Biomedical subjects

Georg Thierbach

Publications and source records attributed to Georg Thierbach.

3 recordsLinked to original sources

PubMed2003

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.

Base Sequence↗

PubMed2002

The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains.

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.

Alanine Racemase↗

PubMed2002

The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100.

We determined the complete nucleotide sequence of the 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum LP-6 which encodes streptomycin, spectinomycin, and tetracycline resistance. The antibiotic resistance determinant of pTET3 comprises an intI1-like gene, which was truncated by the insertion sequence IS6100, and the novel aminoglycoside adenyltransferase gene cassette aadA9. The deduced AADA9 protein showed 61% identity and 71% similarity to AADA6 of integron In51 from Pseudomonas aeruginosa. In addition, pTET3 carries the novel repressor-regulated tetracycline resistance determinant Tet 33 which revealed amino acid sequence homology to group 1 tetracycline efflux systems. The highest level of similarity was observed to the tetracycline efflux protein TetA(Z) from the C. glutamicum plasmid pAG1 with 65% identical and 77% similar amino acids. Each antibiotic resistance region of pTET3 is flanked by identical copies of the widespread insertion sequence IS6100 initially identified in Mycobacterium fortuitum. Transposition assays with a cloned copy of IS6100 revealed that this element is transpositionally active in C. glutamicum. These data suggest a central role of IS6100 in the evolutionary history of pTET3 by mediating the cointegrative assembly of resistance gene-carrying DNA segments.

Amino Acid Sequence↗