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1.
Acta Crystallogr D Struct Biol ; 79(Pt 10): 895-908, 2023 Oct 01.
Article in English | MEDLINE | ID: mdl-37712435

ABSTRACT

4-Amino-4-deoxychorismate synthase (ADCS), a chorismate-utilizing enzyme, is composed of two subunits: PabA and PabB. PabA is a glutamine amidotransferase that hydrolyzes glutamine into glutamate and ammonia. PabB is an aminodeoxychorismate synthase that converts chorismate to 4-amino-4-deoxychorismate (ADC) using the ammonia produced by PabA. ADCS functions under allosteric regulation between PabA and PabB. However, the allosteric mechanism remains unresolved because the structure of the PabA-PabB complex has not been determined. Here, the crystal structure and characterization of PapA from Streptomyces venezuelae (SvPapA), a bifunctional enzyme comprising the PabA and PabB domains, is reported. SvPapA forms a unique dimer in which PabA and PabB domains from different monomers complement each other and form an active structure. The chorismate-bound structure revealed that recognition of the C1 carboxyl group by Thr501 and Gly502 of the 498-PIKTG-502 motif in the PabB domain is essential for the catalytic Lys500 to reach the C2 atom, a reaction-initiation site. SvPapA demonstrated ADCS activity in the presence of Mg2+ when glutamate or NH+4 was used as the amino donor. The crystal structure indicated that the Mg2+-binding position changed depending on the binding of chorismate. In addition, significant structural changes were observed in the PabA domain depending on the presence or absence of chorismate. This study provides insights into the structural factors that are involved in the allosteric regulation of ADCS.


Subject(s)
4-Aminobenzoic Acid , Glutamine , 4-Aminobenzoic Acid/metabolism , Glutamine/metabolism , Ammonia , Glutamates
3.
J Biosci Bioeng ; 134(6): 496-500, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36182634

ABSTRACT

The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110, has three ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) isoenzymes, CbbM, CbbLS-1, and CbbLS-2, which differ in CO2/O2 specificity factor values. Expressions of CbbM and CbbLS-1 are regulated differently by transcriptional regulators of the LysR family, CbbRm and CbbR1, respectively. CbbLS-2 has the highest specificity and is induced under low CO2 conditions, but the regulator for the cbbL2S2 genes encoding CbbLS-2 remains unidentified. In this study, the cbbR2 gene encoding the third CbbR-type regulator was identified in the downstream region of the cbbL2S2 and carboxysome gene cluster via transposon mutagenesis. CO2 depletion induced the cbbR2 gene. The cbbR2 knockout mutant could not grow under low CO2 conditions and did not produce CbbLS-2. Recombinant CbbR2 protein was bound to the promoter region of the cbbL2S2 genes. These results indicate that CbbR2 is the specific regulator for CbbLS-2 expression.


Subject(s)
Hydrogen , Ribulose-Bisphosphate Carboxylase , Ribulose-Bisphosphate Carboxylase/genetics , Carbon Dioxide
4.
Microorganisms ; 10(5)2022 May 10.
Article in English | MEDLINE | ID: mdl-35630445

ABSTRACT

Understanding the metabolic pathways of amino acids and their regulation is important for the rational metabolic engineering of amino acid production. The catabolic pathways of L-asparagine and L-aspartate are composed of transporters for amino acid uptake and asparaginase and aspartase, which are involved in the sequential deamination to fumarate. However, knowledge of the catabolic genes for asparagine in bacteria of the Actinobacteria class has been limited. In this study, we identified and characterized the ans operon required for L-Asn catabolism in Corynebacterium glutamicum R. The operon consisted of genes encoding a transcriptional regulator (AnsR), asparaginase (AnsA2), aspartase (AspA2), and permease (AnsP). The enzymes and permease encoded in the operon were shown to be essential for L-Asn utilization, but another asparaginase, AnsA1, and aspartase, AspA1, were not essential. Expression analysis revealed that the operon was induced in response to extracellular L-Asn and was transcribed as a leaderless mRNA. The DNA-binding assay demonstrated that AnsR acted as a transcriptional repressor of the operon by binding to the inverted repeat at its 5'-end region. The AnsR binding was inhibited by L-Asn. This study provides insights into the functions and regulatory mechanisms of similar operon-like clusters in related bacteria.

5.
ACS Synth Biol ; 10(9): 2308-2317, 2021 09 17.
Article in English | MEDLINE | ID: mdl-34351735

ABSTRACT

The development of microbes for conducting bioprocessing via synthetic biology involves design-build-test-learn (DBTL) cycles. To aid the designing step, we developed a computational technique that suggests next genetic modifications on the basis of relatedness to the user's design history of genetic modifications accumulated through former DBTL cycles conducted by the user. This technique, which comprehensively retrieves well-known designs related to the history, involves searching text for previous literature and then mining genes that frequently co-occur in the literature with those modified genes. We further developed a domain-specific lexical model that weights literature that is more related to the domain of metabolic engineering to emphasize genes modified for bioprocessing. Our technique made a suggestion by using a history of creating a Corynebacterium glutamicum strain producing shikimic acid that had 18 genetic modifications. Inspired by the suggestion, eight genes were considered by biologists for further modification, and modifying four of these genes proved experimentally efficient in increasing the production of shikimic acid. These results indicated that our proposed technique successfully utilized the former cycles to suggest relevant designs that biologists considered worth testing. Comprehensive retrieval of well-tested designs will help less-experienced researchers overcome the entry barrier as well as inspire experienced researchers to formulate design concepts that have been overlooked or suspended. This technique will aid DBTL cycles by feeding histories back to the next genetic design, thereby complementing the designing step.


Subject(s)
Corynebacterium glutamicum/genetics , Synthetic Biology/methods , Corynebacterium glutamicum/metabolism , Glucose/metabolism , Metabolic Engineering/methods , Metabolic Networks and Pathways/genetics , Multigene Family , Research Design , Shikimic Acid/metabolism
6.
Microorganisms ; 9(3)2021 Mar 06.
Article in English | MEDLINE | ID: mdl-33800875

ABSTRACT

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.

8.
Appl Microbiol Biotechnol ; 104(15): 6719-6729, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32556410

ABSTRACT

Cell proliferation is achieved through numerous enzyme reactions. Temperature governs the activity of each enzyme, ultimately determining the optimal growth temperature. The synthesis of useful chemicals and fuels utilizes a fraction of available metabolic pathways, primarily central metabolic pathways including glycolysis and the tricarboxylic acid cycle. However, it remains unclear whether the optimal temperature for these pathways is correlated with that for cell proliferation. Here, we found that wild-type Corynebacterium glutamicum displayed increased glycolytic activity under non-growing anaerobic conditions at 42.5 °C, at which cells do not proliferate under aerobic conditions. At this temperature, glucose consumption was not inhibited and increased by 28% compared with that at the optimal growth temperature of 30 °C. Transcriptional analysis revealed that a gene encoding glucose transporter (iolT2) was upregulated by 12.3-fold compared with that at 30 °C, with concomitant upregulation of NCgl2954 encoding the iolT2-regulating transcription factor. Deletion of iolT2 decreased glucose consumption rate at 42.5 °C by 28%. Complementation of iolT2 restored glucose consumption rate, highlighting the involvement of iolT2 in the accelerating glucose consumption at an elevated temperature. This study shows that the optimal temperature for glucose metabolism in C. glutamicum under anaerobic conditions differs greatly from that for cell growth under aerobic conditions, being beyond the upper limit of the growth temperature. This is beneficial for fuel and chemical production not only in terms of increasing productivity but also for saving cooling costs. KEY POINTS: • C. glutamicum accelerated anaerobic glucose consumption at elevated temperature. • The optimal temperature for glucose consumption was above the upper limit for growth. • Gene expression involved in glucose transport was upregulated at elevated temperature. Graphical abstract.


Subject(s)
Corynebacterium glutamicum/genetics , Glucose Transport Proteins, Facilitative/genetics , Glucose/metabolism , Hot Temperature , Metabolic Networks and Pathways , Anaerobiosis , Biological Transport , Corynebacterium glutamicum/metabolism , Gene Expression , Gene Expression Profiling , Glucose Transport Proteins, Facilitative/metabolism , Up-Regulation
9.
J Biosci Bioeng ; 126(6): 730-735, 2018 Dec.
Article in English | MEDLINE | ID: mdl-29960861

ABSTRACT

The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110 has three ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isoenzymes, designated CbbLS-1, CbbLS-2, and CbbM, which are encoded by the cbbL1S1, cbbL2S2, and cbbM genes, respectively. Functions of these isoenzymes at different CO2 concentrations were investigated using deletion mutants of their genes. Deletion of cbbL1 had no effect on cell growth under any of the test growth conditions. The cbbL2 mutant was unable to grow under lower (≤0.15%) CO2 conditions, though it grew normally under higher (≥2%) CO2 conditions. Growth of the cbbM mutant was retarded under higher CO2 conditions but was not affected by lower CO2 conditions. These results indicate that CbbLS-2 and CbbM specifically function under lower and higher CO2 conditions, respectively. The growth retardation of the cbbL2 and cbbM mutants was not restored by complementation with plasmids carrying the cbbL2S2 and cbbM genes, respectively. The cbbL2S2 and cbbM genes are followed by the carboxysome genes and the cbbQmOm genes, respectively. Co-expression of these downstream genes was probably necessary for the in vivo function of CbbLS-2 and CbbM. CbbLS-1 was upregulated in the cbbL2 and cbbM mutants under the lower and higher CO2 conditions, respectively, indicating that the expression of cbbL1S1 was controlled to compensate the deficiency of the other RuBisCO isoenzymes.


Subject(s)
Carbon Dioxide/pharmacology , Moritella/enzymology , Ribulose-Bisphosphate Carboxylase/drug effects , Ribulose-Bisphosphate Carboxylase/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbon Dioxide/chemistry , Enzyme Activation/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Genes, Bacterial , Moritella/drug effects , Moritella/genetics , Organisms, Genetically Modified , Ribulose-Bisphosphate Carboxylase/metabolism
10.
Mol Microbiol ; 107(3): 312-329, 2018 02.
Article in English | MEDLINE | ID: mdl-29148103

ABSTRACT

Mycolates are α-branched, ß-hydroxylated, long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. They form an outer membrane, which functions as a permeability barrier and confers pathogenic mycobacteria to resistance to antibiotics. Although the mycolate biosynthetic pathway has been intensively studied, knowledge of transcriptional regulation of genes involved in this pathway is limited. Here, we report that the extracytoplasmic function sigma factor σD is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σD -binding regions in the genome, establishing a consensus promoter sequence for σD recognition. The σD regulon comprised acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase and mycolyltransferases; they were involved in mycolate synthesis. Indeed, deletion or overexpression of sigD encoding σD modified the extractable mycolate amount. Immediately downstream of sigD, rsdA encoded anti-σD and was under the control of a σD -dependent promoter. Another σD regulon member, l,d-transpeptidase, conferred lysozyme resistance. Thus, σD modifies peptidoglycan cross-linking and enhances mycolate synthesis to provide resistance to environmental stress.


Subject(s)
Corynebacterium glutamicum/metabolism , Sigma Factor/metabolism , Bacterial Proteins/metabolism , Carbon-Carbon Ligases , Cell Wall/metabolism , Chromatin Immunoprecipitation , Corynebacterium glutamicum/genetics , Fatty Acid Synthases/metabolism , Gene Deletion , Gene Expression Regulation, Bacterial/genetics , Operon/genetics , Peptidoglycan/metabolism , Promoter Regions, Genetic/genetics , Regulon/genetics , Stress, Physiological/genetics
11.
Mol Microbiol ; 100(3): 486-509, 2016 05.
Article in English | MEDLINE | ID: mdl-26789738

ABSTRACT

Bacteria modify their expression of different terminal oxidases in response to oxygen availability. Corynebacterium glutamicum, a facultative anaerobic bacterium of the phylum Actinobacteria, possesses aa3 -type cytochrome c oxidase and cytochrome bd-type quinol oxidase, the latter of which is induced by oxygen limitation. We report that an extracytoplasmic function σ factor, σ(C) , is responsible for the regulation of this process. Chromatin immunoprecipitation with microarray analysis detected eight σ(C) -binding regions in the genome, facilitating the identification of a consensus promoter sequence for σ(C) recognition. The promoter sequences were found upstream of genes for cytochrome bd, heme a synthesis enzymes and uncharacterized membrane proteins, all of which were upregulated by sigC overexpression. However, one consensus promoter sequence found on the antisense strand upstream of an operon encoding the cytochrome bc1 complex conferred a σ(C) -dependent negative effect on expression of the operon. The σ(C) regulon was induced by cytochrome aa3 deficiency without modifying sigC expression, but not by bc1 complex deficiency. These findings suggest that σ(C) is activated in response to impaired electron transfer via cytochrome aa3 and not directly to a shift in oxygen levels. Our results reveal a new paradigm for transcriptional regulation of the aerobic respiratory system in bacteria.


Subject(s)
Bacterial Proteins/metabolism , Corynebacterium glutamicum/metabolism , DNA-Binding Proteins/metabolism , Electron Transport Complex IV/metabolism , Gene Expression Regulation, Bacterial/genetics , Sigma Factor/metabolism , Transcription, Genetic/genetics , Bacterial Proteins/genetics , Binding Sites/genetics , Chromatin Immunoprecipitation , Corynebacterium glutamicum/genetics , Cytochrome b Group/genetics , DNA-Binding Proteins/genetics , Electron Transport Complex III/genetics , Electron Transport Complex III/metabolism , Electron Transport Complex IV/antagonists & inhibitors , Electron Transport Complex IV/genetics , Gene Deletion , Hydroquinones/metabolism , Membrane Proteins/genetics , Oxidation-Reduction , Oxygen/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Sigma Factor/genetics
12.
Appl Microbiol Biotechnol ; 100(1): 45-60, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26496920

ABSTRACT

Corynebacterium glutamicum, a high GC content gram-positive soil bacterium in Actinobacteria, has been used for the industrial production of amino acids and engineered to produce various compounds, including polymer building blocks and biofuels. Since its genome sequence was first published, its versatile metabolic pathways and their genetic components and regulatory mechanisms have been extensively studied. Previous studies on transcriptional factors, including two-component systems and σ factors, in the bacterium have revealed transcriptional regulatory links among the metabolic pathways and those among the stress response systems, forming a complex transcriptional regulatory network. The regulatory links are based on knowledge of the transcription factors, such as their target genes (regulons), DNA sequence motifs for recognition, and effector molecules controlling their activities, all of which are fundamental for understanding their physiological functions. Recent advances in chromatin immunoprecipitation (ChIP)-based genome-wide analyses provide an opportunity to comprehensively identify the transcription factor regulon, composed of its direct target genes, and its precise consensus binding motif. A common feature among the regulon constituents may provide clues to identify an effector molecule targeting the factor. In this mini-review, we summarize the current knowledge of the regulons of the C. glutamicum transcription factors that have been analyzed via ChIP-based technologies. The regulons consisting of direct target genes revealed new physiological roles of the transcription factors and new regulatory interactions, contributing to refinement and expansion of the transcriptional regulatory network and the development of guidelines and genetic tools for metabolic engineering of C. glutamicum.


Subject(s)
Corynebacterium glutamicum/genetics , Regulon , Transcription Factors/genetics , Transcription Factors/metabolism , Chromatin Immunoprecipitation , Gene Regulatory Networks
13.
J Bacteriol ; 197(3): 483-96, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25404703

ABSTRACT

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.


Subject(s)
Corynebacterium glutamicum/genetics , Corynebacterium glutamicum/metabolism , Gene Expression Regulation, Bacterial , Gene Regulatory Networks , Sigma Factor/genetics , Sigma Factor/metabolism , Chromatin Immunoprecipitation , Gene Deletion , Metabolic Networks and Pathways/genetics , Microarray Analysis , Multigene Family , Operon , Promoter Regions, Genetic , Protein Binding
14.
J Bacteriol ; 195(8): 1718-26, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23396909

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , Corynebacterium glutamicum/metabolism , Gene Expression Regulation, Bacterial/physiology , Bacterial Proteins/genetics , Binding Sites , Corynebacterium glutamicum/genetics , DNA, Bacterial , DNA, Intergenic , Down-Regulation , Escherichia coli/genetics , Escherichia coli/metabolism , Mutation , Plasmids , Promoter Regions, Genetic , Protein Binding , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic
15.
J Bacteriol ; 193(16): 4123-33, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21665967

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , Corynebacterium glutamicum/metabolism , Gene Expression Regulation, Bacterial/physiology , Genome, Bacterial , Receptors, Cyclic AMP/metabolism , Bacterial Proteins/genetics , Binding Sites , Cell Division , Corynebacterium glutamicum/cytology , Corynebacterium glutamicum/genetics , Gene Deletion , Gene Expression Profiling , Protein Array Analysis , Protein Binding
16.
Microbiology (Reading) ; 157(Pt 1): 21-28, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20864477

ABSTRACT

The Corynebacterium glutamicum anaerobic nitrate reductase operon narKGHJI is repressed by a transcriptional regulator, ArnR, under aerobic conditions. A consensus binding site of the cAMP receptor protein (CRP)-type regulator, GlxR, was recently found upstream of the ArnR binding site in the narK promoter region. Here we investigated the involvement of GlxR and cAMP in expression of the narKGHJI operon in vivo. Electrophoretic mobility shift assays showed that the putative GlxR binding motif in the narK promoter region is essential for the cAMP-dependent binding of GlxR. Promoter-reporter assays showed that mutation in the GlxR binding site resulted in significant reduction of narK promoter activity. Furthermore, a deletion mutant of the adenylate cyclase gene cyaB, which is involved in cAMP synthesis, exhibited a decrease in both narK promoter activity and nitrate reductase activity. These results demonstrated that C. glutamicum GlxR positively regulates narKGHJI expression in a cAMP-dependent manner.


Subject(s)
Bacterial Proteins/metabolism , Corynebacterium glutamicum/physiology , Cyclic AMP/metabolism , Gene Expression Regulation, Bacterial , Nitrate Reductase/biosynthesis , Operon , Artificial Gene Fusion , Corynebacterium glutamicum/genetics , DNA, Bacterial/metabolism , Electrophoretic Mobility Shift Assay , Gene Deletion , Gene Expression Profiling , Genes, Reporter , Nitrate Reductase/genetics , Promoter Regions, Genetic , Protein Binding
17.
J Bacteriol ; 191(13): 4251-8, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19429617

ABSTRACT

Corynebacterium glutamicum ldhA encodes L-lactate dehydrogenase, a key enzyme that couples L-lactate production to reoxidation of NADH formed during glycolysis. We previously showed that in the absence of sugar, SugR binds to the ldhA promoter region, thereby repressing ldhA expression. In this study we show that LldR is another protein that binds to the ldhA promoter region, thus regulating ldhA expression. LldR has hitherto been characterized as an L-lactate-responsive transcriptional repressor of L-lactate utilization genes. Transposon mutagenesis of a reporter strain carrying a chromosomal ldhA promoter-lacZ fusion (PldhA-lacZ) revealed that ldhA disruption drastically decreased expression of PldhA-lacZ. PldhA-lacZ expression in the ldhA mutant was restored by deletion of lldR, suggesting that LldR acts as a repressor of ldhA in the absence of L-lactate and the LldR-mediated repression is not relieved in the ldhA mutant due to its inability to produce L-lactate. lldR deletion did not affect PldhA-lacZ expression in the wild-type background during growth on either glucose, acetate, or L-lactate. However, it upregulated PldhA-lacZ expression in the sugR mutant background during growth on acetate. The binding sites of LldR and SugR are located around the -35 and -10 regions of the ldhA promoter, respectively. C. glutamicum ldhA expression is therefore primarily repressed by SugR in the absence of sugar. In the presence of sugar, SugR-mediated repression of ldhA is alleviated, and ldhA expression is additionally enhanced by LldR inactivation in response to L-lactate produced by LdhA.


Subject(s)
Bacterial Proteins/genetics , Corynebacterium glutamicum/enzymology , Corynebacterium glutamicum/metabolism , Gene Expression Regulation, Bacterial , L-Lactate Dehydrogenase/genetics , Bacterial Proteins/physiology , Corynebacterium glutamicum/genetics , DNA Footprinting , DNA Transposable Elements/genetics , Deoxyribonuclease I , Electrophoretic Mobility Shift Assay , L-Lactate Dehydrogenase/physiology , Mutagenesis/genetics , Promoter Regions, Genetic/genetics , Protein Binding/genetics
18.
Appl Microbiol Biotechnol ; 83(2): 315-27, 2009 May.
Article in English | MEDLINE | ID: mdl-19221735

ABSTRACT

This paper reports on the transcriptional regulation mechanism of the Corynebacterium glutamicum ldhA gene encoding L: -lactate dehydrogenase responsible for production of L: -lactate. DNA affinity purification allowed us to identify SugR, a global repressor of genes involved in sugar uptake and glycolysis, as a protein binding to the ldhA promoter region. Whereas ldhA gene expression and ldhA promoter activity were completely repressed during growth of wild-type cells in the absence of sugar, no such repression was observed in sugR mutant cells, indicating that SugR represses ldhA transcription. Electrophoretic mobility shift assays and DNase I footprint analyses revealed that two direct repeats, centered at position-17 with respect to the transcriptional start point, are required for SugR binding to the ldhA promoter and that fructose-1-phosphate (F-1-P) is the strongest negative effector of repressor activity of SugR. Furthermore, the ldhA promoter activity during growth on either fructose or sucrose, under which F-1-P is generated, is higher than on glucose, supporting the results of the in vitro binding assays. Thus, C. glutamicum ldhA is repressed by SugR in the absence of sugar and the expression level is dependent on the extent of derepression, which varies in response to sugars provided.


Subject(s)
Bacterial Proteins/metabolism , Corynebacterium glutamicum/genetics , Hexoses/metabolism , L-Lactate Dehydrogenase/genetics , Repressor Proteins/genetics , Up-Regulation , Bacterial Proteins/genetics , Corynebacterium glutamicum/enzymology , Gene Expression Regulation, Bacterial , L-Lactate Dehydrogenase/metabolism , Promoter Regions, Genetic , Protein Binding , Repressor Proteins/metabolism
19.
J Bacteriol ; 191(3): 968-77, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19047347

ABSTRACT

SugR, RamA, GlxR, GntR1, and a MarR-type transcriptional regulator bind to the promoter region of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), essential for glycolysis in Corynebacterium glutamicum. We previously showed that SugR, a transcriptional repressor of phosphotransferase system genes for the sugar transport system, is involved in the downregulation of gapA expression in the absence of sugar. In this study, the role of RamA in the expression of the gapA gene was examined. Comparing the gapA expression and GAPDH activity of a ramA mutant with those of the wild type revealed that RamA is involved in upregulation of gapA expression in glucose-grown cells. DNase I footprint analyses and electrophoretic mobility shift assays revealed that RamA binds with different affinities to three sites in the gapA promoter. lacZ reporter assays with mutated RamA binding sites in the gapA promoter showed that the middle binding site is the most important for RamA to activate gapA expression and that binding of RamA to the gapA promoter activates the gene expression not only in glucose-grown cells but also in acetate-grown cells. Furthermore, RamA also directly activates sugR expression, indicating that two global regulators, RamA and SugR, are coordinately involved in the complex regulation of gapA expression in C. glutamicum.


Subject(s)
Bacterial Proteins/metabolism , Corynebacterium glutamicum/metabolism , Gene Expression Regulation, Bacterial , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Transcription Factors/metabolism , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Corynebacterium glutamicum/genetics , DNA Footprinting , Electrophoresis, Polyacrylamide Gel , Electrophoretic Mobility Shift Assay , Glucose/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transcription, Genetic
20.
Appl Microbiol Biotechnol ; 81(2): 291-301, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18791709

ABSTRACT

Regulation of expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase essential for glycolysis in Corynebacterium glutamicum was studied. We applied DNA affinity beads to isolate proteins binding to the promoter region of the gapA gene and obtained SugR, which has been shown to be a repressor of pts genes involved in sugar transport system. The results of electrophoretic mobility shift assays revealed that SugR specifically bound to the gapA promoter and the consensus sequence TGTTTG in the promoter region was required for its binding. We examined expression of the gapA gene in a sugR deletion mutant. Effect of mutation in the SugR binding site on gapA-lacZ fusion expression was also examined. These assays revealed that SugR acts as a negative transcriptional regulator of the gapA gene in the absence of sugar, and repression by SugR is alleviated in the presence of sugar, i.e., fructose and sucrose. Fructose-1-phosphate and fructose-1,6-bisphosphate revealed negative effects on binding of SugR to the gapA promoter, indicating that the sugar metabolites are involved in the derepression of gapA expression.


Subject(s)
Corynebacterium glutamicum/enzymology , Genes, Regulator , Glyceraldehyde-3-Phosphate Dehydrogenases/biosynthesis , Repressor Proteins/metabolism , Artificial Gene Fusion , Bacterial Proteins/biosynthesis , Binding Sites , DNA, Bacterial/metabolism , Electrophoretic Mobility Shift Assay , Fructose/metabolism , Gene Deletion , Genes, Bacterial , Genes, Reporter , Promoter Regions, Genetic , Protein Binding , Repressor Proteins/genetics , Sucrose/metabolism , beta-Galactosidase/biosynthesis , beta-Galactosidase/genetics
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