scholarly journals Weak Acid Resistance A (WarA), a novel transcription factor required for regulation of weak-acid resistance and spore-spore heterogeneity in Aspergillus niger

2019 ◽  
Author(s):  
Ivey A. Geoghegan ◽  
Malcolm Stratford ◽  
Mike Bromley ◽  
David B. Archer ◽  
Simon V. Avery
Keyword(s):  
Transcription Factor ◽  
Aspergillus Niger ◽  
Sorbic Acid ◽  
Acid Resistance ◽  
Resistance Mechanisms ◽  
Weak Acid ◽  
Benzoic Acids ◽  
Food Spoilage ◽  
Weak Acids ◽  
Food Preservatives

ABSTRACTPropionic, 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 moulds. By screening a library of 401 transcription-factor deletion strains in Aspergillus fumigatus for sorbic acid hypersensitivity, a previously uncharacterised transcription factor was identified, and named as WarA (Weak Acid Resistance A). The orthologous gene in the spoilage mould 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 characterise 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 therefore named as PdrA. Lastly, 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 to inform and improve future food spoilage prevention strategies.IMPORTANCEWeak acids are widely used as food preservatives, as they are very effective at preventing growth of most species of bacteria and fungi. However, some species of moulds 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 characterised gene functions in the spoilage mould 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 moulds will help to design new strategies to reduce food spoilage in the future.

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

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Ivey A. Geoghegan ◽  
Malcolm Stratford ◽  
Mike Bromley ◽  
David B. Archer ◽  
Simon V. Avery
Keyword(s):  
Transcription Factor ◽  
Aspergillus Niger ◽  
Sorbic Acid ◽  
Acid Resistance ◽  
Resistance Mechanisms ◽  
Weak Acid ◽  
Benzoic Acids ◽  
Weak Acids ◽  
Food Preservatives ◽  
Content Type

ABSTRACT 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.

scholarly journals The SPI1 Gene, Encoding a Glycosylphosphatidylinositol-Anchored Cell Wall Protein, Plays a Prominent Role in the Development of Yeast Resistance to Lipophilic Weak-Acid Food Preservatives

2006 ◽  
Vol 72 (11) ◽  
pp. 7168-7175 ◽  
Author(s):  
T. Simões ◽  
N. P. Mira ◽  
A. R. Fernandes ◽  
Isabel Sá-Correia
Keyword(s):  
Cell Wall ◽  
Transcription Factor ◽  
Acetic Acid ◽  
Benzoic Acid ◽  
Acid Stress ◽  
Weak Acid ◽  
Cell Wall Protein ◽  
Weak Acids ◽  
Food Preservatives ◽  
Cell Wall Porosity

ABSTRACT The Saccharomyces cerevisiae SPI1 gene encodes a member of the glycosylphosphatidylinositol-anchored cell wall protein family. In this work we show results indicating that SPI1 expression protects the yeast cell from damage caused by weak acids used as food preservatives. This is documented by a less extended period of adaptation to growth in their presence and by a less inhibited specific growth rate for a parental strain compared with a mutant with SPI1 deleted. Maximal protection exerted by Spi1p against equivalent concentrations of the various weak acids tested was registered for the more lipophilic acids (octanoic acid, followed by benzoic acid) and was minimal for acetic acid. Weak-acid adaptation was found to involve the rapid activation of SPI1 transcription, which is dependent on the presence of the Msn2p transcription factor. Activation of SPI1 transcription upon acetic acid stress also requires Haa1p, whereas this recently described transcription factor has a negligible role in the adaptive response to benzoic acid. The expression of SPI1 was found to play a prominent role in the development of yeast resistance to 1,3-β-glucanase in benzoic acid-stressed cells, while its involvement in acetic acid-induced resistance to the cell wall-lytic enzyme is slighter. The results are consistent with the notion that Spi1p expression upon weak-acid stress leads to cell wall remodeling, especially for the more lipophilic acids, decreasing cell wall porosity. Decreased cell wall porosity, in turn, reduces access to the plasma membrane, reducing membrane damage, intracellular acidification, and viability loss.

scholarly journals The Weak Acid Preservative Sorbic Acid Inhibits Conidial Germination and Mycelial Growth of Aspergillus niger through Intracellular Acidification

2004 ◽  
Vol 70 (6) ◽  
pp. 3506-3511 ◽  
Author(s):  
Andrew Plumridge ◽  
Stephan J. A. Hesse ◽  
Adrian J. Watson ◽  
Kenneth C. Lowe ◽  
Malcolm Stratford ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Mycelial Growth ◽  
Sorbic Acid ◽  
Weak Acid ◽  
Inoculum Size ◽  
Food Preservation ◽  
Rapid Decline ◽  
Intracellular Acidification ◽  
Ph Homeostasis ◽  
Cytosolic Ph

ABSTRACT The growth of the filamentous fungus Aspergillus niger, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (trans-trans-2,4-hexadienoic acid). Conidia inoculated at 105/ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size. A. niger is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pHcyt) by more than 1 pH unit and a depression of vacuolar pH (pHvac) in A. niger occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pHcyt. NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of A. niger mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth.

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

2007 ◽  
Vol 74 (2) ◽  
pp. 550-552 ◽  
Author(s):  
Andrew Plumridge ◽  
Malcolm Stratford ◽  
Kenneth C. Lowe ◽  
David B. Archer
Keyword(s):  
Aspergillus Niger ◽  
Spore Germination ◽  
Sorbic Acid ◽  
Weak Acid ◽  
Acid Decarboxylase ◽  
Cinnamic Acids ◽  
Inhibit Spore Germination

ABSTRACT 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.

scholarly journals Weak acid and alkali stress regulate phosphatidylinositol bisphosphate synthesis in Saccharomyces cerevisiae

2006 ◽  
Vol 395 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Mehdi Mollapour ◽  
John P. Phelan ◽  
Stefan H. Millson ◽  
Peter W. Piper ◽  
Frank T. Cooke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Responses ◽  
Acid Stress ◽  
Weak Acid ◽  
Weak Acids ◽  
Extracellular Medium ◽  
Food Preservatives ◽  
Active Efflux ◽  
Signalling Molecules ◽  
Spoilage Yeasts

Weak organic acids are used as food preservatives to inhibit the growth of spoilage yeasts, including Saccharomyces cerevisiae. Long-term adaptation to weak acids requires the increased expression of the ATP-binding cassette transporter Pdr12p, which catalyses the active efflux of the weak acids from the cytosol; however, very little is known about the signalling events immediately following application of weak acid stress. We have investigated the effects of weak acids on two stress-responsive signalling molecules, PtdIns(3,5)P2 and PtdIns(4,5)P2, which in S. cerevisiae are synthesized by Fab1p and Mss4p respectively. At low extracellular pH, benzoic acid, sorbic acid and acetic acid all cause a transient reduction in PtdIns(3,5)P2 accumulation and a more persistent rise in PtdIns(4,5)P2 levels. The increase in PtdIns(4,5)P2 levels is accompanied by a reorganization of the actin cytoskeleton. However, changes in PtdInsP2 levels are independent of weak acid-induced Pdr12p expression. In contrast, changing the extracellular medium to alkaline pH provokes a prolonged and substantial rise in PtdIns(3,5)P2 levels. As PtdIns(3,5)P2 synthesis is required for correct vacuole acidification, it is possible that levels of this molecule are modulated to maintain intracellular pH homoeostasis in response to weak acid and alkali stresses. In conclusion, we have expanded the repertoire of stress responses that affect PtdInsP2 levels to include weak acid and alkali stresses.

scholarly journals The preservative sorbic acid targets respiration, explaining the resistance of fermentative spoilage-yeast species

2020 ◽  
Author(s):  
M. Stratford ◽  
C. Vallières ◽  
I.A. Geoghegan ◽  
D.B. Archer ◽  
S.V. Avery
Keyword(s):  
Sorbic Acid ◽  
Yeast Species ◽  
Sugar Content ◽  
Acid Resistance ◽  
Rhodotorula Glutinis ◽  
Weak Acid ◽  
Soft Drinks ◽  
Effective Control ◽  
Spoilage Yeast ◽  
Spoilage Yeasts

ABSTRACTA small number (10-20) of yeast species cause major spoilage in foods. Spoilage yeasts of soft drinks are resistant to preservatives like sorbic acid and they are highly fermentative, generating large amounts of carbon dioxide gas. Conversely, many yeast species derive energy from respiration only and most of these are sorbic acid-sensitive, so prevented from causing spoilage. This led us to hypothesize that sorbic acid may specifically inhibit respiration. Tests with respiro-fermentative yeasts showed that sorbic acid was more inhibitory to both Saccharomyces cerevisiae and Zygosaccharomyces bailii during respiration (of glycerol) compared with fermentation (of glucose). The respiration-only species Rhodotorula glutinis was equally sensitive when growing on either carbon source, suggesting that ability to ferment glucose specifically enables sorbic acid-resistant growth. Sorbic acid inhibited the respiration process more strongly than fermentation. We present a dataset supporting a correlation between the level of fermentation and sorbic acid resistance across 191 yeast species. Other weak acids, C2 – C8, inhibited respiration in accordance with their partition coefficients, suggesting that effects on mitochondrial respiration were related to membrane localization rather than cytosolic acidification. Supporting this, we present evidence that sorbic acid causes production of reactive oxygen species, the formation of petite (mitochondria-defective) cells, and Fe-S cluster defects. This work rationalises why yeasts that can grow in sorbic acid-preserved foods tend to be fermentative in nature. This may inform more-targeted approaches for tackling these spoilage organisms, particularly as the industry migrates to lower-sugar drinks, which could favour respiration over fermentation in many spoilage yeasts.IMPORTANCESpoilage by yeasts and moulds is a major contributor to food and drink waste, which undermines food security. Weak acid preservatives like sorbic acid help to stop spoilage but some yeasts, commonly associated with spoilage, are resistant to sorbic acid. Different yeasts generate energy for growth by the processes of respiration and/or fermentation. Here we show that sorbic acid targets the process of respiration, so fermenting yeasts are more resistant. Fermentative yeasts are also those usually found in spoilage incidents. This insight helps to explain the spoilage of sorbic acid-preserved foods by yeasts and can inform new strategies for effective control. This is timely as sugar content of products like soft drinks is being lowered, which may favour respiration over fermentation in key spoilage yeasts.

scholarly journals Mapping the structural requirements of inducers and substrates for decarboxylation of weak acid preservatives by the food spoilage mould Aspergillus niger

2012 ◽  
Vol 157 (3) ◽  
pp. 375-383 ◽  
Author(s):  
Malcolm Stratford ◽  
Andrew Plumridge ◽  
Mike W. Pleasants ◽  
Michaela Novodvorska ◽  
Charles A.G. Baker-Glenn ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Weak Acid ◽  
Food Spoilage ◽  
Structural Requirements

scholarly journals The Aspergillus niger MADS-box transcription factor RlmA is required for cell wall reinforcement in response to cell wall stress

2005 ◽  
Vol 58 (1) ◽  
pp. 305-319 ◽  
Author(s):  
Robbert A. Damveld ◽  
Mark Arentshorst ◽  
Angelique Franken ◽  
Patricia A. VanKuyk ◽  
Frans M. Klis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Transcription Factor ◽  
Aspergillus Niger ◽  
Wall Stress ◽  
Mads Box ◽  
Cell Wall Stress ◽  
Cell Wall Reinforcement

scholarly journals Evaluation of amoxicillin-clavulanic acid resistance in the Bifidobacterium genus

2021 ◽  
Author(s):  
Leonardo Mancabelli ◽  
Walter Mancino ◽  
Gabriele Andrea Lugli ◽  
Chiara Argentini ◽  
Giulia Longhi ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Clavulanic Acid ◽  
Acid Resistance ◽  
Resistance Mechanisms ◽  
Western World ◽  
Beneficial Effects ◽  
Amoxicillin Clavulanic Acid ◽  
High Level ◽  
The Impact

Amoxicillin-Clavulanic acid (AMC) is one of the most frequently prescribed antibiotic formulations in the Western world. Extensive oral use of this antimicrobial combination influences the gut microbiota. One of the most abundant early colonizers of the human gut microbiota is represented by different taxa of the Bifidobacterium genus, which include many members that are considered to bestow beneficial effects upon their host. In the current study, we investigated the impact of AMC administration on the gut microbiota composition, comparing the gut microbiota of 23 children that had undergone AMC antibiotic therapy to that of 19 children that had not been treated with antibiotics during the preceding six months. Moreover, we evaluated AMC sensitivity by Minimal Inhibitory Concentration (MIC) test of 261 bifidobacterial strains, including reference strains for the currently recognized 64 bifidobacterial (sub)species, as well as 197 bifidobacterial isolates of human origin. These assessments allowed the identification of four bifidobacterial strains, which exhibit a high level of AMC insensitivity, and which were subjected to genomic and transcriptomic analyses to identify the putative genetic determinants responsible for this AMC insensitivity. Furthermore, we investigated the ecological role of AMC-resistant bifidobacterial strains by in vitro batch-cultures. Importance Based on our results, we observed a drastic reduction in gut microbiota diversity of children treated with antibiotics, also affecting the abundance of Bifidobacterium, a bacterial genus commonly found in the infant gut. MIC experiments revealed that more than 98% of bifidobacterial strains tested were shown to be inhibited by the AMC antibiotic. Isolation of four insensitive strains and sequencing of their genome revealed the identity of possible genes involved in AMC resistance mechanisms. Moreover, gut-simulating in-vitro experiments revealed that one strain, i.e. B. breve PRL2020, is able to persist in the presence of a complex microbiota combined with AMC antibiotic.

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