Identification of plasmid partition function in coryneform bacteria.

We have identified and characterized a partition function that is required for stable maintenance of plasmids in the coryneform bacteria Brevibacterium flavum MJ233 and Corynebacterium glutamicum ATCC 31831. This function is localized to a HindIII-NspV fragment (673 bp) adjacent to the replication region of the plasmid, named pBY503, from Brevibacterium stationis IFO 12144. The function was independent of copy number control and was not associated directly with plasmid replication functions. This fragment was able to stabilize the unstable plasmids in cis but not in trans.     

Identification of plasmid partition function in coryneform bacteria

We have identified and characterized a partition function that is required for stable maintenance of plasmids in the coryneform bacteria Brevibacterium flavum MJ233 and Corynebacterium glutamicum ATCC 31831. This function is localized to a HindIII-NspV fragment (673 bp) adjacent to the replication region of the plasmid, named pBY503, from Brevibacterium stationis IFO 12144. The function was independent of copy number control and was not associated directly with plasmid replication functions. This fragment was able to stabilize the unstable plasmids in cis but not in trans.     

Potential vectors for molecular cloning in Brevibacterium flavum]

Construction of the shuttle cloning vectors for Escherichia coli-Brevibacterium flavum system is described. Expression of the Sp/Sm resistance determinant derived from the Corynebacterium plasmid pCG4 was registered in Escherichia coli cells. The genetic determinant for Sp/Sm resistance was shown to be located in a 2.2 kb PstI-SphI fragment by the deletion analysis mapping in Escherichia coli cells. Using Escherichia coli as a host we cloned the unique 0.8 kb EcoRI-EcoRI fragment of Brevibacterium flavum bacteriophage phi BSh6 in the plasmids with dual replication origins. Blocking of the shuttle vector transfer to Brevibacterium flavum by the insertion of bacteriophage phi BSh6 DNA was observed. The deletion of entire phage fragment or a specific part of it made it possible introduction of plasmids harboured by Escherichia coli cells into Brevibacterium flavum. A potential vector for homologous DNA cloning in Brevibacterium flavum was constructed.     

A host-vector system for an Arthrobacter species

An efficient host-vector system has been developed for an industrial strain of Arthrobacter sp. (NRRL B3728)used for glucose isomerase production. Protoplasts of Arthrobacter were generated by treating the cells with 0.5 mg lysozyme ml(-1) for 60 min in a solution containing 0.5 M-sucrose. Around 30% of the protoplasts regenerated on agar containing 0.5 M-sodium succinate as osmotic stabilizer. Three hybrid vectors, PBL2100, pCG1100 and pCG2100, were constructed by combining the Escherichia coli plasmid pBR322, a kanamycin- resistance gene from pNCAT4 and a cryptic plasmid from either Brevibacterium lactofermentum NCIB 9567 or Corynebacterium glutamicum NCIB 10026. These vectors transformed the protoplasts and expressed the kanamycin-resistance gene for screening. They contain a number of unique restrictions sites for cloning of foreign DNA. The transformation frequency of this system was 10(5)-10(6) transformants per micrograms of input plasmid and ws constant up to 5 micrograms of DNA. the probability of a plasmid transforming a plasmid transforming a protoplast was in the range 10(-5)-10(-6). The copy number of pBL2100 was around 5 per cell and those of pCG1100 and pCG2100 were around 33 per cell. Deletion mutants were generated from pCG2100. One of them, pCG2120, was able to transform protoplasts of strain NRRL B3728. Plasmids pBL2100 and pCG2100 were structurally stable in cells of NRRL B3728 but could not be maintained in non-selective medium. They segregated at a rate of 12.2 and 2.2% per generation respectively.     

Protoplast transformation of glutamate-producing bacteria with plasmid DNA

A method for polyethylene glycol-induced protoplast transformation of glutamate-producing bacteria with plasmid DNA was established. Protoplasts were prepared from cells grown in the presence of penicillin by treatment with lysozyme in a hypertonic medium. The concentration of penicillin during growth affected the efficiency of formation, regeneration, and polyethylene glycol-induced DNA uptake of protoplasts. Regeneration of protoplasts was accomplished on a hypertonic agar medium containing sodium succinate and yeast extract. The spectinomycin and streptomycin resistance plasmid pCG4, originally from Corynebacterium glutamicum T250, could transform various glutamate-producing bacteria such as C. glutamicum, Corynebacterium herculis, Brevibacterium flavum, and Microbacterium ammoniaphilum. The plasmid was structurally unchanged and stably maintained in new hosts. The transformation frequency of most competent protoplasts with pCG4 DNA isolated from primary transformants was high (ca. 10(6) transformants per microgram of covalently closed circular DNA) but was still two orders of magnitude below the frequency of transfection with modified DNA of the bacteriophage phi CGI. The difference was ascribed to the involvement of regeneration in transformation.     

Construction of novel shuttle vectors and a cosmid vector for the glutamic acid-producing bacteria Brevibacterium lactofermentum and Corynebacterium glutamicum

Novel cloning vectors for glutamic acid-producing bacteria have been constructed. Two cryptic plasmids, pAM330 from Brevibacterium lactofermentum and pHM1519 from Corynebacterium glutamicum, were used as precursors, and recombined with pBR325 or pUB110. Resultant composite plasmids were able to propagate and to express the CmR or KmR phenotype in B. lactofermentum and C. glutamicum. A smaller, high-copy-number plasmid, pAJ43, was also isolated following deletion of a part of the pAM330-pBR325 composite plasmid. Furthermore, a cosmid vector, which can be packaged and transduced through phage infection, has been developed using a cohesive-end fragment of the f1A phage and plasmid pAJ43. These plasmids are suitable for use as cloning vectors in the glutamic acid-producing bacteria.     

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 region of plasmid pBL1 of Brevibacterium lactofermentum involved in replication via the rolling circle model.

The minimal region for autonomous replication of pBL1, a 4.5-kb cryptic plasmid of Brevibacterium lactofermentum ATCC 13869 that has been used to construct a variety of corynebacterium vectors, was shown to be contained on a 1.8-kb HindII-SphI DNA fragment. This region contains two open reading frames (ORFs) (ORF1 and ORF5) which are essential for pBL1 replication in B. lactofermentum. Accumulation of single-strand intermediates in some of the constructions indicates that plasmid pBL1 replicates via the rolling circle replication model; its plus strand and minus strand were identified by hybridization with two synthetic oligonucleotide probes complementary to each pBL1 strand. ORF1 seems to encode the Rep protein and showed partial homology with sequences for Rep proteins from Streptomyces plasmids which replicate via rolling circle replication such as pIJ101, pSB24, and pJV1.     

Development of the vector-host system in Corynebacterium. Cloning and expression of homoserine dehydrogenase and homoserine kinase genes in Corynebacterium cells

Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.     

Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics

Endogenous plasmids and selectable resistance markers are a fundamental prerequisite for the development of efficient recombinant DNA techniques in industrial microorganisms. In this article, we therefore summarize the current knowledge about endogenous plasmids in amino acid-producing Corynebacterium glutamicum isolates. Screening studies identified a total of 24 different plasmids ranging in size from 2.4 to 95 kb. Although most of the C. glutamicum plasmids were cryptic, four plasmids carried resistance determinants against the antibiotics chloramphenicol, tetracycline, streptomycin-spectinomycin, and sulfonamides. Considerable information is now available on the molecular genetic organization of 12 completely sequenced plasmid genomes from C. glutamicum. The deduced mechanism of plasmid DNA replication and the degree of amino acid sequence similarity among replication initiator proteins was the basis for performing a classification of the plasmids into four distinct C. glutamicum plasmid families.     

Temperature-sensitive cloning vector for Corynebacterium glutamicum

We constructed a temperature-sensitive form of the Corynebacterium glutamicum ATCC13869 cryptic plasmid, pBL1. The C. glutamicum/Escherichia coli shuttle vector pSFK6, which is composed of pBL1 and the E. coli cloning vector pK1, was mutagenized in vitro by treatment with hydroxylamine, and introduced into C. glutamicum cells. A mutant plasmid, which was stably maintained at 25 degrees C but not at 34 degrees C, was isolated from the cells. Sequencing the plasmid, which was named p48K, revealed four substitutions in the Rep protein coding region. Moreover, site-directed single-nucleotide substitutions showed that a G to A transition at position 2,920, which resulted in a Pro-47 to Ser substitution in the Rep protein, was responsible for its temperature-sensitive replication. Pro-47 is conserved among the Rep proteins of the pIJ101/pJV1 family of plasmids. This temperature-sensitive cloning vector will be useful for disrupting genes in this industrially important bacterium.     

Discrimination of Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum by restriction pattern analysis of DNA adjacent to the hom gene

Different strains of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum were analysed for restriction fragment length polymorphism using the homoserine dehydrogenase gene (hom) as a probe. The hybridization patterns obtained PvuII- or Asp700-restriction of chromosomal DNA were specific and distinguishable for each of the three species and identical for the different strains of each species. Thus, the method employed allows rapid distinction of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum. The former species could also be discriminated from the latter two by its resistance to 0.5 g/l of the methionine analog ethionine.     

Cloning vectors and antibiotic-resistance markers for Brevibacterium sp. R312

Replication of several cryptic plasmids from coryneform strains was investigated in Brevibacterium sp. R312. Only the Corynebacterium glutamicum pSR1 replicon was found to be suitable for establishing a host-vector system. Two pSR1 derivatives, pRPCG200 and pHYCG1, were used as cloning vectors. They carry a neomycin-resistance-encoding and a tetracycline-resistance-encoding gene, respectively.     

Cloning and study of Corynebacterium glutamicum genes complementing ilvA and thrA2 mutations in Escherichia coli

Molecular cloning and expression of Corynebacterium glutamicum genes complementing Escherichia coli mutations thrA2 and ilvA was performed. It was demonstrated that the thrA2 gene of C. glutamicum is located close to thrB on EcoRI DNA fragment 4.1 kb long. The fragment was cloned in pUC18 vector. The thrA2 gene is expressed in the recombinant plasmid pOBT3 under control of the vector pUC18 Plac promoter. In E. coli minicells, the genes thrA2 and thrB determined synthesis of proteins of Mr 43kD and 25 kD, respectively. A gene complementing ilvA mutation of E. coli was identified in a library of EcoRI C. glutamicum DNA fragments. This library was constructed using plasmid vector. It was shown that the ilvA gene of C. glutamicum is located inside the 3.6 kb EcoRI fragment and is expressed using its own promoter.     

Localization of an origin of replication in Corynebacterium diphtheriae broad host range plasmid pNG2 that also functions in Escherichia coli

Subcloning and protoplast transformation studies identified a 2.6 kb fragment of Corynebacterium diphtheriae plasmid pNG2 which contains an origin of replication (oriR). Molecular combination of the 2.6 kb oriR cartridge with Escherichia coli plasmid pUC18CmR enabled the E. coli cloning vector to replicate within several species of Corynebacterium host cells. A 2.6 kb plasmid formed from the oriR cartridge alone is capable of replicating in E. coli. This suggests that a single origin could be used in vectors shuttling between Corynebacterium spp. and E. coli.     

Plasmid copy number mutation in repA gene encoding RepA replication initiator of cryptic plasmid pHM1519 in Corynebacterium glutamicum

ABSTRACT

Cryptic plasmid pHM1519 is a rolling-circular replication mode plasmid of the pCG1 plasmid family in coryneform bacteria. The derived shuttle vector pPK4 is maintained at about 40–50 copies per chromosome in Corynebacterium glutamicum 2256 (ATCC 13869). We found that a mutation (designated copA1) within the repA gene encoding essential initiator protein RepA of the pHM1519-replicon increased the copy number of the mutant plasmid to about 800 copies per chromosome. The mutation was a single G to A base transition, which changed Gly to Glu at position 429 of the amino acid sequence of RepA. In silico secondary structure prediction of RepA suggested that Gly429 is situated in a disordered region in a helix-turn-helix motif, which is a typical DNA-binding domain. This study shows the first example of a high copy number of a C. glutamicum cryptic plasmid caused by an altered replication initiator protein.     

Isolation of ftsI and murE genes involved in peptidoglycan synthesis from Corynebacterium glutamicum

Corynebacterium glutamicum is known to excrete large amounts of L-glutamic acid upon treatment by penicillin. However, the mechanism of L-glutamate overproduction by penicillin treatment is still unknown. A 5.3-kb HindIII fragment was isolated by directly introducing the C. glutamicum (Brevibacterium lactofermentum) ATCC 13869 gene library into the temperature-sensitive Escherichia coli murE mutant and selecting temperature resistant clones. Two open reading frames (ORFs) were found in this fragment: (1) murE, encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase, and (2)ftsI, encoding septum-peptidoglycan synthetase, one of the targets of penicillin (penicillin-binding protein 3). Both ORFs were involved in peptidoglycan synthesis. Proteins were synthesized from the C. glutamicum murE and ftsI genes, 55 kDa and 73 kDa respectively, in an in vitro protein synthesis system, using E. coli S30 extracts.     

Cloning and structural-functional analysis in Escherichia coli of genes of glutamate-producing corynebacteria controlling biosynthesis of amino acids of aspartic acid series

A library of EcoRI DNA fragments from Brevibacterium flavum was constructed using plasmid vector. The genes complementing ThrA2 and ThrB mutations in Escherichia coli were identified in the library. The gene thrA2 of B. flavum codes for mutant enzyme homoserine dehydrogenase insensitive to inhibition by threonine. The genes thrA2 and thrB are localized wihtin the EcoRI fragment 4.1 kb long and are expressed under the control of their own promoters in E. coli cells. Structural and functional analysis of cloned C. glutamicum gene ilvA was performed. The gene of C. glutamicum complemented ilvA mutation in E. coli and appeared to be localized within the EcoRI--SacI DNA fragment 1.6 kb in size. Using E. coli minicells we have demonstrated that the gene ilvA of C. glutamicum controls the synthesis of polypeptide of relative molecular mass 50 kD.     

Characterization of the cryptic plasmid pCC1 from Corynebacterium callunae and its use for vector construction

The complete nucleotide sequence of the cryptic plasmid pCC1 from Corynebacterium callunae (4109 bp) was determined. DNA sequence analysis revealed five open reading frames longer than 200 bp. One of the deduced polypeptides showed homology with the Rep proteins encoded by plasmids of the pIJ101/pJV1 family of plasmids replicating by the rolling-circle (RC) mechanism. Within this plasmid family, the Rep protein of pCC1 showed the highest degree of similarity to the Rep proteins of corynebacterial plasmids pAG3 and pBL1. These data suggest that the plasmid pCC1 replicates by the RC mechanism. The Escherichia coli/Corynebacterium glutamicum shuttle cloning vector pSCCD1, carrying the pCC1 rep gene on the 2.1-kb DNA fragment and the streptomycin/spectinomycin resistance determinant, was constructed. This vector is stably maintained in population of C. glutamicum cells grown in the absence of selection pressure and it is compatible with plasmid vectors based on corynebacterial plasmids pBL1 and pSR1.     

Cloning and characterization of a DNA region encoding a stress-sensitive restriction system from Corynebacterium glutamicum ATCC 13032 and analysis of its role in intergeneric conjugation with Escherichia coli.

RP4-mediated transfer of mobilizable plasmids in intergeneric conjugation of Escherichia coli donors with Corynebacterium glutamicum ATCC 13032 is severely affected by a restriction system in the recipient that can be inactivated by a variety of exogenous stress factors. In this study a rapid test procedure based on intergeneric conjugal plasmid transfer that permitted the distinction between restriction-negative and restriction-positive C. glutamicum clones was developed. By using this procedure, clones of the restriction-deficient mutant strain C. glutamicum RM3 harboring a plasmid library of the wild-type chromosome were checked for their restriction properties. A complemented clone with a restriction-positive phenotype was isolated and found to contain a plasmid with a 7-kb insertion originating from the wild-type chromosome. This plasmid, termed pRES806, is able to complement the restriction-deficient phenotype of different C. glutamicum mutants. Sequence analysis revealed the presence of two open reading frames (orf1 and orf2) on the complementing DNA fragment. The region comprising orf1 and orf2 displayed a strikingly low G+C content and was present exclusively in C. glutamicum strains. Gene disruption experiments with the wild type proved that orf1 is essential for complementation, but inactivation of orf2 also resulted in a small but significant increase in fertility. These results were confirmed by infection assays with the bacteriophage CL31 from Corynebacterium lilium ATCC 15990.