Diversifying Anaerobic Respiration Strategies to Compete in the Rhizosphere

The rhizosphere is the interface between plant roots and soil where intense, varied interactions between plants and microbes influence plants' health and growth through their influence on biochemical cycles, such as the carbon, nitrogen, and iron cycles. The rhizosphere is also a changing environment where oxygen can be rapidly limited and anaerobic zones can be established. Microorganisms successfully colonize the rhizosphere when they possess specific traits referred to as rhizosphere competence. Anaerobic respiration flexibility contributes to the rhizosphere competence of microbes. Indeed, a wide range of compounds that are available in the rhizosphere can serve as alternative terminal electron acceptors during anaerobic respiration such as nitrates, iron, carbon compounds, sulfur, metalloids, and radionuclides. In the presence of multiple terminal electron acceptors in a complex environment such as the rhizosphere and in the absence of O2, microorganisms will first use the most energetic option to sustain growth. Anaerobic respiration has been deeply studied, and the genes involved in anaerobic respiration have been identified. However, aqueous environment and paddy soils are the most studied environments for anaerobic respiration, even if we provide evidence in this review that anaerobic respiration also occurs in the plant rhizosphere. Indeed, we provide evidence by performing a BLAST analysis on metatranscriptomic data that genes involved in iron, sulfur, arsenate and selenate anaerobic respiration are expressed in the rhizosphere, underscoring that the rhizosphere environment is suitable for the establishment of anaerobic respiration. We thus focus this review on current research concerning the different types of anaerobic respiration that occur in the rhizosphere. We also discuss the flexibility of anaerobic respiration as a fundamental trait for the microbial colonization of roots, environmental and ecological adaptation, persistence and bioremediation in the rhizosphere. Anaerobic respiration appears to be a key process for the functioning of an ecosystem and interactions between plants and microbes

Genomic Species Are Ecological Species as Revealed by Comparative Genomics in Agrobacterium tumefaciens

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome—one on the circular chromosome and six on the linear chromosome—suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species

Efficiency and sensitivity of the digital droplet PCR for the quantification of antibiotic resistance genes in soils and organic residues

Droplet digital PCR (ddPCR) allows absolute quantification and tolerance to inhibitors and has been proposed as the method of choice to overcome limitations of qPCR. The aim of this study was to evaluate ddPCR and qPCR performances to detect low copy number and copy number variation of antibiotic resistance genes (sul1 and qnrB genes encoding for resistance to sulfonamides and quinolones, respectively) using bacterial genomic DNA (gDNA) and metagenomic DNA extracted from soil and organic residue samples. With gDNA, qPCR showed a better range of quantification but the lower limit of quantification was at 15 copies of qnrB target vs. 1.6 in ddPCR. In the presence of background DNA or inhibitors, we observed a high loss of sensitivity in qPCR and an overestimation of target sequences. When using high amount of environmental DNA templates (70 ng per reaction), ddPCR was still allowing accurate quantification without adding PCR facilitator (i.e., T4 Gene 32 protein). Sensitivity to detect copy number variation was tenfold higher in ddPCR than in qPCR. Finally, the advantages of using ddPCR in environmental studies were confirmed with the quantification of sul1 and qnrB in soils, manures, or urban wastes

The proteogenome of symbiotic Frankia alniFrankia\ alni in Alnus glutinosaAlnus\ glutinosa nodules

International audienceOmics are the most promising approaches to investigate microbes for which no genetic tools exist such as the nitrogen-fixing symbiotic Frankia. A proteogenomic analysis of symbiotic Frankia alni was done by comparing those proteins more and less abundant in Alnus glutinosa nodules relative to N2-fixing pure cultures with propionate as the carbon source. There were 250 proteins that were significantly overabundant in nodules at a fold change (FC) ≥ 2 threshold, and 1429 with the same characteristics in in vitro nitrogen-fixing pure culture. Nitrogenase, SuF (Fe–Su biogenesis) and hopanoid lipids synthesis determinants were the most overabundant proteins in symbiosis. Nitrogenase was found to constitute 3% of all Frankia proteins in nodules. Sod (superoxide dismutase) was overabundant, indicating a continued oxidative stress, while Kats (catalase) were not. Several transporters were overabundant including one for dicarboxylates and one for branched amino acids. The present results confirm the centrality of nitrogenase in the actinorhizal symbiosis

Genomic Insights of <i>Alnus</i>-Infective <i>Frankia</i> Strains Reveal Unique Genetic Features and New Evidence on Their Host-Restricted Lifestyle

The present study aimed to use comparative genomics to explore the relationships between Frankia and actinorhizal plants using a data set made of 33 Frankia genomes. The determinants of host specificity were first explored for “Alnus-infective strains” (i.e., Frankia strains belonging to Cluster Ia). Several genes were specifically found in these strains, including an agmatine deiminase which could possibly be involved in various functions as access to nitrogen sources, nodule organogenesis or plant defense. Within “Alnus-infective strains”, Sp+ Frankia genomes were compared to Sp− genomes in order to elucidate the narrower host specificity of Sp+ strains (i.e., Sp+ strains being capable of in planta sporulation, unlike Sp− strains). A total of 88 protein families were lost in the Sp+ genomes. The lost genes were related to saprophytic life (transcriptional factors, transmembrane and secreted proteins), reinforcing the proposed status of Sp+ as obligatory symbiont. The Sp+ genomes were also characterized by a loss of genetic and functional paralogs, highlighting a reduction in functional redundancy (e.g., hup genes) or a possible loss of function related to a saprophytic lifestyle (e.g., genes involved in gas vesicle formation or recycling of nutrients)

Correction: Pujic et al. The Proteogenome of Symbiotic <i>Frankia alni</i> in <i>Alnus glutinosa</i> Nodules. <i>Microorganisms</i> 2022, <i>10</i>, 651

The authors wish to make the following corrections to this paper [...

Whole-genome sequence of a pantoea sp. strain isolated from an olive (Olea europaea L.) knot

Here, we present the total genome sequence of Pantoea sp. strain paga, a plant-associated bacterium isolated from knots present on olive trees grown on the Adriatic Coast. The genome size of Pantoea sp. paga is 5.08 Mb, with a G+C content of 54%. The genome contains 4,776 predicted coding DNA sequences (CDSs), including 70 tRNA genes and 1 ribosomal operon. Obtained genome sequence data will provide insight on the physiology, ecology, and evolution of Pantoea spp

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

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

Taxonomic assignment of uncultured prokaryotes with long range PCR targeting the spectinomycin operon

International audienceThe taxonomic assignment of uncultured prokaryotes to known taxa is a major challenge in microbial systematics. This relies usually on the phylogenetic analysis of the ribosomal small subunit RNA or a few housekeeping genes. Recent works have disclosed ribosomal proteins as valuable markers for system-atics and, due to the boom in complete genome sequencing, their use has become widespread. Yet, in the case of uncultured strains, for which complete genome sequences cannot be easily obtained, sequencing many markers is complicated and time consuming. Taking the advantage of the organization of ribo-somal protein coding genes in large gene clusters, we amplified a 32 kb conserved region encompassing the spectinomycin (spc) operon using long range PCR from isolated and from uncultured nodular endophytic Frankia strains. The phylogenetic analysis of the 27 ribosomal protein genes contained in this region provided a robust phylogenetic tree consistent with phylogenies based on larger set of markers, indicating that this subset of ribosomal proteins contains enough phylogenetic signal to address systematic issues. This work shows that using long range PCR could break down the barrier preventing the use of ribosomal proteins as phylogenetic markers when complete genome sequences cannot be easily obtained

Effect of Inoculation Level on the Impact of the PGPR Azospirillum lipoferum CRT1 on Selected Microbial Functional Groups in the Rhizosphere of Field Maize

International audienceThe impact of inoculated plant growth-promoting rhizobacteria (PGPR) on its host physiology and nutrition depends on inoculum level. Whether the impact of the inoculated PGPR on the indigenous rhizosphere microbiota also varies with the PGPR inoculum level is unclear. Here, we tested this issue using the PGPR Azospirillum lipoferum CRT1—maize model system, where the initial seed inoculation is known to enhance maize growth and germination, and impacts the maize rhizomicrobiota, including microbial functional groups modulating plant growth. A. lipoferum CRT1 was added to the seeds at standard (105–6 cells.seed−1) or reduced (104–5 cells.seed−1) inoculation levels, in three fields. The effect of the two PGPR formulations was assessed on maize growth and on the nifH (nitrogen fixation), acdS (ACC deaminase activity) and phlD (2,4-diacetylphloroglucinol production) microbial functional groups. The size of the three functional groups was monitored by qPCR at the six-leaf stage and the flowering stage, and the diversity of the nifH and acdS functional groups (as well as the bacterial community) were estimated by MiSeq metabarcoding at the six-leaf stage. The results showed that the benefits of the reduced inoculant formulation were significant in two out of three fields, but different (often lower) than those of the standard formulation. The effects of formulations on the size of the three functional groups differed, and depended on field site and functional group. The reduced formulation had an impact on the diversity of nifH and acdS groups at one site, whereas the standard formulation had an impact at the two other sites. Inoculation significantly impacted the total bacterial community in the three fields, but only with the reduced formulation. In conclusion, the reduced inoculant formulation impacted the indigenous rhizosphere microbiota differently, but not less efficiently, than the standard formulation.L'impact des rhizobactéries favorisant la croissance des plantes (PGPR) inoculées sur la physiologie et la nutrition de leur hôte dépend du niveau d'inoculum. Il n'est pas clair si l'impact du PGPR inoculé sur le microbiote indigène de la rhizosphère varie également avec le niveau d'inoculum du PGPR. Ici, nous avons testé ce problème en utilisant le système de modèle de maïs PGPR Azospirillum lipoferum CRT1, où l'inoculation initiale des graines est connue pour améliorer la croissance et la germination du maïs, et a un impact sur le rhizomicrobiote du maïs, y compris les groupes fonctionnels microbiens modulant la croissance des plantes. A. lipoferum CRT1 a été ajouté aux graines au niveau standard (10 5–6 cellules.graine −1 ) ou réduit (10 4–5 cellules.graine −1) niveaux d'inoculation, dans trois champs. L'effet des deux formulations de PGPR a été évalué sur la croissance du maïs et sur les groupes fonctionnels microbiens nifH (fixation de l'azote), acdS (activité ACC désaminase) et phlD (production de 2,4-diacétylphloroglucinol). La taille des trois groupes fonctionnels a été suivie par qPCR au stade six feuilles et au stade floraison, et la diversité des nifH et acdSles groupes fonctionnels (ainsi que la communauté bactérienne) ont été estimés par métabarcoding MiSeq au stade six feuilles. Les résultats ont montré que les bénéfices de la formulation réduite d'inoculant étaient significatifs dans deux champs sur trois, mais différents (souvent inférieurs) de ceux de la formulation standard. Les effets des formulations sur la taille des trois groupes fonctionnels différaient et dépendaient du site de terrain et du groupe fonctionnel. La formulation réduite a eu un impact sur la diversité des nifH et des acdSgroupes sur un site, alors que la formulation standard avait un impact sur les deux autres sites. L'inoculation a eu un impact significatif sur la communauté bactérienne totale dans les trois champs, mais uniquement avec la formulation réduite. En conclusion, la formulation réduite d'inoculant a eu un impact différent, mais pas moins efficace, sur le microbiote indigène de la rhizosphère que la formulation standard