Funktionelle Analyse des essentiellen Zweikomponenten-Signaltransduktionssystems CgtSR4 aus <i>Corynebacterium glutamicum</i>

Corynebacterium glutamicum is an aerobic gram-positive soil bacterium used for large scale production of amino acids, mainly L-glutamate and L-lysine. Genome sequence analysis revealed the presence of 13 two-component regulatory systems. In this thesis, one of these systems, composed of the sensor kinase CgtS$_{4}$ and the response regulator CgtR$_{4}$, was characterized in order to get information about its function. The following results were obtained: 1. Hydrophobicity plots indicated that the N-terminus (amino acids 4-21) ofthe sensor kinase CgtS$_{4}$ is very hydrophobic and possibly forms a transmembrane helix. A second region of lower hydrophobicity (amino acids 43-63) has a strongly amphiphilic character and might form a membrane-associated helix. Using peptide antibodies it was confirmed that CgtS$_{4}$ is located in the membrane fraction of C. glutamicum cells. 2. The characteristic features of two-component systems could be demonstrated in vitro for CgtS$_{4}$/CgtR$_{4}$ : CgtS$_{4}$ purified by means of a carboxyterminal His- or Strep-tag showed autokinase activity and transferred the y-phosphoryl group of ATP rapidly to the purified response regulator CgtR$_{4}$. 3. A deletion of the cgtSR$_{4}$ genes from the chromosome was only possible in the presence of plasmid-borne functional cgtSR$_{4}$ or cgtR$_{4}$. The conclusion derived from these results that cgtR$_{4}$ but not cgtS$_{4}$ is essential for C. glutamicum could be confirmed by the successful deletion of the chromosomal cgtS$_{4}$ gene. The fact that a deletion of the chromosomal cgtSR$_{4}$ genes was possible in the presence of a plasmid-encoded CgtR$_{4}$ protein with an D52N exchange led to the conclusion that CgtR$_{4}$ is active in its unphosphorylated state. 4. The genes cgtSR$_{4}$ are located directly downstream ofthe pgm gene encoding the glycolysis enzyme phosphoglycerate mutase, indicating that pgm expression might be regulated by the CgtS$_{4}$/CgtR$_{4}$ system. However, transcriptome analyses failed to reveal such a regulation. 5. Genome-wide transcriptome analyses using DNA microarrays indicated that CgtSR$_{4}$ is involved in the response to two different types of stress, i.e. phosphate starvation and oxidative stress. Possibly, CgtS$_{4}$/CgtR$_{4}$ functions as a global regulatory system in different stress situations or even triggers a general stress response. So far, no direct target gene of the response regulator CgtR$_{4}$ could be identified

Narberhaus F: Virulence of Agrobacterium tumefaciens requires phosphatidylcholine in the bacterial membrane

Summary Phosphatidylcholine (PC, lecithin) has long been considered a solely eukaryotic membrane lipid. Only a minority of all bacteria is able to synthesize PC. The plant-transforming bacterium Agrobacterium tumefaciens encodes two potential PC forming enzymes, a phospholipid N-methyltransferase (PmtA) and a PC synthase (Pcs). We show that PC biosynthesis and tumour formation on Kalanchoë plants was impaired in the double mutant. The virulence defect was due to a complete lack of the type IV secretion machinery in the Agrobacterium PC mutant. Our results strongly suggest that PC in bacterial membranes is an important determinant for the establishment of hostmicrobe interactions

Lrp of Corynebacterium glutamicum controls expression of the brnFE operon encoding the export system for L-methionine and branched-chain amino acids

Lange C, Mustafi N, Frunzke J, et al. Lrp of Corynebacterium glutamicum controls expression of the brnFE operon encoding the export system for L-methionine and branched-chain amino acids. Journal of Biotechnology. 2012;158(4):231-241

Expression and Physiological Relevance of Agrobacterium tumefaciens Phosphatidylcholine Biosynthesis Genes▿

Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only 10% of all bacteria are predicted to synthesize PC. In Rhizobiaceae, including the phytopathogenic bacterium Agrobacterium tumefaciens, PC is essential for the establishment of a successful host-microbe interaction. A. tumefaciens produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The responsible genes, pmtA (coding for a phospholipid N-methyltransferase) and pcs (coding for a PC synthase), are located on the circular chromosome of A. tumefaciens C58. Recombinant expression of pmtA and pcs in Escherichia coli revealed that the individual proteins carry out the annotated enzyme functions. Both genes and a putative ABC transporter operon downstream of PC are constitutively expressed in A. tumefaciens. The amount of PC in A. tumefaciens membranes reaches around 23% of total membrane lipids. We show that PC is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased biofilm formation, two processes known to be involved in virulence. Our work documents the critical importance of membrane lipid homeostasis for diverse cellular processes in A. tumefaciens