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).     

Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum

A corynebacterial clone, previously isolated by scoring repression of lacZYA fused to the aceB promoter of Corynebacterium glutamicum, was analyzed further. In the clone, an open reading frame designated glxR, consisting of 681 nucleotides and encoding a 24,957-Da protein, was found. The molecular mass of a native GlxR protein was estimated by gel filtration column chromatography to be 44,000 Da, suggesting that the protein formed dimers. The predicted amino acid sequence contained both cyclic AMP (cAMP)- and DNA-binding motifs and was homologous with the cAMP receptor protein family of proteins. The aceB-repressing activity of the glxR clone was markedly relieved in an Escherichia coli cya mutant, but the activity was restored in growth medium containing cAMP. In glucose medium, the intracellular cAMP concentration of C. glutamicum reached 22 nmol/mg of protein in the early exponential phase and then decreased further; but in acetate medium, the intracellular cAMP concentration was only 5 nmol/mg of protein and remained low throughout the growth phase. The expression of glxR was not affected by the carbon source. Binding of purified GlxR to the promoter region of aceB could be demonstrated only in the presence of cAMP. These data suggest that GlxR may form dimers which bind to the aceB promoter region in the presence of cAMP and repress the glyoxylate bypass genes.     

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.     

Molecular analysis of the Corynebacterium glutamicum gene involved in lysine uptake

Two Corynebacterium glutamicum mutants defective in lysine uptake were identified by analysing mutants resistant to S-(2-aminoethyl)-cysteine (AEC). A 5.6 kb genomic DNA fragment restoring AEC sensitivity and lysine uptake was isolated. A 4.2 kb subfragment was sequenced and three open reading frames were identified. Subcloning and gene disruption experiments showed that only the first open reading frame, termed lysl, is involved in lysine uptake. Lysl consists of 501 amino acids with a Mr of 53600. The hydrophobicity profile suggests that the lysl gene product is an integral membrane protein with 13 transmembrane segments. The amino acid sequence of lysl displays strong homology to that of the arcD gene product of Pseudomonas aeruginosa, which is proposed to act as an arginine-ornithine antiporter. Investigation of the influence of the lysl gene on lysine secretion suggests the existence of a separate lysine efflux system in C. glutamicum.     

Characterization of a novel plasmid pXZ608 from Corynebacterium glutamicum

BspA is a non-covalently anchored cystine-binding protein from Lactobacillus fermentum BR11. It has previously been used to present antigens derived from infectious organisms on the L. fermentum BR11 cell surface. In this study, the capacity of BspA to present a very large polypeptide was tested. A temperature sensitive plasmid was constructed that encodes a 175-kDa chimeric protein consisting of a fusion between BspA and an N-terminally truncated derivative of the Streptococcus salivarius ATCC 25975 glucosyltransferase GtfJ. This plasmid was introduced into the L. fermentum genome. Integrants were able to incorporate 20-40 nmol sucrose derived glucose into glucan per ml culture per optical density unit. The glucosyltransferase activity was external to the cytoplasmic membrane and bound to the cell. Unlike native BspA, the BspA-GtfJ fusion could not be removed from the cell by 5 M LiCl wash.     

The amrG1 gene is involved in the activation of acetate in Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic, Gram-positive microorganism, well known as a pro-ducer of several amino acids. Amino acid products are used on a large scale for food industry flavouring, feed additive, pharmaceutical and cosmetic purpose[1,2]. The organism is able to grow not only on glucose, fructose and lactose, but also on acetate, lactate as its sole carbon source. The growth on acetate requires its activation to acetyl-CoA. In C. glutamicum, acetate is activated in a two-step …     

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...     

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.     

Corynebacterium glutamicum whcB, a stationary phase-specific regulatory gene

The function of whcB, one of the four whiB homologues of Corynebacterium glutamicum, was assessed. Cells carrying the P(180)-whcB clone, and thus overexpressing the whcB gene, showed retarded growth, probably due to increased sensitivity to oxidants, whereas cells lacking whcB (ΔwhcB) did not. However, growth retardation was not observed in cells with additionally whcE deleted. Furthermore, the ΔwhcE phenotype, characterized by slow growth and sensitivity to oxidants, was reversed in cells carrying P(180)-whcB. Like the whcE gene, which is also known as a whiB homologue, the whcB gene was preferentially expressed in stationary phase. Determination of the genes under regulation of whcB using two-dimensional polyacrylamide gel electrophoresis identified several genes involved in electron transfer reactions that were regulated in cells carrying P(180)-whcB. Collectively, these findings indicate that whcB function requires whcE. Furthermore, whcB and whcE are paralogues but perform distinct regulatory roles during growth under oxidative stress.     

Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number.

The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode.     

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.     

Sequence of the Corynebacterium glutamicum pyruvate carboxylase gene

Pyruvate carboxylase is an important anaplerotic enzyme replenishing oxaloacetate consumed for biosynthesis during growth, or lysine and glutamic acid production in industrial fermentations. We used regions of homology from pyruvate carboxylase sequences of 12 different species (corresponding to the ATP- and pyruvate-binding sites), to design polymerase chain reaction (PCR) primers for amplifying a fragment of the pyruvate carboxylase (pc) gene from C. glutamicum genomic DNA. This 850-base-pair fragment was used to probe a C. glutamicum cosmid library and four candidate pc cosmids were identified. The fragment was sequenced and the sequence of the complete gene was obtained by several rounds of primer synthesis, PCR on one of the positive cosmids, and sequencing. The C. glutamicumpc sequence shows 64% homology with the pc gene of Mycobacterium tuberculosis and 44% homology with the human pc gene. Regions of ATP, pyruvate and biotin binding have also been identified. Received: 16 December 1997 / Received revision: 31 March 1998 / Accepted: 19 April 1998     

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.