In the process of reconstructing the metabolic network we identified several multi-enzyme complexes (for example, the F0F1 ATP synthase complex or the pyruvate dehydrogenase complex, which consist of several different proteins), and we used the transcriptome data to assess whether there was coordinated control of the expression of genes encoding the proteins of these complexes. Thus, for each enzyme complex included in the metabolic reconstruction of A. nidulans, we investigated whether the corresponding subunits had similar expression profiles. This was checked by verifying whether the genes encoding proteins within each enzyme complex were assigned to the same clusters. Furthermore, we calculated the Pearson correlations for all possible combinations within each enzyme complex (data not shown), in order to evaluate how well the corresponding expression levels correlated to each other. Calculation of Pearson correlations also enabled analysis of genes whose expression did not change significantly in the conditions studied. Based on the clustering and Pearson correlation analyses, we observed that, for about 30% (8/27) of the enzyme complexes considered, the expression profiles of the genes encoding all of the subunits of each enzyme complex were similar. Furthermore, in 11% (3/27) of the cases, the transcription of at least 50% (and
We performed the same analyses for S. cerevisiae using transcription data for similar conditions . Here we observed that for about 21% (4/19) of the enzyme complexes included in the metabolic model for yeast , all of the corresponding subunits had similar expression patterns. Moreover, for 11% (2/19) of the enzyme complexes there was high correlation for at least 50% (and A. nidulans and yeast, there does not appear to be any conservation in terms of transcriptional regulation of enzyme complexes, because only 7% (2/27) of enzyme complexes in A. nidulans with co-regulation on different carbon sources (either all components or 50% of the components) were also found to be co-regulated in yeast.
The catabolism of ethanol, as well as regulation of the genes involved in this process, is presumably one of the best studied systems in A. nidulans (see Felenbok and coworkers  for a recent review). Two genes are responsible for the breakdown of ethanol into acetate via acetaldehyde, namely the genes encoding alcohol dehydrogenase I (alcA; AN8979.2) and aldehyde dehydrogenase (aldA; AN0554.2). The activation of this catabolic pathway is dependent on the transcriptional activator alcR (AN8978.2) . Interestingly, a whole gene cluster composed of seven genes that are responsive to ethanol (or, more specifically, the gratuitous inducer methyl ethyl ketone) has previously been reported . This cluster includes alcA and alcR, as well as five other transcripts (alcP [AN8977.2], alcO, alcM [AN8980.2], alcS [AN8981.2], and alcU [AN8982.2]), whose molecular functions have not yet been identified. In particular, one of these genes (alcO) has not been annotated in the genome sequence of A. nidulans, and similarity searches or gene prediction programs using the DNA sequence of the putative location of this gene were unsuccessful. Because our array design was based on annotated ORFs in the genome, this putative gene was not included in our analysis. However, all of the other genes of this cluster were found to be significantly upregulated on ethanol (alcP, alcR, alcA, alcM, and alcS were found in cluster 7, and alcU was found in cluster 6). Further positional analysis showed that there were no other gene clusters that were significantly regulated under any of the conditions studied (data not shown).
The subnetwork analysis clearly pointed to a coordinated expression of genes involved in ethanol metabolism upon shift from glucose to ethanol (Figure 3), and the response was to a large extent the same in the shift from glycerol to ethanol (Table 5). Ethanol is converted to acetate and is further catabolyzed to acetyl-coenzyme A (CoA), which then enters the mitochondria where it is oxidized (Figure 3). The subnetwork identified (Table 5) includes methylcitrate synthase (encoded by mcsA; AN6650.2), which was upregulated during growth on ethanol. This may point to a role of this enzyme in the catabolism of acetyl-CoA, in addition to the mitochondrial citrate synthase (encoded by citA; AN8275.2), which is expressed during growth both on glucose and ethanol. This is consistent with earlier reports in which it was found that this enzyme also possesses some citrate synthase activity .
The list of reporter metabolites (Table 4) is consistent with the identified subnetwork, because several components of the subnetwork are identified as reporter metabolites (CoA, acetyl-CoA, glyoxylate, oxaloacetate, carnitine, and O-acetyl-carnitine).
Besides alcA or ADH I (AN8979.2), A. nidulans has two additional alcohol dehydrogenases, namely alcB or ADH II (AN3741.2) and ADH III (AN2286.2). The former was assigned to cluster 6, whereas the latter did not appear to be significantly regulated in our analysis. It is interesting to observe that several genes in the identified subnetwork are also part of the metabolism of acetate, which is positively regulated by FacB (AN0689.2). Furthermore, facB was found to be significantly upregulated during growth on ethanol and assigned to cluster 7. FacB has been shown to induce directly the transcription of genes that are involved in the catabolism of acetate (acetyl-CoA synthetase, facA [AN5626.2]; carnitine acetyl transferase, facC [AN1059.2]; isocitrate lyase, acuD [AN5634.2]; malate synthase, acuE [AN6653.2]; and acetamidase, amdS [AN8777.2]) [5,6]. All of these genes were found to be significantly upregulated during growth on ethanol (assigned to cluster 7), and several of them are part of the subnetwork identified from the pair-wise comparison between glucose and ethanol (Table 5).
The subnetwork also included ATP:citrate oxaloacetate-lyase, which catalyzes the formation of acetyl-CoA and oxaloacetate from the reaction of citrate and CoA, with concomitant hydrolysis of ATP to AMP and phosphate. This enzyme represents a major source of cytosolic acetyl-CoA during growth on glucose, which is a precursor for lipid biosynthesis. In A. nidulans, ATP:citrate oxaloacetate-lyase appears to be regulated by the carbon source present in the medium, with high activity in glucose-grown cells and low activity in acetate-grown cells . This may be due to the fact that, during growth on C2 carbon sources, acetyl-CoA is formed directly in the cytosol in connection with the catabolism of the carbon source. The genes encoding the enzyme complex for ATP:citrate oxaloacetate-lyase (AN2435.2 and AN2436.2) were among the most significantly downregulated genes upon shift from glucose to ethanol (decreases of 22.6-fold and 22.2-fold, respectively; Additional data file 9 [Table S6]). Moreover, the genes encoding ATP:citrate oxaloacetate-lyase fell into cluster 2, together with another group of genes that were downregulated upon a shift from glucose to ethanol, namely the major part of the enzymes in the pentose phosphate (PP) pathway (Additional data file 12 [Table S13]). The subnetwork also captured changes in the expression of genes participating in gluconeogenesis, glycolysis, and the PP pathway. It was observed that genes involved in gluconeogenesis (PEP carboxykinase and fructose 1,6-bisphosphatase) were upregulated during growth on ethanol (assigned to clusters 7 and 6, respectively), whereas many of the genes of the PP pathway were downregulated (assigned to cluster 2). This suggests that an energetically more favorable route for supply of NADPH (nicotinamide adenine dinucleotide phosphatase, reduced form) is used during growth on ethanol, namely through the malic enzyme (encoded by maeA [AN6168.2]), which was found to be upregulated during growth on ethanol and was identified in the subnetwork for the glycerol versus ethanol comparison. This is consistent with earlier findings that the activity of malic enzyme is low on glucose and high on ethanol , and that maeA may be weakly regulated by carbon catabolite repression .
From the above, it is clear that there is coordinated regulation of genes in very different parts of the metabolism, which is important for the cell to maintain homeostasis during growth on different carbon sources. The strength of our analysis based on the metabolic network is that these coordinated expression patterns are clearly captured using a nonsupervised algorithm.
For the ethanol versus glucose comparison, it was interesting to note that the gene with the greatest fold change (151 times) was that of alcS. This is relevant considering that no molecular function has been suggested for this gene so far. In silico analysis suggests that AlcS might be a membrane bound transporter protein (six transmembrane-helix domains; conserved domain [PFAM01184]), indicating that AlcS could be an acetate transporter.
Regulation of transcription factors
As mentioned above, we observed that the gene facB was upregulated during growth on ethanol. However, we also found that several other transcription factors were regulated during growth on ethanol. Thus, we observed that creA (AN6195.2), which is the major mediator of carbon catabolite repression in A. nidulans, was located in cluster 6 and hence was upregulated during growth on ethanol. This might seem surprising, considering that CreA is assumed to be a transcriptional repressor and most active on glucose, but our findings corroborate findings reported by Strauss  and Sims  and their coworkers, who showed that creA is regulated at the transcriptional level when the mycelium is shifted to or from ethanol. The low expression of creA on glucose could be due to autoregulation, which is presumably elevated on the de-repressing carbon source ethanol, and on the intermediate repressing carbon source glycerol. However, our findings clearly showed that this regulation of creA not only occurs after changing the carbon source but is also reflected in the mRNA abundance of creA, during balanced growth conditions (it is not a transient phenomenon).
Besides the two transcriptional regulators AlcR and FacB, another known positive regulator was found in cluster 7, namely AreA (AN8667.2). AreA was probably the first regulatory gene described in A. nidulans , and it is a wide-domain regulator necessary for the activation of genes for the utilization of nitrogen sources. To our knowledge, it has not been reported that AreA is upregulated during growth on ethanol as compared with glucose or glycerol (cluster 7). Our results could indicate crosstalk between carbon repression and nitrogen repression pathways in A. nidulans. Supporting our findings on AreA regulation, we identified the gene uapC (AN6730.2) in cluster 7. This gene encodes a purine permease and has been shown to be regulated by AreA . Another transcription factor assigned to cluster 7, namely metR, encodes a transcriptional activator for sulfur metabolism in A. nidulans , and it thereby links yet another branch of central metabolism to the regulatory network that is controlled by the nature of the carbon source.
Glycerol utilization and polyol metabolism
Regulation of the biosynthesis and breakdown of glycerol are less studied in comparison with the metabolism of ethanol, but from our analysis we identified more than 200 genes that were significantly upregulated and another 200 genes that were significantly downregulated only during growth on glycerol as compared with growth on glucose and ethanol (clusters 4 and 8). It was previously described that there are two metabolic pathways that lead to glycerol, from the glycolytic intermediate dihydroxyacetone 3-phosphate. One of these pathways proceeds via dihydroxyacetone kinase to dihydroxyacetone, which is then converted into glycerol, by the action of a glycerol dehydrogenase (NADH [nicotinamide adenine dinucleotide] or NADPH dependent). The alternative route, which has been suggested to be responsible for the catabolism of glycerol , includes the formation of glycerol 3-phosphate (catalyzed by glycerol 3-phosphate dehydrogenase), and subsequently its conversion into glycerol, by the action of glycerol 3-phosphate phosphatase.
Several of the genes encoding these enzymes have previously been characterized, and we identified alternative candidates, as well as the missing ones, in our reconstruction of the metabolic network. The data obtained from the transcriptome analysis confirmed that the catabolic pathway via glycerol 3-phosphate is a major route for glycerol catabolism, because a gene putatively encoding the glycerol kinase (AN5589.2), as well as the gene putatively encoding a FADH-dependent glycerol 3-phosphate dehydrogenase (AN1396.2), were both significantly upregulated on glycerol as compared with ethanol and glucose. Moreover, both genes were assigned to cluster 4, which represents genes that are specifically upregulated during growth on glycerol, and were identified in the subnetworks of glycerol comparisons with the two other carbon sources. However, the transcriptome data also showed that the alternative pathway might be involved in the catabolism of glycerol. In fact, a gene that was identified in the metabolic reconstruction process as putatively encoding a NADPH-dependent glycerol dehydrogenase (AN7193.2) was upregulated on glycerol (cluster 3), as well as a gene that was identified as a putative dihydroxyacetone kinase (AN0034.2; cluster 4). Therefore, it seems likely that both pathways are actually involved in the utilization of glycerol. Interestingly, a previously characterized gene encoding a NADPH-dependent glycerol dehydrogenase (gldB; AN5563.2)  was also found to be significantly regulated, but exhibited a very different expression pattern from the putative gene encoding NADPH-dependent glycerol dehydrogenase (AN7193.2). Thus, because gldB was downregulated on glycerol, it was assigned to cluster 8.
The biosynthesis of mannitol occurs through routes that are similar to the two metabolic pathways that lead to glycerol. It has been reported that mannitol is implicated in the stress response to heat  and that it is the most abundant polyol in conidia of A. nidulans . One of the pathways that lead to mannitol proceeds via mannitol 1-phosphate, from the glycolytic intermediate fructose 6-phosphate, and another one, which has fructose as an intermediate. The metabolic reactions interconverting these four metabolites open the possibility for a cyclic reaction pathway within the cell that allows the conversion of NADH into NADPH at the expense of one molecule of ATP . None of the genes encoding enzymes involved in the mannitol cycle have previously been characterized. However, by applying the comparative genomics approach for the reconstruction of the metabolism, we identified putative candidate ORFs for all the reactions of the mannitol cycle, with the exception of the mannitol 1-phosphate phosphatase. Interestingly, most of these ORFs identified (6/8) showed lower expression levels on ethanol, at least when compared with glycerol (assigned to clusters 2, 3, and 4), and this could point to a role for the mannitol cycle in the formation of NADPH during growth on glycerol. Moreover, the gene that encodes the glucose 6-phosphate dehydrogenase (AN2981.2), which has been shown to be positively correlated with the formation of mannitol, was also assigned to cluster 2 and significantly downregulated on ethanol. This enzyme was identified in the subnetwork for the glucose versus glycerol comparison, and transcription of the corresponding gene was lower during growth on glycerol than during growth on glucose. This could indicate a partial shift from the PP pathway, as the main route for NADPH supply for biosynthesis, to the mannitol cycle.
Glycerol has also been shown to be involved in the response to different osmotic conditions in A. nidulans , and it has also been reported that all of the components of the high-osmolarity glycerol (HOG) response pathway that are known in yeast have orthologs in A. nidulans [43,44]. The analysis of the transcriptional responses of these components to the different growth conditions considered in the present study revealed that only the gene that encodes the sensor protein SlnA (slnA; AN1800.2) was significantly regulated and this was assigned to cluster 4 (slnA seemed to be induced when glycerol was the sole carbon source, as compared with glucose or ethanol).
Metabolism of reserve compounds and cell wall polysaccharides
Another metabolite that has been reported to be related to glycerol metabolism is trehalose. In fact, it has been shown that trehalose, which is stored in the conidiospores, is converted into glycerol upon germination .
The biosynthesis of trehalose occurs, via trehalose 6-phosphate, from glucose 6-phosphate and UDP-glucose, whereas it is degraded directly to glucose. Our reconstruction of the metabolic network includes six genes that might be involved in these metabolic pathways, of which four have been confirmed experimentally [45-48]. The cluster analysis showed that the transcription of three of these six genes was significantly changed, with higher levels on glucose, compared with ethanol and glycerol (genes assigned to clusters 1 and 2). Because these three genes encode each of the different steps in the biosynthesis as well as degradation of trehalose, these observations suggest that there may be a higher turnover of trehalose during growth on glucose.
Glycogen is another reserve carbohydrate, similar to trehalose, and interestingly the two genes putatively assigned to its biosynthesis and degradation exhibited their highest expression levels on glycerol (clusters 3 and 4, respectively), which might suggest an effect of this carbon source on glycogen turnover. In this regard, it was also interesting to verify that the GO term analysis for the pair-wise comparisons showed that genes associated with cell wall metabolism were significantly over-represented in the upregulated gene set as well as in the downregulated gene set.
More detailed analysis of the genes that were upregulated on glycerol compared with glucose, and that resulted in the over-representation of the GO terms, revealed that all of them putatively encode enzymes with β-1,3-glucosidase activity, which suggests that specially the β-1,3-glucan fraction of the fungal cell wall undergoes major rearrangements depending on the carbon source. On the other hand, the genes that were downregulated on glycerol and associated with GO terms for the cell wall biosynthesis encoded α-glucosidases (AN8953.2, AN0941.2, and AN4843.2) and were assigned to cluster 5. These enzymes are responsible for the breakdown of α-linked glucans into glucose, and it is therefore surprising that three genes encoding α-glucosidases (one putatively [AN0941.2] and two experimentally confirmed [agdA (AN2017.2) and agdB (AN8953.2)] ) exhibited their highest expression levels on glucose, which means that they are not repressed by glucose. It could be speculated that these genes are also involved in the remodeling of the α-glucan fraction of the cell wall, depending on the available carbon source.
One of the α-glucosidases (AN2017.2) is part of a gene cluster that encodes proteins responsible for the breakdown of α-glucans (such as starch). This cluster contains a putative glycosyl transferase (AN2015.2) that was significantly downregulated on ethanol compared with glucose and glycerol (assigned to cluster 3); the previously mentioned agdA, which encodes an α-glucosidase; the regulatory protein amyR (AN2016.2), which appears to be regulated in the same way as agdA (also found in cluster 2 and significantly downregulated on ethanol compared with glucose); and, finally, amyA (AN2018.2), which encodes an α-amylase but which does not appear to be significantly regulated under the conditions investigated in the present study.
AmyR directly controls the expression of agdA by binding to its promoter  and the direct correlation between the two mRNA levels suggests that solely the quantity of AmyR within the cell might be responsible for the regulation of agdA, without any further requirement for post-translational activation of AmyR. It is also interesting to note that it has previously been shown that amyR is controlled by CreA , which is in agreement with our findings (compare the expression pattern of cluster 6, containing creA, with that of cluster 2, containing amyR).
It has been reported that the specific growth rate influences the expression of genes encoding ribosomal proteins in S. cerevisiae, and that the transcription of these genes increases with increasing specific growth rates . Similarly, we observed that the expression profiles of genes encoding ribosomal proteins followed the same trend as the maximal specific growth rate. In fact, in the batch cultivations carried out with A. nidulans on the different carbon sources the cells grew at unlimited conditions, and hence at their maximal specific growth rate possible on the given carbon source. The specific growth rates were highest for growth on glucose and about the same on ethanol and glycerol (Figure 1). According to the GO term analysis, cluster 1 is mainly characterized by genes whose functions are related to ribosome biogenesis, as can be observed in Table 3. Indeed, 59 out of the 280 genes assigned to cluster 1 are associated with the GO term 'ribosome biogenesis'. It is interesting that this cluster includes genes that have higher expression levels during growth on glucose, and this indicates - as observed for yeast - that these genes might indeed be involved in the ribosome biogenesis and that they are possibly regulated in a growth-rate dependent manner.