gacS/gacA two-component system and that the MgoA product acts as a positive regulator of mangotoxin biosynthesis.

A Pseudomonas syringae diversity survey reveals a differentiated phylotype of the pathovar syringae associated with the mango host and mangotoxin production.

Pseudomonas syringae pv. syringae, the causal agent of bacterial apical necrosis (BAN) in mango crops, has been isolated in different mango-producing areas worldwide. An extensive collection of 87 P. syringae pv. syringae strains isolated from mango trees affected by BAN from different countries, but mainly from Southern Spain, were initially examined by repetitive sequence-based polymerase chain reaction (rep-PCR) to analyze the genetic diversity with an epidemiological aim. rep-PCR was powerful in assessing intrapathovar distribution and also allowing clustering of the P. syringae pv. syringae strains isolated from mango, depending on the isolation area. A clear pattern of clustering was observed for all the P. syringae pv. syringae strains isolated from mango distinct from strains from other hosts, including strains for the same geographical regions as the mango isolates. For this reason, a representative group of 51 P. syringae pv. syringae strains isolated from mango and other hosts, as well as some P. syringae strains from other pathovars, were further characterized to determine their possible genetic, phenotypic, and phylogenetic relationships. Similar to the rep-PCR results, the randomly amplified polymorphic DNA PCR (RAPD-PCR) and catabolic diversity analysis using the Biolog GN2 profile grouped 90% of the mango isolates together in a unique cluster. Interestingly, the majority of P. syringae pv. syringae strains isolated from mango produced mangotoxin. The analysis of the phylogenetic distribution using the multilocus sequence typing analysis strongly supports the existence of a differentiated phylotype of the pathovar syringae mainly associated with the mango host and characterized by the mangotoxin production.

The mangotoxin biosynthetic operon (mbo) is specifically distributed within Pseudomonas syringae genomospecies 1 and was acquired only once during evolution.

Mangotoxin production was first described in Pseudomonas syringae pv. syringae strains. A phenotypic characterization of 94 P. syringae strains was carried out to determine the genetic evolution of the mangotoxin biosynthetic operon (mbo). We designed a PCR primer pair specific for the mbo operon to examine its distribution within the P. syringae complex. These primers amplified a 692-bp DNA fragment from 52 mangotoxin-producing strains and from 7 non-mangotoxin-producing strains that harbor the mbo operon, whereas 35 non-mangotoxin-producing strains did not yield any amplification. This, together with the analysis of draft genomes, allowed the identification of the mbo operon in five pathovars (pathovars aptata, avellanae, japonica, pisi, and syringae), all of which belong to genomospecies 1, suggesting a limited distribution of the mbo genes in the P. syringae complex. Phylogenetic analyses using partial sequences from housekeeping genes differentiated three groups within genomospecies 1. All of the strains containing the mbo operon clustered in groups I and II, whereas those lacking the operon clustered in group III; however, the relative branching order of these three groups is dependent on the genes used to construct the phylogeny. The mbo operon maintains synteny and is inserted in the same genomic location, with high sequence conservation around the insertion point, for all the strains in groups I and II. These data support the idea that the mbo operon was acquired horizontally and only once by the ancestor of groups I and II from genomospecies 1 within the P. syringae complex.

Recruitment and rearrangement of three different genetic determinants into a conjugative plasmid increase copper resistance in Pseudomonas syringae.

We describe the genetic organization of a copper-resistant plasmid containing copG and cusCBA genes in the plant pathogen Pseudomonas syringae. Chromosomal variants of czcCBA and a plasmid variant of cusCBA were present in different P. syringae pathovar strains. Transformation of the copper-sensitive Pseudomonas syringae pv. syringae FF5 strain with copG or cusCBA conferred copper resistance, and quantitative real-time PCR (qRT-PCR) experiments confirmed their induction by copper.