German Title: CsgA, ein putatives Signalmolekül der Fruchtkörperbildung in dem Myxobakterium Stigmatella aurantiaca : Charakterisierung des csgA-Gens und des Einflusses seiner Inaktivierung auf die Entwicklung

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Abstract

Cell-cell interaction is a prerequisite for multi-cellular development and cellular differentiation of the Gram-negative bacterium Stigmatella aurantiaca. For the elucidation of the temporal and spatial coordination of the physiology and motility of the cells during development, isolation and characterization of molecules involved in cell-cell signalling is needed. The best studied intercellular signal of Myxococcus xanthus is the C signal, which is the gene product of the csgA gene. Using the M. xanthus csgA gene as probe a homologous gene was previously isolated from S. aurantiaca (Butterfass, 1992). Inactivation of the gene by insertional mutagenesis caused alterations in S. aurantiaca fruiting. A XhoI fragment harbouring the csgA gene and flanking regions was isolated. Sequence analysis revealed additional putative start codons located upstream of the proposed csgA GTG translational start. Based on the homology data of the M. xanthus csgA gene the best reading frame indicates that the csgA translational start ATG codon is located 189 bp upstream of GTG. It specifies a protein of 236 amino acids with an estimated molecular mass of 26 kDa. The CsgA protein appears to be a member of the SRD family. The putative catalytic site (Ser139, Tyr158 and Lys162) is highly conserved in CsgA, as well as the putative coenzyme binding domain of the protein. Further, a new ORF, the protoporphyrinogene oxidase gene, was found upstream of csgA in the opposite orientation, 242 bp apart from csgA. The deduced amino acid sequence of this ORF has significant similarity with protoporphyrinogene oxidase from M. xanthus. An unknown ORF, orf2, was found upstream of csgA in same orientation and 251 bp apart from csgA, which encodes a polypeptide of 247 amino acids. No similarity was found between the deduced amino acid sequence of orf2 product and known proteins. An ORF, fprA, was found downstream of csgA in the opposite orientation. It overlaps with csgA (55 bp). The fprA gene specifies a protein of 223 amino acids with significant similarity to the flavin associated protein from M. xanthus. Due to the strong evidences supporting the role of CsgA in M. xanthus intercellular signalling and the close phylogenetic relationship between Stigmatella and Myxococcus it was speculated that the CsgA protein plays a role in communication between S. aurantiaca cells during development. A S. aurantiaca csgA insertion-mutant was constructed. CsgA mutant cells show an altered developmental behaviour as compared with wild type cells. The motility behaviour of the cells during development was changed and their ability to stay more closely together in the early stages of development. Inactivation of the csgA gene completely abolished rippling of the cells. This indicates the crucial role of the CsgA protein in regulating this rhythmic behaviour. S. aurantiaca csgA mutant cells do not produce CsgA but they are able to respond to it when mixed with wild type cells. Mixing the cells of the S.aurantiaca csgA mutant with those of a mutant that expresses the green fluorescence protein, resulted in wild-type fruiting body with an intermediate colour (orange / green). The csgA promoter seems to be very weak. Promoter activity of csgA was studied using a promoterless DtrpA-lacZ gene as reporter gene. b_galactosidase activity was very low and increased weakly at the beginning of the starvation induced development. A 0,6 kbp putative promoter region is sufficient for csgA expression. The concentration of the CsgA protein is low. This is a consequence of weak expression of the csgA gene. CsgA probably acts in the pM range as a signal per se or has an enzymatic function to convert a substrate into the signal molecule.