Advance online publication:

This section includes articles accepted for publication in Microbial Cell, which have not been released in a regular issue, yet. Please note that the PDF versions of advance publication articles are generally paginated starting with page 1. This does not correspond to the final pagination upon release of the issue it will appear in.

 

Barcode sequencing and a high-throughput assay for chronological lifespan uncover ageing-associated genes in fission yeast

Catalina A. Romila, StJohn Townsend, Michal Malecki, Stephan Kamrad, María Rodríguez-López, Olivia Hillson, Cristina Cotobal, Markus Ralser and Jürg Bähler

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Ageing-related processes are largely conserved, with simple organisms remaining the main platform to discover and dissect new ageing-associated genes. Yeasts provide potent model systems to study cellular ageing owing their amenability to systematic functional assays under controlled conditions. Even with yeast cells, however, ageing assays can be laborious and resource-intensive. Here we present improved experimental and computational methods to study chronological lifespan in Schizosaccharomyces pombe. We decoded the barcodes for 3206 mutants of the latest gene-deletion library, enabling the parallel profiling of ~700 additional mutants compared to previous screens. We then applied a refined method of barcode sequencing (Bar-seq), addressing technical and statistical issues raised by persisting DNA in dead cells and sampling bottlenecks in aged cultures, to screen for mutants showing altered lifespan during stationary phase. This screen identified 341 long-lived mutants and 1246 short-lived mutants which point to many previously unknown ageing-associated genes, including 46 conserved but entirely uncharacterized genes. The ageing-associated genes showed coherent enrichments in processes also associated with human ageing, particularly with respect to ageing in non-proliferative brain cells. We also developed an automated colony-forming unit assay to facilitate medium- to high-throughput chronological-lifespan studies by saving time and resources compared to the traditional assay. Results from the Bar-seq screen showed good agreement with this new assay. This study provides an effective methodological platform and identifies many new ageing-associated genes as a framework for analysing cellular ageing in yeast and beyond.

PDF | Published online: 10/05/2021 | In press

DNA polymerase III protein, HolC, helps resolve replication/transcription conflicts

Susan T. Lovett

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In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the processivity clamp and clamp loader complex. The holC gene encodes an accessory protein (known as χ) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability although mutants show growth impairment, genetic instability and sensitivity to DNA damaging agents. In this study we isolate spontaneous suppressor mutants in a holC∆ strain and identify these by whole genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, and of sspA, stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (β) and RpoC (β’) RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA, rpoC and sspA. An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose that transcription complexes block replication in holC mutants and Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.

PDF | Published online: 06/05/2021 | In press

Proanthocyanidin-enriched cranberry extract induces resilient bacterial community dynamics in a gnotobiotic mouse model

Catherine C. Neto, Benedikt M. Mortzfeld, John R. Turbitt, Shakti K. Bhattarai, Vladimir Yeliseyev, Nicholas DiBenedetto, Lynn Bry and Vanni Bucci

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Cranberry consumption has numerous health benefits, with experimental reports showing its anti-inflammatory and anti-tumor properties. Importantly, microbiome research has demonstrated that the gastrointestinal bacterial community modulates host immunity, raising the question of whether the cranberry-derived effect may be related to its ability to modulate the microbiome. Only a few studies have investigated the effect of cranberry products on the microbiome to date. Especially because cranberries are rich in dietary fibers, the extent of microbiome modulation by polyphenols, particularly proanthocyanidins (PACs), remains to be shown. Since previous work has only focused on long-term effects of cranberry extracts, in this study we investigated the effect of a water-soluble, PAC-rich cranberry juice extract (CJE) on the short-term dynamics of a human-derived bacterial community in a gnotobiotic mouse model. CJE characterization revealed a high enrichment in PACs (57%), the highest ever utilized in a microbiome study. In a 37-day experiment with a ten-day CJE intervention and 14-day recovery phase, we profiled the microbiota via 16S rRNA sequencing and applied diverse time-series analytics methods to identify individual bacterial responses. We show that daily administration of CJE induces distinct dynamic patterns in bacterial abundances during and after treatment, before recovering resiliently to pre-treatment levels. Specifically, we observed an increase of Akkermansia muciniphila and Clostridium hiranonis at the expense of Bacteroides ovatus after the offset of the selection pressure imposed by the PAC-rich CJE. This demonstrates that termination of an intervention with a cranberry product can induce changes of a magnitude as high as the intervention itself.

PDF | Supplemental Information | Published online: 29/04/2021 | In press

Mechanisms underlying lactic acid tolerance and its influence on lactic acid production in Saccharomyces cerevisiae

Arne Peetermans, María R. Foulquié-Moreno and Johan M. Thevelein

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One of the major bottlenecks in lactic acid production using microbial fermentation is the detrimental influence lactic acid accumulation poses on the lactic acid producing cells. The accumulation of lactic acid results in many negative effects on the cell such as intracellular acidification, anion accumulation, membrane perturbation, disturbed amino acid trafficking, increased turgor pressure, ATP depletion, ROS accumulation, metabolic dysregulation and metal chelation. In this review, the manner in which Saccharomyces cerevisiae deals with these issues will be discussed extensively not only for lactic acid as a singular stress factor but also in combination with other stresses. In addition, different methods to improve lactic acid tolerance in S. cerevisiae using targeted and non-targeted engineering methods will be discussed.

PDF | Published online: 14/04/2021 | In press

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