IN-VITRO TRANSCRIPTION OF MYXOCOCCUS-XANTHUS GENES WITH RNA-POLYMERASE CONTAINING SIGMA(A), THE MAJOR SIGMA-FACTOR IN GROWING CELLS

Myxococcus xanthus is a Gram-negative bacterium that undergoes multice llular development upon starvation. We have developed a simple and rap id procedure for partial purification of RNA polymerase from growing M . xanthus cells, using heparin-agarose and DNA-cellulose chromatograph ies. In addition to core subunits, the enzyme contains one fairly abun dant polypeptide of approximately 105 kDa. We have shown by Western bl ot analysis and protein sequencing that the 105-kDa polypeptide is sig ma(A), the product of the M. xanthus sigA gene, Partially purified sig ma(A) RNA polymerase, or holoenzyme reconstituted from sigma(A) and co re RNA polymerase, transcribed in vitro the vegA and aphII genes that are known to be expressed in growing M. xanthus cells. Reconstituted s igma(A) RNA polymerase produced vegA mRNA in vitro with the same 5' en d as vegA mRNA produced in vivo, demonstrating that initiation of tran scription was accurate in vitro. These results provide biochemical evi dence that sigma(A) is the major vegetative sigma factor of M. xanthus . To our knowledge, this is the first report of in vitro transcription of M. xanthus chromosomal genes, providing a foundation for further b iochemical analysis of transcriptional regulatory mechanisms in a micr obe that relies extensively on cell-cell interactions.