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Displaying One Session

Session Type
Workshop Session
Date
07/20/2022
Session Time
07:00 PM - 08:30 PM
Room
Hall 3
Chair(s)
  • B. Duim (Netherlands)

Gene Expression, Gene Regulation and Development

Session Type
Workshop Session
Date
07/20/2022
Session Time
07:00 PM - 08:30 PM
Room
Hall 3
Presenter
  • D. Mazel (France)
Lecture Time
07:00 PM - 07:25 PM

Abstract

Abstract Body

Genetic Drivers of Chromosomal Integrons Stability

Integrons are mainly known as the genetic agents responsible for the capture and spread of antibiotic resistance determinants among Gram-negative pathogens. They are also are found in the genomes of hundreds of environmental bacterial species, where cassettes convey much broader adaptive functions. These chromosomal integrons are the sources of both the antibiotics resistance cassettes and the integron platforms that convey these cassettes among bacterial pathogens. We are now tackling one central question linked to the integron functioning:

- why is the number of cassettes carried in their arrays so different (hundreds in sedentary integrons vs less than ten in mobile ones)?

I will present the recent results we obtained which allow to give convincing answers to these two questions.

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The Promises and Challenges of Programmable RNA Antibiotics

Session Type
Workshop Session
Date
07/20/2022
Session Time
07:00 PM - 08:30 PM
Room
Hall 3
Presenter
  • J. Vogel
Lecture Time
07:25 PM - 07:50 PM

Abstract

Abstract Body

Our body is colonized by a vast array of bacteria the sum of which forms our microbiota. The gut alone harbors >1,000 bacterial species. An understanding of their individual or synergistic contributions to human health and disease demands means to interfere with their functions on the species level. Most of the currently available antibiotics are broad-spectrum, thus too unspecific for a selective depletion of a single species of interest from the microbiota. Programmable RNA antibiotics in the form of short antisense oligomers (ASOs) promise to achieve precision manipulation of bacterial communities. These ASOs are coupled to small peptides that carry them inside the bacteria to silence mRNAs of essential genes, for example, to target antibiotic-resistant pathogens as an alternative to standard antibiotics. There is already proof-of-principle with diverse bacteria, but many open questions remain with respect to true species specificity, potential off-targeting, choice of peptides for delivery, bacterial resistance mechanisms and the host response. While there is unlikely a one-fits-all solution for all microbiome species, I will discuss how recent progress in bacterial RNA biology may help to accelerate the development of programmable RNA antibiotics for microbiome editing and other applications.

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INVESTIGATION OF THE GLUCOSE-MEDIATED REGULATORY MECHANISM OF TRANSCRIPTIONAL ACTIVATION OF FRUR.

Session Type
Workshop Session
Date
07/20/2022
Session Time
07:00 PM - 08:30 PM
Room
Hall 3
Presenter
  • S. Lee (Korea, Republic of)
Lecture Time
07:50 PM - 08:00 PM

Abstract

Background and Aims

In most bacteria, efficient use of carbohydrates is primarily mediated by the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) which concomitantly phosphorylates the substrates during import. Therefore, transcription of the PTS-encoding genes is precisely regulated by transcriptional regulators, depending on the availability of the substrate. Fructose is transported mainly through the fructose-specific PTS (PTSFru) and simultaneously converted into fructose 1-phosphate (F1P). The PTS-dependent utilization of fructose is inhibited by glucose in Vibrio cholerae, but the underlying mechanism remains unknown.

Methods

RNA extraction and quantitative real-time reverse transcription-PCR (qRT-PCR), Determination of the amount of sugars in the culture medium using High-Performance Liquid Chromatography system, Protein ligand fishing experiment, Protein binding assay using native polyacrylamide gel electrophoresis (native-PAGE), Electrophoretic mobility shift assay (EMSA), DNase I footprinting

Results

We previously show that, FruR acts as a transcriptional activator of the fru operon and is indispensable for the growth of Vibrio cholerae on fructose. Several lines of evidence suggest that binding of the FruR-F1P complex to an operator which is located between the –35 and –10 promoter elements changes the DNA structure to facilitate RNA polymerase binding to the fru promoter. Here, we investigate the regulatory mechanism by which glucose inhibits the FruR-mediated transcriptional activation of the fru operon.

Conclusions

We found that the fructose operon transcription was inhibited when cultured in glucose and fructose. This results in aberrant transcription of the genes encoding the fructose-specific PTS components. Through this results, we were able to confirm the carbon catabolite repression mechanism between PTS sugars.

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INTERACTIONS BETWEEN STREPTOCOCCUS THERMOPHILUS AND LACTOCOCCUS LACTIS STRAINS SHAPE THE FLAVOR PROFILE OF CHEESE

Session Type
Workshop Session
Date
07/20/2022
Session Time
07:00 PM - 08:30 PM
Room
Hall 3
Presenter
  • C. Melkonian (Netherlands)
Lecture Time
08:00 PM - 08:10 PM

Abstract

Background and Aims

The extent to which microbial interactions play a role in the production of cheese is still an open question.

Methods

We used a strategy where one of the components of the original culture have been left out. We monitored the microbial community dynamics and metabolic changes during a year-long Cheddar cheese-making experiment. Additionally, we performed a controlled milk fermentation experiment using the same culture. We devised an approach that combined: the analysis of the microbial genomes, the construction and analysis of their genome-scale metabolic models, the analysis of the metatranscriptomes across the leave out conditions and the quantification of key metabolites.

Results

A significant benefit was observed on the overall L. lactis community population when S. thermophilus was present. Also, the presence of S. thermophilus strain was found to have an effect on the final metabolic profile of the cheeses. We obtained evidence that S. thermophilus may relieve the nitrogen limitation of the L. lactis community, which is necessary for de novo nucleotide biosynthesis. Interestingly, closely related strains of L. lactis subspecies lactis exhibited different interaction patterns with S. thermophilus, highlighting the importance of strain specificity. Also, we observed increased accumulation of key metabolites, such as diacetyl and acetoin, when the major L. lactis ssp. cremoris strain was left out. Based on experimental evidence, we hypothesize that this is due to competition between L. lactis strains for the available citrate.

Conclusions

We show a range of novel interactions both competitive and cooperative that take place to shape the flavor profile of the cheese.

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POSITIVE FEEDBACK, AND NOT INDUCER EXCLUSION, DRIVES MOST OF THE GLUCOSE-MEDIATED CATABOLITE REPRESSION OF THE LAC OPERON OF ESCHERICHIA COLI

Session Type
Workshop Session
Date
07/20/2022
Session Time
07:00 PM - 08:30 PM
Room
Hall 3
Presenter
  • R. K. Aggarwal (United States of America)
Lecture Time
08:10 PM - 08:20 PM

Abstract

Background and Aims

Glucose-mediated lac repression in Escherichia coli is a classical problem in bacterial physiology. It is widely believed this repression is due to CRP-mediated transcriptional inhibition and PTS-mediated LacY inhibition (inducer exclusion). In 1996, Aiba and coworkers showed that CRP-mediated effect is weak, and hypothesised that the repression must be due to inducer exclusion (1).

Methods

To test this hypothesis, we measured the magnitude of inducer exclusion in E. coli cells induced to various levels. We found that LacY was never repressed more than ~5-fold, which is ~200-fold smaller than the observed ~1000-fold lac repression observed in presence of glucose. To find the source of this discrepancy, we measured the LacZ activities and intracellular inducer levels observed upon addition of glucose to E. coli cultures growing in the presence of lactose as well as TMG (2, 3).

Results

These transient measurements show that inducer exclusion accounts only for the initial ~5-fold repression that occurs within the first 5 min of glucose addition. The subsequent ~200-fold repression that occurs over several hours is due to amplification of the repression by positive feedback that is generated because the lactose enzymes promote the accumulation of the inducer, which in turn induces the synthesis of even more enzymes.

Conclusions

References

1. Inada, T., Kimata, K. and Aiba, H. (1996), Genes to Cells, 1(3), pp. 293–301.

2. Aggarwal, R. K. and Narang, A. (2022), Biophysical Journal. Cell Press, 121(5), pp. 820–829.

3. Aggarwal, R. K. and Narang, A. (2022), Biophysical Journal. Cell Press, 121(5), pp. 808–819.

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THE IMPACT OF CIPROFLOXACIN TREATMENT ON MICROBIAL AND METABOLIC REGIME SHIFTS IN A FERMENTATIVE MIXED CULTURE

Session Type
Workshop Session
Date
07/20/2022
Session Time
07:00 PM - 08:30 PM
Room
Hall 3
Presenter
  • D. G. Weissbrodt (Netherlands)
Lecture Time
08:20 PM - 08:30 PM

Abstract

Background and Aims

Little is known about the effects of antibiotics on microbial communities. We analysed the impact of the broad-spectrum antibiotic ciprofloxacin on the microbial composition and metabolic functionality of a fermentative mixed culture.

Methods

A 0.75-L chemostat was sterilized, inoculated with standardized healthy-donor human faeces, and operated (dilution rate 0.16 h-1) for glucose fermentation in mixed culture. After establishing the fermentative baseline, ciprofloxacin was administered over 3 days, prior to letting the bioreactor recover after treatment. Switches in product spectrum and rates, microbial selection, and metabolic regulation were tracked by on-line monitoring, liquid chromatography, metagenomics, and metaproteomics.

Results

The short-term impact of ciprofloxacin was profound. Fermentative bacteria (Lactobacillus, Enterobacter, Enterococcus) were outcompeted by the yeast Candida albicans (30% of DNA and protein read counts). Bacterial growth was not completely inhibited. The treatment resulted in a metabolic switch in fermentation product fluxes from mainly lactate (0.20±0.04 mol·h-1·C-molx-1) to ethanol and CO2 (both 0.22 mol·h-1·C-molx-1). This matched with the emergence of Candida and the increased expression of key proteins associated with ethanol production (e.g., alcohol and acetaldehyde dehydrogenases, pyruvate decarboxylase). The bacterial community and lactate production recovered to baseline level within 3 days after treatment, although instabilities persisted over the two-week recovery period.

Conclusions

Quantitative biotechnology and multi-omics helped identify the predominant regime shifts under ciprofloxacin administration in fermentative mixed culture derived from human faeces. Our findings provide a deeper understanding of microbial and metabolic perturbations, and antibiotic resistance development, upon antibiotic treatment of fermentative mixed cultures like gut microbiota.

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