Research Article

GntR Family Regulator DasR Controls Acetate Assimilation by Directly Repressing the acsA Gene in Saccharopolyspora erythraea

Di You, Bai-Qing Zhang, Bang-Ce Ye
Anke Becker, Editor
DOI: 10.1128/JB.00685-17

ABSTRACT

The GntR family regulator DasR controls the transcription of genes involved in chitin and N-acetylglucosamine (GlcNAc) metabolism in actinobacteria. GlcNAc is catabolized to ammonia, fructose-6-phosphate (Fru-6P), and acetate, which are nitrogen and carbon sources. In this work, a DasR-responsive element (dre) was observed in the upstream region of acsA1 in Saccharopolyspora erythraea. This gene encodes acetyl coenzyme A (acetyl-CoA) synthetase (Acs), an enzyme that catalyzes the conversion of acetate into acetyl-CoA. We found that DasR repressed the transcription of acsA1 in response to carbon availability, especially with GlcNAc. Growth inhibition was observed in a dasR-deleted mutant (ΔdasR) in the presence of GlcNAc in minimal medium containing 10 mM acetate, a condition under which Acs activity is critical to growth. These results demonstrate that DasR controls acetate assimilation by directly repressing the transcription of the acsA1 gene and performs regulatory roles in the production of intracellular acetyl-CoA in response to GlcNAc.

IMPORTANCE Our work has identified the DasR GlcNAc-sensing regulator that represses the generation of acetyl-CoA by controlling the expression of acetyl-CoA synthetase, an enzyme responsible for acetate assimilation in S. erythraea. The finding provides the first insights into the importance of DasR in the regulation of acetate metabolism, which encompasses the regulatory network between nitrogen and carbon metabolism in actinobacteria, in response to environmental changes.

INTRODUCTION

The carbohydrates N-acetylglucosamine (GlcNAc) and its β-1,4-linked polymer chitin are abundant in soil, originating mainly from the cuticle of arthropods and the cell wall of filamentous fungi (1), and are used by soil microorganisms as carbon and nitrogen sources. The transcription of genes involved in the degradation of chitin and in GlcNAc metabolism in actinobacteria is under the control of the GntR family regulator DasR, which performs an important function in primary and secondary metabolism (2–4). In Streptomyces coelicolor, DasR displays either activating or repressing functions with respect to expression of the genes involved in chitin degradation (chi genes) or in GlcNAc utilization, respectively (5, 6). In Saccharopolyspora erythraea, all putative chi genes as well as those related to GlcNAc utilization are repressed by DasR (1). DasR is also involved in controlling the energy flux and metabolic rate of the tricarboxylic acid (TCA) cycle, by directly controlling the transcription of the citrate synthase (CS) gene, the gatekeeper of the TCA cycle (7). Moreover, DasR is a pleiotropic transcriptional regulator, acting as a master switch for antibiotic production, by regulating all genes related to GlcNAc catabolism (4).

GlcNAc is a preferred carbon and nitrogen source for streptomycetes, and the related metabolite glutamate is preferred over glucose (8, 9). GlcNAc was degraded into three products, including (i) ammonia, the preferred nitrogen source for bacteria; (ii) fructose-6-phosphate (Fru-6P), which directly participates in glycolysis; and (iii) acetate, an important short-chain fatty acid that prokaryotes use as a carbon and energy source. Acetate can be converted into acetyl coenzyme A (acetyl-CoA), a two-carbon metabolite used in various biological processes. During growth at low concentrations of acetate (<10 mM), prokaryotic cells convert acetate into acetyl-CoA before utilization, a reaction catalyzed by acetyl-CoA synthetase (Acs) (10). The Acs enzyme belongs to the acyl-CoA synthetase family (Pfam 00501), which is ubiquitous in almost all domains of life (11, 12). The critical role of Acs has been recognized for decades, and Acs activity is under the control of careful and complex regulation. The expression of the acs gene is strictly regulated by many regulatory factors (such as the carbon regulator cyclic AMP receptor protein CRP, the oxygen regulator FNR, nucleoid proteins FIS and IHF, and the glyoxylate shunt repressor IclR and its activator FadR) in the Gram-negative bacterium Escherichia coli (13–16). In Bacillus subtilis, the CcpA carbon regulator and the CodY GTP-sensing transcription factor are responsible for the activation of acs transcription (17). Nevertheless, the regulation of acs expression in actinobacteria is not well understood.

More recently, our previous work showed that in the high-G+C-content Gram-positive bacterium S. erythraea, three Acs enzymes (AcsA1, AcsA2, and AcsA3 [encoded by S. erythraea E_0337 {SACE_0337}, SACE_2375, and SACE_4729, respectively]) are regulated at the transcriptional and posttranslational levels by the GlnR nitrogen regulator, which mediates the interplay between nitrogen metabolism and acetate metabolism (18). In this study, we further characterized the expression of one acetyl-CoA synthetase (AcsA1 [SACE_0337], the major Acs enzyme in acetate assimilation) in S. erythraea and found that GntR-like regulator DasR directly repressed acsA1 transcription, while GlcNAc could relieve that repression. Additionally, a dasR-deleted mutant (ΔdasR) showed a growth defect in 10 mM acetate minimal medium, a condition under which appropriate Acs activity is critical for normal growth. These results demonstrate that DasR controls assimilation of acetate (one of the degradation products of GlcNAc) by directly repressing the transcription of the acsA1 gene and exerts regulatory effects on the production of intracellular acetyl-CoA in response to GlcNAc.

RESULTS

DasR directly represses transcription of acsA1.DasR-responsive elements (dre) in actinobacteria were investigated in previous studies (1, 3, 4). We have identified a consensus sequence, TGGTCTAGACCA, as the dre motif of S. erythraea; it is highly similar to the one previously observed in S. coelicolor (1). Interestingly, using MAST/MEME tools and PREDetector software, a putative dre site (TGGTCTAAACCA) was found in the upstream region of acsA1, a gene encoding an AMP-forming acetyl-CoA synthetase in S. erythraea (Fig. 1A). To determine whether DasR binds directly to the promoter regions of the acsA1 gene, electrophoretic mobility shift assays (EMSAs) were performed with a 200-fold excess of unlabeled specific probe (S) and nonspecific competitor DNA (sperm DNA) (N) as controls. Obvious shift bands were observed when the purified His-DasR protein was added (Fig. 1B), indicating that DasR specifically bound to the acsA1 promoter. The results suggest that the acsA1 gene is subjected to transcriptional regulation by DasR. To investigate the effect of DasR on acsA1 expression in vivo, the transcription levels of acsA1 in S. erythraea wild-type (WT), ΔdasR, and dasR-complemented (ΔdasR::dasR) strains grown in Evans medium containing glucose were quantified by real-time reverse transcription-PCR (RT-PCR). As shown in Fig. 1C, deletion of dasR resulted in a 4-fold increase in acsA1 expression, and complementation of the mutant strain with the dasR gene resulted in transcription levels similar to those of the WT strain. No significant differences were observed in the growth levels of the WT and dasR-complemented strains. The growth curves showed a slightly lagged exponential phase for the dasR mutant (Fig. 1D). A longer doubling time than that of the WT strain indicated that DasR slightly induced the utilization of glucose (Table 1). Taken together, these observations indicate that DasR is a direct repressor of acsA1 transcription and that it plays an important role in the assimilation of acetate, one of the products of GlcNAc catabolism in S. erythraea.

FIG 1
FIG 1

DasR directly controls transcription of acsA1 in S. erythraea. (A) DasR binding site (dre) in the promoter regions of acsA1. (B) EMSAs of His-DasR protein with upstream promoter regions of acsA1. The DNA probe (10 nM) was incubated with a concentration gradient of His-DasR (0, 0.5, and 2.5 μM). EMSAs with a 200-fold excess of unlabeled specific probe (S) or nonspecific competitor DNA (sperm DNA; N) were performed as controls. (C) Transcriptional analysis of acsA1 in S. erythraea wild-type (WT), ΔdasR, and dasR-complemented (ΔdasR::dasR) strains grown at 30°C in Evans medium containing glucose. The strains were grown for 34 h (exponential phase) prior to RNA isolation. Transcription levels were normalized to the internal reference gene (16S rRNA). Fold changes represent the level of expression compared to the expression of acsA1 in the WT. Error bars show standard deviations of the mean values determined in three independent experiments. (D) Growth curves of S. erythraea WT, ΔdasR, and dasR-complemented (ΔdasR::dasR) strains grown at 30°C in Evans medium (solid lines) and glucose consumption levels (dotted lines). OD600, optical density at 600 nm. ***, P < 0.001.

View this table:
TABLE 1

Growth analysis of S. erythraea WT and ΔdasR strains

Expression of acsA1 is induced by GlcNAc through the relief of DasR-mediated repression.DasR directly controls GlcNAc uptake and chitin metabolism in S. erythraea in response to GlcNAc signal (1). The idea that DasR-mediated regulation of acetyl-CoA synthetase and, thereby, the assimilation of acetate for generation of acetyl-CoA take place under GlcNAc-rich conditions is reasonable, as such activity leads to the complete utilization of GlcNAc as a carbon source. Deletion of the dasR gene resulted in an increase in the growth rate of S. erythraea in Evans medium with GlcNAc as the sole carbon source, reaching 2-fold of the cell density of the WT strain (Fig. 2A). The dasR-complemented strain showed a growth rate similar to that of the WT strain. As shown in Fig. 2A and Table 1, curves of GlcNAc consumption indicated that DasR inhibited the utilization of GlcNAc. The difference in growth compared to that shown in Fig. 1D might have been a consequence of the fact that glucose is the favorite carbon source and that the GlcNAc utilization is under the control of complex forms of regulation such as DasR inhibition. To investigate the regulatory effects of GlcNAc on acsA1 transcription, WT, ΔdasR, and dasR-complemented strains grown in Evans medium containing 0.5% glucose or GlcNAc as the sole carbon source were harvested at the exponential phase. As shown in Fig. 2B, the acsA1 transcript level of the WT strain grown in medium with GlcNAc revealed a 6-fold increase compared to that of the WT strain grown in medium with glucose. The result indicated that DasR inhibits acetate assimilation, whereas GlcNAc relieves the repression of acsA1 transcription mediated by DasR. However, transcription of acsA1 decreased 40% in the ΔdasR strain grown with GlcNAc compared to the same strain grown with glucose. The WT and dasR-complemented strains displayed similar expression patterns (Fig. 2B). GlcNAc is degraded into acetate, Fru-6P, and ammonia and serves as both carbon and nitrogen sources. Enhanced degradation of GlcNAc in the ΔdasR strain might result in an increase in the levels of intracellular ammonium. GlnR (SACE_7101) is the nitrogen-sensing regulator which plays an important role in ammonium assimilation (19) and directly activates the expression of three acs genes in S. erythraea (18). We speculated that the downregulation of acsA1 expression in the ΔdasR mutant grown with GlcNAc was due to GlnR-mediated regulation. To test this hypothesis, we investigated the ammonium levels and glnR expression in three strains (the WT, ΔdasR, and ΔdasR::dasR strains) cultured in Evans medium with GlcNAc as the sole carbon source. As shown in Fig. 3A, the NH4 concentration was increased 1.3-fold in the ΔdasR strain compared with the WT strain. The ΔdasR::dasR strain showed levels similar to those determined for the WT strain. The observations indicated that ammonium was present in excess in the ΔdasR strain due to efficient degradation of GlcNAc. Furthermore, we found that deletion of dasR also resulted in a 3-fold decrease in glnR expression and that complementation of the dasR gene led to recovery of the transcription of the glnR gene in GlcNAc (Fig. 3B). Taken together, the results described above suggested that the enhanced degradation of GlcNAc led to low levels of expression of glnR, which repressed acsA1 expression in the dasR mutant grown on GlcNAc (Fig. 2B).

FIG 2
FIG 2

Transcription of acsA1 in response to GlcNAc. (A) Growth curves of S. erythraea WT, ΔdasR, and dasR-complemented (ΔdasR::dasR) strains grown at 30°C in Evans medium with GlcNAc as the sole carbon source (solid lines) and GlcNAc consumption levels (dotted lines). (B) S. erythraea WT, ΔdasR, and dasR-complemented strains were grown in Evans medium with 0.5% glucose or GlcNAc as the sole carbon source. Cells were harvested at 42 h during the exponential phase and subjected to RNA extraction. Transcription levels determined under each condition were normalized to the internal reference gene (16S rRNA gene). The relative level of expression of the WT strain grown in glucose was set to 1.0. Error bars indicate standard deviations of the mean values determined in three independent experiments. **, P < 0.01; ***, P < 0.001.

FIG 3
FIG 3

The presence of GlcNAc resulted in accumulation of ammonium, repressing expression of glnR in the ΔdasR strain. (A) Analysis of ammonium levels in culture medium for S. erythraea WT, ΔdasR, and dasR-complemented strains grown for 42 h in Evans medium with GlcNAc as the sole carbon source. Results represent averages from three independent experiments. (B) Transcription of glnR in response to GlcNAc. S. erythraea WT, ΔdasR, and dasR-complemented strains were grown in Evans medium with GlcNAc as the sole carbon source. Cells were harvested at 42 h (exponential phase) and subjected to RNA extraction. Transcription levels determined under each condition were normalized to the internal reference gene (16S rRNA). The relative level of expression of the WT strain grown in GlcNAc was set to 1.0. Error bars indicate standard deviations of the mean values determined in three independent experiments. **, P < 0.01.

Deletion of dasR represses the growth of S. erythraea in medium containing 10 mM acetate.Previous studies showed that an appropriate level of activity of acetyl-CoA synthetase is required for optimal microorganism growth in medium containing 10 mM acetate and that high levels of activity of the enzyme have a deleterious effect on growth (18, 20, 21). Those previous studies (18, 20, 21) and our previous work (18, 22) demonstrated that lack of the acs gene (acsA1) also caused a growth defect on 10 mM acetate. To investigate whether DasR affects the growth of S. erythraea strains in acetate-containing medium by controlling its assimilation, WT, ΔdasR, and dasR-complemented strains were grown on 10 mM acetate as the sole carbon source. As shown in Fig. 4A, compared to that of the WT strain, growth of the ΔdasR strain was inhibited by acetate; however, complementation of the ΔdasR mutant with dasR restored the growth, with the levels of acetate consumption shown in Fig. 4A indicating that DasR indeed has an impact on the growth and acetate assimilation of S. erythraea. No difference was observed when the strains were grown in the presence of glucose (Fig. 1C). Our previous studies demonstrated that excessive Acs activity (>0.4 μmol NADH min−1 mg−1) caused poor growth of S. erythraea in acetate-containing medium (18). Thus, we determined the transcript levels of acsA1 and the total levels of acetyl-CoA synthetase activity in WT, ΔdasR, and dasR-complemented strains grown in the presence of 10 mM acetate. A 3-fold increase in the acsA1 mRNA level of the ΔdasR strain in comparison to those determined for the WT strain was observed at the early stationary phase (T2 in Fig. 4A) (Fig. 4B). The total level of acetyl-CoA synthetase activity in cell extracts at T2 was then analyzed using a coupled NADH consumption spectrophotometric assay, and the results were expressed in micromoles of NADH per minute per milligram of protein. The total Acs activity of the ΔdasR strain showed a >2-fold increase from that of the WT strain (Fig. 4C). This result suggests that the low growth rate of the ΔdasR strain in medium with acetate might have been caused by excessive Acs activity (approximately 0.48 μmol NADH min−1 mg−1), which resulted from the absence of the DasR-mediated repression of acsA1 transcription.

FIG 4
FIG 4

DasR influenced the growth of S. erythraea in minimal medium containing 10 mM acetate. (A) Growth curves of S. erythraea WT, ΔdasR, and dasR-complemented (ΔdasR::dasR) strains grown at 30°C in Evans medium with 10 mM acetate as the sole carbon source (solid lines) and acetate consumption levels (dotted lines). “T0,” “T1,” and “T2” denote the early exponential, exponential, and early stationary phases, respectively. Vertical bars show standard deviations of the mean values determined in three replicate cultures. (B) Cells were harvested at the early stationary phase (T2) for RNA extraction. Transcription levels determined under each condition were normalized to the internal reference gene (16S rRNA gene). The relative level of expression of the WT strain was set to 1.0. Error bars indicate standard deviations of the mean values determined in three independent experiments. (C) Total activities of Acs in cell extracts of S. erythraea WT, ΔdasR, and dasR-complemented strains grown in minimal medium containing 10 mM acetate. **, P < 0.01.

GlcNAc exerts an effect on acetate assimilation and on the growth of S. erythraea in the presence of 10 mM acetate.To examine the effect of GlcNAc on acetate assimilation in S. erythraea grown in the presence of 10 mM acetate, WT, ΔdasR, and dasR-complemented strains were cultured in Evans medium with 10 mM acetate as the sole carbon source. As shown in Fig. 4A, GlcNAc was added (final concentration of 7.5 mM) (4, 23) at three time points: 36, 60, and 72 h (corresponding to the early exponential phase [T0], the exponential phase [T1], and the early stationary phase [T2], respectively). Equal amounts of double-distilled water (ddH2O) was added as controls. Samples were collected 45 min after the addition of GlcNAc or ddH2O (4, 23). At T0, acetate was used as the main carbon source in the WT strain grown in 10 mM acetate, and GlcNAc addition had no effect on acs transcription. Addition of GlcNAc induced the transcription of the acsA1 gene in the WT strain grown in 10 mM acetate at T1 and T2 (acetate was consumed, and GlcNAc was used as the carbon source) (Fig. 5A). In the ΔdasR strain, lack of the DasR repression led to high levels of acs transcription, and no obvious changes in acsA1 transcription levels were observed in response to GlcNAc addition at T0 and T1 (with acetate as the carbon source) (Fig. 5A). At T2, the degradation of GlcNAc (acetate was consumed, and GlcNAc was used as the carbon source) resulted in an increase in the levels of intracellular ammonium and in low glnR expression levels, which repressed acsA1 expression in the dasR mutant. The variations in the levels of acsA1 expression in the different phases might have been due to utilization of different carbon sources (acetate or GlcNAc) and different DasR/GlnR-mediated controls. Regarding the total levels of Acs activity, addition of GlcNAc had no effect on the activity at phase T0 in the WT strain, but levels of Acs activities were increased at phases T1 and T2 (Fig. 5B). No significant differences among the levels of Acs activities were observed at the three time points in the ΔdasR strain (Fig. 5B). These results indicate that GlcNAc exerts an effect on acetate assimilation by affecting expression of acsA1 via the presence of DasR. Next, the effect of GlcNAc on the growth of S. erythraea in the presence of 10 mM acetate was investigated by analyzing growth curves of WT and ΔdasR strains after GlcNAc addition at 42 h (mid-exponential phase), and the strains grown in Evans medium with 7.5 mM GlcNAc as the sole carbon source were used as a background control. As shown in Fig. 5C, GlcNAc addition inhibited the growth of the WT strain slightly, whereas no effect on the growth of the ΔdasR strain was observed. Further studies revealed that the levels of acsA1 transcription and Acs activity increased with rising GlcNAc concentrations in the WT strain, while the growth was inhibited accordingly (see Fig. S1 in the supplemental material). These observations confirmed that GlcNAc-induced excess activity of Acs caused growth inhibition.

FIG 5
FIG 5

GlcNAc exerts an effect on acetate assimilation and the growth of S. erythraea in minimal medium containing 10 mM acetate. (A) Effect of GlcNAc addition on the expression of acsA1. RNAs were extracted at the indicated times in Evans medium 45 min after GlcNAc induction. Relative transcript levels were normalized to 16S rRNA at the corresponding time points. The relative value determined for acsA1 in the WT strain without GlcNAc addition at T0 was set to 1.0. Error bars indicate standard deviations of the mean values determined in three independent experiments. (B) Effect of GlcNAc addition on total activity of Acs. The growth conditions and timings were as described for panel A. Error bars show standard deviations from the mean values determined in three independent experiments. (C) Growth curves of WT and ΔdasR strains grown in Evans medium containing 10 mM acetate as the sole carbon source (solid lines). +GlcNAc, GlcNAc was added at 42 h. Growth curves of the WT and ΔdasR strains grown at 30°C in Evans medium with 7.5 mM GlcNAc as the sole carbon source (dotted lines) are also shown. Vertical bars show standard errors of the mean values determined in three replicate cultures.

DISCUSSION

GlcNAc is an abundant source of both carbon and nitrogen nutrients for soil-dwelling microorganisms. The intracellular concentration of GlcNAc is critical for the metabolism, and its signal is transmitted through DasR, which controls the GlcNAc regulon and GlcNAc utilization (2). GlcNAc is also a signaling molecule involved in several cellular processes such as morphogenesis (24, 25); siderophore biosynthesis, antibiotic production, and sporulation (2, 4, 5, 26–29); and virulence (30, 31). DasR is a pleiotropic carbon response regulator mainly involved in GlcNAc catabolism and links carbon availability to morphogenesis of streptomycetes (2). GlcNAc is of great metabolic importance due to its three degradation products. The first is ammonia, the important component of nitrogen metabolism controlled by the GlnR nitrogen-sensing regulator (19). The second is acetate, which can be converted into acetyl-CoA by the activity of the Acs enzyme (12, 32). In bacteria, complex regulatory systems control acs gene expression and the acetylation level of the Acs enzyme to keep an appropriate level of Acs activity for maintaining the supply of acetyl-CoA and energy homeostasis during growth on acetate. Studies in Salmonella enterica have found that elevated Acs activity caused growth arrest of Pat-deficient S. enterica bacteria that were unable to acetylate Acs on acetate due to a drop in the energy charge (24, 25). Our recent work showed that there are three genes (acsA1, acsA2, and acsA3) encoding the AMP-forming acetyl-CoA synthetases (AcsA) in S. erythraea and that the AcsA1 enzyme plays a dominant role in the activation of acetate (18). Three acetyl-CoA synthetases are regulated by the GlnR nitrogen response regulator at both the transcriptional and posttranslational levels in S. erythraea. GlnR directly activated the expression of all three acs genes (18). In the present study, we found that DasR also binds directly to the promoter region of acsA1, exerting regulatory effects on the transcriptional level of acsA1 and on the total activity of acetyl-CoA synthetases as a strong negative regulator, which further influences the production of acetyl-CoA and acetate assimilation. No direct regulatory effect of DasR on acsA2 and acsA3 was observed. Acetyl-CoA is fed into the TCA cycle by the activity of citrate synthase (CS), the gatekeeper mediating the rate of the TCA cycle, providing important substrates and energy for various biosynthetic reactions. Since DasR directly represses the transcription of three S. erythraea CS genes (7), acetyl-CoA synthesis and its feeding into the TCA cycle are controlled by DasR. Acetyl-CoA can also be generated via decarboxylation of pyruvate, the end product of glycolysis. Interestingly, the third degradation product of GlcNAc, fructose-6-phosphate, is a critical component of glycolysis. Thus, there is a comprehensive network of DasR-mediated GlcNAc metabolism in S. erythraea. Regulation of the acsA1 gene in S. erythraea is subject to strict transcriptional control by GntR family regulator DasR, which links GlcNAc catabolism to acetate assimilation and reveals a possible catabolite repression link between acetate metabolism and sugar metabolism.

Nitrogen regulator GlnR and GlcNAc-sensing regulator DasR control production of acetyl-CoA and acetate assimilation in response to nitrogen and GlcNAc signals (18), indicating the regulatory mechanism underlying the integration of nitrogen metabolism and carbon metabolism under GlcNAc-rich conditions (Fig. 6). GlnR is a global regulator that controls genes involved in nitrogen metabolism in which glutamine synthetases are its important targets in S. erythraea. Intracellular nitrogen limitation induced GlnR expression and enhanced the production of acetyl-CoA through acetate assimilation. GlnR was shown to be a strong positive regulator of acsA1, while DasR performs negative regulation of acsA1, which may represent a form of cooperation in responding to different nutrient environments and of collaboration in maintaining the nitrogen/carbon metabolism balance. GlcNAc, which is used as both a carbon source and a nitrogen source, actually represents a critical connection in the acsA1 regulation by GlnR and DasR, enabling a comprehensive tentative model of Acs regulation in S. erythraea. The regulons of GlnR (33) and DasR (3, 4) have been identified in Streptomyces coelicolor, a close relative of S. erythraea. Interestingly, the typical DasR-responsive element (dre) and GlnR-binding motif (GlnR box) were both found in the promoter region of acsA gene (SCO3563) in S. coelicolor, indicating that its expression might be directly regulated by a nitrogen/GlcNAc-related signaling pathway, similarly to S. erythraea. However, this complex regulation of DasR and GlnR in Streptomyces needs to be further investigated in the future.

FIG 6
FIG 6

The DasR/GlnR-mediated network of GlcNAc degradation and acetate assimilation in S. erythraea. The blue lines indicate transcriptional regulation; arrows indicate the positive control; dots are indicative of the negative control. The black solid lines with arrows indicate the metabolic pathway. The black dashed lines with dots indicate negative effect of metabolites on regulators. The GlnR box indicates a GlnR-binding motif.

In summary, we found that GntR family regulator DasR controls acetate assimilation in S. erythraea by directly repressing expression of the acsA1 gene, which encodes acetyl-CoA synthetase. As shown in Fig. 6, acetate assimilation can be considered the last metabolic step of GlcNAc utilization. Previous work (18) and the present work have demonstrated that acetate assimilation is under the control of nitrogen-sensing regulator GlnR and of GlcNAc-sensing regulator DasR in GlcNAc-rich soil. These findings provide the first insights into the importance of DasR in the regulation of acetate metabolism, which encompasses the regulatory network between nitrogen and carbon metabolism in actinobacteria in response to the environmental changes.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.Strains and plasmids used in this work are listed in Table 2. Three S. erythraea strains, wild-type NRRL2338 (from DSM 40517), a ΔdasR mutant, and a dasR-complemented (ΔdasR::dasR) mutant, were used in this study. S. erythraea strains were grown in minimal medium (Evans) (18) with 0.5% (mass/vol) glucose or GlcNAc as the sole carbon source. Aerobic 100-ml batch cultures were grown in 1-liter flasks at 30°C on a rotary shaker at 250 rpm.

View this table:
TABLE 2

Strains and plasmids used in this work

Growth analysis.S. erythraea strains were grown in triplicate, at 30°C, in Evans medium with different carbon sources. Growth was analyzed by cell density measurements at 600 nm, every 8 h, in a microplate reader (BioTek Instruments, Winooski, VT). Data were analyzed using the Prism 5 software package (GraphPad Software).

Analysis of substrate consumption.The glucose concentration was measured using a glucose assay kit (Sigma), the acetate concentration was measured using an acetate colorimetric assay kit (Sigma), and the GlcNAc concentration was measured as described previously (34).

Overexpression and purification of DasR.In vitro production and purification of His-DasR protein were performed as described previously (23). The purified protein was analyzed by SDS-PAGE, and the concentration was determined by the bicinchoninic acid (BCA) method, using bovine serum albumin as the standard.

Electrophoretic mobility shift assay (EMSA).For EMSAs of the promoter regions (350 bp [from −300 to +50]), His-tagged DasR and biotin-labeled PCR-amplified DNA probes were used. EMSAs were performed according to the protocol described for a chemiluminescent EMSA kit (Beyotime Biotechnology, China) and previous work (19).

RNA preparation and real-time RT-PCR.RNA was isolated from the mycelia of S. erythraea and collected at the indicated time, using an RNeasy minikit (Qiagen, Valencia, CA). RT-PCR analyses were performed using a PrimeScript RT reagent kit (TaKaRa, Shiga, Japan) and a SYBR premix Ex Taq GC kit (Perfect Real Time; TaKaRa), according to previous work (19). The primers used in this study had been described in previous work (16, 17). 16S rRNA was used as internal control, while the reaction buffer was used without template DNA as a negative control. Data from three independent experiments were analyzed. Quantitative RT-PCR (qRT-PCR) validation information corresponding to amplification efficiency, calibration curves with slope and R2, specificity (melt), and data analyses are shown in Data Sets S1 to S5 in the supplemental material. Three independent experiments were performed for statistical tests.

Total intracellular Acs activity assays.For total Acs activity measurement, mycelia grown in minimal medium were collected at the indicated phase. Cultures (100 ml) were harvested by centrifugation at 8,000 × g performed for 15 min at 4°C. Cells were washed twice in 15 ml phosphate-buffered saline (PBS) buffer, and the cell pellet was subjected to liquid nitrogen grinding and resuspended in 2 ml PBS buffer containing protease inhibitors. After centrifugation at 8,000 × g for 45 min, the protein concentration in the supernatant was determined using a BCA protein assay kit (Beyotime Biotechnology, China), and equal amounts (10 mg) of protein from different strains were immediately used for Acs activity assays. The assays were performed as described previously (18, 22). Three independent experiments were performed for statistical tests.

Ammonium determination.Ammonium levels were measured in the culture medium using a commercial kit (K-Amiar Ammonia Rapid; Megazyme).

ACKNOWLEDGMENTS

This work was supported by grants from the National Natural Science Foundation of China (31730004, 31700058, and 21575089), the China Postdoctoral Science Foundation (2017M610232), and the Fundamental Research Funds for the Central Universities (222201714025).

We have no conflict of interest to declare.

FOOTNOTES

    • Received 14 November 2017.
    • Accepted 16 April 2018.
    • Accepted manuscript posted online 23 April 2018.
  • Address correspondence to Bang-Ce Ye, bcye{at}ecust.edu.cn.

  • Citation You D, Zhang B-Q, Ye B-C. 2018. GntR family regulator DasR controls acetate assimilation by directly repressing the acsA gene in Saccharopolyspora erythraea. J Bacteriol 200:e00685-17. https://doi.org/10.1128/JB.00685-17.

  • Supplemental material for this article may be found at https://doi.org/10.1128/JB.00685-17.

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KEYWORDS

GntR family regulator DasR
acetylglucosamine metabolism
acetyl-CoA synthetase
acetate assimilation

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