Hans Ulrich Schairer

A physical and genetic map of the Stigmatella aurantiaca DW4/3.1 chromosome

A physical map of the myxobacterium Stigmatella aurantiaca DW4/3.1 chromosome was constructed by pulsed‐field gel (PFG) long‐range mapping. One‐and two‐dimensional pulsed‐field gel analyses were used together with reciprocal double‐restriction, cross‐hybridization and hybridization fingerprint analysis. These PFG results were confirmed by Smith‐Birnstiel analysis, by Southern hybridization using linking clones and clones of a λ genomic library for the determination of adjacent restriction fragments and by transposon insertion mapping using defined genomic sequences for hybridization. It was thus possible to construct a circular restriction map of the single 9.35 Mbp chromosome of S. aurantiaca based on the endonucleases Asel and Spel. Genetic loci as well as the replication origin were located on the physical map by Southern hybridization using heterologous (derived from Myxococcus xanthus, Escherichia coli and Streptomyces lividans) and homologous probes that are mainly involved in development and ceil motility.

Transfer of incP plasmids into Stigmatella aurantiaca leading to insertional mutants affected in spore development

SummaryDerivatives of the broad-host-range plasmid RP4, containing the wild-type or modified transposon Tn5 were transferred by conjugation to various Stigmatella aurantiaca isolates. The transposons and in some cases fragments of the plasmid as well were integrated into the chromosome. Thus, insertional mutants have been obtained affected in spore formation in liquid culture.

Intercellular signalling in Stigmatella aurantiaca

The myxobacterium Stigmatella aurantiaca is a prokaryotic model used to study intercellular signalling and the genetic determination of morphogenesis. Signalling factors and genes required for the generation of the elaborate multicellular fruiting body are to be identified. Recently, the structure of stigmolone, which is the pheromone necessary for fruiting body formation, was elucidated, and genes involved in development were characterised. Progress has also been made in the genetic accessibility of S. aurantiaca.

Detection of developmentally regulated genes of the myxobacterium Stigmatella aurantiaca with the transposon Tn5lacZ

Stigmatella aurantiaca is a prokaryotic organism that undergoes a multicellular cycle of development resulting in the formation of a fruiting body. Insertional mutations were introduced at random sites into the Stigmatella aurantiaca genome with the promotor probe Tn5lacZ derived from Tn5lac by deleting non-essential sequences. 638 transconjugants were obtained with a frequency of 1×10-7. In 260 of the transconjugants isolated the β-glactosidase gene of Tn5lacZ is fused to vegetative promotors of Stigmatella aurantiaca. In 65 of the strains β-galactosidase is induced by starvation; in 14 of the transconjugants β-galactosidase activity is observed after chemical induction of sporulation by 3-methyl-indole. Thirtytwo of the mutants are affected in fruiting body formation and morphology.

Cloning and characterization of the gene encoding the major sigma factor of Stigmatella aurantiaca

The gene (sigA) encoding the major sigma factor of the myxobacterium, Stigmatella aurantiaca, was cloned and sequenced. The deduced polypeptide contains 706 amino acids (aa) and has a deduced M(r) of 79,910. It exhibits four different aa sequence motifs which correlate with the conserved domains of the major sigma factors of Myxococcus xanthus (sigma 80), Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). The sigma factor (sigma A) was detected in crude lysates of vegetative cells and in cells of different developmental stages from S. aurantiaca with an antiserum to M. xanthus sigma 80 by Western blot analysis. The SigA polypeptide copurified with RNA polymerase from vegetative S. aurantiaca cells. The aa sequence of its N terminus matches a sequence located 25 codons downstream from the proposed start codon. The sigA gene was expressed in E. coli and the corresponding gene product cross-reacted with the SigA antiserum as a polypeptide of 100 kDa, which is identical in size to the sigma A detected in vegetative cells of S. aurantiaca.

SigB, an alternative sigma factor of the myxobacterium Stigmatella aurantiaca, is synthesized during development and heat shock

Alternative sigma factors have been detected in the myxobacterium Stigmatella aurantiaca during indole-induced sporulation, fruiting body formation and heat shock using an antiserum raised against sigma factor SigB. The time course of sigB gene expression was analysed by RT-PCR and by determining beta-galactosidase activity during development in a merodiploid strain that harboured a sigB-lacZ fusion gene. Inactivation of the sigB gene by insertion of the neo gene resulted in the loss of one sigma factor as shown by Western analysis. Neither fruiting body formation nor sporulation, nor the production of possible SigB targets, such as DnaK, GroEL or HspA, were affected.