A highly conserved ligand-binding site for AccA transporters of antibiotic and quorum-sensing regulator in Agrobacterium leads to a different specificity.

Plants genetically modified by the pathogenic Agrobacterium strain C58 synthesize agrocinopines A and B, whereas those modified by the pathogenic strain Bo542 produce agrocinopines C and D. The four agrocinopines (A, B, C and D) serve as nutrients by agrobacteria and signaling molecule for the dissemination of virulence genes. They share the uncommon pyranose-2-phosphate motif, represented by the l-arabinopyranose moiety in agrocinopines A/B and the d-glucopyranose moiety in agrocinopines C/D, also found in the antibiotic agrocin 84. They are imported into agrobacterial cytoplasm via the Acc transport system, including the solute-binding protein AccA coupled to an ABC transporter. We have previously shown that unexpectedly, AccA from strain C58 (AccAC58) recognizes the pyranose-2-phosphate motif present in all four agrocinopines and agrocin 84, meaning that strain C58 is able to import agrocinopines C/D, originating from the competitor strain Bo542. Here, using agrocinopine derivatives and combining crystallography, affinity and stability measurements, modeling, molecular dynamics, in vitro and vivo assays, we show that AccABo542 and AccAC58 behave differently despite 75% sequence identity and a nearly identical ligand binding site. Indeed, strain Bo542 imports only compounds containing the d-glucopyranose-2-phosphate moiety, and with a lower affinity compared with strain C58. This difference in import efficiency makes C58 more competitive than Bo542 in culture media. We can now explain why Agrobacterium/Allorhizobium vitis strain S4 is insensitive to agrocin 84, although its genome contains a conserved Acc transport system. Overall, our work highlights AccA proteins as a case study, for which stability and dynamics drive specificity.

Dimethylpolysulfides production as the major mechanism behind wheat fungal pathogen biocontrol, by Arthrobacter and Microbacterium actinomycetes.

IMPORTANCE: As the management of wheat fungal diseases becomes increasingly challenging, the use of bacterial agents with biocontrol potential against the two major wheat phytopathogens, Fusarium graminearum and Zymoseptoria tritici, may prove to be an interesting alternative to conventional pest management. Here, we have shown that dimethylpolysulfide volatiles are ubiquitously and predominantly produced by wheat-associated Microbacterium and Arthrobacter actinomycetes, displaying antifungal activity against both pathogens. By limiting pathogen growth and DON virulence factor production, the use of such DMPS-producing strains as soil biocontrol inoculants could limit the supply of pathogen inocula in soil and plant residues, providing an attractive alternative to dimethyldisulfide fumigant, which has many non-targeted toxicities. Notably, this study demonstrates the importance of bacterial volatile organic compound uptake by inhibited F. graminearum, providing new insights for the study of volatiles-mediated toxicity mechanisms within bacteria-fungus signaling crosstalk.

Microbiome analysis in Lascaux Cave in relation to black stain alterations of rock surfaces and collembola.

Anthropization of Palaeolithic caves open for tourism may favour collembola invasion and result in the formation of black stains attributed to pigmented fungi. However, ecological processes underpinning black stain formation are not fully understood. Here, we tested the hypotheses that black stains from the Apse room of Lascaux Cave display a specific microbiota enriched in pigmented fungi, and that collembola thriving on the stains have the potential to consume and disseminate these black fungi. Metabarcoding showed that the microbiota of black stains and neighbouring unstained parts strongly differed, with in black stains a higher prevalence of Ochroconis and other pigmented fungi and the strong regression of Pseudomonas bacteria (whose isolates inhibited in vitro the growth of pigmented fungi). Isotopic analyses indicated that Folsomia candida collembola thriving on stains could feed on black stain in situ and assimilate the pigmented fungi they were fed with in vitro. They could carry these fungi and disseminate them when tested with complex black stains from Lascaux. This shows that black stain formation is linked to the development of pigmented fungi, which coincides with the elimination of antagonistic pseudomonads, and points towards a key role of F. candida collembola in the dynamics of pigmented fungi.

Datamining and functional environmental genomics reassess the phylogenetics and functional diversity of fungal monosaccharide transporters.

Sugar transporters are essential components of carbon metabolism and have been extensively studied to control sugar uptake by yeasts and filamentous fungi used in fermentation processes. Based on published information on characterized fungal sugar porters, we show that this protein family encompasses phylogenetically distinct clades. While several clades encompass transporters that seemingly specialized on specific "sugar-related" molecules (e.g., myo-inositol, charged sugar analogs), others include mostly either mono- or di/oligosaccharide low-specificity transporters. To address the issue of substrate specificity of sugar transporters, that protein primary sequences do not fully reveal, we screened "multi-species" soil eukaryotic cDNA libraries for mannose transporters, a sugar that had never been used to select transporters. We obtained 19 environmental transporters, mostly from Basidiomycota and Ascomycota. Among them, one belonged to the unusual "Fucose H+ Symporter" family, which is only known in Fungi for a rhamnose transporter in Aspergillus niger. Functional analysis of the 19 transporters by expression in yeast and for two of them in Xenopus laevis oocytes for electrophysiological measurements indicated that most of them showed a preference for D-mannose over other tested D-C6 (glucose, fructose, galactose) or D-C5 (xylose) sugars. For the several glucose and fructose-negative transporters, growth of the corresponding recombinant yeast strains was prevented on mannose in the presence of one of these sugars that may act by competition for the binding site. Our results highlight the potential of environmental genomics to figure out the functional diversity of key fungal protein families and that can be explored in a context of biotechnology. KEY POINTS: • Most fungal sugar transporters accept several sugars as substrates. • Transporters, belonging to 2 protein families, were isolated from soil cDNA libraries. • Environmental transporters featured novel substrate specificities.

Development of a DNA-Based Real-Time PCR Assay To Quantify Allorhizobium vitis Over Time in Grapevine (Vitis vinifera L.) Plantlets.

Allorhizobium vitis is the primary causal pathogen of grapevine crown gall disease. Because this endophytic bacterium can survive as a systemic latent (symptomless) infection in grapevine, detecting and monitoring its development in planta is of great importance. In plant bacteria studies, plate counting is routinely used as a simple and reliable method to evaluate the bacterial population level in planta. However, isolation techniques are time-consuming and present some disadvantages such as the risk of contamination and the need for fresh samples for research. In this study, we developed a DNA-based real-time PCR assay that can replace the classical method to monitor the development of Allorhizobium vitis in grapevine plantlets. Primers targeting Allorhizobium vitis chromosomic genes and the virulent tumor-inducing plasmid were validated. The proposed quantitative real-time PCR technique is highly reliable and reproducible to assess Allorhizobium vitis numeration at the earliest stage of infection until tumor development in grapevine plantlets. Moreover, this low-cost technique provides rapid and robust in planta quantification of the pathogen and is suitable for fundamental research to monitor bacterial development over time.

Characterization of the first tetrameric transcription factor of the GntR superfamily with allosteric regulation from the bacterial pathogen Agrobacterium fabrum.

A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs) degradation is important for the transition between the two lifestyles (rhizospheric versus pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either used as trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. In contrast to HcaR, Atu1419 remains so far uncharacterized. The high-resolution crystal structures of two fortuitous citrate complexes, two DNA complexes and the apoform revealed that the tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729 subfamily of the large GntR superfamily. Until now, GntR regulators were described as dimers. Here, we showed that Atu1419 represses three genes of the HCAs catabolic pathway. We characterized both the effector and DNA binding sites and identified key nucleotides in the target palindrome. From promoter activity measurement using defective gene mutants, structural analysis and gel-shift assays, we propose N5,N10-methylenetetrahydrofolate as the effector molecule, which is not a direct product/substrate of the HCA degradation pathway. The Zn2+ ion present in the effector domain has both a structural and regulatory role. Overall, our work shed light on the allosteric mechanism of transcription employed by this GntR repressor.

Agrobacterium fabrum C58 involved nitrate reductase NapA and antisense RNA NorR to denitrify.

Agrobacterium fabrum C58 is a plant-associated bacterium that is able to denitrify under anoxic conditions. The cluster of denitrification genes harbored by this strain has been well characterized. It includes nir and nor operons encoding nitrite and nitric oxide reductases, respectively. However, the reductase involved in nitrate reduction has not yet been studied and little information is available on denitrification regulators in A. fabrum C58. In this study, we aimed to (i) characterize the nitrate reductase, (ii) determine its role in A. fabrum C58 fitness and root colonization and (ii) reveal the contribution of small RNA on denitrification regulation. By constructing a mutant strain defective for napA, we demonstrated that the reduction of nitrate to nitrite was catalyzed by the periplasmic nitrate reductase, NapA. We evidenced a positive role of NapA in A. fabrum C58 fitness and suggested that A. fabrum C58 is able to use components exuded by plant roots to respire anaerobically. Here, we showed that NorR small RNA increased the level of norCBQ mRNA and a decrease of NorR is correlated with a decrease in N2O emission. Together, our results underscore the importance of understanding the denitrification pathway at the strain level in order to develop strategies to mitigate N2O production at the microbial community level.

Import pathways of the mannityl-opines into the bacterial pathogen Agrobacterium tumefaciens: structural, affinity and in vivo approaches.

Agrobacterium tumefaciens pathogens use specific compounds denoted opines as nutrients in their plant tumor niche. These opines are produced by the host plant cells genetically modified by agrobacteria. They are imported into bacteria via solute-binding proteins (SBPs) in association with ATP-binding cassette transporters. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. Structural and affinity data on mannopinic acid bound to SBPs are currently lacking while those of the three others mannityl opines are available. We investigated the molecular basis of two pathways for mannopinic acid uptake. MoaA was proposed as the specific SBP for mannopinic acid import in mannityl opines-assimilating agrobacteria, which was validated here using genetic studies and affinity measurements. We structurally characterized the mannopinic acid-binding mode of MoaA in two crystal forms at 2.05 and 1.57 Šresolution. We demonstrated that the non-specific SBP MotA, so far characterized as mannopine and Amadori compound importer, was also able to transport mannopinic acid. The structure of MotA bound to mannopinic acid at 2.2 Šresolution defines a different mannopinic acid-binding signature, similar to that of mannopine. Combining in vitro and in vivo approaches, this work allowed us to complete the characterization of the mannityl-opines assimilation pathways, highlighting the important role of two dual imports of agropinic and mannopinic acids. Our data shed new light on how the mannityl-opines contribute to the establishment of the ecological niche of agrobacteria from the early to the late stages of tumor development.

Identification of a small set of genes commonly regulated in rice roots in response to beneficial rhizobacteria.

Rhizosphere bacteria, whether phytopathogenic or phytobeneficial, are thought to be perceived by the plant as a threat. Plant Growth-Promoting Rhizobacteria (PGPR), such as many strains of the Azospirillum genus known as the main phytostimulator of cereals, cooperate with host plants and favorably affect their growth and health. An earlier study of rice root transcriptome, undertaken with two rice cultivars and two Azospirillum strains, revealed a strain-dependent response during the rice-Azospirillum association and showed that only a few genes, including some implicated in plant defense, were commonly regulated in all tested conditions. Here, a set of genes was selected from previous studies and their expression was monitored by qRT-PCR in rice roots inoculated with ten PGPR strains isolated from various plants and belonging to various genera (Azospirillum, Herbaspirillum, Paraburkholderia). A common expression pattern was highlighted for four genes that are proposed to be markers of the rice-PGPR interaction: two genes involved in diterpenoid phytoalexin biosynthesis (OsDXS3 and OsDTC2) and one coding for an uncharacterized protein (Os02g0582900) were significantly induced by PGPR whereas one defense-related gene encoding a pathogenesis-related protein (PR1b, Os01g0382000) was significantly repressed. Interestingly, exposure to a rice bacterial pathogen also triggered the expression of OsDXS3 while the expression of Os02g0582900 and PR1b was down-regulated, suggesting that these genes might play a key role in rice-bacteria interactions. Integration of these results with previous data led us to propose that the jasmonic acid signaling pathway might be triggered in rice roots upon inoculation with PGPR.

Anthropization level of Lascaux Cave microbiome shown by regional-scale comparisons of pristine and anthropized caves.

Limestone areas across the world develop karstic caves, which are populated by a wide range of macro- and microorganisms. Many of these caves display Paleolithic art or outstanding speleothems, and in the last century they have been subjected to anthropization due to touristic management and intense human frequentation. Despite their cultural importance and associated conservation issues, the impact of anthropization on cave biodiversity is not known. Here, we show that anthropization is associated with specific cave biota modifications. We compared diversity in four pristine caves, four anthropized show caves, and the iconic Lascaux Cave with even stronger anthropization. The predominant microbial higher taxa were the same in all caves, but the most anthropized cave (Lascaux) was unique as it differed from the eight others by a higher proportion of Bacteroidetes bacteria and the absence of Euryarchaeota and Woesearchaeota archaea. Anthropization resulted in lower diversity and altered community structure for bacteria and archaea on cave walls, especially in Lascaux, but with a more limited effect on microeukaryotes and arthropods. Our findings fill a key gap in our understanding of the response of karstic communities to anthropization, by revealing that tourism-related anthropization impacts on the prokaryotic microbiome rather than on eukaryotic residents, and that it shapes cave biota irrespective of cave natural features.