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Molecular characterization of AsgA, an unusual signal transduction protein required for multicellular development in Myxococcus xanthus
Abstract
asga gene is one of three known genes required for A-signal production in Myxococcus xanthus. The A-signal is a cell density signal required during early development. We have determined the DNA sequence of asga. The deduced amino acid sequence of AsgA exhibits a remarkable similarity to those of members of the histidine protein kinase family and the response regulator family of the so-called two-component regulatory systems. Gene replacement studies using a copy of asga that contains a kanamycin resistance gene (Kmr) inserted at various positions within or near asga indicate that asga is required for fruiting, but is not essential for growth. The mutation within asgA476 was found to cause a leucine-to-proline substitution within a conserved stretch of hydrophobic residues in the N-ten-ninal receiver domain. Cells containing either the Kmr insertion within asga or the asgA476 allele have similar phenotypes with respect to development, colony color, and expression of an asg-dependent gene. An analysis of expression of a translational asgA-lacZ fusion confirms that asga is expressed during growth and early development. We have overexpressed asga in E. coli and purified AsgA protein. Purified AsgA was used as antigen to raise polyclonal antibodies in rabbits. Western immunoblotting with the AsgA antibody detected a 42 kD protein corresponding to the predicted size of AsgA. This protein is present during both vegetative growth and development of the wild type strain but is absent in an asga disruption strain. Phosphorylation assays with purified AsgA demonstrate that AsgA protein has autokinase activity, and the autophosphorylation of AsgA occurs very rapidly. This reaction is dependent on the presence of divalent cations such as Mn2+ or Mg2+. The phospholinkage in phosphorylated AsgA displays acid liability and base stability which is characteristic of phosphoramidates such as phosphohistidines. No evidence for transfer of the phosphoryl group from the histidine protein kinase domain to the response regulator domain of AsgA has been found in vitro. We propose that AsgA functions within a signal transduction pathway that is required to sense starvation and to respond with the production of extracellular A-signal.
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Citation
Li, Yonghui (1995). Molecular characterization of AsgA, an unusual signal transduction protein required for multicellular development in Myxococcus xanthus. Master's thesis, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /ETD -TAMU -1995 -THESIS -L559.
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