Mosquito sex and mycobiota contribute to fructose metabolism in the Asian tiger mosquito Aedes albopictus.

BACKGROUND: Plant floral nectars contain natural sugars such as fructose, which are a primary energy resource for adult mosquitoes. Despite the importance of carbohydrates for mosquito metabolism, a limited knowledge is available about the pathways involved in sugar assimilation by mosquitoes and their associated microbiota. To this end, we used 13C-metabolomic and stable isotope probing approaches coupled to high-throughput sequencing to reveal fructose-related mosquito metabolic pathways and the dynamics of the active gut microbiota following fructose ingestion. RESULTS: Our results revealed significant differences in metabolic pathways between males and females, highlighting different modes of central carbon metabolism regulation. Competitive and synergistic interactions of diverse fungal taxa were identified within the active mycobiota following fructose ingestion. In addition, we identified potential cross-feeding interactions between this. Interestingly, there is a strong correlation between several active fungal taxa and the presence of fructose-derived metabolites. CONCLUSIONS: Altogether, our results provide novel insights into mosquito carbohydrate metabolism and demonstrate that dietary fructose as it relates to mosquito sex is an important determinant of mosquito metabolism; our results also further highlight the key role of active mycobiota interactions in regulating the process of fructose assimilation in mosquitoes. This study opens new avenues for future research on mosquito-microbiota trophic interactions related to plant nectar-derived sugars. Video abstract.

Implications of carbon catabolite repression for plant-microbe interactions.

Carbon catabolite repression (CCR) plays a key role in many physiological and adaptive responses in a broad range of microorganisms that are commonly associated with eukaryotic hosts. When a mixture of different carbon sources is available, CCR, a global regulatory mechanism, inhibits the expression and activity of cellular processes associated with utilization of secondary carbon sources in the presence of the preferred carbon source. CCR is known to be executed by completely different mechanisms in different bacteria, yeast, and fungi. In addition to regulating catabolic genes, CCR also appears to play a key role in the expression of genes involved in plant-microbe interactions. Here, we present a detailed overview of CCR mechanisms in various bacteria. We highlight the role of CCR in beneficial as well as deleterious plant-microbe interactions based on the available literature. In addition, we explore the global distribution of known regulatory mechanisms within bacterial genomes retrieved from public repositories and within metatranscriptomes obtained from different plant rhizospheres. By integrating the available literature and performing targeted meta-analyses, we argue that CCR-regulated substrate use preferences of microorganisms should be considered an important trait involved in prevailing plant-microbe interactions.

Effects of the Denitrification Inhibitor "Procyanidins" on the Diversity, Interactions, and Potential Functions of Rhizosphere-Associated Microbiome.

Some plant secondary metabolites, such as procyanidins, have been demonstrated to cause biological denitrification inhibition (BDI) of denitrifiers in soils concomitantly with a gain in plant biomass. The present work evaluated whether procyanidins had an impact on the diversity of nontarget microbial communities that are probably involved in soil fertility and ecosystem services. Lettuce plants were grown in two contrasting soils, namely Manziat (a loamy sand soil) and Serail (a sandy clay loam soil) with and without procyanidin amendment. Microbial diversity was assessed using Illumina sequencing of prokaryotic 16S rRNA gene and fungal ITS regions. We used a functional inference to evaluate the putative microbial functions present in both soils and reconstructed the microbial interaction network. The results showed a segregation of soil microbiomes present in Serail and Manziat that were dependent on specific soil edaphic variables. For example, Deltaproteobacteria was related to total nitrogen content in Manziat, while Leotiomycetes and Firmicutes were linked to Ca2+ in Serail. Procyanidin amendment did not affect the diversity and putative activity of microbial communities. In contrast, microbial interactions differed according to procyanidin amendment, with the results showing an enrichment of Entotheonellaeota and Mucoromycota in Serail soil and of Dependentiae and Rozellomycetes in Manziat soil.

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.

Does Biological Denitrification Inhibition (BDI) in the Field Induce an Increase in Plant Growth and Nutrition in Apium graveolens L. Grown for a Long Period?

Intensive agriculture uses a lot of nitrogen fertilizers to increase crop productivity. These crops are in competition with soil-denitrifying microorganisms that assimilate nitrogen in the form of nitrate and transform it into N2O, a greenhouse gas, or N2. However, certain plant species exude secondary metabolites, called procyanidins, which inhibit denitrifiers and increase the nitrate pool in the soil available for plant nutrition. This phenomenon is called biological denitrification inhibition. Previously, we showed that the addition of exogenous procyanidins to a lettuce crop induces denitrifier inhibition and increases nitrate content in the soil, affecting lettuce morphological traits. Here, the effects of procyanidin amendments in the field on a more long-term and nitrogen-consuming crop species such as celery were tested. The effects of procyanidin amendment on celery growth with those of conventional ammonium nitrate amendments were, therefore, compared. Denitrification activity, nitrate concentration, the abundance of denitrifying bacteria in the soil, and traits related to celery growth were measured. It was shown that the addition of procyanidins inhibits denitrifiers and increases the soil nitrate level, inducing an improvement in celery morphological traits. In addition, procyanidin amendment induces the lowest nitrogen concentration in tissues and reduces N2O emissions.

Who is eating fructose within the Aedes albopictus gut microbiota?

The Asian tiger mosquito Aedes albopictus is a major public health concern because of its invasive success and its ability to transmit pathogens. Given the low availability of treatments against mosquito-borne diseases, vector control remains the most suitable strategy. The methods used thus far are becoming less effective, but recent strategies have emerged from the study of mosquito-associated microorganisms. Although the role of the microbiota in insect biology does not require further proof, much remains to be deciphered in mosquitoes, especially the contribution of the microbiota to host nutrient metabolism. Mosquitoes feed on plant nectar, composed of mostly fructose. We used stable isotope probing to identify bacteria and fungi assimilating fructose within the gut of Ae. albopictus. Mosquitoes were fed a 13 C-labelled fructose solution for 24 h. Differences in the active microbial community according to the sex of mosquitoes were highlighted. The bacterium Lelliottia and the fungi Cladosporium and Aspergillus dominated the active microbiota in males, whereas the bacterium Ampullimonas and the yeast Cyberlindnera were the most active in females. This study is the first to investigate trophic interactions between Ae. albopictus and its microbiota, thus underscoring the importance of the microbial component in nectar feeding in mosquitoes.

Stable Isotope Probing of Microbiota Structure and Function in the Plant Rhizosphere.

Stable isotope probing of microbial nucleic acids applied in the rhizosphere enables (a) the identification of the active microbial community involved in root exudate assimilation and those involved in soil organic matter degradation, and (b) the study of the impact of plants via root exudates on the in situ expression of microbial functions. By incubating plants under 13CO2, fresh carbon exuded by the plant will be labeled and hence the microbial community assimilating 13C-root exudates will incorporate 13C into their cellular macromolecules. Labeled DNA, RNA, and proteins can be used to identify microorganisms that assimilated the root exudates. We provide a step-by-step protocol on how to apply stable isotope probing of DNA and RNA in the plant rhizosphere to identify the active microbial communities and analyze their gene expression.

Plant hosts control microbial denitrification activity.

In the rhizosphere, complex and dynamic interactions occur between plants and microbial networks that are primarily mediated by root exudation. Plants exude various metabolites that may influence the rhizosphere microbiota. However, few studies have sought to understand the role of root exudation in shaping the functional capacities of the microbiota. In this study, we aimed to determine the impact of plants on the diversity of active microbiota and their ability to denitrify via root exudates. For that purpose, we grew four plant species, Triticum aestivum, Brassica napus, Medicago truncatula and Arabidopsis thaliana separately in the same soil. We extracted RNA from the root-adhering soil and the root tissues, and we analysed the bacterial diversity by using 16S rRNA metabarcoding. We measured denitrification activity and denitrification gene expression (nirK and nirS) from each root-adhering soil sample and the root tissues using gas chromatography and quantitative PCR, respectively. We demonstrated that plant species shape denitrification activity and modulate the diversity of the active microbiota through root exudation. We observed a positive effect of T. aestivum and A. thaliana on denitrification activity and nirK gene expression on the root systems. Together, our results underscore the potential power of host plants in controlling microbial activities.

Determination of Root Exudate Concentration in the Rhizosphere Using 13C Labeling.

One of the most remarkable metabolic features of plant roots is their ability to secrete a wide range of compounds into the rhizosphere, defined as the volume of soil around living roots. Around 5%-21% of total photosynthetically fixed carbon is transferred into the rhizosphere through root exudates. Until recently, studies on the quantity and quality of root exudates were conducted mostly under axenic or monoxenic in vitro conditions. Today, in situ assays are required to provide a better understanding of root exudates dynamics and role in plant-microbe interactions. By incubating plants with 13CO2 in situ for one week and quantifying 13C enrichment from the root-adhering soil using mass spectrometry, we were able to determine root exudate levels. Indeed, labeled substrate 13CO2 is converted into organic carbon via plant photosynthesis and transferred into the soil through root exudation. We assume that all 13C increases above natural abundance are mainly derived from exudates produced by 13C-labeled plants.

Plant Nutrient Resource Use Strategies Shape Active Rhizosphere Microbiota Through Root Exudation.

Plant strategies for soil nutrient uptake have the potential to strongly influence plant-microbiota interactions, due to the competition between plants and microorganisms for soil nutrient acquisition and/or conservation. In the present study, we investigate whether these plant strategies could influence rhizosphere microbial activities via root exudation, and contribute to the microbiota diversification of active bacterial communities colonizing the root-adhering soil (RAS) and inhabiting the root tissues. We applied a DNA-based stable isotope probing (DNA-SIP) approach to six grass species distributed along a gradient of plant nutrient resource strategies, from conservative species, characterized by low nitrogen (N) uptake, a long lifespans and low root exudation level, to exploitative species, characterized by high rates of photosynthesis, rapid rates of N uptake and high root exudation level. We analyzed their (i) associated microbiota composition involved in root exudate assimilation and soil organic matter (SOM) degradation by 16S-rRNA-based metabarcoding. (ii) We determine the impact of root exudation level on microbial activities (denitrification and respiration) by gas chromatography. Measurement of microbial activities revealed an increase in denitrification and respiration activities for microbial communities colonizing the RAS of exploitative species. This increase of microbial activities results probably from a higher exudation rate and more diverse metabolites by exploitative plant species. Furthermore, our results demonstrate that plant nutrient resource strategies have a role in shaping active microbiota. We present evidence demonstrating that plant nutrient use strategies shape active microbiota involved in root exudate assimilation and SOM degradation via root exudation.

Root exudation rate as functional trait involved in plant nutrient-use strategy classification.

Plants adopt a variety of life history strategies to succeed in the Earth's diverse environments. Using functional traits which are defined as "morphological, biochemical, physiological, or phonological" characteristics measurable at the individual level, plants are classified according to their species' adaptative strategies, more than their taxonomy, from fast growing plant species to slower-growing conservative species. These different strategies probably influence the input and output of carbon (C)-resources, from the assimilation of carbon by photosynthesis to its release in the rhizosphere soil via root exudation. However, while root exudation was known to mediate plant-microbe interactions in the rhizosphere, it was not used as functional trait until recently. Here, we assess whether root exudate levels are useful plant functional traits in the classification of plant nutrient-use strategies and classical trait syndromes? For this purpose, we conducted an experiment with six grass species representing along a gradient of plant resource-use strategies, from conservative species, characterized by low biomass nitrogen (N) concentrations and a long lifespans, to exploitative species, characterized by high rates of photosynthesis and rapid rates of N acquisition. Leaf and root traits were measured for each grass and root exudate rate for each planted soil sample. Classical trait syndromes in plant ecology were found for leaf and root traits, with negative relationships observed between specific leaf area and leaf dry matter content or between specific root length and root dry matter content. However, a new root trait syndrome was also found with root exudation levels correlating with plant resource-use strategy patterns, specifically, between root exudation rate and root dry matter content. We therefore propose root exudation rate can be used as a key functional trait in plant ecology studies and plant strategy classification.

Plant host habitat and root exudates shape fungal diversity.

The rhizospheric microbiome is clearly affected by plant species and certain of their functional traits. These functional traits allow plants to adapt to their environmental conditions by acquiring or conserving nutrients, thus defining different ecological resource-use plant strategies. In the present study, we investigated whether plants with one of the two nutrient-use strategies (conservative versus exploitative) could influence fungal communities involved in soil organic matter degradation and root exudate assimilation, as well as those colonizing root tissues. We applied a DNA-based, stable-isotope probing (DNA-SIP) approach to four grass species distributed along a gradient of plant nutrient resource strategies, ranging from conservative to exploitative species, and analyzed their associated mycobiota composition using a fungal internal transcribed spacer (ITS) and Glomeromycotina 18S rRNA gene metabarcoding approach. Our results demonstrated that fungal taxa associated with exploitative and conservative plants could be separated into two general categories according to their location: generalists, which are broadly distributed among plants from each strategy and represent the core mycobiota of soil organic matter degraders, root exudate consumers in the root-adhering soil, and root colonizers; and specialists, which are locally abundant in one species and more specifically involved in soil organic matter degradation or root exudate assimilation on the root-adhering soil and the root tissues. Interestingly, for arbuscular mycorrhizal fungi analysis, all plant roots were mainly colonized by Glomus species, whereas an increased diversity of Glomeromycotina genera was observed for the exploitative plant species Dactylis glomerata.

The effects of plant nutritional strategy on soil microbial denitrification activity through rhizosphere primary metabolites.

The aim of this study was to determine (i) whether plant nutritional strategy affects the composition of primary metabolites exuded into the rhizosphere and (ii) the impact of exuded metabolites on denitrification activity in soil. We answered this question by analysing primary metabolite content extracted from the root-adhering soil (RAS) and the roots of three grasses representing different nutrient management strategies: conservative (Festuca paniculata), intermediate (Bromus erectus) and exploitative (Dactylis glomerata). We also investigated the impact of primary metabolites on soil microbial denitrification enzyme activity without carbon addition, comparing for each plant RAS and bulk soils. Our data show that plant nutritional strategy impacts on primary metabolite composition of root extracts or RAS. Further we show, for the first time, that RAS-extracted primary metabolites are probably better indicators to explain plant nutrient strategy than root-extracted ones. In addition, our results show that some primary metabolites present in the RAS were well correlated with soil microbial denitrification activity with positive relationships found between denitrification and the presence of some organic acids and negative ones with the presence of xylose. We demonstrated that the analysis of primary metabolites extracted from the RAS is probably more pertinent to evaluate the impact of plant on soil microbial community functioning.

Stable isotope probing of carbon flow in the plant holobiont.

Microbial communities associated with a plant host, constituting a holobiont, affect the physiology and growth of the plant via metabolites that are mainly derived from their photosynthates. The structure and function of active microbial communities that assimilate root exudates can be tracked by using stable isotope probing (SIP) approaches. This article reviews results from ongoing SIP research in plant-microbe interactions, with a specific focus on investigating the fate of fresh and recalcitrant carbon in the rhizosphere with 13C enriched-root exudates, in addition to identifying key players in carbon cycling. Finally, we discuss new SIP applications that have the potential to identify novel enzymes implicated in rhizoremediation or plant genes dedicated to root exudation by combining SIP approaches and genome wide associations studies.

Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI.

Identification of B-type procyanidins in Fallopia spp. involved in biological denitrification inhibition.

Nitrogen (N) is considered as a main limiting factor in plant growth, and nitrogen losses through denitrification can be responsible for severe decreases in plant productivity. Recently, it was demonstrated that Fallopia spp. is responsible for biological denitrification inhibition (BDI) through the release of unknown secondary metabolites. Here, we investigate the secondary metabolites involved in the BDI of Fallopia spp. The antioxidant, protein precipitation capability of Fallopia spp. extracts was measured in relation to the aerobic respiration and denitrification of two bacteria (Gram positive and Gram negative). Proanthocyanidin concentrations were estimated. Proanthocyanidins in extracts were characterized by chromatographic analysis, purified and tested on the bacterial denitrification and aerobic respiration of two bacterial strains. The effect of commercial procyanidins on denitrification was tested on two different soil types. Denitrification and aerobic respiration inhibition were correlated with protein precipitation capacity and concentration of proanthocyanidins but not to antioxidant capacity. These proanthocyanidins were B-type procyanidins that inhibited denitrification more than the aerobic respiration of bacteria. In addition, procyanidins also inhibited soil microbial denitrification. We demonstrate that procyanidins are involved in the BDI of Fallopia spp. Our results pave the way to a better understanding of plant-microbe interactions and highlight future applications for a more sustainable agriculture.

Small regulatory RNAs and the fine-tuning of plant­bacteria interactions.

Small regulatory RNAs (sRNAs) play a key role in many physiological and adaptive responses in bacteria. Faced with rapidly changing environments, it is more advantageous for bacteria to use sRNA-mediated responses than regulation by protein transcriptional factors, as sRNAs act at the post-transcriptional level and require less energy and time for their synthesis and turnover. The use of RNA deep sequencing has provided hundreds of sRNA candidates in different bacterial species that interact with plants. Here, we review the most recent results for the involvement of bacterial sRNAs in beneficial as well as deleterious plant­bacteria interactions. We describe the current view for the role of sRNAs, which are suggested to improve competition for both niches and resources in plant-interacting bacteria. These sRNAs also help plant-associated bacteria individually adapt to the rapidly changing conditions to which they are exposed, during different stages of this interaction.

Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites.

Previous studies on the effect of secondary metabolites on the functioning of rhizosphere microbial communities have often focused on aspects of the nitrogen (N) cycle but have overlooked biological denitrification inhibition (BDI), which can affect plant N-nutrition. Here, we investigated the BDI by the compounds of Fallopia spp., an invasive weed shown to be associated with a low potential denitrification of the soil. Fallopia spp. extracts were characterized by chromatographic analysis and were used to test the BDI effects on the metabolic and respiratory activities of denitrifying bacteria, under aerobic and anaerobic (denitrification) conditions. The BDI of Fallopia spp. extracts was tested on a complex soil community by measuring denitrification enzyme activity (DEA), substrate induced respiration (SIR), as well as abundances of denitrifiers and total bacteria. In 15 strains of denitrifying bacteria, extracts led to a greater BDI (92%) than respiration inhibition (50%). Anaerobic metabolic activity reduction was correlated with catechin concentrations and the BDI was dose dependent. In soil, extracts reduced the DEA/SIR ratio without affecting the denitrifiers: total bacteria ratio. We show that secondary metabolite(s) from Fallopia spp. inhibit denitrification. This provides new insight into plant-soil interactions and improves our understanding of a plant's ability to shape microbial soil functioning.

Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa.

The opportunistic human pathogen Pseudomonas aeruginosa is able to utilize a wide range of carbon and nitrogen compounds, allowing it to grow in vastly different environments. The uptake and catabolism of growth substrates are organized hierarchically by a mechanism termed catabolite repression control (Crc) whereby the Crc protein establishes translational repression of target mRNAs at CA (catabolite activity) motifs present in target mRNAs near ribosome binding sites. Poor carbon sources lead to activation of the CbrAB two-component system, which induces transcription of the small RNA (sRNA) CrcZ. This sRNA relieves Crc-mediated repression of target mRNAs. In this study, we have identified novel targets of the CbrAB/Crc system in P. aeruginosa using transcriptome analysis in combination with a search for CA motifs. We characterized four target genes involved in the uptake and utilization of less preferred carbon sources: estA (secreted esterase), acsA (acetyl-CoA synthetase), bkdR (regulator of branched-chain amino acid catabolism) and aroP2 (aromatic amino acid uptake protein). Evidence for regulation by CbrAB, CrcZ and Crc was obtained in vivo using appropriate reporter fusions, in which mutation of the CA motif resulted in loss of catabolite repression. CbrB and CrcZ were important for growth of P. aeruginosa in cystic fibrosis (CF) sputum medium, suggesting that the CbrAB/Crc system may act as an important regulator during chronic infection of the CF lung.