Role of extracellular matrix components in biofilm formation and adaptation of Pseudomonas ogarae F113 to the rhizosphere environment.

Regulating the transition of bacteria from motile to sessile lifestyles is crucial for their ability to compete effectively in the rhizosphere environment. Pseudomonas are known to rely on extracellular matrix (ECM) components for microcolony and biofilm formation, allowing them to adapt to a sessile lifestyle. Pseudomonas ogarae F113 possesses eight gene clusters responsible for the production of ECM components. These gene clusters are tightly regulated by AmrZ, a major transcriptional regulator that influences the cellular levels of c-di-GMP. The AmrZ-mediated transcriptional regulation of ECM components is primarily mediated by the signaling molecule c-di-GMP and the flagella master regulator FleQ. To investigate the functional role of these ECM components in P. ogarae F113, we performed phenotypic analyses using mutants in genes encoding these ECM components. These analyses included assessments of colony morphology, dye-staining, static attachment to abiotic surfaces, dynamic biofilm formation on abiotic surfaces, swimming motility, and competitive colonization assays of the rhizosphere. Our results revealed that alginate and PNAG polysaccharides, along with PsmE and the fimbrial low molecular weight protein/tight adherence (Flp/Tad) pilus, are the major ECM components contributing to biofilm formation. Additionally, we found that the majority of these components and MapA are needed for a competitive colonization of the rhizosphere in P. ogarae F113.

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.

An Orphan VrgG Auxiliary Module Related to the Type VI Secretion Systems from Pseudomonas ogarae F113 Mediates Bacterial Killing.

The model rhizobacterium Pseudomonas ogarae F113, a relevant plant growth-promoting bacterium, encodes three different Type VI secretion systems (T6SS) in its genome. In silico analysis of its genome revealed the presence of a genetic auxiliary module containing a gene encoding an orphan VgrG protein (VgrG5a) that is not genetically linked to any T6SS structural cluster, but is associated with genes encoding putative T6SS-related proteins: a possible adaptor Tap protein, followed by a putative effector, Tfe8, and its putative cognate immunity protein, Tfi8. The bioinformatic analysis of the VgrG5a auxiliary module has revealed that this cluster is only present in several subgroups of the P. fluorescens complex of species. An analysis of the mutants affecting the vgrG5a and tfe8 genes has shown that the module is involved in bacterial killing. To test whether Tfe8/Tfi8 constitute an effector-immunity pair, the genes encoding Tfe8 and Tfi8 were cloned and expressed in E. coli, showing that the ectopic expression of tfe8 affected growth. The growth defect was suppressed by tfi8 ectopic expression. These results indicate that Tfe8 is a bacterial killing effector, while Tfi8 is its cognate immunity protein. The Tfe8 protein sequence presents homology to the proteins of the MATE family involved in drug extrusion. The Tfe8 effector is a membrane protein with 10 to 12 transmembrane domains that could destabilize the membranes of target cells by the formation of pores, revealing the importance of these effectors for bacterial interaction. Tfe8 represents a novel type of a T6SS effector present in pseudomonads.

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.

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.

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.

Field scale biodegradation of total petroleum hydrocarbons and soil restoration by Ecopiles: microbiological analysis of the process.

Ecopiling is a method for biodegradation of hydrocarbons in soils. It derives from Biopiles, but phytoremediation is added to biostimulation with nitrogen fertilization and bioaugmentation with local bacteria. We have constructed seven Ecopiles with soil heavily polluted with hydrocarbons in Carlow (Ireland). The aim of the study was to analyze changes in the microbial community during ecopiling. In the course of 18 months of remediation, total petroleum hydrocarbons values decreased in 99 and 88% on average for aliphatics and aromatics, respectively, indicating a successful biodegradation. Community analysis showed that bacterial alfa diversity (Shannon Index), increased with the degradation of hydrocarbons, starting at an average value of 7.59 and ending at an average value of 9.38. Beta-diversity analysis, was performed using Bray-Curtis distances and PCoA ordination, where the two first principal components (PCs) explain the 17 and 14% of the observed variance, respectively. The results show that samples tend to cluster by sampling time instead of by Ecopile. This pattern is supported by the hierarchical clustering analysis, where most samples from the same timepoint clustered together. We used DSeq2 to determine the differential abundance of bacterial populations in Ecopiles at the beginning and the end of the treatment. While TPHs degraders are more abundant at the start of the experiment, these populations are substituted by bacterial populations typical of clean soils by the end of the biodegradation process. Similar results are found for the fungal community, indicating that the microbial community follows a succession along the process. This succession starts with a TPH degraders or tolerant enriched community, and finish with a microbial community typical of clean soils.

Adaption of Pseudomonas ogarae F113 to the Rhizosphere Environment-The AmrZ-FleQ Hub.

Motility and biofilm formation are two crucial traits in the process of rhizosphere colonization by pseudomonads. The regulation of both traits requires a complex signaling network that is coordinated by the AmrZ-FleQ hub. In this review, we describe the role of this hub in the adaption to the rhizosphere. The study of the direct regulon of AmrZ and the phenotypic analyses of an amrZ mutant in Pseudomonas ogarae F113 has shown that this protein plays a crucial role in the regulation of several cellular functions, including motility, biofilm formation, iron homeostasis, and bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) turnover, controlling the synthesis of extracellular matrix components. On the other hand, FleQ is the master regulator of flagellar synthesis in P. ogarae F113 and other pseudomonads, but its implication in the regulation of multiple traits related with environmental adaption has been shown. Genomic scale studies (ChIP-Seq and RNA-Seq) have shown that in P. ogarae F113, AmrZ and FleQ are general transcription factors that regulate multiple traits. It has also been shown that there is a common regulon shared by the two transcription factors. Moreover, these studies have shown that AmrZ and FleQ form a regulatory hub that inversely regulate traits such as motility, extracellular matrix component production, and iron homeostasis. The messenger molecule c-di-GMP plays an essential role in this hub since its production is regulated by AmrZ and it is sensed by FleQ and required for its regulatory role. This regulatory hub is functional both in culture and in the rhizosphere, indicating that the AmrZ-FleQ hub is a main player of P. ogarae F113 adaption to the rhizosphere environment.

Regulation of extracellular matrix components by AmrZ is mediated by c-di-GMP in Pseudomonas ogarae F113.

The AmrZ/FleQ hub has been identified as a central node in the regulation of environmental adaption in the plant growth-promoting rhizobacterium and model for rhizosphere colonization Pseudomonas ogarae F113. AmrZ is involved in the regulation of motility, biofilm formation, and bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) turnover, among others, in this bacterium. The mutants in amrZ have a pleiotropic phenotype with distinguishable colony morphology, reduced biofilm formation, increased motility, and are severely impaired in competitive rhizosphere colonization. Here, RNA-Seq and qRT-PCR gene expression analyses revealed that AmrZ regulates many genes related to the production of extracellular matrix (ECM) components at the transcriptional level. Furthermore, overproduction of c-di-GMP in an amrZ mutant, by ectopic production of the Caulobacter crescentus constitutive diguanylate cyclase PleD*, resulted in increased expression of many genes implicated in the synthesis of ECM components. The overproduction of c-di-GMP in the amrZ mutant also suppressed the biofilm formation and motility phenotypes, but not the defect in competitive rhizosphere colonization. These results indicate that although biofilm formation and motility are mainly regulated indirectly by AmrZ, through the modulation of c-di-GMP levels, the implication of AmrZ in rhizosphere competitive colonization occurs in a c-di-GMP-independent manner.

Transcriptomic analysis of Pseudomonas ogarae F113 reveals the antagonistic roles of AmrZ and FleQ during rhizosphere adaption.

Rhizosphere colonization by bacteria involves molecular and cellular mechanisms, such as motility and chemotaxis, biofilm formation, metabolic versatility, or biosynthesis of secondary metabolites, among others. Nonetheless, there is limited knowledge concerning the main regulatory factors that drive the rhizosphere colonization process. Here we show the importance of the AmrZ and FleQ transcription factors for adaption in the plant growth-promoting rhizobacterium (PGPR) and rhizosphere colonization model Pseudomonas ogarae F113. RNA-Seq analyses of P. ogarae F113 grown in liquid cultures either in exponential and stationary growth phase, and rhizosphere conditions, revealed that rhizosphere is a key driver of global changes in gene expression in this bacterium. Regarding the genetic background, this work has revealed that a mutation in fleQ causes considerably more alterations in the gene expression profile of this bacterium than a mutation in amrZ under rhizosphere conditions. The functional analysis has revealed that in P. ogarae F113, the transcription factors AmrZ and FleQ regulate genes involved in diverse bacterial functions. Notably, in the rhizosphere, these transcription factors antagonistically regulate genes related to motility, biofilm formation, nitrogen, sulfur, and amino acid metabolism, transport, signalling, and secretion, especially the type VI secretion systems. These results define the regulon of two important bifunctional transcriptional regulators in pseudomonads during the process of rhizosphere colonization.

Comparative Analysis of Three Bradyrhizobium diazoefficiens Genomes Show Specific Mutations Acquired during Selection for a Higher Motility Phenotype and Adaption to Laboratory Conditions.

Microbial genomes are being extensively studied using next-generation sequencing technologies in order to understand the changes that occur under different selection regimes. In this work, the number and type of mutations that have occurred in three Bradyrhizobium diazoefficiens USDA 110T strains under laboratory conditions and during selection for a more motile phenotypic variant were analyzed. Most of the mutations found in both processes consisted of single nucleotide polymorphisms, single nucleotide deletions or insertions. In the case of adaptation to laboratory conditions, half of the changes occurred within intergenic regions, and around 80% were insertions. When the more motile phenotypic variant was evaluated, eight single nucleotide polymorphisms and an 11-bp deletion were found, although none of them was directly related to known motility or chemotaxis genes. Two mutants were constructed to evaluate the 11-bp deletion affecting the alpha subunit of 2-oxoacid:acceptor oxidoreductase (AAV28_RS30705-blr6743). The results showed that this single deletion was not responsible for the enhanced motility phenotype. IMPORTANCE The genetic and genomic changes that occur under laboratory conditions in Bradyrhizobium diazoefficiens genomes remain poorly studied. Only a few genome sequences of this important nitrogen-fixing species are available, and there are no genome-wide comparative analyses of related strains. In the present work, we sequenced and compared the genomes of strains derived from a parent strain, B. diazoefficiens USDA 110, that has undergone processes of repeated culture in the laboratory environment, or phenotypic selection toward antibiotic resistance and enhanced motility. Our results represent the first analysis in B. diazoefficiens that provides insights into the specific mutations that are acquired during these processes.

Comparative genomics of the Pseudomonas corrugata subgroup reveals high species diversity and allows the description of Pseudomonas ogarae sp. nov.

Pseudomonas corrugata constitute one of the phylogenomic subgroups within the Pseudomonas fluorescens species complex and include both plant growth-promoting rhizobacteria (PGPR) and plant pathogenic bacteria. Previous studies suggest that the species diversity of this group remains largely unexplored together with frequent misclassification of strains. Using more than 1800 sequenced Pseudomonas genomes we identified 121 genomes belonging to the P. corrugata subgroup. Intergenomic distances obtained using the genome-to-genome blast distance (GBDP) algorithm and the determination of digital DNA-DNA hybridization values were further used for phylogenomic and clustering analyses, which revealed 29 putative species clusters, of which only five correspond to currently named species within the subgroup. Comparative and functional genome-scale analyses also support the species status of these clusters. The search for PGPR and plant pathogenic determinants showed that approximately half of the genomes analysed could have a pathogenic behaviour based on the presence of a pathogenicity genetic island, while all analysed genomes possess PGPR traits. Finally, this information together with the characterization of phenotypic traits, allows the reclassification proposal of Pseudomonas fluorescens F113 as Pseudomonas ogarae sp. nov., nom rev., type strain F113T (=DSM 112162T=CECT 30235T), which is substantiated by genomic, functional genomics and phenotypic differences with their closest type strains.

Pseudomonas fluorescens F113 type VI secretion systems mediate bacterial killing and adaption to the rhizosphere microbiome.

The genome of Pseudomonas fluorescens F113, a model rhizobacterium and a plant growth-promoting agent, encodes three putative type VI secretion systems (T6SSs); F1-, F2- and F3-T6SS. Bioinformatic analysis of the F113 T6SSs has revealed that they belong to group 3, group 1.1, and group 4a, respectively, similar to those previously described in Pseudomonas aeruginosa. In addition, in silico analyses allowed us to identify genes encoding a total of five orphan VgrG proteins and eight putative effectors (Tfe), some with their cognate immunity protein (Tfi) pairs. Genes encoding Tfe and Tfi are found in the proximity of P. fluorescens F113 vgrG, hcp, eagR and tap genes. RNA-Seq analyses in liquid culture and rhizosphere have revealed that F1- and F3-T6SS are expressed under all conditions, indicating that they are active systems, while F2-T6SS did not show any relevant expression under the tested conditions. The analysis of structural mutants in the three T6SSs has shown that the active F1- and F3-T6SSs are involved in interbacterial killing while F2 is not active in these conditions and its role is still unknown.. A rhizosphere colonization analysis of the double mutant affected in the F1- and F3-T6SS clusters showed that the double mutant was severely impaired in persistence in the rhizosphere microbiome, revealing the importance of these two systems for rhizosphere adaption.

In Silico Characterization and Phylogenetic Distribution of Extracellular Matrix Components in the Model Rhizobacteria Pseudomonas fluorescens F113 and Other Pseudomonads.

Biofilms are complex structures that are crucial during host-bacteria interaction and colonization. Bacteria within biofilms are surrounded by an extracellular matrix (ECM) typically composed of proteins, polysaccharides, lipids, and DNA. Pseudomonads contain a variety of ECM components, some of which have been extensively characterized. However, neither the ECM composition of plant-associated pseudomonads nor their phylogenetic distribution within the genus has been so thoroughly studied. In this work, we use in silico methods to describe the ECM composition of Pseudomonas fluorescens F113, a plant growth-promoting rhizobacteria and model for rhizosphere colonization. These components include the polysaccharides alginate, poly-N-acetyl-glucosamine (PNAG) and levan; the adhesins LapA, MapA and PsmE; and the functional amyloids in Pseudomonas. Interestingly, we identified novel components: the Pseudomonas acidic polysaccharide (Pap), whose presence is limited within the genus; and a novel type of Flp/Tad pilus, partially different from the one described in P. aeruginosa. Furthermore, we explored the phylogenetic distribution of the most relevant ECM components in nearly 600 complete Pseudomonas genomes. Our analyses show that Pseudomonas populations contain a diverse set of gene/gene clusters potentially involved in the formation of their ECMs, showing certain commensal versus pathogen lifestyle specialization.

Comparative Genomics of the Rhodococcus Genus Shows Wide Distribution of Biodegradation Traits.

The genus Rhodococcus exhibits great potential for bioremediation applications due to its huge metabolic diversity, including biotransformation of aromatic and aliphatic compounds. Comparative genomic studies of this genus are limited to a small number of genomes, while the high number of sequenced strains to date could provide more information about the Rhodococcus diversity. Phylogenomic analysis of 327 Rhodococcus genomes and clustering of intergenomic distances identified 42 phylogenomic groups and 83 species-level clusters. Rarefaction models show that these numbers are likely to increase as new Rhodococcus strains are sequenced. The Rhodococcus genus possesses a small "hard" core genome consisting of 381 orthologous groups (OGs), while a "soft" core genome of 1253 OGs is reached with 99.16% of the genomes. Models of sequentially randomly added genomes show that a small number of genomes are enough to explain most of the shared diversity of the Rhodococcus strains, while the "open" pangenome and strain-specific genome evidence that the diversity of the genus will increase, as new genomes still add more OGs to the whole genomic set. Most rhodococci possess genes involved in the degradation of aliphatic and aromatic compounds, while short-chain alkane degradation is restricted to a certain number of groups, among which a specific particulate methane monooxygenase (pMMO) is only found in Rhodococcus sp. WAY2. The analysis of Rieske 2Fe-2S dioxygenases among rhodococci genomes revealed that most of these enzymes remain uncharacterized.

Analysis of the biodegradative and adaptive potential of the novel polychlorinated biphenyl degrader Rhodococcus sp. WAY2 revealed by its complete genome sequence.

The complete genome sequence of Rhodococcus sp. WAY2 (WAY2) consists of a circular chromosome, three linear replicons and a small circular plasmid. The linear replicons contain typical actinobacterial invertron-type telomeres with the central CGTXCGC motif. Comparative phylogenetic analysis of the 16S rRNA gene along with phylogenomic analysis based on the genome-to-genome blast distance phylogeny (GBDP) algorithm and digital DNA-DNA hybridization (dDDH) with other Rhodococcus type strains resulted in a clear differentiation of WAY2, which is likely a new species. The genome of WAY2 contains five distinct clusters of bph, etb and nah genes, putatively involved in the degradation of several aromatic compounds. These clusters are distributed throughout the linear plasmids. The high sequence homology of the ring-hydroxylating subunits of these systems with other known enzymes has allowed us to model the range of aromatic substrates they could degrade. Further functional characterization revealed that WAY2 was able to grow with biphenyl, naphthalene and xylene as sole carbon and energy sources, and could oxidize multiple aromatic compounds, including ethylbenzene, phenanthrene, dibenzofuran and toluene. In addition, WAY2 was able to co-metabolize 23 polychlorinated biphenyl congeners, consistent with the five different ring-hydroxylating systems encoded by its genome. WAY2 could also use n-alkanes of various chain-lengths as a sole carbon source, probably due to the presence of alkB and ladA gene copies, which are only found in its chromosome. These results show that WAY2 has a potential to be used for the biodegradation of multiple organic compounds.

Metagenomic Insights into the Bacterial Functions of a Diesel-Degrading Consortium for the Rhizoremediation of Diesel-Polluted Soil.

Diesel is a complex pollutant composed of a mixture of aliphatic and aromatic hydrocarbons. Because of this complexity, diesel bioremediation requires multiple microorganisms, which harbor the catabolic pathways to degrade the mixture. By enrichment cultivation of rhizospheric soil from a diesel-polluted site, we have isolated a bacterial consortium that can grow aerobically with diesel and different alkanes and polycyclic aromatic hydrocarbons (PAHs) as the sole carbon and energy source. Microbiome diversity analyses based on 16S rRNA gene showed that the diesel-degrading consortium consists of 76 amplicon sequence variants (ASVs) and it is dominated by Pseudomonas, Aquabacterium, Chryseobacterium, and Sphingomonadaceae. Changes in microbiome composition were observed when growing on specific hydrocarbons, reflecting that different populations degrade different hydrocarbons. Shotgun metagenome sequence analysis of the consortium growing on diesel has identified redundant genes encoding enzymes implicated in the initial oxidation of alkanes (AlkB, LadA, CYP450) and a variety of hydroxylating and ring-cleavage dioxygenases involved in aromatic and polyaromatic hydrocarbon degradation. The phylogenetic assignment of these enzymes to specific genera allowed us to model the role of specific populations in the diesel-degrading consortium. Rhizoremediation of diesel-polluted soil microcosms using the consortium, resulted in an important enhancement in the reduction of total petroleum hydrocarbons (TPHs), making it suited for rhizoremediation applications.

Phylogenomic Analyses of Bradyrhizobium Reveal Uneven Distribution of the Lateral and Subpolar Flagellar Systems, Which Extends to Rhizobiales.

Dual flagellar systems have been described in several bacterial genera, but the extent of their prevalence has not been fully explored. Bradyrhizobium diazoefficiens USDA 110T possesses two flagellar systems, the subpolar and the lateral flagella. The lateral flagellum of Bradyrhizobium displays no obvious role, since its performance is explained by cooperation with the subpolar flagellum. In contrast, the lateral flagellum is the only type of flagella present in the related Rhizobiaceae family. In this work, we have analyzed the phylogeny of the Bradyrhizobium genus by means of Genome-to-Genome Blast Distance Phylogeny (GBDP) and Average Nucleotide Identity (ANI) comparisons of 128 genomes and divided it into 13 phylogenomic groups. While all the Bradyrhizobium genomes encode the subpolar flagellum, none of them encodes only the lateral flagellum. The simultaneous presence of both flagella is exclusive of the B. japonicum phylogenomic group. Additionally, 292 Rhizobiales order genomes were analyzed and both flagellar systems are present together in only nine genera. Phylogenetic analysis of 150 representative Rhizobiales genomes revealed an uneven distribution of these flagellar systems. While genomes within and close to the Rhizobiaceae family only possess the lateral flagellum, the subpolar flagellum is exclusive of more early-diverging families, where certain genera also present both flagella.

Genome-wide analysis of the FleQ direct regulon in Pseudomonas fluorescens F113 and Pseudomonas putida KT2440.

Bacterial motility plays a crucial role in competitiveness and colonization in the rhizosphere. In this work, Chromatin ImmunoPrecipitation Sequencing (ChIP-seq) analysis has been used to identify genes putatively regulated by the transcriptional regulatory protein FleQ in Pseudomonas fluorescens F113 and Pseudomonas putida KT2440. This protein was previously identified as a master regulator of flagella and biofilm formation in both strains. This work has demonstrated that FleQ from both bacteria are conserved and functionally equivalent for motility regulation. Furthermore, the ChIP-seq analysis has shown that FleQ is a global regulator with the identification of 121 and 103 FleQ putative binding sites in P. fluorescens F113 and P. putida KT2440 respectively. Putative genes regulated by FleQ included, as expected, flagellar and motility-related genes and others involved in adhesion and exopolysaccharide production. Surprisingly, the ChIP-seq analysis also identified iron homeostasis-related genes for which positive regulation was shown by RT-qPCR. The results also showed that FleQ from P. fluorescens F113 shares an important part of its direct regulon with AmrZ, a global regulator also implicated in environmental adaption. Although AmrZ also regulates motility and iron uptake, the overlap occurred mostly with the iron-related genes, since both regulators control a different set of motility-related genes.

Metagenomic Analysis of a Biphenyl-Degrading Soil Bacterial Consortium Reveals the Metabolic Roles of Specific Populations.

Polychlorinated biphenyls (PCBs) are widespread persistent pollutants that cause several adverse health effects. Aerobic bioremediation of PCBs involves the activity of either one bacterial species or a microbial consortium. Using multiple species will enhance the range of PCB congeners co-metabolized since different PCB-degrading microorganisms exhibit different substrate specificity. We have isolated a bacterial consortium by successive enrichment culture using biphenyl (analog of PCBs) as the sole carbon and energy source. This consortium is able to grow on biphenyl, benzoate, and protocatechuate. Whole-community DNA extracted from the consortium was used to analyze biodiversity by Illumina sequencing of a 16S rRNA gene amplicon library and to determine the metagenome by whole-genome shotgun Illumina sequencing. Biodiversity analysis shows that the consortium consists of 24 operational taxonomic units (≥97% identity). The consortium is dominated by strains belonging to the genus Pseudomonas, but also contains betaproteobacteria and Rhodococcus strains. whole-genome shotgun (WGS) analysis resulted in contigs containing 78.3 Mbp of sequenced DNA, representing around 65% of the expected DNA in the consortium. Bioinformatic analysis of this metagenome has identified the genes encoding the enzymes implicated in three pathways for the conversion of biphenyl to benzoate and five pathways from benzoate to tricarboxylic acid (TCA) cycle intermediates, allowing us to model the whole biodegradation network. By genus assignment of coding sequences, we have also been able to determine that the three biphenyl to benzoate pathways are carried out by Rhodococcus strains. In turn, strains belonging to Pseudomonas and Bordetella are the main responsible of three of the benzoate to TCA pathways while the benzoate conversion into TCA cycle intermediates via benzoyl-CoA and the catechol meta-cleavage pathways are carried out by beta proteobacteria belonging to genera such as Achromobacter and Variovorax. We have isolated a Rhodococcus strain WAY2 from the consortium which contains the genes encoding the three biphenyl to benzoate pathways indicating that this strain is responsible for all the biphenyl to benzoate transformations. The presented results show that metagenomic analysis of consortia allows the identification of bacteria active in biodegradation processes and the assignment of specific reactions and pathways to specific bacterial groups.