Stigmatella aurantiaca, a Prokaryotic Organism for Studying Intercellular Signalling and Morphogenesis

 


The organism

 

Stigmatella aurantiaca belongs to the myxobacteria, which are Gram-negative soil bacteria. Their nearest prokaryotic relatives are sulphate reducing purple bacteria and Bdellovibrio sp. as shown by 16S RNA analysis. Myxobacteria show both, features of unicellular and multicellular organisms. As the eukaryotic organism Dictyostelium dicoideum they are thought to lie on the boundary between unicellular and multicellular organisms. Myxobacteria grow and divide as separate cells. But they may be regarded as a multicellular organisms whose cells feed in swarms and which under conditions of starvation, assemble to well defined regular three dimensional structures called fruiting bodies which enclose about 105 dormant cells, the myxospores. The shape of the fruiting bodies is species specific and is genetically determined. The fruiting body of S.aurantiaca consists of a stalk bearing several sporangioles on branches at its top.

Myxobacteria secrete hydrolytic enzymes together with slime with which they degrade particulate organic matter of the soil. It has been shown that the growth rate increases with cell density if myxobacteria were grown on a macromolecular substrate as sole nutrient, such as casein. This suggests that cells feed co-operatively and the association in a swarm allows them to feed more efficiently. The advantage of co-operative feeding may have driven the evolution of fruiting body formation. When nutrients are again available after a period of starvation, myxospores germinate and form vegetative cells. The multicellular nature of the fruiting body ensures that a swarm of cells is formed for a new growth cycle.

Myxobacteria move by gliding on solid surfaces. This facilitates the stabilisation of a swarm and of fruiting body formation. Gliding permits tight cell-cell contact and efficient signal transduction between the cells by diffusible molecules. Both features are a prerequisite for the transmission of positional information of the single cell necessary for the co-ordination of the metabolism and movement of the cell in the course of fruiting body formation.

Apart from their ability to form fruiting bodies, myxobacteria form a broad range of secondary metabolites. All these unique features are reflected in the size of the genome and its organisation. The size of the myxobacterial genome has been shown to be about 9.5 Mbp.


A pheromone is produced during starvation

W. Plaga and I. Stamm (Land)

 

Starving cells of Stigmatella aurantiaca secrete a signalling factor. This factor, the pheromone, seems to be essential for the first steps of fruiting body formation during which cells form aggregates. Continuous removal of the factor by dialysis prevents aggregation of the cells and thus fruiting. Low concentrations of starving cells lead to low levels of the pheromone. The consequence is a highly delayed fruiting body formation or this developmental process may be even suppressed. Addition of factor preparations speeds up aggregation and thus fruiting.

Stigmatella cells stick tightly to a cellulose matrix. Therefore the pheromone can be continuously removed from the starving cells sticking on the surface of chromatography paper by descending elution with a starvation buffer. Steam distillation, reversed phase HPLC and a normal phase chromatography on a aminopropyl resin results in a most probably pure compound as shown by chiral GC.

High resolution MS suggests the sum formula to be C12H24O2. By 2-D 1H-NMR a branched alkane derived backbone seems to be most probable. For the elucidation of the complete chemical structure 13C-NMR studies have to be performed. But this needs much material which is produced at present. A rough estimation of the signal intensities of the NMR spectra indicates, the pheromone to be biologically active in the 1 to 10 nM range.


Polypeptides and Genes Involved in Development

 

The fbfA gene

B. Silakowski (DFG)

 

Mutants defective in fruiting body formation have been obtained after insertional mutagenesis using the Tn5 derived promoter probe Tn5 lacZ. When starved, cells of mutant AP182 form only non structured clumps. If cells of this mutant are mixed with cells of mutant AP191, which do not aggregate during starvation, and subsequently starved, a fruiting body is formed which has a mushroom like structure. In mutant AP182 the promoter probe was inserted about 800 bp upstream of an open reading frame which encodes a polypeptide with high homology to the rhizobial polypeptide NodC, which catalyses the synthesis of tetramers or pentamers of N-acetyl-glucoseamine, oligomers forming the backbone of the rhizobial nodulation factors.

To prove that this open reading frame, named fbfA, is essential for fruiting body formation the gene was inactivated in vitro by insertion of the neomycin-phoshotransferase gene. The gene replacement mutants obtained showed the same phenotype as mutant AP182.

It was not possible to determine the time range of the gene's transcription by Northern hybridisation. This may be due to low concentrations of the transcript or the transcript may be very unstable. To prove the transcription of the gene, a DNA sequence of the puf operon of Rhodobacter capsulatus which encodes a highly stable RNA was inserted into fbfA. After about eight hours of starvation, the transcript of the inserted DNA sequence was detected but no fbfA transcript was found. For a more accurate determination of the fbfA expression, a merodiploid strain was constructed, which contains trpE-lacZ fused to fbfA in addition to the wild type gene. b-galactosidase activity was measurable after about eight hours of starvation and increased up to 30 hours. This clearly indicates, that fbfA is expressed during starvation. The replacement of fbfA by the rhizobial nodC did not result in a strain competent in the formation of the wild type fruiting body. This may have various reasons: the FbfA polypeptide catalyses a reaction which differs from that catalysed by NodC, or FbfA may be a receptor involved in cell-cell signalling recognising N-acetyl-glucosamine derived substances, or down stream of fbfA may be a gene whose expression is impaired by the insertion of the rhizobial nodC-Gene.


AP191, a mutant defective in aggregate formation

F. Fathinejad

 

It has been mentioned above that mixing of cells of the mutants AP191 and AP182 defective in fruiting body formation leads to a phenotypic complementation of fruiting. A fruiting body form which corresponds to an intermediate state of fruiting body formation of the wildtype is found. Cloning of the genes which have been affected in mutant AP191 is in progress.


SP21, a stress induced polypeptide of the a-crystallin type

H. Shen (DFG)

 

We reported previously, that stress such as starvation, anoxia, indole treatment or heat induce a polypeptide with a molecular mass of 21000 Da (SP21) in Stigmatella aurantiaca. SP21 belongs to the a-crystallin family. It has been shown for some of these peptides that they protect proteins such as alcohol dehydrogenase or a-glucanase from gross structural changes by heat.

For studying these properties, SP21 was produced in E. coli. But a way, how to renaturate this polypeptide, has still to be developed.

So far it is not known, whether development and heat dependent transcription of the hspA gene which encodes SP21 is dependent on the same promoter or whether transcription starts from different promoters. A prerequisite to study this problem is the sequence determination of the promoter region and the construction of hspA-lacZ fusions. This work is in progress.


Alternate sigmafactors involved in development

M.-P. Coudart (DFG)

 

The genes of two alternate sigmafactors sigB and sigC had been isolated previously. Analysis of merodiploids countering wild type sigB and a truncated sigB gene fused to lacZ proved that sigB is expressed very late in development. To detect developmental polypeptides, whose synthesis depends on the functional sigmafactor B, gene replacement mutants have to be constructed in which sigB is inactivated. As the recombination frequency seems to be low in S. aurantiaca, the sigB gene with long distal DNA sequences was cloned. In these clones, which harbour the sigB gene and about further 10-12 kbp of S. aurantiaca DNA sequences, wild type sigB will be replaced by an inactivated sigB.

The sequence of the sigC gene was determined. The aminoacid sequence of this sigmafactor is very similar to that of SigB. For studying the development dependent expression of this sigmafactor, merodiploid S. aurantiaca strains will be constructed containing the wild type gene and a truncated gene fused to lacZ.


Indole binding proteins

W. Plaga and I. Stamm

 

Addition of indole and some of its derivatives to growing cultures induces the formation of myxospores in S. aurantiaca without the formation of fruiting bodies. Thus sporulation can be uncoupled from fruiting body formation. To elucidate the mechanism of and the signal transfer during this process, indole binding proteins were isolated. Whereas from the cytoplasmic membrane no indole binding proteins could be isolated, two indole binding proteins have been isolated from the cytoplasm by ammoniumsulfate fractionation, metalchelate-affinitychromatography and indole-affinitychromatography using a resin, to which indole propionic acid was bound via the carboxylic group. Both proteins have a mw of about 55000. The sequence of eight and nine N-terminal amino acid residues respectively was determined of which degenerate oligonucleotides have been derived. Using these probes one strong genomic DNA fragment for each protein was identified and cloned. The DNA sequence analysis of these fragments is in progress.


Developmental Proteolysis

 

A high molecular mass proteinase complex from S. aurantiaca

A. Schindzielorz

 

In the course of development certain polypeptides, which are no more needed are degraded, some new are synthesised. The question arises how does the cells discriminate between polypeptides which are degraded and those, which are still needed. One way would be that polypeptides which should be degraded are somehow labelled and subsequently degraded by defined proteases which recognise the label. Such a system is well known in eukaryotes as the proteasome / ubiquitin system. Proteasomes are high molecular mass multicatalytic proteases, which are resistant to inhibitors of most other proteases.

Cytoplasmic proteins from S. aurantiaca were fractionated by sucrose gradient centrifugation, DEAE-Sephacel and Mono Q chromatography. The proteolytic activity was tested in the presence of inhibitors of known proteases and in the absence of ATP to suppress Clp protease activity. A proteinase complex with a molecular mass of 6-800 kDa was enriched. But the probable subunits of this complex, which cross reacted with antisera raised against the Thermoplasma acidophilum proteasomes, showed a molecular mass of more than 40 kDa. This is a much higher molecular mass than that of proteasomal subunits. Some rare structures detected in electron micrographs let us speculate that S. aurantiaca may have a proteasome like protease.


Polypeptides of the cold shock family

A. Leclerque and W. Plaga

 

During our search for development dependent proteolytic systems in S, aurantiaca, we became aware of heat stable polypeptides with a molecular mass of about 5500 Da as determined by SDS-PAGE. By mass spectrometry a molecular mass of 7200 Da was found. The results obtained by Edman degradation suggested that the preparation contained a mixture of at least two peptides which are very similar to the E. coli cold shock protein family. Using polypeptide derived degenerate oligonucleotides, various signals were obtained in a Southern analysis of restricted S. aurantiaca DNA. One of the fragments was cloned and sequenced. The DNA derived polypeptide sequence proved unequivocally, that the cloned gene encodes a polypeptide of the cold shock family.



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