Taxonomic resolution of different 16S rRNA variable regions varies strongly across plant-associated bacteria.

Plant-microbiome research plays a pivotal role in understanding the relationships between plants and their associated microbial communities, with implications for agriculture and ecosystem dynamics. Metabarcoding analysis on variable regions of the 16S ribosomal RNA (rRNA) gene remains the dominant technology to study microbiome diversity in this field. However, the choice of the targeted variable region might affect the outcome of the microbiome studies. In our in silico analysis, we have evaluated whether the targeted variable region has an impact on taxonomic resolution in 16 plant-related microbial genera. Through a comparison of 16S rRNA gene variable regions with whole-genome data, our findings suggest that the V1-V3 region is generally a more suitable option than the widely used V3-V4 region for targeting microbiome analysis in plant-related genera. However, sole reliance on one region could introduce detection biases for specific genera. Thus, we are suggesting that while transitioning to full-length 16S rRNA gene and whole-genome sequencing for plant-microbiome analysis, the usage of genus-specific variable regions can achieve more precise taxonomic assignments. More broadly, our approach provides a blueprint to identify the most discriminating variable regions of the 16S rRNA gene for genera of interest.

A fast method to distinguish between fermentative and respiratory metabolisms in single yeast cells.

Saccharomyces cerevisiae adjusts its metabolism based on nutrient availability, typically transitioning from glucose fermentation to ethanol respiration as glucose becomes limiting. However, our understanding of the regulation of metabolism is largely based on population averages, whereas nutrient transitions may cause heterogeneous responses. Here we introduce iCRAFT, a method that couples the ATP Förster resonance energy transfer (FRET)-based biosensor yAT1.03 with Antimycin A to differentiate fermentative and respiratory metabolisms in individual yeast cells. Upon Antimycin A addition, respiratory cells experienced a sharp decrease of the normalized FRET ratio, while respiro-fermentative cells showed no response. Next, we tracked changes in metabolism during the diauxic shift of a glucose pre-grown culture. Following glucose exhaustion, the entire cell population experienced a progressive rise in cytosolic ATP produced via respiration, suggesting a gradual increase in respiratory capacity. Overall, iCRAFT is a robust tool to distinguish fermentation from respiration, offering a new single-cell opportunity to study yeast metabolism.

Microbial interactions shape cheese flavour formation.

Cheese fermentation and flavour formation are the result of complex biochemical reactions driven by the activity of multiple microorganisms. Here, we studied the roles of microbial interactions in flavour formation in a year-long Cheddar cheese making process, using a commercial starter culture containing Streptococcus thermophilus and Lactococcus strains. By using an experimental strategy whereby certain strains were left out from the starter culture, we show that S. thermophilus has a crucial role in boosting Lactococcus growth and shaping flavour compound profile. Controlled milk fermentations with systematic exclusion of single Lactococcus strains, combined with genomics, genome-scale metabolic modelling, and metatranscriptomics, indicated that S. thermophilus proteolytic activity relieves nitrogen limitation for Lactococcus and boosts de novo nucleotide biosynthesis. While S. thermophilus had large contribution to the flavour profile, Lactococcus cremoris also played a role by limiting diacetyl and acetoin formation, which otherwise results in an off-flavour when in excess. This off-flavour control could be attributed to the metabolic re-routing of citrate by L. cremoris from diacetyl and acetoin towards α-ketoglutarate. Further, closely related Lactococcus lactis strains exhibited different interaction patterns with S. thermophilus, highlighting the significance of strain specificity in cheese making. Our results highlight the crucial roles of competitive and cooperative microbial interactions in shaping cheese flavour profile.

Ethanol-lactate transition of Lachancea thermotolerans is linked to nitrogen metabolism.

Climate change increases sugar content in grapes, resulting in unwanted increase in ethanol content of wine. Lachancea thermotolerans ferments glucose and fructose into both ethanol and lactate, decreasing final ethanol content and positively affecting wine acidity. Reported Lachancea thermotolerans strains show big variation in lactate production during fermentation. However, a mechanistic understanding of this lactate producing phenotype is currently lacking. Through a combination of metabolomics, transcriptomics, genomics and computational methods we show that the lactate production is induced by amino acid limitation in a high lactate producing strain. We found in fermentations in synthetic grape juice media that lactate production starts in the last stages of growth, marked by decreased growth rate and increased expression levels of stress related genes. This onset of lactate production is specific for the high lactate producing strain and independent of oxygen availability. The onset of lactate production was changed by increased amino acid content of the media, and it is shown by both computational methods and amino acid measurements that at the onset of lactate production amino acids become limiting for growth. This study shows that lactate production of Lachancea thermotolerans is directly linked to nitrogen availability in the media, an insight that can further aid in the improvement of wine quality.

Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem.

So far, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to grow methanotrophically under aerobic conditions. Here we report that this metabolic trait is also observed within the Actinobacteria. We enriched and cultivated a methanotrophic Mycobacterium from an extremely acidic biofilm growing on a cave wall at a gaseous chemocline interface between volcanic gases and the Earth's atmosphere. This Mycobacterium, for which we propose the name Candidatus Mycobacterium methanotrophicum, is closely related to well-known obligate pathogens such as M. tuberculosis and M. leprae. Genomic and proteomic analyses revealed that Candidatus M. methanotrophicum expresses a full suite of enzymes required for aerobic growth on methane, including a soluble methane monooxygenase that catalyses the hydroxylation of methane to methanol and enzymes involved in formaldehyde fixation via the ribulose monophosphate pathway. Growth experiments combined with stable isotope probing using 13C-labelled methane confirmed that Candidatus M. methanotrophicum can grow on methane as a sole carbon and energy source. A broader survey based on 16S metabarcoding suggests that species closely related to Candidatus M. methanotrophicum may be abundant in low-pH, high-methane environments.

Metabolic interactions shape a community's phenotype.

Metabolic interactions between auxotrophs and prototrophs in microbial communities are understudied. Yu et al. showed how intracellular as well as intercellular metabolism affects community fitness in the absence and presence of abiotic stress, that is, drugs.

Using Functional Annotations to Study Pairwise Interactions in Urinary Tract Infection Communities.

The behaviour of microbial communities depends on environmental factors and on the interactions of the community members. This is also the case for urinary tract infection (UTI) microbial communities. Here, we devise a computational approach that uses indices of complementarity and competition based on metabolic gene annotation to rapidly predict putative interactions between pair of organisms with the aim to explain pairwise growth effects. We apply our method to 66 genomes selected from online databases, which belong to 6 genera representing members of UTI communities. This resulted in a selection of metabolic pathways with high correlation for each pairwise combination between a complementarity index and the experimentally derived growth data. Our results indicated that Enteroccus spp. were most complemented in its metabolism by the other members of the UTI community. This suggests that the growth of Enteroccus spp. can potentially be enhanced by complementary metabolites produced by other community members. We tested a few putative predicted interactions by experimental supplementation of the relevant predicted metabolites. As predicted by our method, folic acid supplementation led to the increase in the population density of UTI Enterococcus isolates. Overall, we believe our method is a rapid initial in silico screening for the prediction of metabolic interactions in microbial communities.

High biodiversity in a benzene-degrading nitrate-reducing culture is sustained by a few primary consumers.

A key question in microbial ecology is what the driving forces behind the persistence of large biodiversity in natural environments are. We studied a microbial community with more than 100 different types of species which evolved in a 15-years old bioreactor with benzene as the main carbon and energy source and nitrate as the electron acceptor. Using genome-centric metagenomics plus metatranscriptomics, we demonstrate that most of the community members likely feed on metabolic left-overs or on necromass while only a few of them, from families Rhodocyclaceae and Peptococcaceae, are candidates to degrade benzene. We verify with an additional succession experiment using metabolomics and metabarcoding that these few community members are the actual drivers of benzene degradation. As such, we hypothesize that high species richness is maintained and the complexity of a natural community is stabilized in a controlled environment by the interdependencies between the few benzene degraders and the rest of the community members, ultimately resulting in a food web with different trophic levels.

New Insights Into Cinnamoyl Esterase Activity of Oenococcus oeni.

Some strains of Oenococcus oeni possess cinnamoyl esterase activity that can be relevant in the malolactic stage of wine production liberating hydroxycinnamic acids that are precursors of volatile phenols responsible for sensory faults. The objective of this study was to better understand the basis of the differential activity between strains. After initial screening, five commercial strains of O. oeni were selected, three were found to exhibit cinnamoyl esterase activity (CE+) and two not (CE-). Although the use of functional annotation of genes revealed genotypic variations between the strains, no specific genes common only to the three CE+ strains could explain the different activities. Pasteurized wine was used as a natural source of tartrate esters in growth and metabolism experiments conducted in MRS medium, whilst commercial trans-caftaric acid was used as substrate for enzyme assays. Detoxification did not seem to be the main biological mechanism involved in the activity since unlike its phenolic cleavage products and their immediate metabolites (trans-caffeic acid and 4-ethylcatechol), trans-caftaric acid was not toxic toward O. oeni. In the case of the two CE+ strains OenosTM and CiNeTM, wine-exposed samples showed a more rapid degradation of trans-caftaric acid than the unexposed ones. The CE activity was present in all cell-free extracts of both wine-exposed and unexposed strains, except in the cell-free extracts of the CE- strain CH11TM. This activity may be constitutive rather than induced by exposure to tartrate esters. Trans-caftaric acid was totally cleaved to trans-caffeic acid by cell-free extracts of the three CE+ strains, whilst cell-free extracts of the CE- strain CH16TM showed significantly lower activity, although higher for the strains in experiments with no prior wine exposure. The EstB28 esterase gene, found in the genomes of the 5 strains, did not reveal any difference on the upstream regulation and transport functionality between the strains. This study highlights the complexity of the basis of this activity in wine related O. oeni population. Variable cinnamoyl esterases or/and membrane transport activities in the O. oeni strains analyzed and a possible implication of wine molecules could explain this phenomenon.

Finding Functional Differences Between Species in a Microbial Community: Case Studies in Wine Fermentation and Kefir Culture.

Microbial life usually takes place in a community where individuals interact, by competition for nutrients, cross-feeding, inhibition by end-products, but also by their spatial distribution. Lactic acid bacteria are prominent members of microbial communities responsible for food fermentations. Their niche in a community depends on their own properties as well as those of the other species. Here, we apply a computational approach, which uses only genomic and metagenomic information and functional annotation of genes, to find properties that distinguish a species from others in the community, as well as to follow individual species in a community. We analyzed isolated and sequenced strains from a kefir community, and metagenomes from wine fermentations. We demonstrate how the distinguishing properties of an organism lead to experimentally testable hypotheses concerning the niche and the interactions with other species. We observe, for example, that L. kefiranofaciens, a dominant organism in kefir, stands out among the Lactobacilli because it potentially has more amino acid auxotrophies. Using metagenomic analysis of industrial wine fermentations we investigate the role of an inoculated L. plantarum in malolactic fermentation. We observed that L. plantarum thrives better on white than on red wine fermentations and has the largest number of phosphotransferase system among the bacteria observed in the wine communities. Also, L. plantarum together with Pantoea, Erwinia, Asaia, Gluconobacter, and Komagataeibacter genera had the highest number of genes involved in biosynthesis of amino acids.

Taxonomic and Functional Characterization of the Microbial Community During Spontaneous in vitro Fermentation of Riesling Must.

Although there is an extensive tradition of research into the microbes that underlie the winemaking process, much remains to be learnt. We combined the high-throughput sequencing (HTS) tools of metabarcoding and metagenomics, to characterize how microbial communities of Riesling musts sampled at four different vineyards, and their subsequent spontaneously fermented derivatives, vary. We specifically explored community variation relating to three points: (i) how microbial communities vary by vineyard; (ii) how community biodiversity changes during alcoholic fermentation; and (iii) how microbial community varies between musts that successfully complete alcoholic fermentation and those that become 'stuck' in the process. Our metabarcoding data showed a general influence of microbial composition at the vineyard level. Two of the vineyards (4 and 5) had strikingly a change in the differential abundance of Metschnikowia. We therefore additionally performed shotgun metagenomic sequencing on a subset of the samples to provide preliminary insights into the potential relevance of this observation, and used the data to both investigate functional potential and reconstruct draft genomes (bins). At these two vineyards, we also observed an increase in non-Saccharomycetaceae fungal functions, and a decrease in bacterial functions during the early fermentation stage. The binning results yielded 11 coherent bins, with both vineyards sharing the yeast bins Hanseniaspora and Saccharomyces. Read recruitment and functional analysis of this data revealed that during fermentation, a high abundance of Metschnikowia might serve as a biocontrol agent against bacteria, via a putative iron depletion pathway, and this in turn could help Saccharomyces dominate the fermentation. During alcoholic fermentation, we observed a general decrease in biodiversity in both the metabarcoding and metagenomic data. Unexpected Micrococcus behavior was observed in vineyard 4 according to metagenomic analyses based on reference-based read mapping. Analysis of open reading frames using these data showed an increase of functions assigned to class Actinobacteria in the end of fermentation. Therefore, we hypothesize that bacteria might sit-and-wait until Saccharomyces activity slows down. Complementary approaches to annotation instead of relying a single database provide more coherent information true species. Lastly, our metabarcoding data enabled us to identify a relationship between stuck fermentations and Starmerella abundance. Given that robust chemical analysis indicated that although the stuck samples contained residual glucose, all fructose had been consumed, we hypothesize that this was because fructophilic Starmerella, rather than Saccharomyces, dominated these fermentations. Overall, our results showcase the different ways in which metagenomic analyses can improve our understanding of the wine alcoholic fermentation process.