The ldhA Gene Encoding Fermentative l-Lactate Dehydrogenase in Corynebacterium Glutamicum Is Positively Regulated by the Global Regulator GlxR

Bacterial metabolism shifts from aerobic respiration to fermentation at the transition from exponential to stationary growth phases in response to limited oxygen availability. Corynebacterium glutamicum, a Gram-positive, facultative aerobic bacterium used for industrial amino acid production, excretes l-lactate, acetate, and succinate as fermentation products. The ldhA gene encoding l-lactate dehydrogenase is solely responsible for l-lactate production. Its expression is repressed at the exponential phase and prominently induced at the transition phase. ldhA is transcriptionally repressed by the sugar-phosphate-responsive regulator SugR and l-lactate-responsive regulator LldR. Although ldhA expression is derepressed even at the exponential phase in the sugR and lldR double deletion mutant, a further increase in its expression is still observed at the stationary phase, implicating the action of additional transcription regulators. In this study, involvement of the cAMP receptor protein-type global regulator GlxR in the regulation of ldhA expression was investigated. The GlxR-binding site found in the ldhA promoter was modified to inhibit or enhance binding of GlxR. The ldhA promoter activity and expression of ldhA were altered in proportion to the binding affinity of GlxR. Similarly, l-lactate production was also affected by the binding site modification. Thus, GlxR was demonstrated to act as a transcriptional activator of ldhA.


Introduction
Corynebacterium glutamicum is a nonpathogenic, Gram-positive, facultative anaerobic bacterium harboring a high GC content and belonging to the suborder Corynebacterineae within the class Actinobacteria. It is an important industrial bacterium that is widely used for amino acid production, including glutamate and lysine [1,2]. Complete genomic DNA sequencing, understanding of bacterial metabolism and its regulation, and development of genetic tools for metabolic engineering have expanded the portfolio of bioproducts by C. glutamicum and have made this bacterium a promising platform for microbial production of valuable products, including organic acids, biofuel, proteins, commodity chemicals, and healthcare products [3][4][5][6][7][8][9].
The high productivity of several such products is achieved by virtue of the ability of this bacterium to metabolize sugars without growth under oxygen deprivation. On the basis of these properties, we developed a bioprocess in which cells are packed at high cell density to create an oxygen-limiting environment by their own respiration [10]. As proof of concept, we achieved the highest known productivity of organic acids and amino acids via this bioprocess [11][12][13][14].
During the transition from the aerobic exponential growth phase to the oxygenlimiting stationary phase, a metabolic shift from aerobic respiration to mixed acid fermentation occurs for the reoxidation of the reductant NADH generated from central chromosome of the wild type. At the exponential phase (4 h) during cultivation in A medium supplemented with 1% glucose, the promoter activities of all of the fusion constructs were comparably low. Both F2 and F2-1 fusions exhibited an increase in promoter activity at the stationary phase (8 h) (Figure 2). By contrast, the increase in the activity of the F2-2 promoter fusion at the stationary phase was much smaller than that of the other fusions ( Figure 2). This indicates that the GlxR-binding site is involved in the ldhA upregulation at the onset of the stationary phase and that GlxR functions as a transcriptional activator.

Bacterial Strains, Plasmids, and Culture Conditions
The bacterial strains and plasmids used in this study are listed in Table S1. For genetic manipulation, Escherichia coli strains were cultivated in lysogeny broth (LB) at 37 • C. C. glutamicum strains were routinely precultured at 33 • C overnight in nutrient-rich medium (A medium) [16] supplemented with 4% glucose. Cells were resuspended in fresh A medium and inoculated into 100 mL of A medium containing 1% glucose in a 500 mL flask. Growth was monitored by measuring the optical density (OD) at 610 nm. The antibiotic concentration used for E. coli cultures included 50 µg of ampicillin mL −1 and 50 µg of kanamycin mL −1 , and for C. glutamicum, kanamycin (50 µg mL −1 ) was used.

Construction of Plasmids and Mutants
The oligonucleotides used in this study (Table S2) were obtained from FASMAC (Kanagawa, Japan). General recombinant experiments, including transformation of E. coli and plasmid extraction, were performed according to standard protocols [31]. To construct plasmids for the ldhA promoter-lacZ fusions, DNA fragments encompassing various lengths of the ldhA promoter region were amplified using the primers listed in Table S2 and cloned into the integration vector pCRA741 to form translational fusion with the lacZ gene on the vector. To modify the chromosomal GlxR-binding site, the binding site and both flanking regions were amplified by PCR (Table S2). The resulting two fragments were fused via overlapping PCR, digested with SalI, and cloned into the suicide vector pCRA725 [32]. Similarly, the flanking regions of the atlR gene were fused and cloned into pCRA725 to construct the plasmid pCRC664 for the deletion of the atlR gene. For efficient transformation of C. glutamicum, nonmethylated plasmids were obtained from E. coli JM110. C. glutamicum was transformed with the plasmids via electroporation and screened for kanamycin resistance in order to obtain the recombinants harboring chromosomal integration. To obtain marker-free mutants, the transformants were sequentially screened for sucrose resistance and kanamycin sensitivity as described previously [32]. The chromosomal mutations in the resulting mutants were confirmed by PCR amplification of the ldhA promoter region and direct sequencing of the PCR products using appropriate primers. To construct a plasmid for glxR overexpression, the glxR coding region was PCR amplified and cloned into the isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible vector pCRB12iP [33]. The resulting plasmid was electroporated into both wild-type and mutant cells carrying the mutagenized GlxR-binding site within the ldhA promoter region. Overexpression of glxR was induced by supplementing the exponentially growing culture with 0.5 mM IPTG.

β-Galactosidase Assay
β-Galactosidase activity in C. glutamicum strains containing PldhA-lacZ fusion was determined as described previously [16]. Briefly, cells permeabilized with toluene were incubated with o-nitrophenyl-β-galactosidase in Z buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO4, 0.28% (v/v) β-mercaptoethanol, which was added on use) at 30 • C. The absorbance values at 420 and 550 nm were monitored to calculate activity in Miller units as previously described [34]. We confirmed that a strain carrying the vacant vector pCRA741 containing the promoter-less lacZ had no LacZ activity.

Overexpression and Purification of His-Tagged GlxR and AtlR
Overexpression of His-tagged proteins was performed as described previously using the cold-inducible expression system [24]. Briefly, E. coli BL21(DE3) was transformed with pCRC620 or pCRC665 and grown at 37 • C in LB medium to an OD 600 of 0.5. After cooling and incubation at 15 • C, 0.5 mM IPTG was added for induction of His-tagged protein, which was purified by affinity chromatography on Ni-NTA agarose (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The PD-10 column (GE Healthcare Bioscience, Piscataway, NJ, USA) was pre-equilibrated with buffer A (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , and 1 mM EDTA) to desalt the protein sample. The protein concentration was determined by Bio-Rad protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA) using bovine serum albumin as a standard.

Electrophoretic Mobility Shift Assay (EMSA)
EMSA was performed as described previously [24]. Briefly, Cy3-labeled DNA fragments used as probes in EMSA were generated by PCR using pCRC656, pCRC659, and pCRC660, containing the ldhA promoter region with or without the mutated GlxRbinding site as a template. GlxR was incubated with cAMP prior to incubation with the labeled probe. The resulting DNA-protein complexes were loaded onto a 5% polyacrylamide gel and visualized using a Typhoon TRIO Variable Mode Imager (GE Healthcare Bioscience).

RNA Extraction and Quantitative Reverse-Transcription Polymerase Chain (qRT-PCR)
For RNA stabilization, the sampled culture was mixed with an equal volume of RNAprotect bacterial reagent (Qiagen). After incubation at room temperature, cells were collected by centrifugation and stored at −80 • C. Total RNA was isolated using NucleoSpin ® RNA (MACHEREY-NAGEL, Düren, Germany) according to the manufacturer's instructions. Purified RNA was further treated with DNaseI (Takara Bio Inc., Shiga, Japan), ethanol precipitated, and suspended in RNase-free water as described previously [35].
qRT-PCR was performed using the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and Power SYBR ® Green PCR Master Mix with MuLV Reverse Transcriptase and RNase Inhibitor from the GeneAmp RNA PCR Kit (Thermo Fisher Scientific) as described previously [17]. The primers listed in Table S2 were used. The comparative threshold cycle method (Thermo Fisher Scientific) was used to quantify relative expression, and the relative expression ratios of each gene were normalized using the values for 16S rRNA.

Analytical Method
Organic acids in culture supernatants were quantified using the Prominence 20A highperformance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan) equipped with TSKgel OApack-A and OApack-P columns (Tosoh Corporation, Tokyo, Japan) and a photodiode array detector. All organic acids were detected at 210 nm. The flow rate of the mobile phase of 10% acetonitrile with 3.0 mM HClO 4 was 0.8 mL/min, and the column temperature was set at 40 • C.

Involvement of the Putative GlxR-Binding Site in Transcription of ldhA
To examine whether the GlxR-binding site was involved in the transcriptional regulation of ldhA at the transition from the exponential to stationary phase, we constructed a series of PldhA-lacZ fusion constructs. The constructs using primers F2 and F2-1 contained the intact GlxR-binding site, and the construct generated using the primer F2-2 lacked a portion of the site ( Figure 1). The fusion constructs were individually integrated into the chromosome of the wild type. At the exponential phase (4 h) during cultivation in A medium supplemented with 1% glucose, the promoter activities of all of the fusion constructs were comparably low. Both F2 and F2-1 fusions exhibited an increase in promoter activity at the stationary phase (8 h) ( Figure 2). By contrast, the increase in the activity of the F2-2 promoter fusion at the stationary phase was much smaller than that of the other fusions ( Figure 2). This indicates that the GlxR-binding site is involved in the ldhA upregulation at the onset of the stationary phase and that GlxR functions as a transcriptional activator. To investigate the importance of the GlxR-binding site for ldhA promoter activity, we introduced mutations into the site upon the promoter-lacZ fusion. We modified the GlxRbinding site to inhibit (mut1) and enhance the binding of GlxR (mut2) by exchanging nucleotides at the conserved position in the binding site ( Figure 3a). The lacZ fusions were individually integrated into the chromosome of the wild type. The F2 promoter-lacZ fusion carrying the mutation mut1 exhibited lower activity than the native one ( Figure 3b). By contrast, the F2 promoter-lacZ fusion carrying the mutation mut2 showed higher activity than that of the native promoter fusion. The same fusion constructs were also integrated into the chromosome of the sugR-lldR double deletion mutant, in which the ldhA promoter was derepressed. As observed in the wild-type background, the mutation mut1 reduced promoter activity, and the mutation mut2 increased activity in the sugR-lldR deletion mutant background (Figure 3b). These findings indicate that GlxR acts as a transcriptional activator by binding to the site, and its function is independent of the known repressors. To investigate the importance of the GlxR-binding site for ldhA promoter activity, we introduced mutations into the site upon the promoter-lacZ fusion. We modified the GlxR-binding site to inhibit (mut1) and enhance the binding of GlxR (mut2) by exchanging nucleotides at the conserved position in the binding site (Figure 3a). The lacZ fusions were individually integrated into the chromosome of the wild type. The F2 promoter-lacZ fusion carrying the mutation mut1 exhibited lower activity than the native one ( Figure 3b). By contrast, the F2 promoter-lacZ fusion carrying the mutation mut2 showed higher activity than that of the native promoter fusion. The same fusion constructs were also integrated into the chromosome of the sugR-lldR double deletion mutant, in which the ldhA promoter was derepressed. As observed in the wild-type background, the mutation mut1 reduced promoter activity, and the mutation mut2 increased activity in the sugR-lldR deletion mutant background (Figure 3b). These findings indicate that GlxR acts as a transcriptional activator by binding to the site, and its function is independent of the known repressors. Microorganisms 2021, 9, x FOR PEER REVIEW 7 of 17 The effects of the mutations on GlxR binding were investigated using EMSA ( Figure  3c). After incubation with GlxR and cAMP, the same DNA probe encompassing the ldhA promoter region as was used for the F2 promoter-lacZ fusion construct yielded a smeared band shift on the gel, indicating the interaction of GlxR with the ldhA promoter. The DNA fragment carrying the mutation mut1 showed no shifted band. Incubation with the DNA fragment carrying the mutation mut2, in contrast, resulted in a much clearer band shift than the native one. This demonstrated that the mutations mut1 and mut2 inhibited and enhanced GlxR binding, respectively, as expected.

Effects of Chromosomal Mutations in the GlxR-Binding Site in the ldhA Promoter
The GlxR regulon consists of more than 200 genes. Probably because of its large impact on the transcriptome, the glxR deletion mutant shows severely retarded growth compared with the wild type [24]. This prevents the investigation of the physiological function The effects of the mutations on GlxR binding were investigated using EMSA (Figure 3c). After incubation with GlxR and cAMP, the same DNA probe encompassing the ldhA promoter region as was used for the F2 promoter-lacZ fusion construct yielded a smeared band shift on the gel, indicating the interaction of GlxR with the ldhA promoter. The DNA fragment carrying the mutation mut1 showed no shifted band. Incubation with the DNA fragment carrying the mutation mut2, in contrast, resulted in a much clearer band shift than the native one. This demonstrated that the mutations mut1 and mut2 inhibited and enhanced GlxR binding, respectively, as expected.

Effects of Chromosomal Mutations in the GlxR-Binding Site in the ldhA Promoter
The GlxR regulon consists of more than 200 genes. Probably because of its large impact on the transcriptome, the glxR deletion mutant shows severely retarded growth compared with the wild type [24]. This prevents the investigation of the physiological function of GlxR using the knockout mutant. To directly examine the GlxR role in the regulation of ldhA, we introduced the same mutations (i.e., mut1 and mut2) into the binding site on the chromosome and investigated their effects on ldhA expression. The chromosomal mutations had no effect on growth (Figure 4a). As we have shown that ldhA expression is highly upregulated at 6 h compared with that at 3 h [21], RNA was extracted at these time points. The mutant carrying the mutation mut1 in the GlxR-binding site exhibited lower expression of ldhA than the wild type at both the exponential (3 h) and transition (6 h) phases (Figure 4b). By contrast, the presence of mut2 in the GlxR-binding site showed apparently but not significantly increased expression of ldhA compared with the wild type at the transition phase (p = 0.097, 6 h). Thus, the GlxR-binding site was required for the upregulation of ldhA at the stationary phase. The same mutations were introduced into the chromosome of the sugR-lldR deletion mutant. As ldhA expression is fully derepressed in this mutant, ldhA expression can be observed even at the exponential phase. As observed in the wild type, the mutation mut1 reduced ldhA expression, whereas mut2 enhanced it (p = 0.054, 3 h) (Figure 4b). This result showed that GlxR activated ldhA expression at the exponential phase as well as at the stationary phase.

Effects of the Deletion of the cyaB Gene Encoding Adenylate Cyclase
GlxR belongs to a CRP-type transcriptional regulator family and binds to target binding sites in a cAMP-dependent manner in vitro [30,36,37]. In the C. glutamicum genome, a sole adenylate cyclase is encoded by cyaB [25,38]. To examine the effect of cAMP deficiency on ldhA expression, a cyaB deletion mutant was constructed, which grew slightly slower than the wild type ( Figure S1a). The ldhA expression level in the cyaB mutant was comparable to that in the wild type at the exponential and transition phases ( Figure S1b), indicating that the upregulation observed at the transition phase does not require cAMP. The aceA gene encoding isocitrate lyase in the glyoxylate shunt has been shown to be repressed by GlxR and affected by intracellular cAMP levels [26,37]. The expression of aceA in the cyaB deletion mutant was higher than that in the wild type ( Figure S1b), confirming deregulation of GlxR in the mutant. We also deleted the cyaB gene in the background of the sugR deletion mutant. The growth of the cyaB-sugR deletion mutant was slower than that of the wild type and transiently stalled at 12 h of cultivation, but the OD at 24 h reached the level of the wild type ( Figure S1a). The ldhA expression in the mutant was higher than that in either the wild type or sugR-lldR double deletion mutant, in which ldhA expression is derepressed, at the exponential phase (3 h) ( Figure S1b). However, ldhA expression in the cyaB-sugR double mutant was lower than that in the sugR-lldR mutant at the transition phase (6 h). The combination of the sugR deletion with cAMP deficiency by deletion of cyaB likely caused pleiotropic effects on gene expression. This was possibly reflected by the observation that the expression of the aceA gene, derepressed in the cyaB single deletion mutant, was lowered in the cyaB-sugR deletion mutant compared to that in the wild type.

Effects of the Overexpression of GlxR on ldhA Expression
To examine the effects of glxR overexpression on the expression of ldhA, the plasmid containing glxR under control of the IPTG-inducible tac promoter was constructed. The plasmid was introduced into both the wild type and mutants containing mutations (mut1 and mut2) in the GlxR-binding site. The glxR gene was induced with 0.5 mM IPTG, and the expression levels of ldhA were determined via qRT-PCR. Before IPTG supplementation, ldhA expression in the mut1 and mut2 strains was lower and higher than that in the wild type, respectively. The expression of glxR was, as expected, increased by approximately 20-fold following 15 min of IPTG supplementation. However, the overexpression of glxR had almost no effect on the expression of ldhA in the wild type (data not shown).
The glxR overexpression plasmid was introduced into the sugR-lldR deletion mutant as well as the strain containing the mutated GlxR-binding site (mut1 and mut2) on the chromosome. After 30 min of IPTG supplementation, ldhA expression under the control of the native promoter in the sugR-lldR mutant was apparently but not significantly upregulated compared with the control strain carrying the blank vector (p = 0.11, Figure 5). In the mutants with the mutated binding site (mut1 and mut2), the overexpression of glxR had no effect on ldhA expression. Whereas GlxR was unable to bind to the ldhA promoter and affect ldhA expression in the mut1 strain, the wild-type level of GlxR was likely high enough for the activation of ldhA expression in the mut2 strain. In contrast to the deletion of the cyaB gene, the overexpression of glxR resulted in the reduction of aceA expression, confirming that the regulation mediated by GlxR was promoted by IPTG-induced overexpression.

Effects of Chromosomal Mutations in the GlxR-Binding Site on Organic Acid Profiles
The mutations in the GlxR-binding site in the ldhA promoter region altered ldhA expression. The effects of this modification of the expression on organic acid excretion were investigated. Organic acids in the culture supernatant from the transition (6 h) to stationary (10 h) phases were quantified by HPLC. At the exponential phase (4 h), few organic acids were produced. The mutant with the mutations mut1, in which ldhA expression was suppressed, excreted a significantly lower amount of lactate than the wild type ( Figure 6). The consumption of glucose was comparable for all strains. In the sugR-lldR deletion mutant background, in which the repression of ldhA was relieved, the mut1 mutation similarly lowered the amount of excreted lactate, but the effects were smaller than those in the wild-type background and the difference was not significant after 8 h. The reduced lactate production in the mut1 promoter mutant was likely compensated for by the increased production of other organic acids-succinate, acetate, and fumarate-compared with that produced by the native promoter strains. This is consistent with the fact that the excretion levels of these organic acids by the sugR-lldR deletion mutant derivatives were lower than those by the wild-type derivatives. The mutants with the mutations mut2 excreted lactate in an amount comparable to that of the native promoter strains. At the sampling points in this study, the increase in ldhA expression in the mut2 mutants had no effect on organic acid production, suggesting that the LDH expression was not rate limiting for L-lactate production in the wild type and sugR-lldR deletion mutant background.

Effects of the Deletion of the cyaB Gene Encoding Adenylate Cyclase
GlxR belongs to a CRP-type transcriptional regulator family and binds to target binding sites in a cAMP-dependent manner in vitro [30,36,37]. In the C. glutamicum genome, a sole adenylate cyclase is encoded by cyaB [25,38]. To examine the effect of cAMP deficiency on ldhA expression, a cyaB deletion mutant was constructed, which grew slightly slower than the wild type ( Figure S1a). The ldhA expression level in the cyaB mutant was comparable to that in the wild type at the exponential and transition phases ( Figure S1b), indicating that the upregulation observed at the transition phase does not require cAMP. The aceA gene encoding isocitrate lyase in the glyoxylate shunt has been shown to be repressed by GlxR and affected by intracellular cAMP levels [26,37]. The expression of aceA in the cyaB deletion mutant was higher than that in the wild type ( Figure S1b), confirming deregulation of GlxR in the mutant. We also deleted the cyaB gene in the background of

Effects of Chromosomal Mutations in the GlxR-Binding Site on Organic Acid Profiles
The mutations in the GlxR-binding site in the ldhA promoter region altered ldhA expression. The effects of this modification of the expression on organic acid excretion were

Discussion
In this study, we showed that the key fermentative gene ldhA is directly activated by GlxR. GlxR was not obtained by previous DNA affinity purification using the ldhA promoter region as a ligand [20,21], which identified the two repressors SugR and LldR. This . Organic acids and glucose in culture supernatants. The wild type and sugR-lldR deletion mutant with the native (black), mut1 (red), or mut2 (blue) GlxR-binding site in the ldhA promoter were grown in A medium supplemented with 1% glucose. The culture supernatants at 6, 8, and 10 h were analyzed. Mean values obtained from at least three independent cultivations are shown with their standard deviations. p-values were calculated using an unpaired t-test. (*) p < 0.05; (**) p < 0.01 (comparison with the parental wild type).

Discussion
In this study, we showed that the key fermentative gene ldhA is directly activated by GlxR. GlxR was not obtained by previous DNA affinity purification using the ldhA promoter region as a ligand [20,21], which identified the two repressors SugR and LldR. This is probably because the binding site found in the ldhA promoter region incompletely matches the consensus binding motif: 5 -TGTGA-N6-TCACA-3 . Indeed, the shifted band observed in EMSA was smeared, although it was cAMP dependent (Figure 3c). The similar smeared shifted bands have been observed in previous studies on GlxR [30,36]. The mutations of nucleotides at the conserved position in the binding motif (mut1) completely inhibited GlxR binding and reduced ldhA expression. Previous studies have similarly introduced the mismatch mutations in the GlxR-binding site to investigate the regulatory role of GlxR [24,26,39,40]. By contrast, in this study, the exchanging of two nucleotides to match the conserved motif (mut2: TGTGATTTTTTCAACA -> TGTGATTTTTTTCACA) drastically improved GlxR binding, as shown by the clear shifted band. The same mutations increased ldhA promoter activity and ldhA expression. Thus, to the best of our knowledge, this is the first case of a GlxR regulon, whose expression was shown to be modified by the affinity of the target binding site.
The mutations of the GlxR-binding site in the ldhA promoter may affect the binding of other transcription factors, resulting in the alteration of ldhA expression. Previous DNA affinity purification using the ldhA promoter region as a ligand [20,21] identified AtlR, a DeoR-type transcriptional regulator functioning as a repressor of the expression of the arabitol/mannitol catabolic genes [41,42]. Our EMSA using the recombinant AtlR showed that binding of AtlR to the ldhA promoter region was slightly inhibited by the mut1 mutation in vitro, but not affected by mut2 ( Figure S2a). We constructed the atlR deletion mutant based on the sugR-lldR double deletion mutants carrying the native, mut1, or mut2 GlxR-binding site in the ldhA promoter and assessed the effects of the deletion on ldhA expression. The atlR deletion had no effect on ldhA expression in any genetic background, indicating that the mutations mut1 and mut2 had no effect on AtlR function, if any, in ldhA regulation ( Figure S2b). A previous study also showed that single deletion of atlR had no effect on ldhA expression [20].
During growth on acetate, expression of ldhA is repressed by SugR. Moreover, as intracellular cAMP levels during growth on acetate are lower than those on glucose [25], GlxR-mediated activation of ldhA would be alleviated to save cellular resources. Similarly, the glycolytic genes pfkA encoding phosphofructokinase and the gapA-pgk-tpi-ppc operon encoding glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, triose phosphate isomerase, and phosphoenolpyruvate carboxylase were regulated by both SugR and GlxR [17,24,43]. These two global regulators coordinately control the glycolytic pathway and redox balance. In other bacteria, lactate dehydrogenase expression is important during anaerobic metabolism and is regulated by redox-sensing transcription factors [44][45][46]. How the intracellular cAMP levels of C. glutamicum are controlled in response to environmental changes merits further investigation for the understanding of the global regulatory role of GlxR.
Even in strains containing the mut1 mutations, ldhA expression showed more than a threefold increase at the transition phase ( Figure 4). The group 2 sigma factor SigB is likely responsible for this upregulation. We have previously shown that deletion of sigB reduced ldhA expression by twofold [47]. Comprehensive transcriptional start sites (TSSs) determined by RNA-seq revealed another site upstream of the TSS previously identified (shown as +1 in Figure 1) [48,49]. This whole TSS determination by RNA-seq also found characteristic promoter structures. Over 70% of SigB-dependent promoters contain a G at position −5, two nucleotides downstream of the conserved −10 region, with respect to the TSS [49]. Although the ldhA promoter investigated in this study does not contain a G at this position, the upstream novel ldhA promoter does. Therefore, the novel promoter likely functions as the SigB-dependent promoter and may upregulate ldhA expression at the transition phase. SigB also controls the glycolytic genes under aerobic growth conditions and oxygen deprivation [47]. Investigation of the function of the upstream promoter and involvement of SigB in transcription from the promoter will provide further insights into the regulatory mechanism of fermentative genes in C. glutamicum.
Author Contributions: Designing and performing of experiments, K.T.; data analysis, K.T.; writingoriginal draft preparation, K.T.; writing-review and editing, K.T. and M.I. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.