3.1. RNA sampling points were selected according to the dynamics of glycerol consumption in CG or CGHH
To identify genes and metabolic pathways active during glycerol turnover in R. toruloides CBS14, cells were cultivated under different growth conditions. Differential gene expression analysis was performed by bulk RNA-sequencing (RNA-seq) at different time points as explained below: As mentioned above, R. toruloides CBS14 showed different growth rates in cultivation media using either CG or CGHH as the main carbon source [10]. Faster growth, glycerol consumption, and lipid formation were observed in CGHH compared to CG [10]. Thus, sampling timing was selected based on the observed dynamics of glycerol consumption. RNA isolation was done in three independent cultivations for each culture medium (Fig. 1). The first sampling was performed after 10 h to allow the cultures to adapt to the cultivation conditions. In CGHH, the consumption of glycerol was visible after 10 h. However, a physiologically comparable situation was reached in the CG culture after about 30 h, so a further sample was taken from the CG culture at this time. Another sampling point was chosen after 36 h in CGHH and 60 h in CG. In CGHH, about 20 g/L of glycerol was left at this time point and the additional carbon sources from HH were consumed entirely. This culture condition was thus similar to the CG- culture after 60 h, where about 20g/L was also left. In the CGHH culture, another sample was taken after 60 h. At this point, glycerol was still present, but only half as much after 36 h. Thus, the expression profile of this sample may reflect physiological responses to different glycerol concentrations.
3.2. Global gene expression patterns differ clearly between time points and carbon sources.
Prior to differential expression analysis with DESeq2, the quality of the transcriptome reads was checked. Passed reads were mapped and quantified using the annotated genome of R. toruloides CBS14 [23]. The number of TPM was calculated per gene and sample. The expression levels of each condition were thus normalized against gene length and sequencing depth. Weakly expressed genes were filtered out. The density of highly expressed genes within contigs and scaffolds from CBS14 genome assembly is shown in Fig. 2. An expression level of at least 105 TPM is evenly distributed throughout the genome, except for contigs 49 (length 62 kbp) and 64 (length 151 kbp), from which no transcripts were recovered. Differences in the expression profile can be spotted between different time points and media. In addition, gene expression density and transcription levels in the mitochondrial genome were much higher than in the rest of the genome (results not shown). We conduct PCA to analyze differences in the clustering of biological replicates and global gene expression patterns between the samples (Additional file1: Fig. S1-S4). At all investigated time points analyzed, the gene transcription significantly differed. Most of the variances (92%) can be explained by the principal component 1, the sampling time, and 5% (PC2) by medium composition (Fig. S1). The genes with annotated function which contributed most to the differences between conditions were in decreasing order: RHOT147219 (encoding NADH-ubiquinone oxidoreductase chain 1), RHOT147222 (cytochrome c oxidase subunit 1), RHOT142646 (sulfated surface glycoprotein 185), RHOT149100 (putative protein TPRXL) and RHOT149239 (elongation of fatty acids protein 3).
Transcription levels of genes were first compared pairwise between the sampling points of the same growth condition (CG or CGHH). More specifically, we compared each of the two later sampling points with the first after 10 hours of growth and assigned the identified differentially expressed genes to the KEGG metabolic pathways and cellular processes (Fig. S5). The accounted genes summarized in Fig. S5 showed a significant up- or downregulation (p < 0.05) with a log2Fold change > 1.5 or < -1.5, which is also in line with the high variance in gene expression shown by the principal component 1 (PC1 92%, see Fig. S1). In all differential comparisons, the number of down-regulated genes is higher than the number of upregulated genes per KEGG pathway and process. In other words, the highest number of upregulated genes was present in the 10 h-samples. In CGHH, glucose was exhausted at this time point (Fig. 1b), and thus, the significantly higher gene expression at 10 h compared to later time points is probably related to the transition to a broader spectrum of metabolic activities to assimilate other carbon sources [34]. In comparison, the number of differentially expressed genes in CG with no additional carbon sources remained close to zero between 10 h and 30 h of growth for most pathways and processes. This is also indicated by the low expression variance between these samples (Fig. S1).
Changes in transcript abundance were further evaluated to identify differentially expressed genes (adjusted p-value < 0.05) between R. toruloides cell cultures grown on different carbon sources at a similar physiological situation, as illustrated in Volcano plots (Fig. 3, Fig. S2-S4, and Table S1-S3). They correspond to samples after adapting to cultivation conditions in each medium (CGHH 10 h vs. CG 10 h), when glycerol consumption became visible (CGHH 10 h vs. CG 30 h), and when there is about 20 g/l of glycerol left, and the additional carbon sources from HH were completely consumed (CGHH 36 h vs. CG 60 h).
3.3. Increased protein turnover and energy metabolism in CGHH after 10 hours of cultivation
Of 634 differentially expressed genes, 396 were significantly higher transcribed (p < 0.05, log2Fold change < 0) in CGHH 10 h than in CG 10 h (Fig. 3a, and Table S1). Many of these genes were generally higher expressed also when compared to the other sampling points, both in CGHH and CG. Genes encoding enzymes involved in metabolic pathways were most differentially expressed within the assigned KEGG orthologs. An exceptionally high proportion of these genes are involved in amino acid metabolism and were upregulated mainly in cells grown in CGHH as carbon source (Fig. 4a). While genes involved in signal transduction had the highest number of upregulated genes among cellular processes in CG 10 h, genes involved in translation was highest in CGHH 10 h (Fig. 4a).
About 27 ribosomal protein genes were upregulated in CGHH 10 h, including both cytoplasmic and mitochondrial ribosomes (Fig. S6b). Genes involved in ribosome biogenesis and spliceosome formation, as well as translation initiation factors and components of all three DNA-dependent RNA-polymerases were also higher expressed in CGHH. Apart from genes involved in protein synthesis, protein degradation also appeared activated in CGHH 10 h. Transcription of 13 proteasome-related genes was upregulated in CGHH (Fig. S6c), while none were downregulated. Compared to all other measuring points, most of them had TPM values about 2-3-fold higher, both in CGHH and CG.
Besides gene expression, protein synthesis, and protein degradation through proteasome, a high proportion of the upregulated genes in CGHH 10 h compared to CG 10 h were associated with energy metabolism (Fig. 4a). This includes especially genes encoding proteins for oxidative phosphorylation and other mitochondrial enzymes. 21 genes involved in the mitochondrial electron transport chain-complex I (NADH ubiquinone oxidoreductase), III (cytochrome c reductase), IV (cytochrome c oxidase), and F-type ATPase were significantly regulated (p < 0.05), 20 of which were upregulated in CGHH 10 h (Fig. S6a). Two prohibitin genes (RHOT148333 and RHOT147096), involved in the formation of respiratory supercomplexes [35], were regulated similarly. A variety of genes encoding mitochondrial enzymes or accessory components followed the same pattern: They were upregulated in CGHH 10 h compared to CG 10 h, had the highest TPM values in CGHH 10 h, and were at 36 h lower, but still at a similar level as in CG 10 h (Fig. 5g, and Table S4). This includes genes encoding enzymes involved in the tricarboxylic acid (TCA) cycle, such as NADP+-specific isocitrate dehydrogenase (ICDH), succinate-CoA ligase, and fumarate hydratase (RHOT145845, -7009 and − 5604, respectively), or in the synthesis of cofactors of mitochondrial enzymes such as riboflavin (RHOT149252, 2556 and 2045), lipoic acid (by lipoyl synthase, RHOT145711) and thiamine pyrophosphate (TPP, by thiamine pyrophosphokinase, RHOT149040). Lipoic acid and TPP are cofactors of pyruvate dehydrogenase and α-keto glutarate dehydrogenase [36].
On the other hand, transcription of most genes involved in glycolytic reactions and regulation were not significantly different between CG 10 h and CGHH 10 h, when this medium still had xylose and acetic acid as additional carbon sources (Fig. 1b). The exceptions were the genes encoding a phosphofructokinase (PFK2, RHOT143173), which was downregulated in CGHH 10 h, phosphoglyceromutase (GPM1, RHOT146772) and triosephosphate isomerase (TPI1, RHOT141080), which were upregulated in CGHH 10 h. Other glycolytic genes were expressed constitutively. Phosphoglyceromutase (PGM) has been identified as the only glycolytic enzyme significantly differentially expressed in R. toruloides cells depending on the carbon source, glucose, or xylose [37]. It was reported for a recombinant S. cerevisiae strain that the overexpression of a PGM- gene enhanced xylose fermentation [38]. Furthermore, PFK2 was also downregulated on YP (yeast/peptone) medium supplemented with either xylose or glucose when compared to the expression on YP only [19]. In the same study, TPI1 was overexpressed when YP medium was supplemented with acetate [19].
Some of the suggested genes that are involved in xylose assimilation in R. toruloides are NAD(P)H-dependent xylose reductase (XR), NADH-dependent xylitol dehydrogenase (XDH), xylulose kinase (XK), and phosphoketolase [37]. By performing homology analyses, we identified RHOT147125 as XR (Rhto_03963). Its transcription was more than doubled in CGHH (Fig. 5b, and Table S4), although significant upregulation (p < 0.05) was only found compared to CG 30 h and to the later CGHH sample points. In both media, RHOT147125 expression decreased with time. Besides XR, Tjukova et al. [37] identified three aldo/keto reductases that were potentially involved in xylose assimilation. They are annotated in CBS14 as galacturonate reductase (RHOT148868), glycerol 2-dehydrogenase (NADP+) (RHOT14370), and uncharacterized oxidoreductase C2F3.05c (RHOT143299). RHOT148868 and RHOT14370 were upregulated (p < 0.05) in CGHH 10 h compared to CG 10 h. The gene encoding XDH (RHOT149007) was significantly higher transcribed (p < 0.05) at CGHH 10 h and strongly decreased between 10 and 36 h. (Fig. 5b, and Table S4). In CG, XDH was equally low expressed at all sample points. According to homology analysis encoded by RHOT149455, XK was poorly expressed (Table S4) at all sample points in both media without any significant differential expression, as observed in a previous study [19]. Contradictory, a phosphoketolase gene (RHOT147705) was significantly higher transcribed (p < 0.05) in CG 10 h than in CGHH 10 h. Tjukova et al. [37] proposed that with excess carbon, the reaction catalyzed by phosphoketolase might be unnecessary, resulting in most xylulose-5-P entering the pentose phosphate pathway (PPP). However, RHOT146580, encoding ribulose-phosphate 3-epimerase, was also expressed but with no significant differences between media and sample points. Jagtap et al. [19, 39] suggested that R. toruloides might use an alternative route for xylose utilization, in which xylulose is converted to D-arabitol rather than to xylulose-5-phosphate by the activity of XK. An enzyme catalyzing this alternative reaction is D-arabinitol 4-dehydrogenase. The genome harbors a gene coding for arabinitol 4-dehydrogenase (RHOT144154). It was transcribed highest in CGHH 10 h, and the expression decreased with time (Fig. 5b, and Table S4). The differences were also significant between CGHH 10 h and all the time points in CG. However, whether it can act on D- and L-arabinitol remains unclear. ARD1 (RHOT146692), which encodes D-arabinitol 2-dehydrogenase, was also higher transcribed in CGHH 10 h compared to CG 10 h but without statistical significance. Its expression decreased with time in CGHH. ARD1 could be involved in the formation of D-ribulose from D-arabinitol. These results agree with the proposition from Jagtap et al. [19, 39], though Ribulokinase (RHOT145356) was expressed at an even lower level and not significantly different.
Five genes that may be important in the degradation of aromatic compounds were clearly expressed in CGHH 10 h, while there was only weak expression in CG (Fig. 5f, and Table S4). Aromatic monomers could originate from the lignin and thus reside in the hemicellulose portion of CGHH.
The highest number of upregulated genes involved in lipid metabolism was found at 10 h of cultivation, with higher levels in CGHH (Fig. 4a, and Additional file1: Fig. S5). The gene encoding acetyl-CoA synthetase (ACS, RHOT148257) was upregulated in CGHH 10 h compared to CG 10 h. This acetate-converting enzyme is part of the acetate assimilation pathway and was likely upregulated since cells at this point were consuming the acetic acid present in the HH (Fig. 1b) [10, 40]. At later time points, when acetate was also no longer detected in the medium, the transcription of this gene was downregulated compared to 10 h. Interestingly, its expression in CG 10 h was relatively high, about half that in CGHH 10 h, even though no acetate was present in the cultivation medium. Here, too, the gene was downregulated with increasing cultivation time. Acetate could originate as a secondary metabolite from other metabolic pathways associated with glycerol assimilation. ATP-dependent citrate lyase (ACL, RHOT147175), thought to be the main producer of acetyl-CoA in FA synthesis [41, 42], was downregulated in CGHH 10 h compared to CG 10 h. High levels of cytoplasmic acetyl CoA produced by ACS could affect the expression level of ACL. A previous study also showed reduced expression of ACL1 in R. toruloides grown on YP supplemented with acetate compared to when supplemented with glucose [19]. At later time points, higher transcription levels were found in CGHH (Fig. 5d, and Table S4). The expression of acetyl CoA carboxylase (RHOT148968) showed no significant differences between media or time points. The FA synthase genes RtFAS1 (RHOT148939) and RtFAS2 (RHOT146383) were transcribed at low levels in both substrates, particularly in CGHH, upon 10 h of cultivation, but without significant differences between the media. In contrast, two genes involved in fatty acid (FA) biosynthesis, 3-ketoacyl-acyl carrier protein reductase (FabG, RHOT148056) and enoyl carrier protein reductase (RHOT148822), were significantly upregulated in CGHH 10 h compared to CG 10 h. Transcription of these genes declined at later time points in CGHH down to levels comparable to those in CG (Fig. 5d, and Table S4). A diglyceride acyltransferase encoding gene (RHOT149017), involved in triacylglycerol biosynthesis, was also upregulated in CGHH 10 h compared to CG 10 h, and its expression significantly decreased with cultivation time in CGHH. Upregulation of this enzyme on acetate-containing medium has previously been observed elsewhere [19]. RHOT147182, which codes for a putative acyl-CoA desaturase, had very low TPM values in CGHH 10 h compared to the other conditions and was downregulated in CGHH compared to CG 10 h. Additionally, ten genes involved in FA degradation, about half of the genes being mitochondrial and the other half being peroxisomal, were significantly upregulated in CGHH 10 h compared to CG 10 h. FA accumulation is considered higher at later time points when there is nitrogen or phosphate limitation but a surplus of carbon [18, 43]. FA degradation at earlier growth stages could be related to an increase in released FA through autophagy processes triggered by glucose depletion [44]. Transcription of genes encoding enzymes involved in NADPH- generation did not differ significantly between media at 10 h, except for mitochondrial NADP+-specific ICDH, as described above.
The catabolic L-glycerol 3-phosphate (G3P) pathway, involving glycerol kinase (GUT1) and FAD-dependent glycerol-3-phosphate dehydrogenase (GUT2), is used by Saccharomyces cerevisiae as the main assimilation pathway for glycerol as demonstrated by deletion studies targeting GUT1 and GUT2 [45, 46]. Another proposed pathway in yeast is the catabolic dihydroxyacetone (DHA) pathway. It is performed by glycerol dehydrogenase (GDH) and DHA kinase (DAK) [47, 48]. A third pathway, termed the catabolic glyceraldehyde (GA) pathway, has been proposed for Neurospora crassa. Here, the glycerol is first oxidized by an NADP+-dependent GDH to GA, which is then either phosphorylated by a GA kinase to GA-3-P or reduced by an aldehyde dehydrogenase to D-glycerate. A glycerate 3-kinase then converts the D-glycerate to 3-P-D-glycerate [47, 49, 50].
At 10 h of cultivation, transcripts of two putative glycerol transporters (STL1, RHOT147915, and GPU1, RHOT144353) were more abundant in CG than in CGHH. Their transcription was slightly enhanced at 36 h in CGHH, the time when all other carbon sources were depleted (Fig. 5c, and Table S4), however, the differences between media and between time points were not of statistical significance. Enzymes belonging to the catabolic G3P pathway were transcribed under all conditions without significant differences. Enzymes belonging to the catabolic DHA pathway were also expressed, indicating the presence and expression of alternative pathways of glycerol assimilation in R. toruloides. The genome harbors two NADP+-dependent glycerol dehydrogenase genes (GCY1- homologs), RHOT14370 and RHOT144361, that convert glycerol to DHA. They were highest expressed in CGHH 10 h. Transcription levels decreased with time, except for RHOT14370 in CG, where the level increased. This enzyme was previously described as involved mostly in glycerol anabolic reactions [47]. The genome also encodes a DHA kinase 2 homolog, alternative name glycerone kinase 2 (DAK2, RHOT142321), which phosphorylates both DHA and GA, indicating that it may also be involved in the GA pathway in addition to the catabolic DHA pathway. DAK2 expression decreased in the 60 h samples in both media compared to the earlier time points, but there were no significant differences between the conditions. The genome harbors an Alcohol dehydrogenase [NADP(+)] gene (ARI, RHOT147125), whose encoding protein was found to have 54% sequence identity to NADP+-dependent GDH from Trichoderma reesei (ABD83952.1), besides 100% identity to XR from R. toruloides NP11. ARI could have mediated the conversion of glycerol to GA, which represents the first step of the catabolic GA pathway, in addition to its role in xylose metabolism [47]. ARI was transcribed under all conditions, and the transcription levels decreased with time (Fig. 5c). It was higher transcribed in CGHH 10 h than in CG 10 h but without significant differences. A variety of aldehyde dehydrogenases were expressed under both experimental conditions, however, differences in their expression could not be shown. We identified RHOT146637 as D-glycerate 3-kinase (RTG_01831) by performing homology analyses. It was transcribed under all conditions but without significant differences. Summarized, this suggests that R. toruloides can utilize all three glycerol assimilation pathways described in fungi [47]. The expression level of enzymes belonging to the DHA catabolic pathway could account for the differences in glycerol assimilation between cells grown in CGHH and CG after 10 h.
Several genes involved in handling oxidative stress were upregulated in CGHH at 10 h, including three thioredoxin genes (RHOT143685, 7078, and 3176). However, some stress-related genes were also upregulated in CG, including a catalase gene (RHOT141031) (Fig. 5e, and Table S4).
Central metabolic pathways that were differentially regulated at the 10 h sampling point are represented in Fig. 6a.
3.4. The number of differentially expressed genes was highest when glycerol consumption became visible
Of the 787 differentially expressed genes in CGHH 10 h, 585 were upregulated in CG 30 h (Fig. 3b, and Table S2). Central metabolic pathways that were differentially regulated are shown in Fig. 6b. The pathways with a higher number of differentially expressed genes were energy, carbohydrate, and amino acid metabolism, in descending order (Fig. 4b). These mainly were upregulated in CGHH 10 h, similar as when compared to the earlier time point in CG.
About 27 genes associated with oxidative phosphorylation were upregulated in CGHH 10 h (Fig. S7a) compared to CG 30 h. Additionally, the upregulated genes at 10 h cultivations which encoded mitochondrial enzymes or components such as prohibitin, NADP+-specific ICDH, succinate- CoA ligase, fumarate hydratase, riboflavin synthase, lipoyl synthase, and thiamine pyrophosphokinase, were still upregulated in CGHH 10 h when compared to CG 30 h.
Differences in the transcription of glycolytic genes included the downregulation of PFK2 and upregulation of GPM1 and TPI1 in CGHH, similarly as described at 10 h comparison. A gene encoding pyruvate kinase (RHOT144157) was upregulated in CGHH. Phosphoenolpyruvate carboxykinase gene (PCK1, RHOT144519), which can be linked with gluconeogenic reactions, was also upregulated in CGHH, while pyruvate carboxylase gene (PYC1, RHOT149202) was downregulated. PCK1 was previously found to be upregulated during growth of R. toruloides on acetate [19]. Another upregulated gene involved in carbohydrate metabolic processes encodes a putative glucose-6-phosphate 1-epimerase (RHOT149194). Of the described differentially expressed genes involved in xylose assimilation in R. toruloides, ARI, the aldo/keto reductases RHOT148868 and RHOT14370, XYL2, LAD1, and ARD1 were upregulated in CGHH (Fig. 6b), which suggests an increased flux via arabitol.
The processes of folding, sorting and degradation, and translation also had a high number of upregulated genes in CGHH 10 h compared to CG 30 h (Fig. 4b). Transcription of 46 ribosomal proteins (Fig. S7b), five RNA polymerases, and 20 genes associated with the spliceosome was upregulated in CGHH. There were no downregulated genes whose functions are involved in transcription. In addition, the transcription of genes involved in protein degradation was activated in CGHH. Transcription of 19 genes involved in proteasome assembly was upregulated (Fig. S7c), while none was downregulated in CGHH.
The highest number of upregulated genes involved in lipid metabolism was also found on the carbon source CGHH at these time points (Fig. 4b). In a similar pattern to that described in the comparison of the 10 h sample points, transcription of the genes encoding for ACS, FabG, enoyl carrier protein reductase, and diglyceride acyltransferase was upregulated in CGHH. ACL and probable acyl-CoA desaturase were downregulated, and there were no significant differences between media for ACC1, RtFAS1, and RtFAS2. Twelve genes involved in FA degradation were significantly upregulated in CGHH, about six of which were peroxisomal. Transcription of genes encoding enzymes involved in cytosolic NADPH- generation did not differ significantly between media when glycerol consumption became visible.
The genes involved in glycerol assimilation which were differentially expressed when comparing CGHH 10 h with CG30 h were two GCY1 genes and ARI. They might be associated with the catabolic pathways via DHA and GA, respectively.
Several genes related to oxidative stress management were also upregulated in CGHH at 10 h compared to CG 30 h, including the three thioredoxin genes described above and a glutathione-S transferase (RHOT149349).
3.5. The depletion of additional carbon sources induces changes in glycerol utilization and carbohydrate pathways
311 of 632 differentially expressed genes were higher transcribed in CGHH 36 h than in CG 60 h (Fig. 3c, and Table S3). Central metabolic pathways that were differentially expressed are shown in Fig. 6c. The cellular process with the highest number of upregulated genes was signal transduction in CGHH (Fig. 4c). Carbohydrate metabolism was the pathway with a higher number of differentially expressed genes, with a more prominent expression level on the CGHH carbon source.
Transcription of seven genes associated with Glycolysis and Gluconeogenesis was upregulated in CGHH, while none was downregulated. Besides TPI1 and GPM1, the other upregulated genes were an aldehyde dehydrogenase (RHOT148569), glyceraldehyde 3-phosphate dehydrogenase (RHOT147990), pyruvate dehydrogenase E1 component subunit beta (RHOT14206), pyruvate dehydrogenase complex component E2 (RHOT146289), and enolase (RHOT142969). Another upregulated gene involved in carbohydrate metabolic processes was that for glucose-6-phosphate 1-dehydrogenase (RHOT146681), which provides NADPH and pentose phosphates for the synthesis of FA and nucleic acids.
There were few significant differences in gene expression related to lipid metabolism between CGHH 36 h and CG 60 h. The exceptions were diglyceride acyltransferase, involved in triacylglycerol biosynthesis, and the peroxisomal multifunctional beta-oxidation protein (RHOT144031), involved in FA degradation. They were upregulated in CGHH and CG, respectively. Interestingly, RtFAS21 (RHOT146384), which forms an antisense RNA [23], was expressed in CG 60 h.
About 24 genes associated with oxidative phosphorylation were upregulated in CGHH 36 h (Fig. S8a) compared to CG 60 h. Other genes encoding mitochondrial enzymes or associated components still upregulated in CGHH when glycerol was the sole carbon source were prohibitin-2, NAD+-specific ICDH (RHOT14435), fumarate hydratase, DHBP synthase, and lipoyl synthase. A cytoplasmic malate dehydrogenase (RHOT147988) was also upregulated in CGHH.
There were two RNA polymerase genes (DNA-directed RNA polymerase I subunit rpa1 and DNA-directed RNA polymerase II subunit rpb1) that were downregulated and none upregulated in cultivations with CGHH as carbon source. However, the transcription of 22 ribosomal proteins (Fig. S8b) was upregulated. In addition, the transcription of genes involved in protein degradation was activated in CGHH, including seven genes involved in proteasome assembly (Fig. S8c).
At these time points, GCY1 and ARI transcription, possibly involved in glycerol and xylose metabolism, were no longer significantly different between cultivations. However, expression of an NAD+-dependent glycerol-3-phosphate dehydrogenase (RHOT141674) was upregulated in CGHH, generating NADH along with glycerol catabolism. NADH could then be transported to the mitochondria by the malate dehydrogenase shuttle or GUT2.
In general, there were 116 shared ortholog clusters of genes with a significantly different expression between the R. toruloides cell cultures grown on different carbon sources but otherwise at similar physiological conditions (Fig. S9). Besides gene clusters involved in respiration, protein synthesis, and protein degradation, some other functions or pathways that were expressed significantly different between media were vacuole-ER tethering, oxidoreductase activity, unidimensional cell growth and sporulation.