Genetic characterization of the resorcinol catabolic pathway in Corynebacterium glutamicum

Corynebacterium glutamicum grew on resorcinol as a sole source of carbon and energy. By genome-wide data mining, two gene clusters, designated NCgl1110-NCgl1113 and NCgl2950-NCgl2953, were proposed to encode putative proteins involved in resorcinol catabolism. Deletion of the NCgl2950-NCgl2953 gene cluster did not result in any observable phenotype changes. Disruption and complementation of each gene at NCgl1110-NCgl1113, NCgl2951, and NCgl2952 indicated that these genes were involved in resorcinol degradation. Expression of NCgl1112, NCgl1113, and NCgl2951 in Escherichia coli revealed that NCgl1113 and NCgl2951 both coded for hydroxyquinol 1,2-dioxygenases and NCgl1112 coded for maleylacetate reductases. NCgl1111 encoded a putative monooxygenase, but this putative hydroxylase was very different from previously functionally identified hydroxylases. Cloning and expression of NCgl1111 in E. coli revealed that NCgl1111 encoded a resorcinol hydroxylase that needs NADPH as a cofactor. E. coli cells containing Ncgl1111 and Ncgl1113 sequentially converted resorcinol into maleylacetate. NCgl1110 and NCgl2950 both encoded putative TetR family repressors, but only NCgl1110 was transcribed and functional. NCgl2953 encoded a putative transporter, but disruption of this gene did not affect resorcinol degradation by C. glutamicum. The function of NCgl2953 remains unclear.     

Identification of D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Expression of a putative acyltransferase encoded by NCgl- 0350 of Corynebacterium glutamicum is induced by cell-free culture fluids obtained from stationary-phase growth of both C. glutamicum and Pseudomonas aeruginosa, providing evidence for interspecies communication. Here, we further confirmed that such communication occurs by showing that acyltransferase expression is induced by culture fluid obtained from diverse Gram-negative and -positive bacterial strains, including Escherichia coli, Salmonella Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium sp. strain JC1, and Mycobacterium smegmatis. A homologous acyltransferase encoded by PA5238 of P. aeruginosa was also induced by fluids obtained from P. aeruginosa as well as other bacterial strains, as observed for NCgl0350 of C. glutamicum. Because C. glutamicum is difficult to study using molecular approaches, the homologous gene PA5238 of P. aeruginosa was used to identify PA5309 as an upstream regulator of expression. A homologous D-amino acid dehydrogenase encoded by NCgl- 2909 of C. glutamicum was cloned based on amino acid similarity to PA5309, and its role in the regulation of NCgl0350 expression was confirmed. Moreover, NCgl2909 played positive roles in growth of C. glutamicum. Thus, we identified a D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in C. glutamicum.     

Enhancement of l-ornithine production by disruption of three genes encoding putative oxidoreductases in Corynebacterium glutamicum

Recently, Corynebacterium glutamicum has been shown to exhibit gluconate bypass activity, with two key enzymes, glucose dehydrogenase (GDH) and gluconate kinase, that provides an alternate route to 6-phosphogluconate formation. In this study, gene disruption analysis was used to examine possible metabolic functions of three proteins encoded by open reading frames having significant sequence similarity to GDH of Bacillus subtilis. Chromosomal in-frame deletion of three genes (NCgl0281, NCgl2582, and NCgl2053) encoding putative NADP⁺-dependent oxidoreductases led to the absence of GDH activity and correlated with increased specific glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. This finding suggested that enhanced carbon flux from glucose was directed toward the oxidative pentose phosphate (PP) pathway, when the mutant was cultivated with 6 % glucose. Consequently, the mutant showed 72.4 % increased intracellular NADPH and 66.3 % increased extracellular l-ornithine production. The enhanced activities of the oxidative PP pathway in the mutant explain both the increased intracellular NADPH and the high extracellular concentration of l-ornithine. Thus, the observed metabolic changes in this work corroborate the importance of NADPH in l-ornithine production from C. glutamicum.     

Glutamate Is Excreted Across the Cytoplasmic Membrane through the NCgl1221 Channel of Corynebacterium glutamicum by Passive Diffusion

The NCgl1221 gene, which encodes a mechanosensitive channel, has been reported to be critically involved in glutamate (Glu) overproduction by Corynebacterium glutamicum, but direct evidence of Glu excretion through this channel has not yet been provided. In this study, by electrophysiological methods, we found direct evidence of Glu excretion through this channel by passive diffusion. We found that the introduction into Phe-producing Escherichia coli of mutant NCgl1221 genes that induce Glu overproduction by C. glutamicum improved productivity. This suggests a low-substrate preference of this channel, indicates its potential as a versatile exporter, and more broadly, indicates the potential of exporter engineering.     

<Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> RNase E/G-type endoribonuclease encoded by NCgl2281 is involved in the 5′ maturation of 5S rRNA

Corynebacterium glutamicum has one RNase E/G ortholog and one RNase J ortholog but no RNase Y. We previously reported that the C. glutamicum NCgl2281 gene encoding the RNase E/G ortholog complemented the rng::cat mutation in Escherichia coli but not the rne-1 mutation. In this study, we constructed an NCgl2281 knockout mutant and found that the mutant cells accumulated 5S rRNA precursor molecules. The processing of 16S and 23S rRNA, tRNA, and tmRNA was normal. Primer extension analysis revealed that the RNase E/G ortholog cleaved at the −1 site of the 5′ end of 5S rRNA. However, 3′ maturation was essentially unaffected. These findings showed that C. glutamicum NCgl2281 endoribonuclease is involved in the 5′ maturation of 5S rRNA. This is the first report showing the physiological function of the RNase E/G ortholog in bacteria having one RNase E/G and one RNase J but no RNase Y.     

Transaminase encoded by NCgl2515 gene of Corynebacterium glutamicum ATCC13032 is involved in γ-aminobutyric acid decomposition

Corynebacterium glutamicum that expresses an exogenous l-glutamate decarboxylase (GAD) gene can synthesize γ-aminobutyric acid (GABA). GABA is decomposed to succinic semialdehyde (SSA) by GABA transaminase (GABA-T) and to succinate thereafter by SSA dehydrogenase (SSADH). However, deletion of the gabT gene encoding GABA-T could not prevent GABA from decomposing at neutral pH. In this study, an additional transaminase gene, NCgl2515, was deleted in a gabT-deleted GAD strain, and GABA fermentation in this gabT NCgl2515-deleted GAD strain was investigated. GABA concentration remained at 22.5–24.0 g/L when pH was maintained at 7.5–8.0, demonstrating that GABA decomposition was reduced. Activity assay indicated that unlike GabT, which exhibits high GABA-T activity (1.34 ± 0.06 U/mg) and utilizes only α-ketoglutarate as amino acceptor, the purified NCgl2515 protein exhibits very low GABA-T activity (approximately 0.03 U/mg) only when coupled with the SSADH, GabD, but can utilize both α-ketoglutarate and pyruvate as amino acceptor. The optimum pH for coupled NCgl2515–GabD was 8.0, similar to that of GabT (7.8). Therefore, NCgl2515 has weak GABA-T activity and is involved in GABA decomposition in C. glutamicum. Deletion of gabT and NCgl2515 could effectively reduce GABA decomposition at neutral pH.     

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite d,l-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of l-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.     

Conserved residues in the aromatic acid/H symporter family are important for benzoate uptake by NCgl2325 in Corynebacterium glutamicum

Corynebacterium glutamicum is a model organism for genetic and physiological studies in Gram-positive bacteria. NCgl2325 in C. glutamicum, a transporter belonging to the aromatic acid/H⁺ symporter family, has previously been reported to be involved in benzoate assimilation. Here, we showed that this transporter, fused with GFP, was associated with the cell membrane in Escherichia coli and C. glutamicum. Uptake assays with [¹⁴C]-labeled benzoate demonstrated that NCgl2325 transported benzoate into the cells at a V_max of 0.19 ± 0.01 nmol/min/mg of dry weight, and the K_m value was determined to be 1.11 ± 0.24 ??M. Among the competing substrates tested, hydroxyl-substituted benzoates resulted in significant inhibition (>50%) of benzoate uptake. Site-directed mutagenesis of conserved residues in the hydrophilic cytoplasmic loops (Gly-80, Asp-84 and Asp-312) and the hydrophobic transmembrane regions (Asp-35, Arg-119, Glu-139 and Arg-386) resulted in loss of benzoate transport activity. This is the first study to investigate the molecular basis of benzoate transport.     

Enhanced L-cysteine production by overexpressing potential L-cysteine exporter genes in an L-cysteine-producing recombinant strain of Corynebacterium glutamicum

ABSTRACT

We identified L-cysteine exporter candidates of Corynebacterium glutamicum and investigated the effect of overexpression of the potential L-cysteine exporter genes on L-cysteine production in a recombinant strain of C. glutamicum. Overexpression of NCgl2566 and NCgl0580 resulted in enhanced L-cysteine production in an L-cysteine-producing recombinant strain of C. glutamicum.     

Current knowledge on mycolic acids in Corynebacterium glutamicum and their relevance for biotechnological processes

Corynebacterium glutamicum is the world’s largest producer of glutamate and lysine. Industrial glutamate overproduction is induced by empirical processes, such as biotin limitation, supplementation with specific surfactants or addition of sublethal concentration of certain antibiotics to the culture media. Although Gram-positive bacteria, C. glutamicum and related bacterial species and genera contain, in addition to the plasma membrane, an outer permeability membrane similar to that of Gram-negative microorganisms. As the amino acids have to cross both membranes, their integrity, composition and fluidity influence the export process. While the precise mechanism of the export of the amino acids by C. glutamicum is not fully understood, the excretion of amino acids through the inner membrane involved at least a major export system mechanosensitive channel MscS family (MscCG) encoded by NCgl1221. As the various industrial treatments have been shown to affect the lipid content of the bacterial cell, it is strongly believed that defects in the hallmark of the outer membrane, 2-alkyl, 3-hydroxylated long-chain fatty acids (mycolic acids), could be key factors in the glutamate overproduction. This review aims at giving an overview of the current knowledge on mycolic acids structure, biosynthesis and transfer in C. glutamicum and their relevance for amino acid biotechnological production.     

Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum

Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Δncg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.     

Metabolism of resorcinylic compounds by bacteria: alternative pathways for resorcinol catabolism in Pseudomonas putida.

Two strains of Pseudomonas putida isolated by enrichment cultures with orcinol as the sole source of carbon were both found to grow with resorcinol. Data are presented which show that one strain (ORC) catabolizes resorcinol by a metabolic pathway, genetically and mechanistically distinct from the orcinol pathway, via hydroxyquinol and ortho oxygenative cleavage to give maleylacetate, but that the other strain (O1) yields mutants that utilize resorcinol. One mutant strain, designated O1OC, was shown to be constitutive for the enzymes of the orcinol pathway. After growth of this strain on resorcinol, two enzymes of the resorcinol pathway are also induced, namely hydroxyquinol 1,2-oxygenase and maleylacetate reductase. Thus hydroxyquniol, formed from resorcinol, undergoes both ortho and meta diol cleavage reactions with the subsequent formation of both pyruvate and maleylacetate. Evidence was not obtained for the expression of resorcinol hydroxylase in strain O1OC; the activity of orcinol hydroxylase appears to be recruited for this hydroxylation reaction. P. putida ORC, on the other hand, possesses individual hydroxylases for orcinol and resorcinol, which are specifically induced by growth on their respective substrates. The spectral changes associated with the enzymic and nonenzymic oxidation of hydroxyquinol are described. Maleylacetate was identified as the product of hydroxyquinol oxidation by partially purified extracts obtained from P. putida ORC grown with resorcinol. Its further metabolism was reduced nicotinamide adenine dinucleotide dependent.     

Expression of the Escherichia coli Catabolic Threonine Dehydratase in Corynebacterium glutamicum and Its Effect on Isoleucine Production

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1.16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to α-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.     

The Corynebacterium glutamicum cglIM gene encoding a 5-cytosine methyltransferase enzyme confers a specific DNA methylation pattern in an McrBC-deficient Escherichia coli strain

The cglIM gene of the coryneform soil bacterium Corynebacterium glutamicum ATCC 13032 has been cloned and characterized. The coding region comprises 1092 nucleotides and specifies a protein of 363 amino acid residues with a deduced M_r of 40 700. The amino acid sequence showed striking similarities to methyltransferase enzymes generating 5-methylcytosine residues, especially to M·NgoVII from Neisseria gonorrhoeae recognizing the sequence GCSGC. The cglIM gene is organized in an unusual operon which contains, in addition, two genes encoding stress-sensitive restriction enzymes. Using PCR techniques the entire gene including the promoter region was amplified from the wild-type chromosome and cloned in Escherichia coli. Expression of the cglIM gene in E. coli under the control of its own promoter conferred the C. glutamicum-specific methylation pattern to co-resident shuttle plasmids and led to a 260-fold increase in the transformation rate of C. glutamicum. In addition, the methylation pattern produced by this methyltransferase enzyme is responsible for the sensitivity of DNA from C. glutamicum to the modified cytosine restriction (Mcr) system of E. coli.     

3′ Untranslated Region-Dependent Degradation of the aceA mRNA,Encoding the Glyoxylate Cycle Enzyme Isocitrate Lyase,by RNase E/G in Corynebacterium glutamicum

We previously reported that the Corynebacterium glutamicum RNase E/G encoded by the rneG gene (NCgl2281) is required for the 5′ maturation of 5S rRNA. In the search for the intracellular target RNAs of RNase E/G other than the 5S rRNA precursor, we detected that the amount of isocitrate lyase, an enzyme of the glyoxylate cycle, increased in rneG knockout mutant cells grown on sodium acetate as the sole carbon source. Rifampin chase experiments showed that the half-life of the aceA mRNA was about 4 times longer in the rneG knockout mutant than in the wild type. Quantitative real-time PCR analysis also confirmed that the level of aceA mRNA was approximately 3-fold higher in the rneG knockout mutant strain than in the wild type. Such differences were not observed in other mRNAs encoding enzymes involved in acetate metabolism. Analysis by 3′ rapid amplification of cDNA ends suggested that RNase E/G cleaves the aceA mRNA at a single-stranded AU-rich region in the 3′ untranslated region (3′-UTR). The lacZ fusion assay showed that the 3′-UTR rendered lacZ mRNA RNase E/G dependent. These findings indicate that RNase E/G is a novel regulator of the glyoxylate cycle in C. glutamicum.     

Metabolism of phenol and resorcinol in Trichosporon cutaneum.

Trichosporon cutaneum was grown with phenol or resorcinol as the carbon source. The formation of beta-ketoadipate from phenol, catechol, and resorcinol was shown by a manometric method using antipyrine and also by its isolation and crystallization. Metabolism of phenol begins with o-hydroxylation. This is followed by ortho-ring fission, lactonization to muconolactone, and delactonization to beta-ketoadipate. No meta-ring fission could be demonstrated. Metabolism of resorcinol begins with o-hydroxylation to 1,2,4-benzenetriol, which undergoes ortho-ring fission yielding maleylacetate. Isolating this product leads to its decarboxylation and isomerization to trans-acetylacrylic acid. Maleylacetate is reduced by crude extracts to beta-ketoadipate with either reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate as a cosubstrate. The enzyme catalyzing this reaction was separated from catechol 1,2-oxygenase, phenol hydroxylase, and muconate lactonizing enzyme on a diethyl-aminoethyl-Sephadex A50 column. As a result it was purified some 50-fold, as was the muconate-lactonizing enzyme. Methyl-, fluoro-, and chlorophenols are converted to a varying extent by crude extracts and by purified enzymes. None of these derivatives is converted to maleylacetate, beta-ketoadipate, or their derivatives. Cells grown on resorcinol contain enzymes that participate in the degradation of phenol and vice versa.     

Differential Arabinan Capping of Lipoarabinomannan Modulates Innate Immune Responses and Impacts T Helper Cell Differentiation

Toll-like receptors (TLRs) recognize pathogens by interacting with pathogen-associated molecular patterns, such as the phosphatidylinositol-based lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM). Such structures are present in several pathogens, including Mycobacterium tuberculosis, being important for the initiation of immune responses. It is well established that the interaction of LM and LAM with TLR2 is a process dependent on the structure of the ligands. However, the implications of structural variations on TLR2 ligands for the development of T helper (Th) cell responses or in the context of in vivo responses are less studied. Herein, we used Corynebacterium glutamicum as a source of lipoglycan intermediates for host interaction studies. In this study, we have deleted a putative glycosyltransferase, NCgl2096, from C. glutamicum and found that it encodes for a novel α(1→2)arabinofuranosyltransferase, AftE. Biochemical analysis of the lipoglycans obtained in the presence (wild type) or absence of NCgl2096 showed that AftE is involved in the biosynthesis of singular arabinans of LAM. In its absence, the resulting molecule is a hypermannosylated (hLM) form of LAM. Both LAM and hLM were recognized by dendritic cells, mainly via TLR2, and triggered the production of several cytokines. hLM was a stronger stimulus for in vitro cytokine production and, as a result, a more potent inducer of Th17 responses. In vivo data confirmed hLM as a stronger inducer of cytokine responses and suggested the involvement of pattern recognition receptors other than TLR2 as sensors for lipoglycans.     

Benzothiazinones Mediate Killing of Corynebacterineae by Blocking Decaprenyl Phosphate Recycling Involved in Cell Wall Biosynthesis

Benzothiazinones (BTZs) are a new class of sulfur containing heterocyclic compounds that target DprE1, an oxidoreductase involved in the epimerization of decaprenyl-phosphoribose (DPR) to decaprenyl-phosphoarabinose (DPA) in the Corynebacterineae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis. As a result, BTZ inhibition leads to inhibition of cell wall arabinan biosynthesis. Previous studies have demonstrated the essentiality of dprE1. In contrast, Cg-UbiA a ribosyltransferase, which catalyzes the first step of DPR biosynthesis prior to DprE1, when genetically disrupted, produced a viable mutant, suggesting that although BTZ biochemically targets DprE1, killing also occurs through chemical synthetic lethality, presumably through the lack of decaprenyl phosphate recycling. To test this hypothesis, a derivative of BTZ, BTZ043, was examined in detail against C. glutamicum and C. glutamicum::ubiA. The wild type strain was sensitive to BTZ043; however, C. glutamicum::ubiA was found to be resistant, despite possessing a functional DprE1. When the gene encoding C. glutamicum Z-decaprenyl-diphosphate synthase (NCgl2203) was overexpressed in wild type C. glutamicum, resistance to BTZ043 was further increased. This data demonstrates that in the presence of BTZ, the bacilli accumulate DPR and fail to recycle decaprenyl phosphate, which results in the depletion of decaprenyl phosphate and ultimately leads to cell death.