Exploring the resilience and stability of a defined human gut microbiota consortium: An isothermal microcalorimetric study.

The gut microbiota significantly contributes to human health and well-being. The aim of this study was to evaluate the stability and resilience of a consortium composed of three next-generation probiotics (NGPs) candidates originally found in the human gut. The growth patterns of Akkermansia muciniphila, Bacteroides thetaiotaomicron, and Faecalibacterium prausnitzii were studied both individually and consortium. The growth kinetics of Akkermansia muciniphila (A. muciniphila), Bacteroides thetaiotaomicron (B. thetaiotaomicron), and Faecalibacterium prausnitzii (F. prausnitzii) were characterized both individually and in consortium using isothermal microcalorimetry and 16S ribosomal RNA next-generation sequencing. The consortium reached stability after three passages and demonstrated resilience to changes in its initial composition. The concentration of butyrate produced was nearly twice as high in the consortium compared to the monoculture of F. prausnitzii. The experimental conditions and methodologies used in this article are a solid foundation for developing further complex consortia.

The Quantitative Measurement of Peptidoglycan Components Obtained from Acidic Hydrolysis in Gram-Positive and Gram-Negative Bacteria via Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry.

The high throughput in genome sequencing and metabolic model (MM) reconstruction has democratised bioinformatics approaches such as flux balance analysis. Fluxes' prediction accuracy greatly relates to the deepness of the MM curation for a specific organism starting from the cell composition. One component is the cell wall, which is a functional barrier (cell shape, exchanges) with the environment. The bacterial cell wall (BCW), including its thickness, structure, and composition, has been extensively studied in Escherichia coli but poorly described for other organisms. The peptidoglycan (PG) layer composing the BCW is usually thinner in Gram- bacteria than in Gram+ bacteria. In both bacteria groups, PG is a polymeric mesh-like structure of amino acids and sugars, including N-acetylglucosamine, N-acetylmuramic acid, and amino acids. In this study, we propose a high-throughput method to characterise and quantify PG in Gram-positive and Gram-negative bacteria using acidic hydrolysis and hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS). The method showed a relatively short time frame (11 min analytical run), low inter- and intraday variability (3.2% and 4%, respectively), and high sensitivity and selectivity (limits of quantification in the sub mg/L range). The method was successfully applied on two Gram-negative bacteria (Escherichia coli K12 MG1655, Bacteroides thetaiotaomicron DSM 2079) and one Gram-positive bacterium (Streptococcus salivarius ssp. thermophilus DSM20259). The PG concentration ranged from 1.6% w/w to 14% w/w of the dry cell weight. The results were in good correlation with previously published results. With further development, the PG concentration provided by this newly developed method could reinforce the curation of MM.

Absolute quantification of viable bacteria abundances in food by next-generation sequencing: Quantitative NGS of viable microbes.

Next-generation sequencing (NGS) is an important tool for taxonomical bacteria identification. Recent technological developments have led to its improvement and availability. Despite the undeniable advantages of this approach, it has several limitations and shortcomings. The usual outcome of microbiota sequencing is a relative abundance of bacterial taxa. The information about bacteria viability or enumeration is missing. However, this knowledge is crucial for many applications. In the current study, we elaborated the complete workflow for the absolute quantification of living bacteria based on 16S rRNA gene amplicon sequencing. A fluorescent PMAxx reagent penetrating a damaged cell membrane was used to discriminate between the total and viable bacterial population. Bacteria enumeration was estimated by the spike-in technique or qPCR quantification. For method optimization, twenty bacterial species were taken, and the results of the workflow were validated by widely accepted methodologies: flow cytometry, microbiological plating, and viability-qPCR. Despite the minor discrepancy between all methods used, they all showed compatible results. Finally, we tested the workflow with actual food samples and received a good correlation between the methods regarding the estimation of the number of viable bacteria. Overall, the elaborated and integrated NGS approach could be the next step in perceiving a holistic picture of a sample microbiota.

Detailed analysis of metabolism reveals growth-rate-promoting interactions between Anaerostipes caccae and Bacteroides spp.

INTRODUCTION: Human gut microbiota species which are next-generation probiotics (NGPs) candidates are of high interest as they have shown the potential to treat intestinal inflammation and other diseases. Unfortunately, these species are often not robust enough for large-scale cultivation, especially in maintaining diversity in co-culture production. OBJECTIVES: In this study, we describe interactions between human gut microbiota species in the cultivation process with unique substrates. We also demonstrated that it is possible to change the species ratio in co-culture by changing the ratio of carbon sources. METHODS: We screened 25 different bacterial species based on their metabolic capabilities. After evaluating unique substrate possibilities, we chose Anaerostipes caccae (A. caccae), Bacteroides thetaiotaomicron (B. thetaiotaomicron), and Bacteroides vulgatus (B. vulgatus) as subjects for further study. D-sorbitol, D-xylose, and D-galacturonic acid were selected as substrates for A. caccae, B. thetaiotaomicron, and B. vulgatus respectively. All three species were cultivated as both monocultures and in co-cultures in serial batch fermentations in an isothermal microcalorimeter. RESULTS: Positive interactions were detected between the species in both co-cultures (A. caccae + B. thetaiotaomicron; A. caccae + B. vulgatus) resulting in higher heat production compared to the sum of the monocultures. The same positive cross-feeding interactions took place in larger-scale cultivation experiments. We confirmed acetate and lactate cross-feeding between A. caccae and B. thetaiotaomicron with flux balance analysis (FBA). CONCLUSION: Changing the ratio of the selected carbon sources in the medium changed the species ratio accordingly. Such robustness is the basis for developing more efficient industrial co-culture processes including the production of NGPs.

Amino acids are key substrates to Escherichia coli BW25113 for achieving high specific growth rate.

Studying substrate consumption in nutrient-rich conditions is challenging because often the growth medium includes undefined components like yeast extract or peptone. For clear and consistent results, it is necessary to use defined medium, where substrate utilization can be followed. In the present work, Escherichia coli BW25113 batch growth in a medium supplemented with 20 proteinogenic amino acids and glucose was studied. Focus was on the quantitative differences in substrate consumption and proteome composition between minimal and nutrient-rich medium. In the latter, 72% of carbon used for biomass growth came from amino acids and 28% from glucose. Serine was identified as the most consumed substrate with 41% of total carbon consumption. Proteome comparison between nutrient-rich and minimal medium revealed changes in TCA cycle and acetate producing enzymes that together with extracellular metabolite data pointed to serine being consumed mainly for energy generation purposes. Serine removal from the growth medium decreased specific growth rate by 22%. In addition, proteome comparison between media revealed a large shift in amino acid synthesis and translation related proteins. Overall, this work describes in quantitative terms the batch growth carbon uptake profile and proteome allocation of E. coli BW25113 in minimal and nutrient-rich medium.

Avoiding amino acid depletion in a complex medium results in improved Escherichia coli BW25113 growth.

We studied Escherichia coli BW25113 growth in a complex medium with emphasis on amino acid consumption. The aim was to profile amino acid utilization in acid-hydrolysed casein and a defined nutrient-rich medium and based on these measurements modify the medium for better growth performance. Amino acid depletions in both media caused apparent biomass growth stops that prolonged growth duration. Obtained amino acid consumption values enabled a new defined medium to be formulated, where no growth stops were observed, the specific growth rate was constant, and the provided substrates were fully utilized. Similarly, we modified the acid-hydrolysed casein medium by adding pure amino acids that removed the apparent biomass growth stops. Key to our results was the combination of growth medium analysis and process monitoring data, specifically oxygen partial pressure and produced carbon dioxide that were used to track growth changes. Our findings showed the deficiencies of the nutrient-rich medium and how rational medium design, based on consumption values, removed these shortcomings. The resulting balanced medium gives a high specific growth rate and is suitable for studying E. coli physiology at fast growth.

Proteome reallocation in Escherichia coli with increasing specific growth rate.

Cells usually respond to changing growth conditions with a change in the specific growth rate (µ) and adjustment of their proteome to adapt and maintain metabolic efficiency. Description of the principles behind proteome resource allocation is important for understanding metabolic regulation in response to changing µ. Thus, we analysed the proteome resource allocation dynamics of Escherichia coli into different metabolic processes in response to changing µ. E. coli was grown on minimal and defined rich media in steady state continuous cultures at different µ and characterised combining two LC-MS/MS-based proteomics methods: stable isotope labelling by amino acids in cell culture (SILAC) and intensity based label-free absolute quantification. We detected slowly growing cells investing more proteome resources in energy generation and carbohydrate transport and metabolism whereas for achieving faster growth cells needed to devote most resources to translation and processes closely related to the protein synthesis pipeline. Furthermore, down-regulation of energy generation and carbohydrate metabolism proteins with faster growth displayed very similar expression dynamics with the global transcriptional regulator CRP (cyclic AMP receptor protein), pointing to a dominant protein resource allocating role of this protein. Our data also suggest that acetate overflow may be the result of global proteome resource optimisation as cells saved proteome resources by switching from fully respiratory to respiro-fermentative growth. The presented results give a quantitative overview of how E. coli adjusts its proteome to achieve faster growth and in future could contribute to the design of more efficient cell factories through proteome optimisation.

Coordinated activation of PTA-ACS and TCA cycles strongly reduces overflow metabolism of acetate in Escherichia coli.

Elimination of acetate overflow in aerobic cultivation of Escherichia coli would improve many bioprocesses as acetate accumulation in the growth environment leads to numerous negative effects, e.g. loss of carbon, inhibition of growth, target product synthesis, etc. Despite many years of studies, the mechanism and regulation of acetate overflow are still not completely understood. Therefore, we studied the growth of E. coli K-12 BW25113 and several of its mutant strains affecting acetate-related pathways using the continuous culture method accelerostat (A-stat) at various specific glucose consumption rates with the aim of diminishing acetate overflow. Absolute quantitative exo-metabolome and proteome analyses coupled to metabolic flux analysis enabled us to demonstrate that onset of acetate overflow can be postponed and acetate excretion strongly reduced in E. coli by coordinated activation of phosphotransacetylase-acetyl-CoA synthetase (PTA-ACS) and tricarboxylic acid (TCA) cycles. Fourfold reduction of acetate excretion (2 vs. 8 % from total carbon) at fastest growth compared to wild type was achieved by deleting the genes responsible for inactivation of acetyl-CoA synthetase protein (pka) and TCA cycle regulator arcA. The Δpka ΔarcA strain did not accumulate any other detrimental by-product besides acetate and showed identical µ max and only ~5 % lower biomass yield compared to wild type. We conclude that a fine-tuned coordination between increasing the recycling capabilities of acetate in the PTA-ACS node through a higher concentration of active acetate scavenging Acs protein and downstream metabolism throughput in the TCA cycle is necessary for diminishing overflow metabolism of acetate in E. coli and achieving higher target product production in bioprocesses.

Stock culture heterogeneity rather than new mutational variation complicates short-term cell physiology studies of Escherichia coli K-12 MG1655 in continuous culture.

Nutrient-limited continuous cultures in chemostats have been used to study microbial cell physiology for over 60 years. Genome instability and genetic heterogeneity are possible uncontrolled factors in continuous cultivation experiments. We investigated these issues by using high-throughput (HT) DNA sequencing to characterize samples from different phases of a glucose-limited accelerostat (A-stat) experiment with Escherichia coli K-12 MG1655 and a duration regularly used in cell physiology studies (20 generations of continuous cultivation). Seven consensus mutations from the reference sequence and five subpopulations characterized by different mutations were detected in the HT-sequenced samples. This genetic heterogeneity was confirmed to result from the stock culture by Sanger sequencing. All the subpopulations in which allele frequencies increased (betA, cspG/cspH, glyA) during the experiment were also present at the end of replicate A-stats, indicating that no new subpopulations emerged during our experiments. The fact that ~31 % of the cells in our initial cultures obtained directly from a culture stock centre were mutants raises concerns that even if cultivations are started from single colonies, there is a significant chance of picking a mutant clone with an altered phenotype. Our results show that current HT DNA sequencing technology allows accurate subpopulation analysis and demonstrates that a glucose-limited E. coli K-12 MG1655 A-stat experiment with a duration of tens of generations is suitable for studying cell physiology and collecting quantitative data for metabolic modelling without interference from new mutations.

Multi-omics approach to study the growth efficiency and amino acid metabolism in Lactococcus lactis at various specific growth rates.

BACKGROUND: Lactococcus lactis is recognised as a safe (GRAS) microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth. RESULTS: Using glucose limited continuous cultivations, specific growth rate dependent metabolism of L. lactis including utilization of amino acids was studied based on extracellular metabolome, global transcriptome and proteome analysis. A new growth medium was designed with reduced amino acid concentrations to increase precision of measurements of consumption of amino acids. Consumption patterns were calculated for all 20 amino acids and measured carbon balance showed good fit of the data at all growth rates studied. It was observed that metabolism of L. lactis became more efficient with rising specific growth rate in the range 0.10-0.60 h(-1), indicated by 30% increase in biomass yield based on glucose consumption, 50% increase in efficiency of nitrogen use for biomass synthesis, and 40% reduction in energy spilling. The latter was realized by decrease in the overall product formation and higher efficiency of incorporation of amino acids into biomass. L. lactis global transcriptome and proteome profiles showed good correlation supporting the general idea of transcription level control of bacterial metabolism, but the data indicated that substrate transport systems together with lower part of glycolysis in L. lactis were presumably under allosteric control. CONCLUSIONS: The current study demonstrates advantages of the usage of strictly controlled continuous cultivation methods combined with multi-omics approach for quantitative understanding of amino acid and energy metabolism of L. lactis which is a valuable new knowledge for development of balanced growth media, gene manipulations for desired product formation etc. Moreover, collected dataset is an excellent input for developing metabolic models.