Characterization of a 24-kb plasmid pCGR2 newly isolated from Corynebacterium glutamicum

A 24-kb plasmid with 21 open reading frames (ORFs) was newly isolated from Corynebacterium glutamicum ATCC 14997 and named pCGR2. Three of its ORFs were indispensable for stable autonomous replication of pCGR2 in C. glutamicum: in the absence of selective pressure, deletion derivatives of pCGR2 containing the three ORFs showed stability in C. glutamicum for over 50 generations. The first of these ORFs encoded replicase repA whose gene product revealed high amino acid sequence similarity to corresponding gene products of C. glutamicum pCG1-family plasmids in general, and to that of pTET3 plasmid repA in particular. The other two ORFs were located upstream of repA and exhibited high sequence similarity to pTET3 parA and parB, respectively. Interestingly, plasmids based on the pCGR2 were compatible not only with those based on different family plasmids (pBL1, pCASE1) but also with those based on pCG1-family plasmid. Plasmids comprising pCGR2 repA showed a copy number of four in C. glutamicum. The number increased to 240 upon introduction of a mutation within the repA origin of the putative promoter for counter-transcribed RNA. This 60-fold increase in copy number should immensely contribute towards enhanced expression of desired genes in C. glutamicum.     

Characterization of pGA1, a new plasmid from Corynebacterium glutamicum LP-6.

A new plasmid, pGA1, has been isolated from Corynebacterium glutamicum LP-6, and its detailed restriction map has been prepared. The 4.9-kb plasmid has a G + C content of 57%. It replicates in C. glutamicum ATCC13032 and is compatible with the three other plasmids, pCC1, pBL1 and pHM1519, commonly used for vector construction for amino acid-producing corynebacteria. Fusions of pGA1 with different Escherichia coli replicons (transferred from E. coli to Corynebacterium via transformation of spheroplasts or by filter mating experiments with intact cells) are shown to be suitable as shuttle plasmids; some of them are highly stable in C. glutamicum, even when propagated without any selection pressure.     

Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum

Abstract Phosphoenolpyruvate (PEP) carboxykinase is present in crude extracts of Corynebacterium glutamicum grown on both glucose and lactate. Preparation of PEP carboxykinase free from interfering PEP carboxylase and oxaloacetate decarboxylase showed an absolute dependence on divalent manganese and IDP for activity in the oxaloacetate (OAA) formation. Other diphosphate nucleotides could not substitute for IDP. The enzyme activity displayed Michaelis-Menten kinetics for the substrates PEP, IDP, KHCO₃, OAA and ITP with a K _m of 0.7 mM, 0.4 mM, 12 mM, 1.0 mM, and 0.5 mM, respectively. At the optimum pH of 6.6, 850 nmol of OAA were formed per min per mg of protein. ATP inhibited PEP carboxykinase in the OAA forming reaction for 60% at 0.1 mM, indicating that the enzyme mainly functions in gluconeogenesis.     

Identification of a novel gene involved in stable maintenance of plasmid pGA1 from Corynebacterium glutamicum.

The cryptic plasmid pGA1 (4.8 kb) from Corynebacterium glutamicum, replicating in the rolling-circle mode, has been reported to contain four open reading frames longer than 200 bp (ORFA/per, ORFA2, ORFB, ORFC/rep). Here we present another pGA1 gene, ORFE (174 bp), located in the region downstream of the per-ORFA2 gene cluster. The ORFE is transcribed into two RNA species in a direction opposite to that of the per-ORFA2 RNA. Introduction of ORFE in trans into the cells harboring the pGA1 derivatives carrying the main stability determinant, the per gene coding for a product that positively influences the pGA1 copy number and maintenance, increased their segregational stability. Mutation of the putative translational start of the ORFE abolished this observed positive effect in trans. ORFE thus codes for a protein acting as an accessory element involved in stable maintenance of plasmid pGA1 and was hence designated the aes gene (accessory effector of stable maintenance).     

Purification and Characterization of Fumarase from Corynebacterium glutamicum

Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K _eq) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K _eq=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.     

Characterization of novel plasmid p1B146 from Corynebacterium tuberculostearicum

Corynebacterium tuberculostearicum B146, a strain derived from healthy human skin, contains a medium copy plasmid, p1B146. This plasmid was cloned and its complete nucleotide sequence determined. As a result, p1B146 was found to be 4.2 kb in size with a 53% G+C content, plus six open reading frames (ORFs) were distinguished. According to a computer-assisted alignment, two of the ORFs exhibited significant similarities to already-known common plasmid proteins, the first being the RepA gene, responsible for plasmid replication via a rolling-circle mechanism, and the second being an FtsK-like protein, the function of which remains unclear. The presence and quantity of RNA fragments in the putative ORFs were also evaluated.     

Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum

A high L-asparaginase (L-asparagine amidohydrolase: EC 3.5.1.1) activity was found under conditions of lysine overproduction in cultures of Corynebacterium glutamicum. L-Asparaginase was purified 98-fold by protamine sulphate precipitation. DEAE-Sephacel anion exchange, ammonium sulphate precipitation and Sephacryl S-200 gel filtration. The asparaginase protein was subjected to PAGE under non-denaturing conditions, identified by an in situ reaction and eluted from the gel in an active form. The estimated Mr from gel filtration and SDS-PAGE was 80,000. The L-asparaginase activity was inhibited by the L-asparagine analogue 5-diazo-4-oxo-L-norvaline. Neither D-asparagine nor L-glutamine was a substrate for the enzyme. L-Asparaginase was produced constitutively: its role may be that of an overflow enzyme, converting excess asparagine into aspartic acid, the direct precursor of lysine and threonine.     

Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum

Applied Microbiology and Biotechnology - Beyond its traditional role as an L-amino acid producer, Corynebacterium glutamicum has recently received significant attention regarding its use in the...     

Identification of a gene involved in plasmid structural instability in Corynebacterium glutamicum

Expression plasmids that facilitate production of bio-based products are susceptible to toxic effects that frequently affect plasmid structural stability in recombinant microbial cells. In order to enhance plasmid stability in recombinant Corynebacterium glutamicum, an expression plasmid containing genes of the Clostridium acetobutylicum butyryl-CoA synthesis operon with high structural instability within wild-type C. glutamicum was employed. From a total of 133 mutants exhibiting disruptions in 265 suspect genes, only cgR_0322-deficient mutant was able to maintain the expression plasmid intact. The mutant exhibited normal growth under standard laboratory conditions but its transformation efficiency was about one order of magnitude lower than that of wild-type strain. The cgR_0322 gene encodes an endonuclease that is active against single- as well as double-stranded DNA substrates in the presence of Mg²⁺. The cgR_0322-deficient strain should therefore facilitate the development of more robust C. glutamicum strains to be used as microbial production hosts.     

谷氨酸生产菌S9114中的谷氨酸脱氢酶的研究

谷氨酸脱氢酶 (GDH)是谷氨酸生物合成的关键酶 ,谷氨酸棒杆菌S91 1 4是目前我国味精工业应用最广泛的生产菌种 ,其谷氨酸脱氢酶的研究尚未见报道。分离纯化该菌中的谷氨酸脱氢酶 ,研究其辅酶组成 ,对揭示谷氨酸脱氢酶的分子结构和性质 ,提高谷氨酸产率很有必要。将培养至对数期中期的细胞离心收集并用含适量DTT、ED TA的Tris_HCl缓冲液 (pH 7 5 )洗涤 ,用Frenchpressurecellpress破碎 ,离心去除菌体碎片得无细胞抽提液。然后使用 KTA_10 0快速纯化系统经DEAE_纤维素柱、疏水柱 (HIC)、G_2 0 0凝胶过滤柱层析得到纯化大约 70倍的以NAD PH为辅酶的GDH和部分纯化的以NADH辅酶的GDH。这两个酶分别对NADPH、NADH高度专一 ,不能相互代替。经HPLC和SDS_PAGE测得前一种酶的分子量和亚基分子量分别为 188kD和 32kD ,表明该酶为具有相同亚基的六聚体。酶活性测定使用HITACHIU_30 0 0分光光度计利用NAD(P)H在 340nm氧化的初速度进行。蛋白质含量测定利用Bradford方法进行 ,并以牛血清白蛋白为标准蛋白。纯化结果表明S91 1 4中确实存在两种GDH ,其中以NADH为辅酶的GDH尚未见报道。和某些具有两种GDH的微生物一样 ,S91 1 4可能也是以NADPH为辅酶的GDH参与谷氨酸的合成代谢 ,以NADH为辅酶的GDH参与谷氨酸的分解代谢。     

Structural organization of the Corynebacterium glutamicum plasmid pCG100.

pCG100, a 3 kb cryptic plasmid of Corynebacterium glutamicum ATCC 13058, probably identical with pSR1 from C. glutamicum ATCC 19223, was characterized. The minimum region for autonomous replication was shown to be contained on a 1.9 kb BglII-NcoI fragment; a 380 bp HindIII-SphI fragment can replicate in the presence of the parental plasmid, which presumably provides a trans-acting replication factor. Derivatives of pCG100 are able to replicate in several Corynebacterium, Brevibacterium and Arthrobacter strains. pCG100 is compatible with pBL1, a cryptic plasmid of Brevibacterium lactofermentum. Shuttle plasmid vectors, containing the kanamycin-resistance gene from Tn903 or from Streptococcus faecalis as selectable markers and the AmpR, TetR or lacZ alpha genes for insertional inactivation, were constructed using the minimum replication fragment of pCG100.     

Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid

Random chemical mutation of a Corynebacterium glutamicum-Escherichia coli shuttle vector derived from plasmid pCGR2 was done using hydroxylamine. It brought about amino acid substitutions G109D and E180K within the replicase superfamily domain of the plasmid's RepA protein and rendered the plasmid highly unstable, especially at higher incubation temperatures. Colony formation of C. glutamicum was consequently completely inhibited at 37 °C but not at 25 °C. G109 is a semi-conserved residue mutation which resulted in major temperature sensitivity. E180 on the other hand is not conserved even among RepA proteins of closely related C. glutamicum pCG1 family plasmids and its independent mutation caused relatively moderate plasmid instability. Nonetheless, simultaneous mutation of both residues was required to achieve temperature-sensitive colony formation. This new pCGR2-derived temperature-sensitive plasmid enabled highly efficient chromosomal integration in a variety of C. glutamicum wild-type strains, proving its usefulness in gene disruption studies. Based on this, an efficient markerless gene replacement system was demonstrated using a selection system incorporating the temperature-sensitive replicon and Bacillus subtilis sacB selection marker, a system hitherto not used in this bacterium. Single-crossover integrants were accurately selected by temperature-dependent manner and 93% of the colonies obtained by the subsequent sucrose selection were successful double-crossover recombinants.     

Subcellular Localization and Characterization of the ParAB System from Corynebacterium glutamicum

Faithful segregation of chromosomes and plasmids is a vital prerequisite to produce viable and genetically identical progeny. Bacteria use a specialized segregation system composed of the partitioning proteins ParA and ParB to segregate certain plasmids. Strikingly, homologues of ParA and ParB are found to be encoded in many chromosomes. Although mutations in the chromosomal Par system have effects on segregation efficiency, the exact mechanism by which the chromosomes are segregated into the daughter cells is not fully understood. We describe the polar localization of the ParB origin nucleoprotein complex in the actinomycete Corynebacterium glutamicum. ParB and the origin of replication were found to be stably localized to the cell poles. After replication, the origins move toward the opposite pole. Purified ParB was able to bind to the parS consensus sequence in vitro. C. glutamicum possesses two ParA-like partitioning ATPase proteins. Both proteins interact with ParB but show a slightly different subcellular localization and phenotype. While ParA might be part of a conventional partitioning system, PldP seems to play a role in division site selection.Bacterial cell division is a temporally and spatially tightly regulated process (1, 13, 16, 36, 37). Spatial regulation is achieved by division site selection and prevents fatal division across the nucleoids and aberrant division close to the cell poles (3, 40). Temporal control ensures that division does not precede chromosome replication and segregation. Replicated chromosomes are rapidly segregated into the daughter cells. However, the machinery that performs this active segregation is not fully elucidated. In contrast, plasmid segregation is somewhat better understood. Plasmids such as pB171 (8) encode a machinery composed of a tripartite system. Centromere-like DNA sequences, named parS sites, are composed of short inverted repeats. Centromere-binding proteins (ParB) are recruited to the parS sites, forming nucleoprotein complexes. Finally, a partitioning ATPase is recruited to the ParB-parS complex. The hydrolytic activity of ParA oligomers is believed to drive the active segregation process. Strikingly, many bacterial chromosomes encode orthologs of the plasmid partitioning genes parA and parB. A comparatively well-examined chromosomal partitioning system is that of Bacillus subtilis. B. subtilis encodes a ParA ATPase (called Soj) and a ParB protein (called Spo0J). B. subtilis contains eight parS sites that cluster around the oriC region and bind Spo0J. Subsequently Spo0J spreads across the DNA, thereby forming a huge nucleoprotein complex that could serve as a platform for anchoring the segregation machinery. The ParA protein Soj is a DNA-binding protein that dissociates from DNA upon ATP hydrolysis. A direct interaction of Soj and Spo0J has been described (35). Interestingly, analysis of knockout mutations revealed that only the loss of the ParB protein Spo0J increases the amount of anucleate cells slightly, while the loss of Soj has no significant effect on chromosome segregation (17, 18). However, knockout mutations in either parA or parB result only in subtle effects on chromosome segregation. Thus, although the two proteins might act together they have certainly multiple roles during chromosome segregation and cell division. Recently, it was shown that Spo0J (ParB) helps to recruit SMC proteins (for structural maintenance of chromosomes) to the oriC region, thereby ensuring correct chromosome organization, which seems essential for proper segregation (15, 39). The B. subtilis ParA homologue Soj was shown to play an role in the initiation of DNA replication by interacting with DnaA (32). Hence, the ParAB system is a central component connecting replication and segregation. Interestingly, Par proteins have been implicated with different developmental processes in other bacteria. In Caulobacter crescentus ParAB are involved in cell cycle progression and cell division. A ParA-like protein, MipZ, was shown to interact with ParB and directly inhibit FtsZ polymerization (42). Thus, chromosome segregation and cell division are directly coupled. Consequently, null mutations in ParA and ParB are lethal in C. crescentus. In Vibrio cholerae it was shown that ParA and ParB encoded on the large chromosome contribute to active chromosome segregation and anchor the oriC region of the chromosomes to the cell poles (10).Although these diverse properties of the Par system have been studied in some detail in the classical model organisms, the situation in other bacteria remains unknown. Corynebacteria are high GC Gram-positive bacteria and, depending on the growth medium, rod-shaped or club-shaped. A remarkable feature of corynebacteria and their close relatives is a special cell wall that has, in addition to the common peptidoglycan, an arabino-galactan and a mycolic acid layer. Notorious pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, and Corynebacterium diphtheriae are members of this family, and hence an understanding of fundamental cell biological mechanisms might reveal insights how to combat these organisms. We now report the subcellular localization of the chromosome partitioning system and the oriC in the actinomycete Corynebacterium glutamicum. We show localization and phenotypic consequences of the canonical ParAB proteins. Furthermore, we identified a ParA-like division protein (PldP) that plays a role in division site selection.     

Characterization of shikimate dehydrogenase homologues of Corynebacterium glutamicum

The function of three Corynebacterium glutamicum shikimate dehydrogenase homologues, designated as qsuD (cgR_0495), cgR_1216, and aroE (cgR_1677), was investigated. A disruptant of aroE required shikimate for growth, whereas a qsuD-deficient strain did not grow in medium supplemented with either quinate or shikimate as sole carbon sources. There was no discernible difference in growth rate between wild-type and a cgR_1216-deficient strain. Enzymatic assays showed that AroE both reduced 3-dehydroshikimate, using NADPH as cofactor, and oxidized shikimate, the reverse reaction, using NADP⁺ as cofactor. The reduction reaction was ten times faster than the oxidation. QsuD reduced 3-dehydroquinate using NADH and oxidized quinate using NAD⁺ as cofactor. Different from the other two homologues, the product of cgR_1216 displayed considerably lower enzyme activity for both the reduction and the oxidation. The catalytic reaction of QsuD and AroE was highly susceptible to pH. Furthermore, reduction of 3-dehydroshikimate by AroE was inhibited by high concentrations of shikimate, but neither quinate nor aromatic amino acids had any effect on the reaction. Expression of qsuD mRNA was strongly enhanced in the presence of shikimate, whereas that of cgR_1216 and aroE decreased. We conclude that while AroE is the main catalyst for shikimate production in the shikimate pathway, QsuD is essential for quinate/shikimate utilization.     

Functional Characterization of Corynebacterium glutamicum Mycothiol S-Conjugate Amidase

The present study focuses on the genetic and biochemical characterization of mycothiol S-conjugate amidase (Mca) of Corynebacterium glutamicum. Recombinant C. glutamicum Mca was heterologously expressed in Escherichia coli and purified to apparent homogeneity. The molecular weight of native Mca protein determined by gel filtration chromatography was 35 kDa, indicating that Mca exists as monomers in the purification condition. Mca showed amidase activity with mycothiol S-conjugate of monobromobimane (MSmB) in vivo while mca mutant lost the ability to cleave MSmB. In addition, Mca showed limited deacetylase activity with N-acetyl-D-glucosamine (GlcNAc) as substrate. Optimum pH for amidase activity was between 7.5 and 8.5, while the highest activity in the presence of Zn²⁺ confirmed Mca as a zinc metalloprotein. Amino acid residues conserved among Mca family members were located in C. glutamicum Mca and site-directed mutagenesis of these residues indicated that Asp14, Tyr137, His139 and Asp141 were important for activity. The mca deletion mutant showed decreased resistance to antibiotics, alkylating agents, oxidants and heavy metals, and these sensitive phenotypes were recovered in the complementary strain to a great extent. The physiological roles of Mca in resistance to various toxins were further supported by the induced expression of Mca in C. glutamicum under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH.     

DivS, a novel SOS-inducible cell-division suppressor in Corynebacterium glutamicum

DNA damage-induced SOS response elicits the induction of cell-division suppressor as well as DNA repair genes. In Gram-positive bacteria, cell-division suppressor genes, so far characterized from Bacillus subtilis (yneA) and Mycobacterium tuberculosis (rv2719c), share limited homology, but are both located in the vicinity of lexA on their respective genomes. Using this proximity to lexA, Corynebacterium glutamicum R divS (cgR1759) was identified as an SOS-inducible cell-division suppressor in this study. The amino acid sequence of DivS showed no homology to that of YneA and Rv2719c. divS expression was markedly induced by DNA-damaging mitomycin C treatment in wild-type cells, but not in its DeltarecA mutant cells, which are unable to induce the SOS response. Wild-type C. glutamicum R cells exposed to DNA-damaging mitomycin C exhibited elongated morphology that, using green fluorescent protein-FtsZ fusion protein, was attributed to defects in FtsZ ring assembly. Cells defective in FtsZ ring assembly were subsequently incapable of septum wall synthesis. In the presence of mitomycin C, divS mutant cells did not exhibit this elongated morphology, whereas cells overexpressing divS were elongated and abnormally branched.     

Characterization of a secondary uptake system for l-glutamate in Corynebacterium glutamicum

Abstract A new transport system for the uptake of l-glutamate was characterized in Corynebacterium glutamicum strain Δ glu, in which the previously described binding protein-dependent glutamate uptake system is not present. Kinetic characterization revealed a highly specific secondary transport system, dependent on sodium ions. Glutamate uptake showed Michaelis-Menten kinetics, with a K _m of 0.6 mM and a V _max of 15 nmol min⁻¹ (mg dw)⁻¹. For the co-transported sodium ions, a relatively low K _m of 3.3 mM was determined.