Genotypic variations and interspecific interactions modify gene expression and biofilm formation of Xanthomonas retroflexus.

Multispecies biofilms are important models for studying the evolution of microbial interactions. Co-cultivation of Xanthomonas retroflexus (XR) and Paenibacillus amylolyticus (PA) systemically leads to the appearance of an XR wrinkled mutant (XRW), increasing biofilm production. The nature of this new interaction and the role of each partner remain unclear. We tested the involvement of secreted molecular cues in this interaction by exposing XR and XRW to PA or its supernatant and analysing the response using RNA-seq, colony-forming unit (CFU) estimates, biofilm quantification, and microscopy. Compared to wild type, the mutations in XRW altered its gene expression and increased its CFU number. These changes matched the reported effects for one of the mutated genes: a response regulator part of a two-component system involved in environmental sensing. When XRW was co-cultured with PA or its supernatant, the mutations effects on XRW gene expression were masked, except for genes involved in sedentary lifestyle, being consistent with the higher biofilm production. It appears that the higher biofilm production was the result of the interaction between the genetic context (mutations) and the biotic environment (PA signals). Regulatory genes involved in environmental sensing need to be considered to shed further light on microbial interactions.

Artificial selection of stable rhizosphere microbiota leads to heritable plant phenotype changes.

Artificial selection of microbiota opens new avenues for improving plants. However, reported results lack consistency. We hypothesised that the success in artificial selection of microbiota depends on the stabilisation of community structure. In a ten-generation experiment involving 1,800 plants, we selected rhizosphere microbiota of Brachypodium distachyon associated with high or low leaf greenness, a proxy of plant performance. The microbiota structure showed strong fluctuations during an initial transitory phase, with no detectable leaf greenness heritability. After five generations, the microbiota structure stabilised, concomitantly with heritability in leaf greenness. Selection, initially ineffective, did successfully alter the selected property as intended, especially for high selection. We show a remarkable correlation between the variability in plant traits and selected microbiota structures, revealing two distinct sub-communities associated with high or low leaf greenness, whose abundance was significantly steered by directional selection. Understanding microbiota structure stabilisation will improve the reliability of artificial microbiota selection.

Effect of long-term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce.

Long-term agricultural fertilization strategies gradually change soil properties including the associated microbial communities. Cultivated crops recruit beneficial microbes from the surrounding soil environment via root exudates. In this study, we aimed to investigate the effects of long-term fertilization strategies across field sites on the rhizosphere prokaryotic (Bacteria and Archaea) community composition and plant performance. We conducted growth chamber experiments with lettuce (Lactuca sativa L.) cultivated in soils from two long-term field experiments, each of which compared organic versus mineral fertilization strategies. 16S rRNA gene amplicon sequencing revealed the assemblage of a rhizosphere core microbiota shared in all lettuce plants across soils, going beyond differences in community composition depending on field site and fertilization strategies. The enhanced expression of several plant genes with roles in oxidative and biotic stress signalling pathways in lettuce grown in soils with organic indicates an induced physiological status in plants. Lettuce plants grown in soils with different fertilization histories were visibly free of stress symptoms and achieved comparable biomass. This suggests a positive aboveground plant response to belowground plant-microbe interactions in the rhizosphere. Besides effects of fertilization strategy and field site, our results demonstrate the crucial role of the plant in driving rhizosphere microbiota assemblage.

Microbial Diversity and Putative Opportunistic Pathogens in Dishwasher Biofilm Communities.

Extreme habitats are not only limited to natural environments, but also exist in manmade systems, for instance, household appliances such as dishwashers. Limiting factors, such as high temperatures, high and low pHs, high NaCl concentrations, presence of detergents, and shear force from water during washing cycles, define microbial survival in this extreme system. Fungal and bacterial diversity in biofilms isolated from rubber seals of 24 different household dishwashers was investigated using next-generation sequencing. Bacterial genera such as Pseudomonas, Escherichia, and Acinetobacter, known to include opportunistic pathogens, were represented in most samples. The most frequently encountered fungal genera in these samples belonged to Candida, Cryptococcus, and Rhodotorula, also known to include opportunistic pathogenic representatives. This study showed how specific conditions of the dishwashers impact the abundance of microbial groups and investigated the interkingdom and intrakingdom interactions that shape these biofilms. The age, usage frequency, and hardness of incoming tap water of dishwashers had significant impact on bacterial and fungal community compositions. Representatives of Candida spp. were found at the highest prevalence (100%) in all dishwashers and are assumed to be one of the first colonizers in recently purchased dishwashers. Pairwise correlations in tested microbiomes showed that certain bacterial groups cooccur, as did the fungal groups. In mixed bacterial-fungal biofilms, early adhesion, contact, and interactions were vital in the process of biofilm formation, where mixed complexes of bacteria and fungi could provide a preliminary biogenic structure for the establishment of these biofilms.IMPORTANCE Worldwide demand for household appliances, such as dishwashers and washing machines, is increasing, as is the number of immunocompromised individuals. The harsh conditions in household dishwashers should prevent the growth of most microorganisms. However, our research shows that persisting polyextremotolerant groups of microorganisms in household appliances are well established under these unfavorable conditions and supported by the biofilm mode of growth. The significance of our research is in identifying the microbial composition of biofilms formed on dishwasher rubber seals, how diverse abiotic conditions affect microbiota, and which key microbial members were represented in early colonization and contamination of dishwashers, as these appliances can present a source of domestic cross-contamination that leads to broader medical impacts.

Monitoring plasmid-mediated horizontal gene transfer in microbiomes: recent advances and future perspectives.

The emergence of antimicrobial resistant bacteria constitutes an increasing global health concern. Although it is well recognized that the cornerstone underlying this phenomenon is the dissemination of antimicrobial resistance via plasmids and other mobile genetic elements, the antimicrobial resistance transfer routes remain largely uncharted. In this review, we describe different methods for assessing the transfer frequency and host ranges of plasmids within complex microbiomes. The discussion is centered around the critical evaluation of recent advances for monitoring the fate of fluorescently tagged plasmids in bacterial communities through the coupling of fluorescence activated cell sorting and next generation sequencing techniques. We argue that this approach constitutes an exceptional tool for obtaining quantitative data regarding the extent of plasmid transfer, key disseminating taxa, and possible propagation routes. The integration of this information will provide valuable insights on how to develop alternative avenues for fighting the rise of antimicrobial resistant pathogens, as well as the means for constructing more comprehensive risk assessment models.

Deciphering conjugative plasmid permissiveness in wastewater microbiomes.

Wastewater treatment plants (WWTPs) are designed to robustly treat polluted water. They are characterized by ceaseless flows of organic, chemical and microbial matter, followed by treatment steps before environmental release. WWTPs are hotspots of horizontal gene transfer between bacteria via conjugative plasmids, leading to dissemination of potentially hazardous genetic material such as antimicrobial resistance genes (AMRGs). While current focus is on the threat of AMRGs spreading and their environmental maintenance, conjugative plasmid transfer dynamics within and between bacterial communities still remains largely uncharted. Furthermore, current in vitro methods used to assess conjugation in complex microbiomes do not include in situ behaviours of recipient cells, resulting in partial understanding of transfers. We investigated the in vitro conjugation capacities of WWTP microbiomes from inlet sewage and outlet treated water using the broad-host range IncP-1 conjugative plasmid, pKJK5. A thorough molecular approach coupling metagenomes to 16S rRNA DNA/cDNA amplicon sequencing was established to characterize microbiomes using the ecological concept of functional response groups. A broad diversity of recipient bacterial phyla for the plasmid was observed, especially in WWTP outlets. We also identified permissive bacteria potentially able to cross WWTPs and engage in conjugation before and after water treatment. Bacterial activity and lifestyle seem to influence conjugation extent, as treated water copiotrophs were the most represented strategist amongst transconjugants. Correlation analysis highlighted possible plasmid transmission routes into communities between the sewage to the environment, with identification of keystone members (e.g., Arcobacter) potentially involved in cross-border exchanges between distant Gram-positive and Gram-negative phyla.

Comparative Genomics Analysis of Keratin-Degrading Chryseobacterium Species Reveals Their Keratinolytic Potential for Secondary Metabolite Production.

A promising keratin-degrading strain from the genus Chryseobacterium (Chryseobacterium sp. KMC2) was investigated using comparative genomic tools against three publicly available reference genomes to reveal the keratinolytic potential for biosynthesis of valuable secondary metabolites. Genomic features and metabolic potential of four species were compared, showing genomic differences but similar functional categories. Eleven different secondary metabolite gene clusters of interest were mined from the four genomes successfully, including five common ones shared across all genomes. Among the common metabolites, we identified gene clusters involved in biosynthesis of flexirubin-type pigment, microviridin, and siderophore, showing remarkable conservation across the four genomes. Unique secondary metabolite gene clusters were also discovered, for example, ladderane from Chryseobacterium sp. KMC2. Additionally, this study provides a more comprehensive understanding of the potential metabolic pathways of keratin utilization in Chryseobacterium sp. KMC2, with the involvement of amino acid metabolism, TCA cycle, glycolysis/gluconeogenesis, propanoate metabolism, and sulfate reduction. This work uncovers the biosynthesis of secondary metabolite gene clusters from four keratinolytic Chryseobacterium species and shades lights on the keratinolytic potential of Chryseobacterium sp. KMC2 from a genome-mining perspective, can provide alternatives to valorize keratinous materials into high-value bioactive natural products.

Metagenomic analysis of a keratin-degrading bacterial consortium provides insight into the keratinolytic mechanisms.

Keratin is an insoluble fibrous protein from natural environments, which can be recycled to value-added products by keratinolytic microorganisms. A microbial consortium with efficient keratinolytic activity was previously enriched from soil, but the genetic basis behind its remarkable degradation properties was not investigated yet. To identify the metabolic pathways involved in keratinolysis and clarify the observed synergy among community members, shotgun metagenomic sequencing was performed to reconstruct metagenome-assembled genomes. More than 90% genera of the enriched bacterial consortium were affiliated to Chryseobacterium, Stenotrophomonas, and Pseudomonas. Metabolic potential and putative keratinases were predicted from the metagenomic annotation, providing the genetic basis of keratin degradation. Furthermore, metabolic pathways associated with keratinolytic processes such as amino acid metabolism, disulfide reduction and urea cycle were investigated from seven high-quality metagenome-assembled genomes, revealing the potential metabolic cooperation related to keratin degradation. This knowledge deepens the understanding of microbial keratinolytic mechanisms at play in a complex community, pinpointing the significance of synergistic interactions, which could be further used to optimize industrial keratin degradation processes.

Soil microbial legacies differ following drying-rewetting and freezing-thawing cycles.

Climate change alters frequencies and intensities of soil drying-rewetting and freezing-thawing cycles. These fluctuations affect soil water availability, a crucial driver of soil microbial activity. While these fluctuations are leaving imprints on soil microbiome structures, the question remains if the legacy of one type of weather fluctuation (e.g., drying-rewetting) affects the community response to the other (e.g., freezing-thawing). As both phenomenons give similar water availability fluctuations, we hypothesized that freezing-thawing and drying-rewetting cycles have similar effects on the soil microbiome. We tested this hypothesis by establishing targeted microcosm experiments. We created a legacy by exposing soil samples to a freezing-thawing or drying-rewetting cycle (phase 1), followed by an additional drying-rewetting or freezing-thawing cycle (phase 2). We measured soil respiration and analyzed soil microbiome structures. Across experiments, larger CO2 pulses and changes in microbiome structures were observed after rewetting than thawing. Drying-rewetting legacy affected the microbiome and CO2 emissions upon the following freezing-thawing cycle. Conversely, freezing-thawing legacy did not affect the microbial response to the drying-rewetting cycle. Our results suggest that drying-rewetting cycles have stronger effects on soil microbial communities and CO2 production than freezing-thawing cycles and that this pattern is mediated by sustained changes in soil microbiome structures.

Rhizosphere Bacterial Networks, but Not Diversity, Are Impacted by Pea-Wheat Intercropping.

Plant-plant associations, notably cereal-legume intercropping, have been proposed in agroecology to better value resources and thus reduce the use of chemical inputs in agriculture. Wheat-pea intercropping allows to decreasing the use of nitrogen fertilization through ecological processes such as niche complementarity and facilitation. Rhizosphere microbial communities may account for these processes, since they play a major role in biogeochemical cycles and impact plant nutrition. Still, knowledge on the effect of intecropping on the rhizosphere microbiota remains scarce. Especially, it is an open question whether rhizosphere microbial communities in cereal-legume intercropping are the sum or not of the microbiota of each plant species cultivated in sole cropping. In the present study, we assessed the impact of wheat and pea in IC on the diversity and structure of their respective rhizosphere microbiota. For this purpose, several cultivars of wheat and pea were cultivated in sole and intercropping. Roots of wheat and pea were collected separately in intercropping for microbiota analyses to allow deciphering the effect of IC on the bacterial community of each plant species/cultivar tested. Our data confirmed the well-known specificity of the rhizosphere effect and further stress the differentiation of bacterial communities between pea genotypes (Hr and hr). As regards the intercropping effect, diversity and structure of the rhizosphere microbiota were comparable to sole cropping. However, a specific co-occurrence pattern in each crop rhizosphere due to intercropping was revealed through network analysis. Bacterial co-occurrence network of wheat rhizosphere in IC was dominated by OTUs belonging to Alphaproteobacteria, Bacteroidetes and Gammaproteobacteria. We also evidenced a common network found in both rhizosphere under IC, indicating the interaction between the plant species; this common network was dominated by Acidobacteria, Alphaproteobacteria, and Bacteroidetes, with three OTUs belonging to Acidobacteria, Betaproteobacteria and Chloroflexi that were identified as keystone taxa. These findings indicate more complex rhizosphere bacterial networks in intercropping. Possible implications of these conclusions are discussed in relation with the functioning of rhizosphere microbiota in intercropping accounting for its beneficial effects.