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Methanogenic Archaea are of importance at the end of the anaerobic digestion (AD) chain for biomass conversion. They finally produce methane, the end-product of AD. Among this group of microorganisms, members of the genus Methanobacterium are ubiquitously present in anaerobic habitats, such as bioreactors. The genome of a novel methanogenic archaeon, namely Methanobacterium congolense Buetzberg, originally isolated from a mesophilic biogas plant, was completely sequenced to analyze putative adaptive genome features conferring competitiveness of this isolate within the biogas reactor environment. Sequencing and assembly of the M. congolense Buetzberg genome yielded a chromosome with a size of 2,451,457 bp and a mean GC-content of 38.51%. Additionally, a plasmid with a size of 18,118 bp, featuring a GC content of 36.05% was identified. The M. congolense Buetzberg plasmid showed no sequence similarities with the plasmids described previously suggesting that it represents a new plasmid type. Analysis of the M. congolense Buetzberg chromosome architecture revealed a high collinearity with the Methanobacterium paludis chromosome. Furthermore, annotation of the genome and functional predictions disclosed several genes involved in cell wall and membrane biogenesis. Compilation of specific genes among Methanobacterium strains originating from AD environments revealed 474 genetic determinants that could be crucial for adaptation of these strains to specific conditions prevailing in AD habitats. Fil: Torres Tejerizo, Gonzalo Arturo. Universitat Bielefeld. Center For Biotechnology; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Kim, Yong Sung. Hamburg University of Applied Sciences; Alemania Fil: Maus, Irena. Universitat Bielefeld. Center For Biotechnology; Alemania Fil: Wibberg, Daniel. Universitat Bielefeld. Center For Biotechnology; Alemania Fil: Winkler, Anika. Universitat Bielefeld. Center For Biotechnology; Alemania Fil: Off, Sandra. Hamburg University of Applied Sciences; Alemania Fil: Pühler, Alfred. Universitat Bielefeld. Center For Biotechnology; Alemania Fil: Scherer, Paul. Hamburg University of Applied Sciences; Alemania Fil: Schlüter, Andreas. Universitat Bielefeld. Center For Biotechnology; Alemania

Taxonomic and functional profiling based on metagenome analyses frequently revealed that members of the class Clostridia dominate biogas reactor communities and perform different essential metabolic pathways in the biogas fermentation process. Clostridium bornimense strain M2/40T was recently isolated from a mesophilic two-phase lab-scale biogas reactor continuously fed with maize silage and wheat straw. The genome of the strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding carbohydrate active enzyme production and fermentation of organic compounds for consolidated biofuel production from biomass. The C. bornimense M2/40T genome consists of a chromosome (2,917,864 bp in size) containing 2613 protein coding sequences, and a 699,161 bp chromid (secondary replicon) harboring 680 coding sequences. Both replicons feature very similar GC-contents of approximately 29%. The complex genome comprises three prophage regions, two CRISPR-cas systems and a putative cellulosomal gene cluster that is located on the second replicon (chromid) of the strain. The overexpressed glycosyl hydrolases (GH) Ce1K (GH9) and Ce1A (GH48) encoded in the cellulosomal gene cluster were shown to be active on the substrates xylan and xyloglucan whereas XghA (GH74) is highly active on xyloglucan. Reconstruction of fermentation pathways from genome sequence data revealed that strain M2/40T encodes all enzymes for hydrogen, acetate, formate, lactate, butyrate, and ethanol production, leading to the classification of the isolate as acidogenic bacterium. Phylogenetic analyses uncovered that the closest characterized relative of C. bornimense is C. cellulovorans. Comparative analyses of the C. bornimense and C. cellulovorans genomes revealed considerable rearrangements within their chromosomes suggesting that both species evolved separately for a relatively long period of time and adapted to specific tasks within microbial consortia responsible for anaerobic digestion. (C) 2015 Elsevier B.V. All rights reserved.

The alpha-glucosidase inhibitor acarbose is used for treatment of diabetes mellitus type II, and is manufactured industrially with overproducing derivatives of Actinoplanes sp. SE50/110, reportedly obtained by conventional mutagenesis. Despite of high industrial significance, only limited information exists regarding acarbose metabolism, function and regulation of these processes, due to the absence of proper genetic engineering methods and tools developed for this strain. Here, a basic toolkit for genetic engineering of Actinoplanes sp. SE50/110 was developed, comprising a standardized protocol for a DNA transfer through Escherichia coli-Actinoplanes intergeneric conjugation and applied for the transfer of phi C31, phi BT1 and VWB actinophage-based integrative vectors. Integration sites, occurring once per genome for all vectors, were sequenced and characterized for the first time in Actinoplanes sp. SE50/110. Notably, in case of phi C31 based vector pSET152, the integration site is highly conserved, while for phi BT1 and the VWB based vectors pRT801 and pSOK804, respectively, no sequence similarities to those in other bacteria were detected. The studied plasmids were proven to be stable and neutral with respect to strain morphology and acarbose production, enabling future use for genetic manipulations of Actinoplanes sp. SE50/110. To further broaden the spectrum of available tools, a GUS reporter system, based on the pSET152 derived vector, was also established in Actinoplanes sp. SE50/110. (C) 2016 Elsevier B.V. All rights reserved.

Xanthomonas campestris pv. campestris (Xcc) is the major producer of the exopolysaccharide xanthan, the commercially most important natural polysaccharide of microbial origin. The current work provides deeper insights into the yet uncharacterized transcriptomic features of the xanthan producing strain Xcc-B100. Towards this goal, RNA sequencing of a library based on the selective enrichment of the 5' ends of native transcripts was performed. This approach resulted in the genome wide identification of 3067 transcription start sites (TSSs) that were further classified based on their genomic positions. Among them, 1545 mapped upstream of an actively transcribed CDS and 1363 were classified as novel TSSs representing antisense, internal, and TSSs belonging to previously unidentified genomic features. Analyzing the transcriptional strength of primary and antisense TSSs revealed that in some instances antisense transcription seemed to be initiated at a higher level than its sense counterpart. Mapping the exact positions of TSSs aided in the identification of promoter consensus motifs, ribosomal binding sites, and enhanced the genome annotation of 159 in silico predicted translational start (TLS) sites. The global view on length distribution of the 5' untranslated regions (5'-UTRs) deduced from the data pointed to the occurrence of leaderless transcripts and transcripts with unusually long 5'-UTRs, in addition to identifying seven putative riboswitch elements for Xcc-B100. Concerning the biosynthesis of xanthan, we focused on the transcriptional organization of the gum gene cluster. Under the conditions tested, we present evidence for a complex transcription pattern of the gum genes with multiple TSSs and an obvious considerable role of antisense transcription. The gene gumB, encoding an outer membrane xanthan exporter, is presented here as an example for genes that possessed a strong antisense TSS.

The application of genome editing technologies, like CRISPR/Cas9 for industrially relevant microorganisms, is becoming increasingly important. Compared to other methods of genetic engineering the decisive factor is that CRISPR/Cas9 is relatively easy to apply and thus time and effort can be significantly reduced in organisms, which are otherwise genetically difficult to access. Because of its many advantages and opportunities, we adopted the CRISPR/Cas9 technology for Actinoplanes sp. SE50/110, the producer of the diabetes type II drug acarbose. The functionality of genome editing was successfully shown by the starless and antibiotic marker-free deletion of the gene encoding the tyrosinase Me1C, which catalyzes the formation of the dark pigment eumelanin in the wild type strain. The generated AmelC2 mutant of Actinoplanes sp. SE50/110 no longer produces this pigment and therefore the supernatant does not darken. Furthermore, it was shown that the plasmid containing the gene for the Cas9 protein was removed by increasing the temperature due to its temperature-sensitive replication. The precision of the intended mutation was proven and possible off-target effects caused by the genome editing system were ruled out by genome sequencing of several mutants. (C) 2016 Elsevier B.V. All rights reserved.

A novel bacterial species was isolated from an industrial-scale biogas plant. The isolate Herbinix hemicellulosilytica T3/55(T) is able to degrade crystalline cellulose. Comparative 16S rRNA gene sequencing demonstrated that the isolate is closely related to environmental samples forming a hitherto unknown sub-cluster within the family Lachnospiraceae. The draft genome sequence of strain T3/55(T) was established and now provides the genetic basis for application of this microorganism in thermophilic degradation of lignocellulosic biomass. (C) 2015 Published by Elsevier B.V.

Nitrogen is an essential component of nearly all of the complex macromolecules in a bacterial cell, e.g. proteins, nucleic acids, and cell wall components. Accordingly, most prokaryotes have developed elaborate control mechanisms to provide an optimal supply of nitrogen for cellular metabolism and to cope with situations of nitrogen limitation. In this communication, a global analysis of the Corynebacterium glutamicum nitrogen starvation response by transcriptional profiling using DNA microarrays is presented. Our results show that C. glutamicum reacts to nitrogen starvation with a rearrangement of the cellular transport capacity, changes in metabolic pathways concerning nitrogen assimilation and amino acid biosynthesis, and a decreased capacity for protein synthesis. (C) 2005 Elsevier B.V. All rights reserved.

Reference LGCB-ARTICLE-2004-008View record in Web of Science Record created on 2006-02-27, modified on 2016-08-08

As a high throughput technique, microarray experiments produce large data sets, consisting of measured data, laboratory protocols, and experimental settings. We have implemented the open source platform EMMA to store and analyze these data. The system provides automated pipelines for data processing and has a modular architecture that can be easily extended. EMMA features detailed reports about spots and their corresponding measurements. In addition to routine data analysis algorithms, the system can be integrated with other components that contain additional data sources (e.g. genome annotation systems). (C) 2003 Elsevier B.V. All rights reserved.

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. (C) 2003 Elsevier B.V. All rights reserved.

In the "post-genome" era, mass spectrometry (MS) has become an important method for the analysis of proteome data. The rapid advancement of this technique in combination with other methods used in proteomics results in an increasing number of high-throughput projects. This leads to an increasing amount of data that needs to be archived and analyzed. To cope with the need for automated data conversion, storage, and analysis in the field of proteomics, the open source system ProDB was developed. The system handles data conversion from different mass spectrometer software, automates data analysis, and allows the annotation of MS spectra (e.g. assign gene names, store data on protein modifications). The system is based on an extensible relational database to store the mass spectra together with the experimental setup. It also provides a graphical user interface (GUI) for managing the experimental steps which led to the MS data. Furthermore, it allows the integration of genome and proteome data. Data from an ongoing experiment was used to compare manual and automated analysis. First tests showed that the automation resulted in a significant saving of time. Furthermore, the quality and interpretability of the results was improved in all cases. (C) 2003 Elsevier B.V. All rights reserved.

In order to isolate transcriptional regulatory proteins involved in L-methionine-dependent repression in Corynebacterium glutamicum, proteins binding to the putative promoter region upstream of the metY gene were isolated by DNA affinity chromatography. One of the isolated proteins was identified as a putative transcriptional repressor of the TetR-family by a mass spectrometry fingerprint technique based on the complete C glutamicum genome sequence. The respective gene, designated mcbR, was deleted in the mutant strain C glutamicum DR1 Using 2D-PAGE, the protein contents of the C glutamicum wild type and the mutant strain DR1 grown in media with or without L-methionine supplementation were compared and a set of six proteins was identified. Their abundance was drastically enhanced in the mutant strain and no longer influenced by L-methionine added to the growth medium. The corresponding genes were identified by mass spectrometry fingerprint analysis. They included metY encoding O-acetyl-L-homoserine sulfhydrylase, metK encoding S-adenosyl-methionine synthethase, hom encoding homoserine dehydrogenase, cysK encoding L-cysteine synthase, cysI encoding an NADPH dependant sulfite reductase, and ssuD encoding an alkanesulfonate monooxygenase. Evidently, the putative transcriptional repressor McbR is involved in the regulation of the metabolic network directing the synthesis Of L-methionine in C glutamicum. The C glutamicum mcbR mutant can be considered to represent a first step in the construction of an L-methionine production strain. (C) 2003 Elsevier Science B.V. All rights reserved.

A DNA microarray was developed to analyse global gene expression of the amino acid-producing bacterium Corynebacterium glutamicum. PCR products representing 93.4% of the predicted C. glutamicum genes were prepared and spotted in quadruplicate onto 3-aminopropyltrimethoxysilane-coated glass slides. The applicability of the C. glutamicum DNA microarray was demonstrated by co-hybridisation with fluorescently labelled cDNA probes. Analysis of the technical variance revealed that C. glutamicum genes detected with different intensities resulting in ratios greater than 1.52 or smaller than -1.52 can be regarded as differentially expressed with a confidence level of greater than 95%. In a validation example, we measured changes of the mRNA levels during growth of C. glutamicum with acetate and propionate as carbon sources. Acetate-grown C glutamicum cultures were used as reference. At the 95% confidence interval, 117 genes revealed increased transcript levels in the presence of propionate, while 43 genes showed a decreased expression compared with the acetate-grown culture. Global expression profiling confirmed the induction of the prpD2B2C2 gene cluster already known to be essential for propionate degradation via the 2-methylcitrate cycle. Besides many genes of unknown function, the paralogous prpD1B1C1 gene cluster as well as fasI-B (encoding fatty-acid synthase IB), dtsR1 and dtsR2 (components of acyl-CoA carboxylases), gluABCD (glutamate transport system), putP (proline transport system), and pyc (pyruvate carboxylase) showed significantly increased expression levels. Differential expression of these genes was confirmed by real-time reverse transcription (RT) PCR assays. (C) 2003 Elsevier B.V. All rights reserved.

The genome sequence of Corynebacterium glutamicum. a gram-positive soil bacterium widely used as an amino acid producer, was analyzed by a similarity-based approach to elucidate the pathway for the biosynthesis Of L-methionine. The functions of candidate ORFs were derived by gene deletion and, if necessary, by homologous complementation of suitable mutants. Of nine candidate ORFs (four of which were known previously), seven ORFs (cg0754 (metX), cg0755 (met Y), cg1290 (metE), cg1702 (metH), cg2383 (metf), cg2536 (aecD), and eg2687 (metB)) were demonstrated to be part of the pathway while two others (cg0961 and cg3086) could be excluded. C glutamicum synthesizes methionine in three, respectively four steps, starting from homoserine. C glutamicum possesses two genes with similarity to homoserine acetyltransferases but only MetX can act as such while Cg0961 catalyzes a different, unknown reaction. For the incorporation of the sulfur moiety, the known functions of MetY and MetB could be confirmed and AecD was proven to be the only functional cystathionine beta-lyase in C glutamicum, while Cg3086 can act neither as cystathionine gamma-synthase nor as cystathionine beta-lyase. Finally, MetE and MetH, which catalyze the conversion of L-homocysteine to L-methionine, could be newly identified, together with MetF which provides the necessary N-5-methyltetrahydrofolate. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

The potential of the alanine racemase gene alr from Corynebacterium glutamicum ATCC 13032 to substitute for antibiotic resistance determinants in cloning systems has been investigated. The alr gene was identified by a PCR technique and its nucleotide sequence was determined. The deduced protein revealed the highest amino acid sequence similarity to the Alr protein from Mycobacterium smegmatis with 45% identical and 58% similar amino acids. A defined alr deletion mutant of C. glutamicum displayed a strict dependence on the presence of D-alanine for growth on complex and minimal medium. The alr gene was placed on a novel C. glutamicum vector which is completely free of antibiotic resistance genes. In vivo complementation of the chromosomal alr deletion with alr-carrying vectors permitted growth of the mutant strain in the absence of external D-alanine and provided strong selective pressure to maintain the plasmid. The alr gene enabled the selection of C. glutamicum transformants with a similar efficiency as the tetracycline resistance gene tetA (33). These data provided experimental evidence that the alr gene can be applied as an alternative selection marker to antibiotic resistance genes in industrial C. glutamicum strains. In an application example, the novel Deltaalr host-alr(+) vector-system for C. glutamicum was used to overproduce the vitamin D-pantothenic acid. (C) 2002 Elsevier Science B.V. All rights reserved.

The tetracycline resistance region of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium striatum M82B was analyzed in Corynebacterium glutamicum ATCC 13032 and confined to a 4.4-kb SphI-SalI DNA fragment. Nucleotide sequence analysis revealed two open reading frames, termed tetA and tetB, specifying proteins of 513 and 528 amino acids, respectively. The deduced amino acid sequences of tetAB displayed similarity to ATP-binding cassette transporters including StrV and StrW of Streptomyces glaucescens which are proposed to play a role in the export of streptomycin-like aminoglycosides. An antibiotic susceptibility screening in C. glutamicum showed that the tetAB genes confer resistance to tetracycline, oxytetracycline and to the structurally and functionally unrelated beta-lactam antibiotic oxacillin. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

The diversity, growth and symbiotic behaviour of symbiotically efficient alfalfa-nodulating rhizobia isolated from acid soils of Argentina and Uruguay were analysed. Partial sequencing of the 16S rDNA indicated that these isolates belong to Sinorhizobium meliloti species. IS-fingerprinting analysis revealed a high diversity among the isolates but some of them appear related to inoculant strains currently used in the region. The S. meliloti isolates showed a decreased growth rate with increasing acidity. They were, however, able to nodulate alfalfa at pH 5.6, but showed a delayed nodulation and decreased nodule number typical of S. meliloti strains. The impaired nodulation of S. meliloti at pH 5.6 did not result in a reduction of alfalfa dry matter production or nitrogen content. However, significant differences were observed for the relative symbiotic effectiveness of the strains analysed. LPU63 (Argentina) was the most effective among the isolates and exhibited a high nodulation competitiveness at both neutral and acidic pH. These results suggest that the isolate LPU63 may be a potential efficient inoculant for alfalfa in acid soils., Instituto de Biotecnología y Biología Molecular

Using gene replacement and transposon Tn5 mutagenesis, an Escherichia coli ilvC panE double mutant completely lacking ketopantoate reductase activity was isolated. This E. call double mutant was employed to isolate the E, coli panE gene by genetic complementation. The E. coli panE gene is characterized by a 912 bp coding region, which specifies a protein of 303 amino acids with a deduced molecular mass of 33.8 kD. A. panE expression plasmid carrying the panE gene under the control of the tac promotor was constructed. Introduction of the panE expression plasmid into E. coli resulted in a threefold increase in ketopantoate reductase activity. It was also shown that the enhanced panE expression in E. coli K12 led to 3.5-fold increase in pantothenate excretion. Pantothenate excretion could even be more enhanced when the growth medium was supplemented with ketopantoate. (C) 1999 Elsevier Science B.V. All rights reserved.

Greenhouse experiments revealed a reduced viability of a RecA(-) Sinorhizobium meliloti strain in non-sterile soil during a heat period compared with an isogenic RecA(+) strain. The higher sensitivity to heal stress of the S. meliloti RecA(-) strain was demonstrated during short- and long-term heat stress experiments under controlled conditions in a growth cabinet using different microcosm systems. Live/dead staining of the rhizobial cells showed that the increased sensitivity to heat stress of the RecA(-) S. meliloti strain was due to its higher mortality compared to the isogenic RecA(+) strain. These results demonstrate for the first time the importance of the recA function for survival of S. meliloti under heat stress conditions in soil. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

The transfer of genetic information between distantly or even unrelated organisms during evolution had been inferred from nucleotide sequence comparisons. These studies provided circumstantial evidence that in rare cases genes had been laterally transmitted amongst organisms of the domains bacteria, archaea and eukarya. Laboratory-based studies confirmed that the gene pools of the various domains of organisms are linked. Amongst the bacterial gene exchange mechanisms transduction, transformation and conjugation, the latter was identified as the mechanism with potentially the broadest host range of transfer. Previously, the issue of horizontal gene transfer has become important in the context of biosafety. Gene transfer studies carried out under more natural conditions such as in model ecosystems or in the environment established that all gene transfer mechanisms worked under these conditions. Moreover, environmental hot-spots were identified where favourable conditions such as nutrient enrichment increased the probability of genetic exchange among bacteria. In particular, the phytosphere was shown to provide conducive conditions for conjugative gene exchange. Concern has been expressed that transfer of recombinant DNA (e.g. antibiotic resistance genes) from genetically modified organisms (GMOs) such as transgenic plants to phytosphere bacteria may occur and thus contribute to the undesirable spread of antibiotic resistance determinants. Studies which were performed to address this issue clearly showed that such a transfer occurs, if at all, at extremely low frequency. (C) 1998 Elsevier Science B.V. All rights reserved.

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· Ngo VII 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 .

Using a newly developed integration vector, the Escherichia coli gusA gene conferring GUS-activity or the firefly (Photinus pyralis) he gene mediating bioluminescence were integrated into a non-essential site of the chromosome of Rhizobium meliloti 2011. The integration of the constitutively expressed marker genes into the chosen site per se did not affect the strains' ability to perform homologous recombination, its growth characteristics or its symbiotic nitrogen fixation. Comparative microcosm analyses between the bioluminescent, recombination-proficient (RecA(+)) R. meliloti strain L33 whose construction is reported in this paper, and its previously described recombination-deficient (RecA(-)) isogenic counterpart L1 indicate that RecA(-) strains of Rhizobium are safe hosts for deliberate release experiments.

Interspecific complementation of a Xanthomonas campestris pv. campestris phosphomannose isomerase (PMI) mutant was used to isolate a cosmid from a genomic library of Rhizobium meliloti 2011 carrying the pmi gene of this strain. Subcloning experiments localized the coding region to a 2.0-kb SalI-ClaI fragment. Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames (ORFs), coding for 18- and 43-kDa polypeptides. The analysis of the gene function by gene disruption experiments showed that ORF2 codes for pmi. A comparison of the deduced amino acid sequence with the corresponding sequences of the Pseudomonas aeruginosa and Escherichia coli PMIs revealed no significant homology, indicating that the isolated gene encodes a new type of PMI. The construction of a pmi-deficient mutant of R. meliloti using the sacB-sacR cassette technique showed that the loss of PMI activity does not affect the symbiotic properties of this strain.

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.

We have constructed derivatives of the transposon Tn5 carrying the mob site (oriT) of plasmid RP4, and an nptI-sacB-sacR cassette [Ried and Collmer, Gene 57 (1987) 239-246]. The mob site, in conjunction with the antibiotic-resistance markers carried on the transposons, allows identification of transposon inserts in cryptic plasmids by mobilisation to other strains. The sacB-sacR genes allow direct selection for the loss or curing of plasmids, because only strains which no longer contain an active sacB gene are able to grow on media containing sucrose. We have tested these transposons in four strains of Rhizobium leguminosarum and two strains of Rhizobium meliloti, and have been able to demonstrate curing of several large cryptic plasmids, and generation of large deletions in many other plasmids. This method has enabled us to show that the R. leguminosarum plasmids pRL12JI and pR1eVF39f carry auxotrophic markers, and that the plasmid pR1eVF39c carries genes which affect colony morphology.