Andreas Tauch

Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum.

Here we describe small mobilizable vectors based on the Escherichia coli plasmids pK18 and pK19. We combined the useful properties of the pK plasmids (e.g., multiple cloning site, lacZ alpha fragment, sequencing with M13 primers) with the broad-host-range transfer machinery of plasmid RP4 and a modified sacB gene from Bacillus subtilis. The new pK derivatives can be transferred by RP4-mediated conjugation into a wide range of Gram- and Gram+ bacteria, and should facilitate gene disruption and allelic exchange by homologous recombination. As an application example, the generation of a defined deletion of the hom-thrB genes in the chromosome of the Gram+ bacterium Corynebacterium glutamicum is presented.

The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins

The complete genomic sequence of Corynebacterium glutamicum ATCC 13032, well-known in industry for the production of amino acids, e.g. of L-glutamate and L-lysine was determined. The C. glutamicum genome was found to consist of a single circular chromosome comprising 3282708 base pairs. Several DNA regions of unusual composition were identified that were potentially acquired by horizontal gene transfer, e.g. a segment of DNA from C. diphtheriae and a prophage-containing region. After automated and manual annotation, 3002 protein-coding genes have been identified, and to 2489 of these, functions were assigned by homologies to known proteins. These analyses confirm the taxonomic position of C. glutamicum as related to Mycobacteria and show a broad metabolic diversity as expected for a bacterium living in the soil. As an example for biotechnological application the complete genome sequence was used to reconstruct the metabolic flow of carbon into a number of industrially important products derived from the amino acid L-aspartate.

Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum

SUMMARY Actinobacteria constitute one of the largest phyla among Bacteria and represent gram-positive bacteria with a high G+C content in their DNA. This bacterial group includes microorganisms exhibiting a wide spectrum of morphologies, from coccoid to fragmenting hyphal forms, as well as possessing highly variable physiological and metabolic properties. Furthermore, Actinobacteria members have adopted different lifestyles, and can be pathogens (e.g., Corynebacterium, Mycobacterium, Nocardia, Tropheryma, and Propionibacterium), soil inhabitants (Streptomyces), plant commensals (Leifsonia), or gastrointestinal commensals (Bifidobacterium). The divergence of Actinobacteria from other bacteria is ancient, making it impossible to identify the phylogenetically closest bacterial group to Actinobacteria. Genome sequence analysis has revolutionized every aspect of bacterial biology by enhancing the understanding of the genetics, physiology, and evolutionary development of bacteria. Various actinobacterial genomes have been sequenced, revealing a wide genomic heterogeneity probably as a reflection of their biodiversity. This review provides an account of the recent explosion of actinobacterial genomics data and an attempt to place this in a biological and evolutionary context.

The metagenome of a biogas-producing microbial community of a production-scale biogas plant fermenter analysed by the 454-pyrosequencing technology

Composition and gene content of a biogas-producing microbial community from a production-scale biogas plant fed with renewable primary products was analysed by means of a metagenomic approach applying the ultrafast 454-pyrosequencing technology. Sequencing of isolated total community DNA on a Genome Sequencer FLX System resulted in 616,072 reads with an average read length of 230 bases accounting for 141,664,289 bases sequence information. Assignment of obtained single reads to COG (Clusters of Orthologous Groups of proteins) categories revealed a genetic profile characteristic for an anaerobic microbial consortium conducting fermentative metabolic pathways. Assembly of single reads resulted in the formation of 8752 contigs larger than 500 bases in size. Contigs longer than 10kb mainly encode house-keeping proteins, e.g. DNA polymerase, recombinase, DNA ligase, sigma factor RpoD and genes involved in sugar and amino acid metabolism. A significant portion of contigs was allocated to the genome sequence of the archaeal methanogen Methanoculleus marisnigri JR1. Mapping of single reads to the M. marisnigri JR1 genome revealed that approximately 64% of the reference genome including methanogenesis gene regions are deeply covered. These results suggest that species related to those of the genus Methanoculleus play a dominant role in methanogenesis in the analysed fermentation sample. Moreover, assignment of numerous contig sequences to clostridial genomes including gene regions for cellulolytic functions indicates that clostridia are important for hydrolysis of cellulosic plant biomass in the biogas fermenter under study. Metagenome sequence data from a biogas-producing microbial community residing in a fermenter of a biogas plant provide the basis for a rational approach to improve the biotechnological process of biogas production.

Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum.

During the last decades, the gram-positive soil bacterium Corynebacterium glutamicum has been shown to be a very versatile microorganism for the large-scale fermentative production of L-amino acids. Up to now, a vast amount of techniques and tools for genetic engineering and amplification of relevant structural genes have been developed. The objectives of this study are to summarize the published literature on tools for genetic engineering in C. glutamicum and to focus on new sophisticated and highly efficient methods in the fields of DNA transfer techniques, cloning vectors, integrative genetic tools, and antibiotic-free self-cloning. This repertoire of C. glutamicum methodology provides an experimental basis for efficient genetic analyses of the recently completed genome sequence.

Efficient Electrotransformation of Corynebacterium diphtheriae with a Mini-Replicon Derived from the Corynebacterium glutamicum Plasmid pGA1

Efficient transformation of the human pathogen Corynebacterium diphtheriae was achieved with novel cloning vectors consisting of a mini-replicon from the cryptic C. glutamicum plasmid pGA1 as well as of the aph(3′)-IIa or tetA(Z) antibiotic resistance genes. Plasmid-containing transformants of C. diphtheriae were recovered at frequencies ranging from 1.3 × 105 to 4.8 × 106 colony forming units (cfu)/μg of plasmid DNA. Vector DNA was directly transferred from Escherichia coli into C. diphtheriae with frequencies up to 5.6 × 105 cfu/μg of plasmid DNA. On the basis of the pGA1 mini-replicon, an expression vector system was established for C. diphtheriae by means of the Ptac promoter and the green fluorescent reporter protein. In addition, other commonly used vector systems from C. glutamicum, including the pBL1 and pHM1519 replicons, and the sacB conditionally lethal selection marker from Bacillus subtilis, were shown to be functional in C. diphtheriae. Thus, the ability to apply the standard methods of C. glutamicum recombinant DNA technology will greatly facilitate the functional analysis of the recently completed C. diphtheriae genome sequence.

Taxonomic composition and gene content of a methane-producing microbial community isolated from a biogas reactor.

A total community DNA sample from an agricultural biogas reactor continuously fed with maize silage, green rye, and small proportions of chicken manure has recently been sequenced using massively parallel pyrosequencing. In this study, the sample was computationally characterized without a prior assembly step, providing quantitative insights into the taxonomic composition and gene content of the underlying microbial community. Clostridiales from the phylum Firmicutes is the most prevalent phylogenetic order, Methanomicrobiales are dominant among methanogenic archaea. An analysis of Operational Taxonomic Units (OTUs) revealed that the entire microbial community is only partially covered by the sequenced sample, despite that estimates suggest only a moderate overall diversity of the community. Furthermore, the results strongly indicate that archaea related to the genus Methanoculleus, using CO2 as electron acceptor and H2 as electron donor, are the main producers of methane in the analyzed biogas reactor sample. A phylogenetic analysis of glycosyl hydrolase protein families suggests that Clostridia play an important role in the digestion of polysaccharides and oligosaccharides. Finally, the results unveiled that most of the organisms constituting the sample are still unexplored.

The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis

The complete genome sequence of the Xanthomonas campestris pv. campestris strain B100 was established. It consisted of a chromosome of 5,079,003bp, with 4471 protein-coding genes and 62 RNA genes. Comparative genomics showed that the genes required for the synthesis of xanthan and xanthan precursors were highly conserved among three sequenced X. campestris pv. campestris genomes, but differed noticeably when compared to the remaining four Xanthomonas genomes available. For the xanthan biosynthesis genes gumB and gumK earlier translational starts were proposed, while gumI and gumL turned out to be unique with no homologues beyond the Xanthomonas genomes sequenced. From the genomic data the biosynthesis pathways for the production of the exopolysaccharide xanthan could be elucidated. The first step of this process is the uptake of sugars serving as carbon and energy sources wherefore genes for 15 carbohydrate import systems could be identified. Metabolic pathways playing a role for xanthan biosynthesis could be deduced from the annotated genome. These reconstructed pathways concerned the storage and metabolization of the imported sugars. The recognized sugar utilization pathways included the Entner-Doudoroff and the pentose phosphate pathway as well as the Embden-Meyerhof pathway (glycolysis). The reconstruction indicated that the nucleotide sugar precursors for xanthan can be converted from intermediates of the pentose phosphate pathway, some of which are also intermediates of glycolysis or the Entner-Doudoroff pathway. Xanthan biosynthesis requires in particular the nucleotide sugars UDP-glucose, UDP-glucuronate, and GDP-mannose, from which xanthan repeat units are built under the control of the gum genes. The updated genome annotation data allowed reconsidering and refining the mechanistic model for xanthan biosynthesis.

The individual and common repertoire of DNA-binding transcriptional regulators of Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium diphtheriae and Corynebacterium jeikeium deduced from the complete genome sequences

BackgroundThe genus Corynebacterium includes Gram-positive microorganisms of great biotechnologically importance, such as Corynebacterium glutamicum and Corynebacterium efficiens, as well as serious human pathogens, such as Corynebacterium diphtheriae and Corynebacterium jeikeium. Although genome sequences of the respective species have been determined recently, the knowledge about the repertoire of transcriptional regulators and the architecture of global regulatory networks is scarce. Here, we apply a combination of bioinformatic tools and a comparative genomic approach to identify and characterize a set of conserved DNA-binding transcriptional regulators in the four corynebacterial genomes.ResultsA collection of 127 DNA-binding transcriptional regulators was identified in the C. glutamicum ATCC 13032 genome, whereas 103 regulators were detected in C. efficiens YS-314, 63 in C. diphtheriae NCTC 13129 and 55 in C. jeikeium K411. According to amino acid sequence similarities and protein structure predictions, the DNA-binding transcriptional regulators were grouped into 25 regulatory protein families. The common set of DNA-binding transcriptional regulators present in the four corynebacterial genomes consists of 28 proteins that are apparently involved in the regulation of cell division and septation, SOS and stress response, carbohydrate metabolism and macroelement and metal homeostasis.ConclusionThis work describes characteristic features of a set of conserved DNA-binding transcriptional regulators present within the corynebacterial core genome. The knowledge on the physiological function of these proteins should not only contribute to our understanding of the regulation of gene expression but will also provide the basis for comprehensive modeling of transcriptional regulatory networks of these species.

The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum

BackgroundThe knowledge about complete bacterial genome sequences opens the way to reconstruct the qualitative topology and global connectivity of transcriptional regulatory networks. Since iron is essential for a variety of cellular processes but also poses problems in biological systems due to its high toxicity, bacteria have evolved complex transcriptional regulatory networks to achieve an effective iron homeostasis. Here, we apply a combination of transcriptomics, bioinformatics, in vitro assays, and comparative genomics to decipher the regulatory network of the iron-dependent transcriptional regulator DtxR of Corynebacterium glutamicum.ResultsA deletion of the dtxR gene of C. glutamicum ATCC 13032 led to the mutant strain C. glutamicum IB2103 that was able to grow in minimal medium only under low-iron conditions. By performing genome-wide DNA microarray hybridizations, differentially expressed genes involved in iron metabolism of C. glutamicum were detected in the dtxR mutant. Bioinformatics analysis of the genome sequence identified a common 19-bp motif within the upstream region of 31 genes, whose differential expression in C. glutamicum IB2103 was verified by real-time reverse transcription PCR. Binding of a His-tagged DtxR protein to oligonucleotides containing the 19-bp motifs was demonstrated in vitro by DNA band shift assays. At least 64 genes encoding a variety of physiological functions in iron transport and utilization, in central carbohydrate metabolism and in transcriptional regulation are controlled directly by the DtxR protein. A comparison with the bioinformatically predicted networks of C. efficiens, C. diphtheriae and C. jeikeium identified evolutionary conserved elements of the DtxR network.ConclusionThis work adds considerably to our currrent understanding of the transcriptional regulatory network of C. glutamicum genes that are controlled by DtxR. The DtxR protein has a major role in controlling the expression of genes involved in iron metabolism and exerts a dual regulatory function as repressor of genes participating in iron uptake and utilization and as activator of genes responsible for iron storage and DNA protection. The data suggest that the DtxR protein acts as global regulator by controlling the expression of other regulatory proteins that might take care of an iron-dependent regulation of a broader transcriptional network of C. glutamicum genes.