Population heterogeneity and dynamics in starter culture and lag phase adaptation of the spoilage yeast Zygosaccharomyces bailii to weak acid preservatives

The food spoilage yeast Zygosaccharomyces bailii shows great resistance to weak-acid preservatives, including sorbic acid (2, 4-hexadienoic acid). That extreme resistance was shown to be due to population heterogeneity, with a small sub-population of cells resistant to a variety of weak acids, probably caused by a lower internal pH reducing the uptake of all weak acids. In the present paper, it was found that resistant cells were extremely rare in exponential cultures, but increased by up to 8000-fold in stationary phase. Inoculation of media containing sorbic acid with a population of Z. bailii cells gave rise to what appeared to be a prolonged lag phase, suggesting adaptation to the conditions before the cells entered the period of exponential growth. However, the apparent lag phase caused by sorbic acid was largely due to the time required for the resistant sub-population to grow to detectable levels. The slow growth rate of the sub-population was identical to that of the final total population. The non-resistant bulk population remained viable for 3. days but had lost viability by 6. days and, during that time, there was no indication of any development of resistance in the bulk population. The sub-population growing in sorbic acid showed very high population diversity in colony size and internal pH. After removal of sorbic acid, the population rapidly reverted back to the normal, largely non-resistant, population distribution. The data presented suggest that a reevaluation of the lag phase in microbial batch culture is required, at least for the resistance of Z. bailii to sorbic acid. Furthermore, the significance of phenotypic diversity and heterogeneity in microbial populations is discussed more broadly with potential relevance to bacterial "persisters", natural selection and evolution. © 2014

Transcriptional landscape of Aspergillus niger at breaking of conidial dormancy revealed by RNA-sequencing

Background Genome-wide analysis was performed to assess the transcriptional landscape of germinating A. niger conidia using both next generation RNA-sequencing and GeneChips. The metabolism of storage compounds during conidial germination was also examined and compared to the transcript levels from associated genes. Results The transcriptome of dormant conidia was shown to be highly differentiated from that of germinating conidia and major changes in response to environmental shift occurred within the first hour of germination. The breaking of dormancy was associated with increased transcript levels of genes involved in the biosynthesis of proteins, RNA turnover and respiratory metabolism. Increased transcript levels of genes involved in metabolism of nitrate at the onset of germination implies its use as a source of nitrogen. The transcriptome of dormant conidia contained a significant component of antisense transcripts that changed during germination. Conclusion Dormant conidia contained transcripts of genes involved in fermentation, gluconeogenesis and the glyoxylate cycle. The presence of such transcripts in dormant conidia may indicate the generation of energy from non-carbohydrate substrates during starvation-induced conidiation or for maintenance purposes during dormancy. The immediate onset of metabolism of internal storage compounds after the onset of germination, and the presence of transcripts of relevant genes, suggest that conidia are primed for the onset of germination. For some genes, antisense transcription is regulated in the transition from resting conidia to fully active germinants

Extreme osmotolerance and halotolerance in food-relevant yeasts and the role of glycerol-dependent cell individuality

Osmotolerance and halotolerance are used to describe resistance to sugars and salt, respectively. Here, a comprehensive screen of more than 600 different yeast isolates revealed that osmosensitive species were equally affected by NaCl and glucose. However, the relative toxicity of salt became increasingly prominent in more osmoresistant species. We confirmed that growth inhibition by glucose in a laboratory strain of Saccharomyces cerevisiae occurred at a lower water activity (Aw) than by salt (NaCl), and pre-growth in high levels of glucose or salt gave enhanced cross-resistance to either. Salt toxicity was largely due to osmotic stress but with an additive enhancement due to effects of the relevant cation. Almost all of the yeast isolates from the screen were also noted to exhibit hetero-resistance to both salt and sugar, whereby high concentrations restricted growth to a small minority of cells within the clonal populations. Rare resistant colonies required growth for up to 28 days to become visible. This cell individuality was more marked with salt than sugar, a possible further reflection of the ion toxicity effect. In both cases, heteroresistance in S. cerevisiae was strikingly dependent on the GPD1 gene product, important for glycerol synthesis. In contrast, a tps1? deletant impaired for trehalose showed altered MIC but no change in heteroresistance. Effects on heteroresistance were evident in chronic (but not acute) salt or glucose stress, particularly relevant to growth on low Aw foods. The study reports diverse osmotolerance and halotolerance phenotypes and heteroresistance across an extensive panel of yeast isolates, and indicates that Gpd1-dependent glycerol synthesis is a key determinant enabling growth of rare yeast subpopulations at low Aw, brought about by glucose and in particular salt

Weak Acid Resistance A (WarA), a Novel Transcription Factor Required for Regulation of Weak-Acid Resistance and Spore-Spore Heterogeneity in Aspergillus niger

Copyright © 2020 Geoghegan et al. Propionic, sorbic, and benzoic acids are organic weak acids that are widely used as food preservatives, where they play a critical role in preventing microbial growth. In this study, we uncovered new mechanisms of weak-acid resistance in molds. By screening a library of 401 transcription factor deletion strains in Aspergillus fumigatus for sorbic acid hypersensitivity, a previously uncharacterized transcription factor was identified and named weak acid resistance A (WarA). The orthologous gene in the spoilage mold Aspergillus niger was identified and deleted. WarA was required for resistance to a range of weak acids, including sorbic, propionic, and benzoic acids. A transcriptomic analysis was performed to characterize genes regulated by WarA during sorbic acid treatment in A. niger Several genes were significantly upregulated in the wild type compared with a ΔwarA mutant, including genes encoding putative weak-acid detoxification enzymes and transporter proteins. Among these was An14g03570, a putative ABC-type transporter which we found to be required for weak-acid resistance in A. niger We also show that An14g03570 is a functional homologue of the Saccharomyces cerevisiae protein Pdr12p and we therefore name it PdrA. Last, resistance to sorbic acid was found to be highly heterogeneous within genetically uniform populations of ungerminated A. niger conidia, and we demonstrate that pdrA is a determinant of this heteroresistance. This study has identified novel mechanisms of weak-acid resistance in A. niger which could help inform and improve future food spoilage prevention strategies.IMPORTANCE Weak acids are widely used as food preservatives, as they are very effective at preventing the growth of most species of bacteria and fungi. However, some species of molds can survive and grow in the concentrations of weak acid employed in food and drink products, thereby causing spoilage with resultant risks for food security and health. Current knowledge of weak-acid resistance mechanisms in these fungi is limited, especially in comparison to that in yeasts. We characterized gene functions in the spoilage mold species Aspergillus niger which are important for survival and growth in the presence of weak-acid preservatives. Such identification of weak-acid resistance mechanisms in spoilage molds will help in the design of new strategies to reduce food spoilage in the future

Metabolic activity in dormant conidia ofAspergillus nigerand developmental changes during conidial outgrowth

The early stages of development of Aspergillus niger conidia during outgrowth were explored by combining genome-wide gene expression analysis (RNAseq), proteomics, Warburg manometry and uptake studies. Resting conidia suspended in water were demonstrated for the first time to be metabolically active as low levels of oxygen uptake and the generation of carbon dioxide were detected, suggesting that low-level respiratory metabolism occurs in conidia for maintenance. Upon triggering of spore germination, generation of CO2 increased dramatically. For a short period, which coincided with mobilisation of the intracellular polyol, trehalose, there was no increase in uptake of O2 indicating that trehalose was metabolised by fermentation. Data from genome-wide mRNA profiling showed the presence of transcripts associated with fermentative and respiratory metabolism in resting conidia. Following triggering of conidial outgrowth, there was a clear switch to respiration after 25 min, confirmed by cyanide inhibition. No effect of SHAM, salicylhydroxamic acid, on respiration suggests electron flow via cytochrome c oxidase. Glucose entry into spores was not detectable before 1 h after triggering germination. The impact of sorbic acid on germination was examined and we showed that it inhibits glucose uptake. O2 uptake was also inhibited, delaying the onset of respiration and extending the period of fermentation. In conclusion, we show that conidia suspended in water are not completely dormant and that conidial outgrowth involves fermentative metabolism that precedes respiration

Retinoic acid is required for the initiation of outgrowth in the chick limb bud

AbstractBackground Retinoic acid (RA) is present in the chick limb bud, and excess RA induces limb duplications. Here, we have investigated the role of endogenous RA during chick limb development by preventing the synthesis of RA and testing the effect on various genes expressed during limb initiation and outgrowth.Results We demonstrate that the stage 20/21 limb bud synthesizes didehydroretinoic acid (ddRA), and that the posterior half of the limb bud synthesizes ddRA at a higher rate than the anterior half. Disulphiram inhibits this synthesis at micromolar concentrations. Administering disulphiram to embryos prior to limb bud outgrowth (stages 12–18) abolishes outgrowth, and no limb develops in the majority of cases. Disulphiram treatment also prevents the expression of Sonic hedgehog (Shh), but the expression of the fibroblast growth factor-8 gene (Fgf-8) appears as normal in the ectoderm over the prospective limb bud. The application of a bead soaked in RA can rescue Shh expression. Disulphiram treatment of later limb buds (stages 20–23) similarly down-regulates Shh, and also Fgf-4, expression, whereas the expression of Fgf-8, as at earlier stages, is initially unaffected. Again, RA can rescue the expression of Shh in these limb buds.Conclusions RA, in conjunction with Fgf-8, may be needed for the induction of the chick limb bud and the induction of Shh and Fgf-4 expression. The expression of Shh and Fgf-4 remains dependent upon the continued synthesis of RA within the limb bud. Didehydroretinoic acid is the major active retinoid in the stage 20 chick limb bud

The Weak-Acid Preservative Sorbic Acid Is Decarboxylated and Detoxified by a Phenylacrylic Acid Decarboxylase, PadA1, in the Spoilage Mold Aspergillus niger▿ †

Resistance to sorbic and cinnamic acids is mediated by a phenylacrylic acid decarboxylase (PadA1) in Aspergillus niger. A. niger ΔpadA1 mutants are unable to decarboxylate sorbic and cinnamic acids, and the MIC of sorbic acid required to inhibit spore germination was reduced by ∼50% in ΔpadA1 mutants

Understanding the hydrochemical evolution of a coastal dune system in SW England using a multiple tracer technique

An improved knowledge of the hydrology of coastal dune systems is desirable for successful management of their diverse ecology under a changing climate. As a near-pristine coastal dune spit system, Braunton Burrows (SW England) is an ideal location for the study of the natural processes governing recharge to the dune groundwater system and the evolution of its water quality. Whereas previous investigations have tended to focus on inter-dune slacks, this study has also given attention to infiltration through the high dunes. Cores were taken through dunes and the resulting sand samples processed to provide information on grain size distribution and porewater chemistry. Groundwater samples were obtained from beneath dunes and slacks. A variety of geochemical techniques were applied including hydrochemistry, stable isotopes and residence time indicators. The unsaturated zone profiles indicate the existence of piston flow recharge with an infiltration rate of 0.75–1 m/yr, although faster rates probably also occur locally. Groundwater beneath the high dunes gave ages in the range 13–16 yr, compared to the dune slack groundwater ages of 5–7 yr, and an age of 22 yr for groundwater from the underlying mudstone aquifer. The chemistry of waters in both unsaturated and saturated zones is dominated by Ca and HCO3, supplemented by variable amounts of other ions derived from marine aerosols and limited reaction with sand grains and their coatings. The main chemical evolution of the porewaters occurs rapidly through the mobilisation of surface salt crusts and dissolution of shell carbonate. This situation changes little in the underlying groundwater, though an evolution towards reducing conditions increases the concentrations of redox-sensitive species such as Fe and Mn. The rapid chemical evolution of the infiltrating water means that its composition will respond quickly to changes in the supply of shell material and/or marine salts, which are possible consequences of climate change. However, the residence time measurements suggest the dune aquifer has a relatively long turnover time which will to some extent buffer such changes. The results of the present study should be transferable to natural dune systems in similar coastal situations