Metabolic activity in dormant conidia of Aspergillus niger and developmental changes during conidial outgrowth

Abstract

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 25min, 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 1h 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.

Keywords: Aspergillus niger; Conidial development; Manometry; Proteome; RNAseq; Sorbic acid.

Fig. 1

O₂ uptake and CO₂ efflux by dormant conidia in water at 28 °C. The means and standard deviations of duplicate samples are shown. (A) O₂ uptake (open squares) and CO₂ efflux (closed squares) over the period of 5 h. (B) O₂ uptake in the presence (closed squares) and absence (open squares) of cyanide over the period of 1.5 h. (C) O₂ uptake in the presence (closed squares) and absence (open squares) of SHAM over the period of 5 h.

Fig. 2

O₂ uptake and CO₂ efflux by 10⁹ conidia at 28 °C. The means and standard deviations of duplicate samples are shown. (A) Percentage of total CO₂ efflux from fermentation in the presence (closed squares) and absence (open squares) of sorbic acid over the period of 54 min of germination. (B) O₂ uptake (open squares) and CO₂ efflux (open circles) by 10⁹ conidia without and with cyanide (closed squares, closed circles) over the first 39 min of germination. (C) O₂ uptake (open squares) and CO₂ efflux (open circles) by 10⁹ conidia without and with SHAM (closed squares, closed circles) over the first 39 min of germination.

Fig. 3

(A) d-Trehalose levels in conidia over 4 h of germination at 28 °C in the absence (open triangles) and presence (closed triangles) of sorbic acid. The means and standard deviations of duplicate samples are shown. (B) Uptake of d-[U-¹⁴C]glucose over the period of 5 h by 10⁷ conidia at 28 °C in the presence (closed circles) and absence (open circles) of sorbic acid. The means and standard deviations of duplicate samples are shown.

Fig. 4

O₂ uptake (open squares) and CO₂ efflux (open circles) by 10⁸ conidia without and with sorbic acid (closed squares, closed circles) over the period of 120 min of germination at 28 °C. The means and standard deviations of duplicate samples are shown. ACM ± sorbic acid was added into conidia resuspended in water at time 0.