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Abstract Background Plant endophytes, comprising non-pathogenic bacteria, fungi, and archaea, inhabit various plant parts, including roots, stems, leaves, and seeds. These microorganisms play a crucial role in plant development by enhancing germination, growth, and stress resilience. Seed endophytes, in particular, represent the most adapted and conserved segment of plant microbiota, significantly influencing the initial stages of plant growth and microbial community establishment. This study investigates the impact of environmental and genotypic factors on the endophytic communities of Chenopodium quinoa Willd. (quinoa), a crop notable for its adaptability and nutritional value. Results We aimed to characterize the core endophytic communities in quinoa seeds and roots from two distinct genotypes under well-watered (WW) and water-deficit (WD) conditions, utilizing various soil infusions as inoculants to explore potential changes in these endophytes. Our findings reveal distinct changes with quinoa seeds exhibiting a high degree of conservation in their endophytic microbiome, even between maternal and offspring seeds, with specific bacterial taxa showing only minor differences. Tissue specificity emerged as a key factor, with seeds maintaining a stable microbial community, while roots exhibited more pronounced shifts, highlighting the tissue-dependent patterns of microbial enrichment. Conclusions The results highlight the stability and conservation of endophytic communities in quinoa seeds, even under varying water conditions and across different genotypes, emphasizing the role of tissue specificity in shaping microbial associations. These findings suggest that quinoa-associated endophytes, particularly those conserved in seeds, may play a crucial role in enhancing drought resilience. Understanding the dynamics of plant-microbe interactions in quinoa is vital for developing stress-resilient crop varieties, supporting sustainable agricultural practices, and ensuring food security in the face of climate change and environmental challenges.

Abstract Microorganisms are present in snow/ice of the Antarctic Plateau, but their biogeography and metabolic state under extreme local conditions are poorly understood. Here, we show the diversity and distribution of microorganisms in air (1.5 m height) and snow/ice down to 4 m depth at three distant latitudes along a 2578 km transect on the East Antarctic Plateau on board an environmentally friendly, mobile platform. Results demonstrate the widespread distribution of microorganisms in the ice down to at least 4 m depth. Data point to geochemical and bacterial geographic distribution that correlate with wind trajectory and speed, modulated by local gathering and recirculation of microorganisms through snow drifting. Reservoir effects and community selection appear to occur over time, favoring microorganisms best adapted to hypothermal and hyperarid conditions. A new cyanobacterial species (Gloeocapsopsis sp) was isolated from 3 to 4 m depth. Our findings suggest that some microorganisms could exhibit transient, basal metabolic activity when associated to high salt particles, contributing to set biodiversity patterns and biogeographic compartmentalization on Antarctic Plateau ice.

Abstract A bacterial consortium was isolated from a soil in Noblejas (Toledo, Spain) with a long history of mixed hydrocarbons pollution, by enrichment cultivation. Serial cultures of hydrocarbons polluted soil samples were grown in a minimal medium using diesel (1 mL/L) as the sole carbon and energy source. The bacterial composition of the Noblejas Consortium (NC) was determined by sequencing 16S rRNA gene amplicon libraries. The consortium contained around 50 amplicon sequence variants (ASVs) and the major populations belonged to the genera Pseudomonas, Enterobacter, Delftia, Stenotrophomonas, Achromobacter, Acinetobacter, Novosphingobium, Allorhizobium-Neorhizobium-Rhizobium, Ochrobactrum and Luteibacter. All other genera were below 1%. Metagenomic analysis of NC has shown a high abundance of genes encoding enzymes implicated in aliphatic and (poly) aromatic hydrocarbons degradation, and almost all pathways for hydrocarbon degradation are represented. Metagenomic analysis has also allowed the construction of metagenome assembled genomes (MAGs) for the major players of NC. Metatranscriptomic analysis has shown that several of the ASVs are implicated in hydrocarbon degradation, being Pseudomonas, Acinetobacter and Delftia the most active populations.

Abstract Drought is among the main abiotic factors causing agronomical losses worldwide. To minimize its impact, several strategies have been proposed, including the use of plant growth‐promoting bacteria (PGPBs), as they have demonstrated roles in counteracting abiotic stress. This aspect has been little explored in emergent crops such as quinoa, which has the potential to contribute to reducing food insecurity. Thus, here we hypothesize that the genotype, water environment and the type of inoculant are determining factors in shaping quinoa rhizosphere bacterial communities, affecting plant performance. To address this, two different quinoa cultivars (with contrasting water stress tolerance), two water conditions (optimal and limiting water conditions) and different soil infusions were used to define the relevance of these factors. Different bacterial families that vary among genotypes and water conditions were identified. Certain families were enriched under water stress conditions, such as the Nocardioidaceae, highly present in the water‐sensitive cultivar F15, or the Pseudomonadaceae, Burkholderiaceae and Sphingomonadaceae, more abundant in the tolerant cultivar F16, which also showed larger total polyphenol content. These changes demonstrate that the genotype and environment highly contribute to shaping the root‐inhabiting bacteria in quinoa, and they suggest that this plant species is a great source of PGPBs for utilization under water‐liming conditions.

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.

Abstract Quinoa is an Andean crop whose cultivation has been extended to many different parts of the world in the last decade. It shows a great capacity for adaptation to diverse climate conditions, including environmental stressors, and, moreover, the seeds are very nutritious in part due to their high protein content, which is rich in essential amino acids. They are gluten-free seeds and contain good amounts of other nutrients such as unsaturated fatty acids, vitamins, or minerals. Also, the use of quinoa hydrolysates and peptides has been linked to numerous health benefits. Altogether, these aspects have situated quinoa as a crop able to contribute to food security worldwide. Aiming to deepen our understanding of the protein quality and function of quinoa seeds and how they can vary when this crop is subjected to water-limiting conditions, a shotgun proteomics analysis was performed to obtain the proteomes of quinoa seeds harvested from two different water regimes in the field: rainfed and irrigated conditions. Differentially increased levels of proteins determined in seeds from each field condition were analysed, and the enrichment of chitinase-related proteins in seeds harvested from rainfed conditions was found. These proteins are described as pathogen-related proteins and can be accumulated under abiotic stress. Thus, our findings suggest that chitinase-like proteins in quinoa seeds can be potential biomarkers of drought. Also, this study points to the need for further research to unveil their role in conferring tolerance when coping with water-deficient conditions.

Abstract 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.

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.

Abstract 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.

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.

ABSTRACT 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.

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.

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.

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.

The genomic sequence of Pseudomonas fluorescens F113 has shown the presence of a 41 kb cluster of genes that encode the production of a second flagellar apparatus. Among 2535 pseudomonads strains with sequenced genomes, these genes are only present in the genomes of F113 and other six strains, all but one belonging to the P. fluorescens cluster of species, in the form of a genetic island. The genes are homologous to the flagellar genes of the soil bacterium Azotobacter vinelandii. Regulation of these genes is mediated by the flhDC master operon, instead of the typical regulation in pseudomonads, which is through fleQ. Under laboratory conditions, F113 does not produce this flagellum and the flhDC operon is not expressed. However, ectopic expression of the flhDC operon is enough for its production, resulting in a hypermotile strain. This flagellum is also produced under laboratory conditions by the kinB and algU mutants. Genetic analysis has shown that kinB strongly represses the expression of the flhDC operon. This operon is activated by the Vfr protein probably in a c-AMP dependent way. The strains producing this second flagellum are all hypermotile and present a tuft of polar flagella instead of the single polar flagellum produced by the wild-type strain. Phenotypic variants isolated from the rhizosphere produce this flagellum and mutation of the genes encoding it, results in a defect in competitive colonization, showing its importance for root colonization.

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.

The Pseudomonas fluorescens complex includes Pseudomonas strains that have been taxonomically assigned to more than fifty different species, many of which have been described as plant growth-promoting rhizobacteria (PGPR) with potential applications in biocontrol and biofertilization. So far the phylogeny of this complex has been analyzed according to phenotypic traits, 16S rDNA, MLSA and inferred by whole-genome analysis. However, since most of the type strains have not been fully sequenced and new species are frequently described, correlation between taxonomy and phylogenomic analysis is missing. In recent years, the genomes of a large number of strains have been sequenced, showing important genomic heterogeneity and providing information suitable for genomic studies that are important to understand the genomic and genetic diversity shown by strains of this complex. Based on MLSA and several whole-genome sequence-based analyses of 93 sequenced strains, we have divided the P. fluorescens complex into eight phylogenomic groups that agree with previous works based on type strains. Digital DDH (dDDH) identified 69 species and 75 subspecies within the 93 genomes. The eight groups corresponded to clustering with a threshold of 31.8% dDDH, in full agreement with our MLSA. The Average Nucleotide Identity (ANI) approach showed inconsistencies regarding the assignment to species and to the eight groups. The small core genome of 1,334 CDSs and the large pan-genome of 30,848 CDSs, show the large diversity and genetic heterogeneity of the P. fluorescens complex. However, a low number of strains were enough to explain most of the CDSs diversity at core and strain-specific genomic fractions. Finally, the identification and analysis of group-specific genome and the screening for distinctive characters revealed a phylogenomic distribution of traits among the groups that provided insights into biocontrol and bioremediation applications as well as their role as PGPR.

Deciphering the mechanisms leading to symbiotic nitrogen-fixing root nodule organogenesis in legumes resulted in the identification of numerous nodule-specific genes and gene families. Among them, NCR and GRP genes encode short secreted peptides with potential antimicrobial activity. These genes appear to form large multigenic families in Medicago truncatula and other closely related legume species, whereas no similar genes were found in databases of Lotus japonicus and Glycine max. We analyzed the genomic organization of these genes as well as their evolutionary dynamics in the M. truncatula genome. A total of 108 NCR and 23 GRP genes have been mapped that were often clustered in the genome. These included 29 new NCR and 17 new GRP genes. Reverse transcription-polymerase chain reaction analyses of the novel genes confirmed their exclusive nodule-specific expression similar to the previously identified members. Protein alignments and phylogenetic analyses revealed traces of several duplication events in the history of GRP and NCR genes. Moreover, microsyntenic evidences between M. truncatula and L. japonicus validated the hypothesis that these genes are specific for the inverted repeat–lacking clade of hologalegoid legumes, which allowed dating the appearance of these two gene families during the evolution of legume plants.

Samples of Rhizobium bacteroids isolated from pea nodule symbiosomes reacted positively with a monoclonal antibody recognizing N-linked glycan epitopes on plant glycoproteins associated with the peribacteroid membrane and peribacteroid fluid. An antiserum recognizing the symbiosomal lectin-like glycoprotein PsNLEC-1 also reacted positively. Samples of isolated bacteroids also reacted with an antibody recognizing a glycolipid component of the peribacteroid membrane and plasma membrane. Bacterial cells derived from free-living cultures then were immobilized on nitrocellulose sheets and tested for their ability to associate with components of plant extracts derived from nodule fractionation. A positive antibody-staining reaction indicated that both PsNLEC-1 and membrane glycolipid had become associated with the bacterial surface. A range of rhizobial strains with mutants affecting cell surface polysaccharides all showed similar interactions with PsNLEC-1 and associated plant membranes, with the exception of strain B659 (a deep-rough lipopolysaccharide mutant of Rhizobium leguminosarum). However, the presence of a capsule of extracellular polysaccharide apparently prevented interactions between rhizobial cells and these plant components. The importance of a close association between peribacteroid membranes, PsNLEC-1, and the bacterial surface is discussed in the context of symbiosome development.

The sequence of the genome of Pseudomonas fluorescens F113 has shown the presence of multiple traits relevant for rhizosphere colonization and plant growth promotion. Among these traits are denitrification and chemotactic motility. Besides aerobic growth, F113 is able to grow anaerobically using nitrate and nitrite as final electron acceptors. F113 is able to perform swimming motility under aerobic conditions and under anaerobic conditions when nitrate is used as the electron acceptor. However, nitrite can not support swimming motility. Regulation of swimming motility is similar under aerobic and anaerobic conditions, since mutants that are hypermotile under aerobic conditions, such as gacS, sadB, kinB, algU and wspR, are also hypermotile under anaerobic conditions. However, chemotactic behavior is different under aerobic and denitrification conditions. Unlike most pseudomonads, the F113 genome encode three complete chemotaxis systems, Che1, Che2 and Che3. Mutations in each of the cheA genes of the three Che systems has shown that the three systems are functional and independent. Mutation of the cheA1 gene completely abolished swimming motility both under aerobic and denitrification conditions. Mutation of the cheA2 gene, showed only a decrease in swimming motility under both conditions, indicating that this system is not essential for chemotactic motility but is necessary for optimal motility. Mutation of the cheA3 gene abolished motility under denitrification conditions but only produced a decrease in motility under aerobic conditions. The three Che systems proved to be implicated in competitive rhizosphere colonization, being the cheA1 mutant the most affected.

Symbiosome development was studied in pea root nodules from plants growing in the absence of boron (B). Rhizobia released into the host cells of nodules from B-deficient plants developed to abnormal endophytic forms with an altered electrophoretic lipopolysaccharide pattern. Immunostaining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting of nodule homogenates with antibodies that recognize glycoprotein components showed that two previously described lectin-like glycoproteins (PsNLEC-1A and PsNLEC-1B) did not harbor the carbohydrate epitope normally recognized by specific monoclonal antibodies. Material derived from B-deficient nodules, however, still contained three antigenic isoforms with similar electrophoretic mobilities to PsNLEC-1 isoforms A, B, and C. These could be detected following immunoblotting and immunostaining with a specific antiserum originating from the purified PsNLEC protein that had been heterologously expressed in Escherichia coli. Immunogold localization of PsNLEC-1 sugar epitopes in B-deficient nodules showed that they were associated mostly with cytoplasmic vesicles rather than normal localization in the symbiosome compartment of mature infected cells. These results suggest that a modification of the glycosyl-moieties of PsNLEC-1 and an alteration of vesicle targeting occur during the development of pea nodules in the absence of B, and that these changes are associated with the development of aberrant nonfunctional symbiosomes.

Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC) and one phosphodiesterase (BifA) implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ.

Flagella mediated motility in Pseudomonas fluorescens F113 is tightly regulated. We have previously shown that motility is repressed by the GacA/GacS system and by SadB through downregulation of the fleQ gene, encoding the master regulator of the synthesis of flagellar components, including the flagellin FliC. Here we show that both regulatory pathways converge in the regulation of transcription and possibly translation of the algU gene, which encodes a sigma factor. AlgU is required for multiple functions, including the expression of the amrZ gene which encodes a transcriptional repressor of fleQ. Gac regulation of algU occurs during exponential growth and is exerted through the RNA binding proteins RsmA and RsmE but not RsmI. RNA immunoprecipitation assays have shown that the RsmA protein binds to a polycistronic mRNA encoding algU, mucA, mucB and mucD, resulting in lower levels of algU. We propose a model for repression of the synthesis of the flagellar apparatus linking extracellular and intracellular signalling with the levels of AlgU and a new physiological role for the Gac system in the downregulation of flagella biosynthesis during exponential growth.

The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix(-) nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this "nodule-specific transcriptome" were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches.