Verlag des Forschungszentrums Jülich
JUEL-4129
Wessel, Mirja
Funktionelle Analyse des essentiellen Zweikomponenten-Signaltransduktionssystems CgtSr4 aus Corynebacterium glutamicum
VIII, 140 S., 2004
Abstract
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 CgtS4 and the response regulator CgtR4, 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
CgtS4 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 CgtS4
is located in the membrane fraction of C. glutamicum cells.
2. The characteristic features of two-component systems could be demonstrated in vitro for
CgtS4/CgtR4 : CgtS4 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 CgtR4.
3 . A deletion of the cgtSR4 genes from the chromosome was only possible in the presence of
plasmid-borne functional cgtSR4 or cgtR4. The conclusion derived from these results that
cgtR4 but not cgtS4 is essential for C. glutamicum could be confirmed by the successful
deletion of the chromosomal cgtS4 gene. The fact that a deletion of the chromosomal
cgtSR4 genes was possible in the presence of a plasmid-encoded CgtR4 protein with an
D52N exchange led to the conclusion that CgtR4 is active in its unphosphorylated state.
4. The genes cgtSR4 are located directly downstream ofthe pgm gene encoding the glycolysis
enzyme phosphoglycerate mutase, indicating that pgm expression might be regulated by
the CgtS4/CgtR4 system. However, transcriptome analyses failed to reveal such a
regulation.
5. Genome-wide transcriptome analyses using DNA microarrays indicated that CgtSR4 is
involved in the response to two different types of stress, i.e. phosphate starvation and
oxidative stress. Possibly, CgtS4/CgtR4 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 CgtR4 could be identified.
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