Autophagy is important to the acidogenic metabolism of Aspergillus niger.

Significant phenotypic overlaps exist between autophagy and acidogenesis in Aspergillus niger. The possible role of autophagy in the acidogenic growth and metabolism of this fungus was therefore examined and the movement of cytosolic EGFP to vacuoles served to monitor this phenomenon. An autophagy response to typical as well as a metabolic inhibitor-induced nitrogen starvation was observed in A. niger mycelia. The vacuolar re-localization of cytosolic EGFP was not observed upon nitrogen starvation in the A. niger Δatg1 strain. The acidogenic growth of the fungus consisted of a brief log phase followed by an extended autophagy-like state throughout the idiophase of fermentation. Mycelia in the idiophase were highly vacuolated and EGFP was localized to the vacuoles but no autolysis was observed. Both autophagy and acidogenesis are compromised in Δatg1 and Δatg8 strains of A. niger. The acidogenic growth of the fungus thus appears to mimic a condition of nutrient limitation and is associated with an extended autophagy-like state. This crucial role of autophagy in acidogenic A. niger physiology could be of value in improving citric acid fermentation.

Introduction

Aspergillus niger is a filamentous fungus of industrial and commercial importance. It is capable of growing on simple and inexpensive substrates and secretes large amounts of proteins, metabolites and organic acids [1]. This fungus is industrially famous for citric acid production [2]. The acidogenic growth requires a specific set of media conditions with low pH, nitrogen limitation [3] and Mn deficiency [4] as major features. Despite numerous studies addressing the biochemistry and physiology of this fungus, no single, clear cause-effect relation describing acidogenesis has emerged [5]. Acidogenic A. niger mycelia are highly vacuolated and the fraction of vacuolated mycelia increases along with the citrate production [3,6,7]. Many of the biochemical, physiological and morphological features of A. niger acidogenic growth mirror those observed during fungal autophagy (Table 1). Although both conidiation and germination are influenced by the organism’s state in autophagy and acidogenesis, these effects appear to be divergent. The phenotypic overlaps between autophagy and acidogenesis suggest that the two processes may be related and that autophagy may play an important role during A. niger citric acid fermentation.

Table 1

Autophagy and acidogenesis–A comparison.

Acidogenesis	Autophagy
Acidogenesis requires nitrogen-limited medium [3]	Nitrogen starvation induces autophagy [8–10]
Trace metals deficiency [3]	Metal ion starvation induces autophagy [10]
Germination is impaired during acidogenic growth [11]	Delayed germination in the absence of autophagy [12]
Reduced conidiation during acid production [3]	Reduced conidiation in the absence of autophagy [12]
Enhanced intracellular protein degradation [13]	Increased protein degradation during autophagy [14]
Accumulation of intracellular amino acids [15]	Autophagy leads to elevated amino acid pool [14]
Enhanced vacuolization observed during acidogenesis [6]	Number of vacuoles increases during autophagy [16]

Autophagy is a highly conserved process ubiquitous to eukaryotes wherein the cellular constituents are degraded inside their own vacuole/lysosome. Saccharomyces cerevisiae has been studied extensively to define autophagy pathways and organelle specific autophagy [17,18]. Autophagy provides an efficient means to recycle and translocate the contents of ageing hyphae for the benefit of the mycelial entity without losing them into the surrounding environment [19-22]. It is important for normal growth of Aspergilli as well as their survival during starvation and ageing [8-10,12,23-27]. Autophagy is involved in germination and conidiation of Aspergillus oryzae and Aspergillus fumigatus; the autophagy mutants show poor conidiation even under non-autophagic conditions and this could be rescued by supplementing the nitrogen source [10]. The cell organelles like peroxisomes, mitochondria and nuclei from the basal cells of A. oryzae are subjected to autophagy even during nutrient rich growth conditions [21,27]. The secretion of enzymes and metabolites by filamentous fungi is also influenced by autophagy. Blocking autophagy by disrupting key atg genes resulted in a three-fold increase in the production of bovine chymosin by A. oryzae [28]. Impaired degradation of peroxisomes and delayed mycelia deterioration led to a 37% increase in penicillin yields in the atg1 deleted strain of Penicillium chrysogenum [29].

Observations like highly vacuolated state of acidogenic mycelia, increased protein degradation coupled to nutrient-limited growth [4,6,13] suggest a link between citric acid fermentation and autophagy in A. niger. Whether autophagy occurs and is involved in the acidogenic metabolism of this fungus was therefore examined. The movement of cytosolic EGFP to vacuoles was used as a tool to monitor autophagy [9,25,26] and we show that an autophagy-like state exists throughout the acidogenic stages of fermentation and a block in autophagy pathway negatively impacts citric acid production.

Materials and methods

Strains, cultivation conditions and sampling

Various A. niger strains expressing EGFP in their cytoplasm were used. These included C6JT2 strain (NCIM 1380) [30] while AR19#1, BN56.2 (Δatg1), AW24.2 (BN56.2 complemented with atg1), BN57.1 (Δatg8) and AW25.1 (BN57.1 complemented with atg8) [25] were obtained from Fungal Genetics Stock Center, KS, USA. The fungal strains were maintained on yeast dextrose agar [31]. This medium was either supplemented with 1.0 mg mL-1 phosphinothricin (for C6JT2 strain with a bar marker) or 100 μg mL-1 hygromycin (for strains BN56.2, AW24.2, BN57.1 and AW25.1). Whereas A. niger NCIM 565 and AR19#1 (both wild type strains) were cultured on potato dextrose agar [31].

All the experiments were performed under shake flask conditions (at 30°C and 220 rpm). A total of 108 spores (harvested from suitable solid media in Petri plates) were inoculated into 100 ml of respective liquid media in one liter Erlenmeyer flasks. The fungal strains were cultured either on the minimal medium (MM; which contained 20.0 g/l glucose, 3.0 g/l KH2PO4, 6.0 g/l Na2HPO4, 0.5 g/l MgSO4.7H2O, 2.25 g/l NH4NO3, 10 mg/l ZnSO4.7H2O, 3.0 mg/l MnSO4.7H2O, 1.5 mg/l Na2MoO4.H2O, 20.0 mg/l FeCl3.6H2O and 1.0 mg/l CuSO4.H2O. The medium pH was adjusted with 0.1 N HCl to 5.5–6.0) or on the acidogenic medium (AM; which contained 140.0 g/l sucrose, 1.0 g/l KH2PO4, 0.1 g/l Fe(NH4)2(SO4)2, 2.25 g/l NH4NO3 and 0.25 g/l MgSO4.7H2O for eight days) [31]. Since citric acid fermentation is highly sensitive to presence of trace metals (especially Mn2+ ions) all the glassware was treated first with 20% nitric acid and then thoroughly washed with double distilled water. The minimal medium prepared without the addition of NH4NO3 (MM-N) served to create nitrogen starved condition.

The mycelia harvested from shake flask cultures were harvested by drying between filter papers. The harvested biomass was immediately frozen in liquid nitrogen and stored at −20°C till further use. The spent medium was also frozen and stored similarly for further analysis. For biomass dry weight measurements mycelia were dried in hot air oven (70°C) to constant weight (4–5 days).

A. niger morphology

The A. niger was normally grown on MM (200 mL in one L flasks) in shake flasks for 20 h (log phase) and the mycelia were then harvested, washed with cold sterile distilled water and transferred to nitrogen deprived media (MM-N). Parallelly, an equal amount of mycelial biomass (~ 2.0 g wet weight) was transferred back to fresh MM to serve as control. These resuspended mycelia were again incubated for 4 h (at 30°C and 220 rpm) and sampled for microscopy. Washed A. niger mycelia were also transferred to MM containing either dimethyl isophthalate (DMIP; 3.0 mM) or citric acid (10 mM or 100 mM, pH adjusted) to test their effects. Stock DMIP solution was prepared in acetone because of its limited solubility in water and suitable solvent controls were included for such experiments.

A. niger strains were inoculated (108 spores per 100 mL culture medium) in either MM or AM media and grown as shake flask cultures (at 30°C and 220 rpm) for 8 days. The samples of mycelia and corresponding spent media were obtained (separate flasks for each time point) for microscopy and citrate analysis, respectively.

Microscopy and image analysis

Freshly harvested A. niger mycelia were directly observed under the microscope without any fixation step. The Olympus fluorescence inverted microscope model (IX83) equipped with plan-apochromat 100×/1.4NA objective lens and DIC was used for capturing fluorescent images. The GFP 4050 B-000 filter (excitation-466/40-25, emission- 525/50-25) used for capturing EGFP fluorescence and DAPI-5060C filter (excitation-377/50-25, emission- 447/60-25) was used for CMAC visualization. The mitotracker and FM4-64 staining was followed using a combination of TRITC-B-000 filter (excitation-543/22-25) and LF561/LP-C-000 (emission-561R), respectively. A Zeiss Axio-Observer Z1 inverted confocal microscope equipped with iplan-apochromat 63×/1.4NA objective lens was used to visualize EGFP fluorescence (488 nm argon laser), FM4-64 and mitotracker stain (561 DPSS laser) and CMAC (multiphoton laser). The images were deconvoluted to enhance signal to noise ratio [32] and analyzed using respective software (CellSense, Zen black or ImageJ).

The culture media of mycelia at different growth stages were replaced with the respective fresh media containing 5 μM FM4-64. After incubation for 10 min (at 30°C and 220 rpm) the mycelia were quickly harvested, washed twice with respective media without the dye and further incubated for 10 min; these were subjected to microscopy. For vacuolar staining with CMAC, the dye was directly added (at a final concentration of 10 μM) to an aliquot of culture medium. These samples were incubated for 15 min (at 30°C and 220 rpm) before observing under the microscope [33].

Typically, a total of 50 micrograph of each cell type were imaged from five independent experiments. Unless otherwise mentioned, the scale bar in the images corresponds to 2 μm.

A. niger genomic DNA PCR

The frozen A. niger mycelia were crushed in liquid nitrogen to form fine powder; this was followed by all the steps as recommended for QIAGEN DNeasy Plant Mini kit for genomic DNA isolation. The genomic DNA was used for characteristic PCR according to standard molecular biology procedures [34]. In all PCR reactions—3.5 mM MgCl2, 250 μM dNTPs, 0.5 μM primers and 5 units of Pfu polymerase (MBI Fermentas) were present in a final volume of 100 μL. The PCR products were subjected to electrophoresis on 1% agarose gels. The primers used in study are listed in S1 Table.

The three A. niger strains namely, AR19#1 (wild type), BN56.2 (Δatg1) and AW24.2 (atg1 complemented) were subjected to genotypic characterization, in terms of the presence and/or absence of atg1, atg8 and hph (marker used to disrupt atg1) genes, before use. Their respective genomic DNA was subjected to diagnostic PCR using the primer pair diagnRP/diagnatg1FP (for atg1 gene), Atg8FwdP/ATG8cDNARP (for atg8 gene) and HYnested/YGnested (for hph gene). As expected, the characteristic PCR amplification patterns confirmed that the atg1 sequence was deleted in BN56.2 strain, hph gene was absent in AR19#1 strain and that atg8 gene was intact in all three strains.

Citric acid and protein estimations

The concentration of citric acid in the spent medium was determined using citrate lyase and phenylhydrazine as reported before [31]. Citrate lyase (Roche Diagnostics India Pvt. Ltd.) from Aerobacter aerogenes was used in these estimations. The citrate data on day 8 of acidogenesis for all three strains was statistically evaluated. The one way ANOVA was run on the data for citrate concentration in the spent media as well as on the citrate yield per gram mycelial dry weight.

The frozen fungal mycelia were crushed and protein extracted with buffer [31]. Protein from these cell free extracts was estimated by Bradford’s method [35] with crystalline bovine serum albumin as standard.

Results and discussion

The filamentous growth of A. niger comprise variously entangled hyphae to result in the formation of loose or tight pellets and dispersed mycelia [36,37]. The hyphal tubes are differentiated into tip cells, followed by few intermediate cells and inactive basal cells (Fig 1). This heterogenous arrangement of cells makes it interesting yet difficult to study and generalize on the cellular phenomena like autophagy. Autophagy has evolved to recycle and translocate the contents of aging hyphae for the benefit of the mycelial entity without losing them into the surrounding environment [21]. The basal cells or older hyphae are nearly filled with large vacuoles and enter autolysis subsequently [33,38]. Hence the mycelial morphology and subcellular organelles of viable tip cells (and the following few intermediate cells) were monitored to study autophagy in the acidogenic A. niger. The pH sensitivity of tagged-EGFP fluorescence in the A. oryzae vacuoles in vivo is reported [39]. However, the intracellular pH homeostasis of A. niger during acidogenic growth is well documented. Both the vacuolar pH (of 6.2) and cytoplasmic pH (of 7.6) of the fungus do not vary much when the extra-cellular pH was varied between 2.5 and 9.5 (and also in the presence of citrate) [40,41]. The movement of EGFP from the A. niger cytoplasm to its vacuoles could thus be studied independent of the pH effects during fermentation. Relocation of cytosolic EGFP to vacuoles was therefore conveniently exploited and used to mark autophagy in A. niger mycelia.

Fig 1

A. niger mycelia respond to nitrogen starvation.

(A) The mycelia of A. niger C6JT2 strain grown (up to 20 h) on MM were transferred to MM-N. The EGFP fluorescence of mycelia at the time of transfer (MM-N, 0 h) and after 4 h of N starvation (MM-N, 4 h) was monitored. The corresponding non-starved mycelia control is also shown. In each panel, the left frame shows the image of a tip cell and the right frame is of an intermediate cell. The vacuolar membranes of mycelia were stained red with FM4-64 (scale bar = 10 μm). (B) Schematic of A. niger hypha. The nuclei (in dark blue), vacuoles (in light blue) and the septa (dark green lines between cells) are shown. The dashed lines represent the connecting intermediate cells up to the basal cell.

Nitrogen starvation induces EGFP localization to vacuoles in A. niger

Autophagy involves the transport of cytoplasmic components to the lysosome/vacuole for degradation and it is strongly induced by nutrient starvation. The movement of cytosolic EGFP to vacuoles has served well to monitor autophagy in response to carbon/nitrogen starvation in S. cerevisiae [14], A. oryzae [8,9], A. niger [25], Aspergillus nidulans [26] and P. chrysogenum [29]. That autophagy occurs with carbon depletion in submerged A. niger cultures is well established but the study with respect to nitrogen limitation was restricted to surface cultures on solid MM [25]. Also, the Δatg8 strain was shown to be more sensitive to nitrogen limitation than the Δatg1 strain, whereas both deletion strains were comparably affected by carbon limitation. Since acidogenesis is possibly a nitrogen limited state of submerged growth, it was of interest to first demonstrate that nitrogen starvation leads to autophagy in two different A. niger cultures.

Two A. niger strains expressing EGFP in their cytosol [25,30] were used to visualize autophagy upon nitrogen starvation. The log phase mycelia of C6JT2 and AR19#1 strains were transferred to a nitrogen starvation medium (MM-N). Significant EGFP movement to vacuoles could be observed within 2 h after the transfer of mycelia to MM-N. However, this was complete by 4 h and by then the EGFP was localized exclusively in vacuoles, particularly of the intermediate cells (Figs 1 and 2). The vacuoles at this stage were generally more in number and larger in size and were filled with EGFP. The tip cells contained fewer but smaller vacuoles. In the normal (transferred to MM for 4 h) non-starved mycelia EGFP fluorescence remained exclusively outside the vacuoles, both in the tip and the intermediate cells (Fig 1). The results confirmed that nitrogen starvation induces the movement of cytosolic EGFP to vacuoles in A. niger. Both the strains behaved similarly when subjected to carbon starvation as well (not shown).

Fig 2

Autophagy response by nitrogen starved A. niger mycelia.

The mycelia of A. niger strains AR19#1 (wild type), BN56.2 (Δatg1) and AW24.2 (atg1 complemented) grown on MM till 20 h were transferred to MM-N. The EGFP fluorescence of mycelia after 4 h of N starvation is shown along with corresponding non-starved mycelia control. Each frame shows the image of an intermediate cell. The mycelia were stained with CMAC when vacuoles appear blue; the vacuoles containing EGFP appear cyan on merge (scale bar = 2 μm).

The effect of nitrogen starvation on the biomass yield of four A. niger strains (AR19#1, BN56.2, AW24.2 and C6JT2) was also monitored. There was little increase in the biomass after 4 h of transfer to MM or MM-N. But the growth difference was obvious when the mycelia were allowed to grow for 24 h. The fungal biomass increased 3 to 4 fold after transfer on MM while biomass gain on MM-N was marginal (Table 2). On an equal biomass wet weight basis, the total extractable mycelial protein content was substantially lower in the nitrogen starved mycelia (S1 Fig). The 24 h nitrogen starved mycelia generally showed increased vacuolation and cryptic growth in the form of many thinner mycelia was clearly observed (not shown). The cryptic growth and occurrence of thin mycelia (compared to the regular mycelia growing on normal media) are generally observed in starved cultures [42-44].

Table 2

Effect of nitrogen starvation on the biomass yield of A. niger.

A. niger strain	Biomass (wet weight, g)a
	at 0 h	24 h on MM-N	24 h on MM
AR19#1 (wild type)	6.5	6.9	19.5
BN56.2 (Δatg1)	5.3	5.5	15.6
AW24.2 (atg1 complemented)	5.1	6.2	15.0
C6JT2 (NCIM 1380)	2.7	3.6	11.4

a Average of three separate experiments

Autophagy induced by nitrogen starvation in A. niger is atg1 dependent

The autophagy phenomenon is best established by its impairment in the corresponding atg gene disrupted/deleted strains. While our efforts to disrupt atg1 gene in the background of C6JT2 strain were ongoing, an analysis of autophagy by disrupting atg genes (in the background of A. niger N402 strain) was reported [25]. The three relevant A. niger strains (all expressing EGFP in the cytosol) namely, AR19#1 (wild type), BN56.2 (atg1 deletion; Δatg1) and AW24.2 (atg1 complemented) from that study were first used to confirm nitrogen starvation induced autophagy. The log phase grown mycelia of these three strains were subjected to nitrogen starvation (by transfer to MM-N medium; see above) and the mycelia were observed after 4 h. The cytosolic EGFP was localized to vacuoles in AR19#1 strain but remained in the cytosol of atg1 deleted strain BN56.2 (Fig 2). However, the vacuolar relocation of EGFP was restored upon atg1 complementation (in AW24.2 strain). The results clearly demonstrate that autophagy is induced when the fungus experiences nitrogen starvation directly or indirectly by nitrogen metabolic inhibition (see below).

Blocking ammonia assimilation also induces autophagy in A. niger

Besides nutrient starvation, autophagy can be induced by treatment with rapamycin, an inhibitor of Tor (target of rapamycin) kinase [23]. Inhibition of nitrogen metabolism, blocking ammonia assimilation for instance, could also create a nitrogen starvation-like condition. Incubation with dimethyl isophthalate (DMIP) and subsequent inhibition of NADP-glutamate dehydrogenase in vivo resulted in growth inhibition and extensive vacuolation of A. niger mycelia [45]. Treatment with DMIP should mimic nitrogen starvation even when plenty of nitrogen (as ammonium) is available in the growth medium. This was tested by incubating log phase A. niger mycelia with 3 mM DMIP in MM. Autophagy was induced within 3 h in such mycelia and the cytosolic EGFP was translocated to vacuoles. Also, the DMIP induced translocation of EGFP to vacuoles was adversely affected in A. niger BN56.2 (atg1 deleted strain) but was restored by atg1 complementation (in AW24.2 strain) (S2 Fig). The DMIP effect thus offers a unique model to induce autophagy by means of a specific metabolic inhibitor (or an antimetabolite).

Autophagy is integral to acidogenesis in A. niger

It is generally believed that acidogenesis by A. niger represents a nutrient limited growth with a major imbalance in C:N ratio of the fermentation medium [3,4]. Perceived nitrogen starvation (see above) may therefore push the fungus towards autophagy. Significant phenotypic overlaps between autophagy and acidogenesis (Table 1) suggested that the two processes may be interconnected. This was directly tested by growing A. niger on fermentation medium (AM) and monitoring the mycelial morphology. The normal growth of filamentous fungi (such as the growth of A. niger on MM) typically involves distinct stages: the growth phase which later transitions into autolysis of the older mycelia [44]. However, during acidogenic growth A. niger goes through two broad phases namely, the trophophase (the growth phase) and the idiophase (citric acid production phase). The fate of cytosolic EGFP was monitored through these two phases of acidogenesis.

The EGFP fluorescence was in the cytoplasm of the AR19#1 strain (wild type) during the trophophase (up to day 1) but in the subsequent idiophase (day 2 to 8) it was localized with the vacuoles (Fig 3). This idiophase was characterized by highly vacuolated mycelia with EGFP exclusively found in the vacuoles. In contrast, the movement of EGFP to vacuoles was not observed in the autophagy impaired A. niger BN56.2 strain. Upon agt1 complementation (in AW24.2 strain) however, the movement of EGFP to vacuoles was restored (Fig 3). Comparable results were obtained when autophagy was manipulated by agt8 deletion (BN57.1 strain) and its subsequent complementation (AW25.1 strain) (S3 Fig). Clearly, an autophagy-like state of mycelial morphology exists throughout the acidogenic stages (idiophase) of citric acid fermentation. This extended autophagy phase was not followed by autolysis (even after 20 days of fermentation). Whether the observed phenotype could possibly be due to the presence of citrate in the medium was also tested. The mycelial morphology of all three strains remained unaffected even after 8 h of incubation with citrate (added at 10 mM and 100 mM, to log phase A. niger mycelia grown in MM), indicating that the presence of extra-cellular citrate did not cause the fungal autophagy response. A systematic study to unravel the role of other factors (listed in Table 1) on acidogenic metabolism, morphology and associated autophagy response is useful/ underway.

Fig 3

A. niger mycelial morphology during acidogenic growth.

The morphology and EGFP fluorescence of A. niger strains, AR19#1 (wild type), BN56.2 (Δatg1) and AW24.2 (atg1 complemented) was recorded on fermentation medium (AM) for 8 days. In each panel, the left frame shows the image of a tip cell and the right frame is of an intermediate cell. The mycelia were stained with CMAC when vacuoles appear blue; the vacuoles containing EGFP appear cyan on merge (scale bar = 2 μm).

The effect of impaired autophagy on citric acid fermentation was as also studied. The three A. niger strains namely, AR19#1 (wild type), BN56.2 (Δatg1) and AW24.2 (atg1 complemented) were grown on AM and citrate levels in the spent media were measured during the course of fermentation. Acidogenesis was compromised in both Δatg1 (Fig 4) as well as Δatg8 (S4 Fig) strains of A. niger. The earliest time point at which citric acid was detected coincided with the movement of EGFP from cytoplasm to vacuoles in the AR19#1 strain (Fig 4A; day 2 onwards). The citric acid production in the autophagy impaired A. niger (BN56.2) strain was significantly reduced (p < 0.0001) (Fig 4B); the drop in citrate formation was alleviated significantly by atg1 complementation (in the AW24.2 strain). A delay in citrate production in AW24.2 (atg1 complemented) was observed (Fig 4A, top panel). This strain is not strictly comparable to the parent strain AR19#1 for the deletion (in BN56.2 strain) was complemented by random integration of the atg1 cassette and not by homologous gene replacement [25]. All the three strains showed comparable increase in biomass during their growth on AM as well as MM (Fig 4A, bottom panel) and none of them produced citrate when grown on MM. The citrate yield (gram citric acid produced per gram mycelial dry weight) on AM, at each time point was also calculated. On both counts (on the basis of citrate levels in the spent media and yield per mycelial dry weights) it is evident that disrupting autophagy negatively affects acidogenesis. These results clearly implicate an autophagy-like state in the acidogenic metabolism of A. niger and that a block in autophagy pathway negatively impacts citric acid production. This is in contrast to enhanced penicillin yields in Δatg1 strain of P. citrinum [29] and increased ethanol production in Δatg32 strain of yeast [46]. Obviously, we do not yet fully understand the mechanism(s) responsible for the unique acidogenic metabolism in A. niger [5,47,48].

Fig 4

Citric acid production by A. niger.

(A) The A. niger strains AR19#1 (●), BN56.2 (■) and AW24.2 (▲) were grown on fermentation medium (AM) and the citric acid in the spent medium (mM) and citric acid yield (per gram dry mycelia mass) are shown. Mycelial dry weights were measured for the strains AR19#1 (●), BN56.2 (■) and AW24.2 (▲) grown on AM and compared with corresponding data for growth on MM (respective open symbols; bottom graph). The error bars represent the data spread of five independent experiments. (B) Relative citric acid values (in percent) calculated from day 8 data for AR19#1, BN56.2 and AW24.2 strains are shown (*** p < 0.0001 with n = 5).

Conclusions

We have observed autophagy in response to nitrogen starvation and also by metabolically blocking ammonium assimilation in this fungus. Many phenotypic overlaps were noted between autophagy and acidogenesis (Table 1). One key feature being unusual C:N balance and nitrogen limited growth of the fungus during acidogenesis. An extended autophagy-like state was observed throughout the acidogenic stages (idiophase) of fermentation and a block in autophagy pathway negatively impacted citric acid production. Both autophagy and acidogenesis are compromised in the Δatg1 and Δatg8 strains of A. niger. To our knowledge, this is the first report on the association of autophagy with the acidogenic metabolism of A. niger. Despite the extensive accumulated literature on the biochemistry and physiology of this fungus, a clear physiological cause-effect relation describing acidogenesis has not emerged so far [5,48]. There are various possibilities for how autophagy may perhaps support acidogenesis in A. niger. For instance, autophagy may—a) provide important metabolites to sustain acidogenesis, b) the enzymes (and the pathway) for citrate formation may possibly operate from the vacuolar compartment during acidogenesis, or c) simply keep the organism alive for citrate production to proceed. While how the phenomena of acidogenesis and autophagy are linked needs further study, underlying mechanisms could assist in improving citric acid fermentation.

Effect of nitrogen starvation on the protein content of A. niger mycelia.

The total protein from the mycelial extracts (2 g wet weight; collected 24 h after transfer to either MM or MM-N) of A. niger strains AR19#1 (wild type), BN56.2 (Δatg1), AW24.2 (atg1 complemented) and C6JT2 is shown (average of two separate experiments).

(TIF)

Click here for additional data file.

Effect of DMIP treatment on A. niger mycelial morphology.

The mycelia of A. niger strains AR19#1 (wild type) and BN56.2 (Δatg1) expressing EGFP in the cytoplasm were grown on minimal medium (MM) till 20 h and then transferred to MM+ DMIP, MM+acetone (solvent control) and MM alone. The EGFP fluorescence of mycelia recorded after 6 h of DMIP treatment is shown. A total of 50 micrograph of each cell type were imaged (scale bar = 2 μm).

(TIF)

Click here for additional data file.

The morphology and EGFP fluorescence of A. niger strains, AR19#1 (wild type), BN57.1 (Δatg8) and AW25.1 (atg8 complemented) was recorded on fermentation medium (AM) for 8 days. In each panel, the left frame shows the image of a tip cell and the right frame is of an intermediate cell. The mycelia were stained with CMAC when vacuoles appear blue; the vacuoles containing EGFP appear cyan on merge (scale bar = 2 μm).

(TIF)

Click here for additional data file.

The A. niger strains C6JT2 (○), AR19#1 (●), BN57.1 (■) and AW25.1 (▲) were grown on fermentation medium (AM) and the citric acid measured in the spent medium is shown.

(TIF)

Click here for additional data file.

Primers used in this work.

(DOC)

Click here for additional data file.

29 Aug 2019

[EXSCINDED]

PONE-D-19-21095

Autophagy is important to the acidogenic metabolism of Aspergillus niger

PLOS ONE

Dear Dr. Punekar,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please address all of the reviewer comments, most of which are focused upon changes to the manuscript text. Please also pay special attention to the comments of Reviewer 1 regarding a potential revisitation of results from a previous paper.

We would appreciate receiving your revised manuscript by Oct 13 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Cory D. Dunn, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Our internal editors have looked over your manuscript and determined that it is within the scope of our Autophagy and Proteostasis Call for Papers. This collection of papers is headed by a team of Guest Editors: Sharon Tooze, Fulvio Regiori and Thorsten Hoope. The Collection will encompass a diverse range of research articles from early initiation of autophagy, to understand the role other proteostasis pathways play in maintaining cellular homeostasis and the cross talk between the two.

Additional information can be found on our announcement page: https://collections.plos.org/s/autophagy-proteostasis..

If you would like your manuscript to be considered for this collection, please let us know in your cover letter and we will ensure that your paper is treated as if you were responding to this call. If you would prefer to remove your manuscript from collection consideration, please specify this in the cover letter.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors seek to determine whether citrate production (acidogenesis) in Aspergillus niger requires autophagy. They do provide evidence that mutants compromised in autophagy have reduced citrate production, with the best evidence coming from reduced citrate production in the atg1 mutant (Figure 4). However, there are a number of issues that should be addressed before publication.

1. Table 1, which compares aspects of acidogenesis and autophagy, is portrayed as a major motivation for the study, but contains items that do not support the hypothesis that the two processes are related. Specifically, the effects of acidogenesis on germination and conidiation appear to be the same as those seen in the absence of autophagy. This would argue against the overall correlation of the organism’s state in autophagy and acidogenesis and are not “similarities” as indicated in the Table’s title. These differences should be discussed further and/or removed from the table.

2. Reference 25 has already established that autophagy occurs with nitrogen limitation, demonstrated relocation of GFP to the vacuole under these conditions, and showed that the relocation is prevented in an atg1 mutant. The authors largely appear to be reproducing these results in Figures 1 and 2 and should distinguish their studies from those in Ref. 25 more clearly.

3. The data in Figure 4A are the most important and novel in the paper, as they demonstrate that acidogenesis is severely compromised in the atg1 mutant. However, the basis of the error bars is not clear. In addition, the atg1 strain complemented with the wild-type ATG1 gene shows a significant delay in citrate production that is masked in Figure 4B by using a later time point. This delay should be discussed.

4. There is no real support for the statement that because of the vacuolar morphology changes, citrate formation might occur in the vacuole (lines 320-322). This is misleading and should be removed.

Reviewer #2: This manuscript examines the role of autophagy in acidogenesis in A. niger. Based on the observations that both increased acidogenesis and autophagy share a number of similar characteristics such as activation by nitrogen starvation and metals deficiency, impaired proliferation, and enhanced protein degradation, the authors test the hypothesis that autophagy is required for citric acid fermentation in this organism. They investigate this relationship by tracking cytosolic eGFP movement to the vacuole as a marker for autophagy. In parallel, the authors also examine citric acid production, both concentration and yield, to quantify acidogenesis in wild type and autophagy deficient mutants. The authors demonstrate that autophagy does occur in A. niger, and show that loss of autophagy diminishes citric acid production. The experiments are well controlled and executed, and the results are conclusive.

While the authors’ overall conclusion that autophagy is required for citric acid production is useful with respect to improving industrial citric acid fermentation yields, the findings are not particularly revealing about the mechanism by which autophagy supports citric acid production, which the authors acknowledge. Identifying this mechanism is clearly beyond the scope of this study, and the authors should address the following points before publishing.

Major points

1. The authors’ conclusion on lines 200-201 that the movement of eGFP to the vacuole represents autophagy is premature without yet showing the requirement for Atg1 or other autophagy machinery. Prior to the Atg1 experiments in Fig 2, the authors should not refer to eGFP vacuole localization as autophagy in the text or titles. For example, a more appropriate title for the first section would be “nitrogen starvation induces eGFP localization to vacuoles in A. niger.” The same holds true for the title and section starting on line 220 about ammonia assimilation.

2. The section on biomass yield beginning on line 210 should include the data in figure or table form.

3. Starting on line 319 of the conclusion, the authors state that their results suggest that enzymes for citrate production are localized to the vacuole. This statement seems a bit premature, and there are numerous possibilities for how autophagy may support citric acid production. For example, it’s possible that autophagy provides important metabolites to maintain citrate production and/or just keeps the organism alive so citrate production can proceed. The authors should modify this statement to include other possible interpretations of their data.

Minor:

Figure 4A: Remove curve fitting for scatterplot, and use linear interpolation lines instead.

Figures 1-3: Differentiate tip intermediate cell images from basal cell images and add arrows to show example of vacuole in images.

Lines 249-259: Consider moving this section into the introduction.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

23 Sep 2019

Professor Cory D. Dunn, Ph.D.

Academic Editor

PLOS ONE September 23, 2019

Dear Professor Dunn,

We thank you and the reviewers for raising relevant issues that helped improve the quality of this work and presentation.

We would like our manuscript to be considered for the PLoS collection of “Autophagy and Proteostasis” Call for Papers.

We have now revised the manuscript (PONE-D-19-21095) entitled “Autophagy is important to the acidogenic metabolism of Aspergillus niger”, addressing all concerns of the reviewers. Our responses to each point brought up by the academic editor and reviewer(s) are appended below. Since the first submission of this manuscript we have also obtained additional results related to acidogenic metabolism where autophagy was manipulated by agt8 deletion (BN57.1 strain) and its subsequent complementation (AW25.1 strain). We have now included this atg8 related data on cellular morphology (as Figure S3) and citric acid production (as Figure S4) that additionally supports the findings and adds value to this study. The manuscript is accordingly modified at appropriate places.

Reviewer #1:

In this manuscript, the authors seek to determine whether citrate production (acidogenesis) in Aspergillus niger requires autophagy. They do provide evidence that mutants compromised in autophagy have reduced citrate production, with the best evidence coming from reduced citrate production in the atg1 mutant (Figure 4). However, there are a number of issues that should be addressed before publication.

We have now included the data on cellular morphology (as Figure S3) and citric acid production (as Figure S4) with Δatg8 strain of A. niger that additionally supports the findings and adds value to this study.

1. Table 1, which compares aspects of acidogenesis and autophagy, is portrayed as a major motivation for the study, but contains items that do not support the hypothesis that the two processes are related. Specifically, the effects of acidogenesis on germination and conidiation appear to be the same as those seen in the absence of autophagy. This would argue against the overall correlation of the organismʼs state in autophagy and acidogenesis and are not “similarities” as indicated in the Tableʼs title. These differences should be discussed further and/or removed from the table.

The point made by the reviewer is well taken. We have accordingly modified the Table 1 title and also modified the related text (lines 41-43).

2. Reference 25 has already established that autophagy occurs with nitrogen limitation, demonstrated relocation of GFP to the vacuole under these conditions, and showed that the relocation is prevented in an atg1 mutant. The authors largely appear to be reproducing these results in Figures 1 and 2 and should distinguish their studies from those in Ref. 25 more clearly.

That autophagy occurs with carbon depletion in submerged A. niger cultures is well established; but their study with respect to nitrogen limitation was restricted to surface cultures on solid MM [25]. Also, the Δatg8 strain was shown to be more sensitive to nitrogen limitation than the Δatg1 strain, whereas both deletion strains were comparably affected by carbon limitation. Acidogenesis is possibly a nitrogen limited state of submerged growth. It was therefore of interest to first demonstrate that nitrogen starvation leads to autophagy in two different A. niger strains, grown in liquid culture. This justification is now included (lines 202-208).

3. The data in Figure 4A are the most important and novel in the paper, as they demonstrate that acidogenesis is severely compromised in the atg1 mutant. However, the basis of the error bars is not clear. In addition, the atg1 strain complemented with the wild-type ATG1 gene shows a significant delay in citrate production that is masked in Figure 4B by using a later time point. This delay should be discussed.

The basis of error bars is now distinctly mentioned in the Figure 4 legend (line 376). The atg1 complementation was achieved by random integration (and was not by homologous recombination, Ref 25). This could possibly account for the observed delay in acid production. This possibility is clearly stated now (lines 353-357). The atg8 data freshly included as Figures S3 and S4 further corroborates the novelty of this work.

4. There is no real support for the statement that because of the vacuolar morphology changes, citrate formation might occur in the vacuole (lines 320-322). This is misleading and should be removed.

While addressing the mechanism(s) that connect acidogenesis and autophagy are beyond the scope of this study, we wish to provide few possibilities for future study. As suggested by the Reviewer 2, we have now modified the text and included other possible interpretations for this linkage (lines 390-395).

Reviewer #2:

This manuscript examines the role of autophagy in acidogenesis in A. niger. Based on the observations that both increased acidogenesis and autophagy share a number of similar characteristics such as activation by nitrogen starvation and metals deficiency, impaired proliferation, and enhanced protein degradation, the authors test the hypothesis that autophagy is required for citric acid fermentation in this organism. They investigate this relationship by tracking cytosolic eGFP movement to the vacuole as a marker for autophagy. In parallel, the authors also examine citric acid production, both concentration and yield, to quantify acidogenesis in wild type and autophagy deficient mutants. The authors demonstrate that autophagy does occur in A. niger, and show that loss of autophagy diminishes citric acid production. The experiments are well controlled and executed, and the results are conclusive.

While the authorsʼ overall conclusion that autophagy is required for citric acid production is useful with respect to improving industrial citric acid fermentation yields, the findings are not particularly revealing about the mechanism by which autophagy supports citric acid production, which the authors acknowledge. Identifying this mechanism is clearly beyond the scope of this study, and the authors should address the following points before publishing.

Major points

1. The authorsʼ conclusion on lines 200-201 that the movement of eGFP to the vacuole represents autophagy is premature without yet showing the requirement for Atg1 or other autophagy machinery. Prior to the Atg1 experiments in Fig 2, the authors should not refer to eGFP vacuole localization as autophagy in the text or titles. For example, a more appropriate title for the first section would be “nitrogen starvation induces eGFP localization to vacuoles in A. niger.” The same holds true for the title and section starting on line 220 about ammonia assimilation.

The title of the first section is changed to “Nitrogen starvation induces EGFP localization to vacuoles in A. niger” (line 196). Also, the text is modified at line 218. To be consistent with this narrative the section “Blocking ammonia assimilation also induces autophagy in A. niger” is moved to a place after atg experiments.

2. The section on biomass yield beginning on line 210 should include the data in figure or table form.

The biomass data is now included as Table 2 (lines 241-252) and is mentioned on line 235. The data on protein content (lines 235-237) is provided as a supplementary Figure S1.

3. Starting on line 319 of the conclusion, the authors state that their results suggest that enzymes for citrate production are localized to the vacuole. This statement seems a bit premature, and there are numerous possibilities for how autophagy may support citric acid production. For example, itʼs possible that autophagy provides important metabolites to maintain citrate production and/or just keeps the organism alive so citrate production can proceed. The authors should modify this statement to include other possible interpretations of their data.

While addressing the mechanism(s) that connect acidogenesis and autophagy are beyond the scope of this study, we wish to provide few possibilities for future study. As suggested by this reviewer, we have now modified the text and included other possible interpretations for this linkage (lines 390-395).

Minor:

Figure 4A: Remove curve fitting for scatterplot, and use linear interpolation lines instead.

This is done and fresh Figure 4 is uploaded.

Figures 1-3: Differentiate tip intermediate cell images from basal cell images and add arrows to show example of vacuole in images.

The legends to these three figures are re-worded to include this information (and color codes described).

Lines 249-259: Consider moving this section into the introduction.

We believe that this preamble (lines 307-317) is appropriate to the data being presented in the subsequent paragraphs. Since we do have a combined Results and Discussion section, we think it is best to retain the section as is.

2 Oct 2019

Autophagy is important to the acidogenic metabolism of Aspergillus niger

PONE-D-19-21095R1

Dear Dr. Punekar,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Cory D. Dunn, Ph.D.

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript is now acceptable. The authors have satisfied the points raised in my previous review.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

4 Oct 2019

PONE-D-19-21095R1

Autophagy is important to the acidogenic metabolism of Aspergillus niger

Dear Dr. Punekar:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Cory D. Dunn

Academic Editor

PLOS ONE