Construction of a Rhodobacter sphaeroides Strain That Efficiently Produces Hydrogen Gas from Acetate without Poly(ß-Hydroxybutyrate) Accumulation: Insight into the Role of PhaR in Acetate Metabolism.

The purple nonsulfur phototrophic bacterium Rhodobacter sphaeroides produces hydrogen gas (H2) from acetate. An approach to improve the H2 production is preventing accumulation of an intracellular energy storage molecule known as poly(ß-hydroxybutyrate) (PHB), which competes with H2 production for reducing power. However, disruption of PHB biosynthesis has been reported to severely impair the acetate assimilation depending on the genetic backgrounds and/or culture conditions. To solve this problem, we analyzed the relationship between PHB accumulation and acetate metabolism in R. sphaeroides. Gene deletion analyses based on the wild-type strain revealed that among the two polyhydroxyalkanoate synthase genes in the genome, phaC1, but not phaC2, is essential for PHB accumulation, and the phaC1 deletion mutant exhibited slow growth with acetate. On the other hand, a strain with the deletion of phaC1 together with phaR, which encodes a transcriptional regulator capable of sensing PHB accumulation, exhibited growth comparable to that of the wild-type strain despite no accumulation of PHB. These results suggest that PHB accumulation is required for normal growth with acetate by altering the expression of genes under the control of phaR. This hypothesis was supported by a transcriptome sequencing (RNA-seq) analysis revealing that phaR is involved in the regulation of the ethylmalonyl coenzyme A pathway for acetate assimilation. Consistent with these findings, deletion of phaC1 in a genetically engineered H2-producing strain resulted in lower H2 production from acetate due to growth defects, whereas deletion of phaR together with phaC1 restored growth with acetate and increased H2 production from acetate without PHB accumulation. IMPORTANCE This study provides a novel approach for increasing the yield of photofermentative H2 production from acetate by purple nonsulfur phototrophic bacteria. This study further suggests that polyhydroxyalkanoate is not only a storage substance for carbon and energy in bacteria, but may also act as a signaling molecule that mediates bacterial metabolic adaptations to specific environments. This notion will be helpful for understanding the physiology of polyhydroxyalkanoate-producing bacteria, as well as for their metabolic engineering via synthetic biology.

Engineering the transcriptional activator NifA for the construction of Rhodobacter sphaeroides strains that produce hydrogen gas constitutively.

Purple non-sulfur photosynthetic bacteria such as Rhodobacter sphaeroides and Rhodopseudomonas palustris produce hydrogen gas (H2) via proton reduction, which is catalyzed by nitrogenase. Although the expression of nitrogenase is usually repressed under nitrogen-sufficient conditions, a partial deletion of nifA, which encodes a transcriptional activator of nitrogen-fixation genes, has been reported to enable the constitutive expression of nitrogenase in R. palustris. In this study, we evaluated the effects of a similar mutation (nifA* mutation) on H2 production during the photoheterotrophic growth of R. sphaeroides, based on the notion that H2 production by nitrogenase compensates for the loss of CO2 fixation via the Calvin cycle, thereby restoring the redox balance. The chromosomal nifA* mutation resulted in the slight restoration of the photoheterotrophic growth of a mutant strain lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the key enzyme of the Calvin cycle, when the strain was cultured in van Niel's yeast medium. In addition, the strain with the nifA* mutation produced detectable levels of H2 during photoheterotrophic growth with acetate and ammonium; however, the H2 production was considerably lower than that observed during the photoheterotrophic growth of the strain with acetate and L-glutamate, where L-glutamate serves as a poor nitrogen source, thereby causing nitrogenase derepression. On the other hand, introduction of a multicopy plasmid harboring nifA* markedly restored the photoheterotrophic growth of the RubisCO-deletion mutant in van Niel's yeast medium and resulted in efficient H2 production during the photoheterotrophic growth with acetate and ammonium.

Introduction of Glyoxylate Bypass Increases Hydrogen Gas Yield from Acetate and l-Glutamate in Rhodobacter sphaeroides.

Rhodobacter sphaeroides produces hydrogen gas (H2) from organic compounds via nitrogenase under anaerobic-light conditions in the presence of poor nitrogen sources, such as l-glutamate. R. sphaeroides utilizes the ethylmalonyl-coenzyme A (EMC) pathway for acetate assimilation, but its H2 yield from acetate in the presence of l-glutamate has been reported to be low. In this study, the deletion of ccr encoding crotonyl-coenzyme A (crotonyl-CoA) carboxylase/reductase, a key enzyme for the EMC pathway in R. sphaeroides, revealed that the EMC pathway is essential for H2 production from acetate and l-glutamate but not for growth and acetate consumption in the presence of l-glutamate. We introduced a plasmid expressing aceBA from Rhodobacter capsulatus encoding two key enzymes for the glyoxylate bypass into R. sphaeroides, which resulted in a 64% increase in H2 production. However, compared with the wild-type strain expressing heterologous aceBA genes, the strain with aceBA introduced in the genetic background of an EMC pathway-disrupted mutant showed a lower H2 yield. These results indicate that a combination of the endogenous EMC pathway and a heterologously expressed glyoxylate bypass is beneficial for H2 production. In addition, introduction of the glyoxylate bypass into a polyhydroxybutyrate (PHB) biosynthesis-disrupted mutant resulted in a delay in growth along with H2 production, although its H2 yield was comparable to that of the wild-type strain expressing heterologous aceBA genes. These results suggest that PHB production is important for fitness to the culture during H2 production from acetate and l-glutamate when both acetate-assimilating pathways are present.IMPORTANCE As an alternative to fossil fuel, H2 is a promising renewable energy source. Although photofermentative H2 production from acetate is key to developing an efficient process of biohydrogen production from biomass-derived sugars, H2 yields from acetate and l-glutamate by R. sphaeroides have been reported to be low. In this study, we observed that in addition to the endogenous EMC pathway, heterologous expression of the glyoxylate bypass in R. sphaeroides markedly increased H2 yields from acetate and l-glutamate. Therefore, this study provides a novel strategy for improving H2 yields from acetate in the presence of l-glutamate and contributes to a clear understanding of acetate metabolism in R. sphaeroides during photofermentative H2 production.

Functional analysis of arabinofuranosidases and a xylanase of Corynebacterium alkanolyticum for arabinoxylan utilization in Corynebacterium glutamicum.

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and ß-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-L-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-L-arabinofuranoside with k cat values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.

AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions.

UNLABELLED: In Corynebacterium glutamicum ATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression of l-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDA and galM-araR) and two (BSE1 and BSE2) upstream of araE. L-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSB mutation resulted in derepression of both araBDA and galM-araR operons. The effects of BSE1 and/or BSE2 mutation on araE expression revealed that the two sites independently function as the cis elements, but BSE1 plays the primary role. However, AraR was shown to bind to these sites with almost the same affinity in vitro. Taken together, the expression of araBDA and araE is strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the -10 or -35 region of the galM-araR and araE promoters is less effective in repression. Furthermore, downregulation of araBDA and araE dependent on l-arabinose catabolism observed in the BSB mutant and the AraR-independent araR promoter identified within galM-araR add complexity to regulation of the AraR regulon derepressed by L-arabinose. IMPORTANCE: Corynebacterium glutamicum has a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. Most C. glutamicum strains are unable to use a pentose sugar L-arabinose as a carbon source. However, genes for L-arabinose utilization and its regulation have been recently identified in C. glutamicum ATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression by L-arabinose and thereby highlights the complex regulatory feedback loops in combination with l-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner.

Regulation of the Expression of De Novo Pyrimidine Biosynthesis Genes in Corynebacterium glutamicum.

UNLABELLED: Expression of pyrimidine de novo biosynthesis is downregulated by an exogenous uracil in many bacteria. In this study, we show that a putative binding motif sequence of PyrR is required for uracil-mediated repression of pyrR-lacZ translational fusion. However, the uracil response was still observed in the strain with the pyrR gene deleted, implying the existence of a uracil response factor other than PyrR which also acts through the PyrR binding loop region. Deletion of rho, encoding the transcription termination factor Rho, resulted in an increase in the expression of pyrR-lacZ. Moreover, the strain with a double deletion of pyrR and rho showed elimination of the uracil-responsive downregulation of the pyrR-lacZ. Therefore, expression of the pyrimidine biosynthetic gene cluster in Corynebacterium glutamicum is controlled by two different mechanisms mediated by PyrR and Rho. IMPORTANCE: The pyr genes of C. glutamicum are downregulated in the presence of uracil in culture medium. The mRNA binding regulator PyrR represses the expression of pyr genes, as reported previously. However, the uracil response was still observed in the pyrR deletion strain. Deletion of rho in addition to pyrR deletion results in the elimination of the uracil response. Therefore, we identified the factors that are involved in the uracil response. Involvement of Rho in the regulation of pyrimidine de novo biosynthesis genes has not been reported.

Copper homeostasis-related genes in three separate transcriptional units regulated by CsoR in Corynebacterium glutamicum.

In Corynebacterium glutamicum R, CsoR acts as a transcriptional repressor not only of the cognate copA-csoR operon but also of the copZ1-copB-cgR_0126 operon. It is predicted that copA and copB encode P-type ATPases for copper efflux and copZ1 encodes a metallochaperone. Here, a CsoR-binding motif was found upstream of another copZ-like gene, copZ2, and the in vitro binding of the CsoR protein to its promoter was confirmed. The monocistronic copZ2 transcript was upregulated by excess copper in a CsoR-dependent manner. Among the extended CsoR regulon, deletion of copA, but not of copB, copZ1, or copZ2, resulted in decreased resistance to copper, indicating a major role of the CopA copper exporter in the multilayered systems for copper homeostasis. A redundant role of copZ1 and copZ2 in copper resistance was also indicated by double deletion of these genes. The copper-dependent activation of the copA, copZ1, and copZ2 promoters was confirmed by lacZ reporter assays, consistent with the coordinated derepression of the three transcriptional units. The copZ1 promoter activity showed the highest responsiveness to copper and was also induced by excess zinc and nickel. Furthermore, zinc-inducible expression observed for the CsoR-regulated genes was independent of Zur, recently found to uniquely act as a transcriptional repressor of zinc efflux genes. These results implied complicated cross talk between homeostasis of multiple transition metals.

Expanding the regulatory network governed by the extracytoplasmic function sigma factor σH in Corynebacterium glutamicum.

The extracytoplasmic function sigma factor σ(H) is responsible for the heat and oxidative stress response in Corynebacterium glutamicum. Due to the hierarchical nature of the regulatory network, previous transcriptome analyses have not been able to discriminate between direct and indirect targets of σ(H). Here, we determined the direct genome-wide targets of σ(H) using chromatin immunoprecipitation with microarray technology (ChIP-chip) for analysis of a deletion mutant of rshA, encoding an anti-σ factor of σ(H). Seventy-five σ(H)-dependent promoters, including 39 new ones, were identified. σ(H)-dependent, heat-inducible transcripts for several of the new targets, including ilvD encoding a labile Fe-S cluster enzyme, dihydroxy-acid dehydratase, were detected, and their 5' ends were mapped to the σ(H)-dependent promoters identified. Interestingly, functional internal σ(H)-dependent promoters were found in operon-like gene clusters involved in the pentose phosphate pathway, riboflavin biosynthesis, and Zn uptake. Accordingly, deletion of rshA resulted in hyperproduction of riboflavin and affected expression of Zn-responsive genes, possibly through intracellular Zn overload, indicating new physiological roles of σ(H). Furthermore, sigA encoding the primary σ factor was identified as a new target of σ(H). Reporter assays demonstrated that the σ(H)-dependent promoter upstream of sigA was highly heat inducible but much weaker than the known σ(A)-dependent one. Our ChIP-chip analysis also detected the σ(H)-dependent promoters upstream of rshA within the sigH-rshA operon and of sigB encoding a group 2 σ factor, supporting the previous findings of their σ(H)-dependent expression. Taken together, these results reveal an additional layer of the sigma factor regulatory network in C. glutamicum.

Genome-wide analysis of the role of global transcriptional regulator GntR1 in Corynebacterium glutamicum.

The transcriptional regulator GntR1 downregulates the genes for gluconate catabolism and pentose phosphate pathway in Corynebacterium glutamicum. Gluconate lowers the DNA binding affinity of GntR1, which is probably the mechanism of gluconate-dependent induction of these genes. In addition, GntR1 positively regulates ptsG, a gene encoding a major glucose transporter, and pck, a gene encoding phosphoenolpyruvate carboxykinase. Here, we searched for the new target of GntR1 on a genome-wide scale by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analysis. This analysis identified 56 in vivo GntR1 binding sites, of which 7 sites were previously reported. The newly identified GntR1 sites include the upstream regions of carbon metabolism genes such as pyk, maeB, gapB, and icd, encoding pyruvate kinase, malic enzyme, glyceraldehyde 3-phosphate dehydrogenase B, and isocitrate dehydrogenase, respectively. Binding of GntR1 to the promoter region of these genes was confirmed by electrophoretic mobility shift assay. The activity of the icd, gapB, and maeB promoters was reduced by the mutation at the GntR1 binding site, in contrast to the pyk promoter activity, which was increased, indicating that GntR1 is a transcriptional activator of icd, gapB, and maeB and is a repressor of pyk. Thus, it is likely that GntR1 stimulates glucose uptake by inducing the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) gene while repressing pyk to increase PEP availability in the absence of gluconate. Repression of zwf and gnd may reduce the NADPH supply, which may be compensated by the induction of maeB and icd. Upregulation of icd, gapB, and maeB and downregulation of pyk by GntR1 probably support gluconeogenesis.

The LacI-Type transcriptional regulator AraR acts as an L-arabinose-responsive repressor of L-arabinose utilization genes in Corynebacterium glutamicum ATCC 31831.

The Corynebacterium glutamicum ATCC 31831 araBDA operon consists of three l-arabinose catabolic genes, upstream of which the galM, araR, and araE genes are located in opposite orientation. araR encodes a LacI-type transcriptional regulator that negatively regulates the l-arabinose-inducible expression of araBDA and araE (encoding an l-arabinose transporter), through a mechanism that has yet to be identified. Here we show that the AraR protein binds in vitro to three sites: one upstream of araBDA and two upstream of araE. We verify that a 16-bp consensus palindromic sequence is essential for binding of AraR, using a series of mutations introduced upstream of araB in electrophoretic mobility shift assays. Moreover, the DNA-binding activity of AraR is reduced by l-arabinose. We employ quantitative reverse transcription-PCR (qRT-PCR) analyses using various mutant strains deficient in l-arabinose utilization genes to demonstrate that the prominent upregulation of araBDA and araE within 5 min of l-arabinose supplementation is dependent on the uptake but independent of the catabolism of l-arabinose. Similar expression patterns, together with the upregulation by araR disruption without l-arabinose, are evident with the apparent galM-araR operon, although attendant changes in expression levels are much smaller than those realized with the expression of araBDA and araE. The AraR-binding site upstream of araB overlaps the -10 region of the divergent galM promoter. These observations indicate that AraR acts as a transcriptional repressor of araBDA, araE, and galM-araR and that l-arabinose acts as an intracellular negative effector of the AraR-dependent regulation.

Corynebacterium glutamicum ArnR controls expression of nitrate reductase operon narKGHJI and nitric oxide (NO)-detoxifying enzyme gene hmp in an NO-responsive manner.

Corynebacterium glutamicum ArnR is a novel transcriptional regulator that represses expression of the nitrate reductase operon narKGHJI and the nitric oxide (NO)-detoxifying flavohemoglobin gene hmp under aerobic conditions. In a previous study, we showed that ArnR-mediated repression is relieved during anaerobic nitrate respiration, but we could not pinpoint the specific signal that ArnR senses. In this study, we show that in the absence of nitrate, ArnR-mediated repression is maintained under anaerobic conditions. The derepression in response to nitrate is eliminated by disruption of narG, suggesting that ArnR senses nitrate derivatives generated during nitrate respiration. Specifically, the hmp gene is upregulated in the presence of nitrite or nitric oxide (NO) in an ArnR-dependent manner, although the response of narK appears to be greatly affected by ArnR-independent regulation. In vitro binding of ArnR to the narK and hmp promoter regions is more strongly inhibited by NO than by nitrite. We previously showed that the UV-visible spectrum of ArnR is typical of a Fe-S cluster-containing protein. Site-directed mutagenesis of each of three cysteine residues, which are possibly involved in coordination of the cofactor in the ArnR protein, results in loss of the binding of this protein to its target promoters in vitro and eliminates the repression of the target genes in vivo under aerobic conditions. These observations suggest that the cofactor coordinated by these three cysteine residues in the ArnR protein plays a critical role in the NO-responsive expression of the narKGHJI operon and the hmp gene.

OxyR acts as a transcriptional repressor of hydrogen peroxide-inducible antioxidant genes in Corynebacterium glutamicum R.

OxyR, a LysR-type transcriptional regulator, has been established as a redox-responsive activator of antioxidant genes in bacteria. This study shows that OxyR acts as a transcriptional repressor of katA, dps, ftn and cydA in Corynebacterium glutamicum R. katA encodes H2O2-detoxifing enzyme catalase, dps and ftn are implicated in iron homeostasis and cydA encodes a subunit of cytochrome bd oxidase. Quantitative RT-PCR analyses revealed that expression of katA and dps, but not of ftn and cydA, was induced by H2O2. Disruption of the oxyR gene encoding OxyR resulted in a marked increase in katA and dps mRNAs to a level higher than that induced by H2O2, and the oxyR-deficient mutant showed a H2O2-resistant phenotype. This is in contrast to the conventional OxyR-dependent regulatory model. ftn and cydA were also upregulated by oxyR disruption but to a smaller extent. Electrophoretic mobility shift assays revealed that the OxyR protein specifically binds to all four upstream regions of the respective genes under reducing conditions. We observed that the oxidized form of OxyR similarly bound to not only the target promoter regions, but also nonspecific DNA fragments. Based on these findings, we propose that the transcriptional repression by OxyR is alleviated under oxidative stress conditions in a titration mechanism due to the decreased specificity of its DNA-binding activity. DNase I footprinting analyses revealed that the OxyR-binding site in the four target promoters is ~ 50 bp in length and has multiple T-N11-A motifs, a feature of LysR-type transcriptional regulators, but no significant overall sequence conservation.

Involvement of regulatory interactions among global regulators GlxR, SugR, and RamA in expression of ramA in Corynebacterium glutamicum.

The central carbon metabolism genes in Corynebacterium glutamicum are under the control of a transcriptional regulatory network composed of several global regulators. It is known that the promoter region of ramA, encoding one of these regulators, interacts with its gene product, RamA, as well as with the two other regulators, GlxR and SugR, in vitro and/or in vivo. Although RamA has been confirmed to repress its own expression, the roles of GlxR and SugR in ramA expression have remained unclear. In this study, we examined the effects of GlxR binding site inactivation on expression of the ramA promoter-lacZ fusion in the genetic background of single and double deletion mutants of sugR and ramA. In the wild-type background, the ramA promoter activity was reduced to undetectable levels by the introduction of mutations into the GlxR binding site but increased by sugR deletion, indicating that GlxR and SugR function as the transcriptional activator and repressor, respectively. The marked repression of ramA promoter activity by the GlxR binding site mutations was largely compensated for by deletions of sugR and/or ramA. Furthermore, ramA promoter activity in the ramA-sugR double mutant was comparable to that in the ramA mutant but was significantly higher than that in the sugR mutant. Taken together, it is likely that the level of ramA expression is dynamically balanced by GlxR-dependent activation and repression by RamA along with SugR in response to perturbation of extracellular and/or intracellular conditions. These findings add multiple regulatory loops to the transcriptional regulatory network model in C. glutamicum.

Corynebacterium glutamicum CsoR acts as a transcriptional repressor of two copper/zinc-inducible P(1B)-type ATPase operons.

The mechanism of regulation of the expression of copA and copB, encoding putative copper-translocating P(1B)-type ATPases in Corynebacterium glutamicum, was investigated. The levels of copA and copB mRNAs were upregulated in response to excess copper as well as excess zinc. Disruption of csoR, encoding a transcriptional regulator, resulted in constitutive expression of copA and copB. The CsoR protein bound to the promoter regions of the copA-csoR and the cgR_0124-copB-cgR_0126 operon. In vitro DNA binding activity was strongly inhibited by copper, but much less inhibited by zinc. A csoR-deficient mutant showed slightly increased resistance to copper, but slightly decreased resistance to zinc. These findings indicate that CsoR acts as a transcriptional repressor not only of the cognate copA-csoR operon but also of the cgR_0124-copB-cgR_0126 operon, which is not physically linked to csoR on the chromosome, and that CsoR plays a major role in copper homeostasis.

Coordinated regulation of gnd, which encodes 6-phosphogluconate dehydrogenase, by the two transcriptional regulators GntR1 and RamA in Corynebacterium glutamicum.

The transcriptional regulation of Corynebacterium glutamicum gnd, encoding 6-phosphogluconate dehydrogenase, was investigated. Two transcriptional regulators, GntR1 and RamA, were isolated by affinity purification using gnd promoter DNA. GntR1 was previously identified as a repressor of gluconate utilization genes, including gnd. Involvement of RamA in gnd expression had not been investigated to date. The level of gnd mRNA was barely affected by the single deletion of ramA. However, gnd expression was downregulated in the ramA gntR1 double mutant compared to that of the gntR1 single mutant, suggesting that RamA activates gnd expression. Two RamA binding sites are found in the 5' upstream region of gnd. Mutation proximal to the transcriptional start site diminished the gluconate-dependent induction of gnd-lacZ. DNase I footprinting assay revealed two GntR1 binding sites, with one corresponding to a previously proposed site that overlaps with the -10 region. The other site overlaps the RamA binding site. GntR1 binding to this newly identified site inhibits DNA binding of RamA. Therefore, it is likely that GntR1 represses gnd expression by preventing both RNA polymerase and RamA binding to the promoter. In addition, DNA binding activity of RamA was reduced by high concentrations of NAD(P)H but not by NAD(P), implying that RamA senses the redox perturbation of the cell.

Corynebacterium glutamicum Zur acts as a zinc-sensing transcriptional repressor of both zinc-inducible and zinc-repressible genes involved in zinc homeostasis.

Zur is a zinc-dependent transcriptional repressor of zinc uptake systems in bacteria. In the present study, we examined the role of Corynebacterium glutamicum Zur in the zinc-inducible expression of two genes: one encoding a cation diffusion facilitator (zrf) and the other a metal-translocating P-type ATPase (zra). Both genes were shown to be involved in zinc resistance. Disruption of the zur gene encoding Zur resulted in constitutive expression of zrf and zra mRNAs. An electrophoretic mobility shift assay revealed that the Zur protein binds to the zrf and zra promoters, for which the in vivo activities were up-regulated in response to excess zinc. Interestingly, the in vitro DNA binding activity of Zur was inhibited by zinc, in contrast to its zinc-dependent binding to the promoter region of a zinc-repressible ABC transporter gene znuB2. A 21-bp motif found in the Zur binding site overlaps the putative -35 region of both the zrf and zra promoters. This new motif is a 10-1-10 direct repeat sequence distinct from the 10-1-10 inverted repeat sequence of a previously identified Zur box for zinc-dependent binding. Nevertheless, their 10-bp elements share some sequence similarities. Overexpression of zur in the zur deletion mutant background, as well as deletion of zur in the zrf and zra double deletion mutant background, resulted in decreased resistance to zinc. These results suggest that the direct negative control of both zinc uptake and export systems by Zur is central to C. glutamicum zinc homeostasis and is effected in distinct ways.

NdnR is an NAD-responsive transcriptional repressor of the ndnR operon involved in NAD de novo biosynthesis in Corynebacterium glutamicum.

The Corynebacterium glutamicum ndnR gene, which is chromosomally located in a gene cluster involved in NAD de novo biosynthesis, negatively regulates expression of the cluster genes, i.e. nadA, nadC, nadS and ndnR itself. Although ndnR encodes a member of the recently identified NrtR family of transcriptional regulators, whether or not the NdnR protein directly regulates these NAD biosynthesis genes remains to be verified. Here, two NdnR binding sites in the promoter region of the ndnR-nadA-nadC-nadS operon in C. glutamicum were confirmed by in vitro DNA binding assay and analysis of in vivo expression of the chromosomally integrated ndnR promoter-lacZ reporter fusion. Electrophoretic mobility shift assay revealed that the NdnR protein binds to the 5'-upstream region of ndnR, and that the binding is significantly enhanced by NAD. Mutation in two 21 bp NdnR binding motifs in the ndnR promoter region inhibited the binding of NdnR in vitro. The mutation also enhanced the promoter activity in cells cultured in the presence of nicotinate, which is utilized in NAD biosynthesis, resulting in the loss of the repression in response to an exogenous NAD precursor; this is consistent with the effect of deletion of ndnR reported in our previous study. These results indicate that NAD acts as a co-repressor for the NdnR protein that directly regulates the ndnR operon involved in NAD de novo biosynthesis; the NAD-NdnR regulatory system likely plays an important role in the control of NAD homeostasis in C. glutamicum.

Genome-wide identification of in vivo binding sites of GlxR, a cyclic AMP receptor protein-type regulator in Corynebacterium glutamicum.

Corynebacterium glutamicum GlxR is a cyclic AMP (cAMP) receptor protein-type regulator. Although over 200 GlxR-binding sites in the C. glutamicum genome are predicted in silico, studies on the physiological function of GlxR have been hindered by the severe growth defects of a glxR mutant. This study identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR-binding sites. In vitro binding assays and promoter-reporter assays demonstrated that GlxR directly activates expression of genes for aerobic respiration, ATP synthesis, and glycolysis and that it is required for expression of genes for cell separation and mechanosensitive channels. GlxR also directly represses a citrate uptake gene in the presence of citrate. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a mutant with a deletion of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. Thus, GlxR is physiologically functional at the relatively low cAMP levels in the cyaB mutant, allowing the cyaB mutant to grow much better than the glxR mutant.

Transcriptional regulators of multiple genes involved in carbon metabolism in Corynebacterium glutamicum.

Corynebacterium glutamicum, a high-GC Gram-positive soil bacterium, has been used in development of bioprocesses for production of various compounds such as amino acids, organic acids, and alcohols. Recently, several transcriptional regulators, each of which is involved in multiple carbon metabolic pathways in this bacterium, have been identified and characterized. These regulators appear to form a complicated network mediating coordinated expression of a number of metabolic genes for efficient utilization of carbon and energy sources in response to changing environmental conditions. The C. glutamicum regulatory system of carbon metabolism is markely different from well-studied model systems of other bacteria such as Escherichia coli and Bacillus subtilis. Thus, elucidation of the C. glutamicum regulatory system should establish a new model for bacterial control of carbon metabolism, providing a rational basis for metabolic engineering of this industrial important microorganism to efficiently direct carbon flow from available carbon resources into useful compounds. Here, recent findings of studies on C. glutamicum transcriptional regulators of carbon metabolism, namely, SugR, RamB, RamA, GlxR, GntR1/2, and LldR, are summarized.

Gene expression profiling of Corynebacterium glutamicum during Anaerobic nitrate respiration: induction of the SOS response for cell survival.

The gene expression profile of Corynebacterium glutamicum under anaerobic nitrate respiration revealed marked differences in the expression levels of a number of genes involved in a variety of cellular functions, including carbon metabolism and respiratory electron transport chain, compared to the profile under aerobic conditions using DNA microarrays. Many SOS genes were upregulated by the shift from aerobic to anaerobic nitrate respiration. An elongated cell morphology, similar to that induced by the DivS-mediated suppression of cell division upon cell exposure to the DNA-damaging reagent mitomycin C, was observed in cells subjected to anaerobic nitrate respiration. None of these transcriptional and morphological differences were observed in a recA mutant strain lacking a functional RecA regulator of the SOS response. The recA mutant cells additionally showed significantly reduced viability compared to wild-type cells similarly grown under anaerobic nitrate respiration. These results suggest a role for the RecA-mediated SOS response in the ability of cells to survive any DNA damage that may result from anaerobic nitrate respiration in C. glutamicum.