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.info:eu-repo/semantics/publishedVersio

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

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

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

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

A Computational Odyssey into Microbial Communities Across Ecosystems

This thesis uses mainly computational data-driven approaches to study variation, taxonomy and functional characterization, interactions between members and underlying principles that govern microbial communities across ecosystems. The importance to study microbes in the natural context of complex systems of communities was highlighted in Chapter 1. In Chapter 2 we studied the variation of the microbial community composition during spontaneous in vitro wine fermentation of riesling must. We made the following observations: (i) There is a general influence of the vineyard on microbial composition with a striking differential abundance of Metschnikowia. (ii) There is a decrease in biodiversity during alcoholic fermentation. Unexpectedly, the fraction of Micrococcus increased in one vineyard during alcoholic fermentation. (iii) There is a relation between stuck fermentations and the abundance of Starmerella. In Chapter 3, we continued to explore microbial communities during wine fermentation. Specifically, the role of the adjunct Lactobacillus plantarum in the malolactic conversion of industrial wine fermentation was investigated and this species was found to thrive better on white than on red wine fermentations. We obtained experimental evidence to support the hypothesis that a successful introduction of this species in a community was in the case of wine determined by the composition of the must, and possibly by the presence of grape skins during fermentation. In Chapter 5 a simple experimental coloring method is presented which distinguishes colonies of the yeasts Lachancea thermotolerans and Saccharomyces cerevisiae on agar media. It does so by the addition of bromocresol purple which induces Lachancea colonies to develop a brown color, whereas Saccharomyces colonies remain white. In Chapter 3 we also studied a kefir community using genome sequences of isolated and sequenced Bacteria representative strains. We found that Lactobacillus kefiranofaciens, a dominant organism in kefir, stands out among the lactobacilli because it potentially has a high number of amino acid auxotrophies. Also, the only organism in kefir that had genes for flagellar assembly and chemotaxis was Acetobacter. The presence of flagella in Acetobacter was experimentally confirmed. In Chapter 4 we study pairwise interactions between microbes using measures based on genes involved in metabolic processes. In the case of microbes from the urinary tract, a number of putative metabolic interactions were identified that could explain the experimentally obtained pairwise growth effects. We found that members of Enterococcus may be complemented in their metabolism by the other members of the community. In Chapter 6 we investigated the metabolism of a novel Mycobacterium species, which was found to be dominant in a microbial community residing in an acidic biofilm attached to the wall of a sulfur cave in Romania. This Mycobacterium species expresses a full suite of enzymes involved in methanotrophic growth. Growth experiments using methane as the sole carbon- and free energy source verify its methanotrophic niche. To our knowledge, this is the first report about a methanotrophic Mycobacterium of Actinobacteria. In Chapter 7 a key question in microbial ecology is asked - how large biodiversity can be maintained on a few resources. To address this question a highly diverse microbial community was investigated, which grew for 15 years in an anoxic bioreactor on benzene as the main carbon and free energy source and nitrate as an electron acceptor. We found evidence that many different niches are present and while only a few community members seem to degrade benzene, the majority of species seems to feed on metabolic left-overs, microbial necromass or even autotrophically using anaerobic ammonium oxidation for free energy transduction and carbon fixation. An additional succession experiment verified that the same few community members are the actual drivers of benzene degradation

A Computational Odyssey into Microbial Communities Across Ecosystems

This thesis uses mainly computational data-driven approaches to study variation, taxonomy and functional characterization, interactions between members and underlying principles that govern microbial communities across ecosystems. The importance to study microbes in the natural context of complex systems of communities was highlighted in Chapter 1. In Chapter 2 we studied the variation of the microbial community composition during spontaneous in vitro wine fermentation of riesling must. We made the following observations: (i) There is a general influence of the vineyard on microbial composition with a striking differential abundance of Metschnikowia. (ii) There is a decrease in biodiversity during alcoholic fermentation. Unexpectedly, the fraction of Micrococcus increased in one vineyard during alcoholic fermentation. (iii) There is a relation between stuck fermentations and the abundance of Starmerella. In Chapter 3, we continued to explore microbial communities during wine fermentation. Specifically, the role of the adjunct Lactobacillus plantarum in the malolactic conversion of industrial wine fermentation was investigated and this species was found to thrive better on white than on red wine fermentations. We obtained experimental evidence to support the hypothesis that a successful introduction of this species in a community was in the case of wine determined by the composition of the must, and possibly by the presence of grape skins during fermentation. In Chapter 5 a simple experimental coloring method is presented which distinguishes colonies of the yeasts Lachancea thermotolerans and Saccharomyces cerevisiae on agar media. It does so by the addition of bromocresol purple which induces Lachancea colonies to develop a brown color, whereas Saccharomyces colonies remain white. In Chapter 3 we also studied a kefir community using genome sequences of isolated and sequenced Bacteria representative strains. We found that Lactobacillus kefiranofaciens, a dominant organism in kefir, stands out among the lactobacilli because it potentially has a high number of amino acid auxotrophies. Also, the only organism in kefir that had genes for flagellar assembly and chemotaxis was Acetobacter. The presence of flagella in Acetobacter was experimentally confirmed. In Chapter 4 we study pairwise interactions between microbes using measures based on genes involved in metabolic processes. In the case of microbes from the urinary tract, a number of putative metabolic interactions were identified that could explain the experimentally obtained pairwise growth effects. We found that members of Enterococcus may be complemented in their metabolism by the other members of the community. In Chapter 6 we investigated the metabolism of a novel Mycobacterium species, which was found to be dominant in a microbial community residing in an acidic biofilm attached to the wall of a sulfur cave in Romania. This Mycobacterium species expresses a full suite of enzymes involved in methanotrophic growth. Growth experiments using methane as the sole carbon- and free energy source verify its methanotrophic niche. To our knowledge, this is the first report about a methanotrophic Mycobacterium of Actinobacteria. In Chapter 7 a key question in microbial ecology is asked - how large biodiversity can be maintained on a few resources. To address this question a highly diverse microbial community was investigated, which grew for 15 years in an anoxic bioreactor on benzene as the main carbon and free energy source and nitrate as an electron acceptor. We found evidence that many different niches are present and while only a few community members seem to degrade benzene, the majority of species seems to feed on metabolic left-overs, microbial necromass or even autotrophically using anaerobic ammonium oxidation for free energy transduction and carbon fixation. An additional succession experiment verified that the same few community members are the actual drivers of benzene degradation

Chrats-Melkonian/mi_cheese: Microbial interactions shape cheese flavor formation

<p>Updated code and data accompanying the manuscript <strong>Microbial interactions shape cheese flavor formation</strong></p> <h2>Data</h2> <ul> <li>[x] Replace old models (gapfilled on milk) with updated models sets gapfilled on each media variation + no gapfilling (<a href="https://github.com/Chrats-Melkonian/mi_cheese/commit/699c122d125611035e96ea2040a144f44868bf68">699c122</a>)</li> </ul> <pre><code>├── data │ ├── Models │ │ ├── LC.xml │ │ ├── LLm1.xml │ │ ├── LLm2.xml │ │ ├── ST.xml </code></pre> <h2>Code</h2> <ul> <li><p>[x] Add helper scripts (<a href="https://github.com/Chrats-Melkonian/mi_cheese/commit/935dfb7b8a70b22afb09f877034c56e3de4ace96">935dfb7</a>):</p> <ul> <li>[x] <code>model_summary_generate.py</code> to read models and produce individual summaries</li> <li>[x] <code>model_summary_compile.sh</code> script to compile individual summary files into <code>model_summary.tsv</code> file</li> <li>[x] <code>gf_rxns_compile.sh</code> to read through models and extract gapfilled reaction information into <code>gf_rxns.tsv</code> file</li> </ul> </li> </ul&gt

Chrats-Melkonian/mi_cheese: Microbial interactions shape cheese flavor formation 1.1.1

<p>Updated README.md (#3) and created CITATION.bib file (#4)</p&gt

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