H-061. Identification and Characterization of Target Developmental Genes for the Nla6 and Nla28 Enhancer Binding Proteins
In response to amino acid starvation, Myxococcus xanthus cells initiate a complex developmental cycle. Large scale changes in gene expression direct the formation of multicellular fruiting bodies, and the differentiation of cells within these structures into stress-resistant spores. Recent studies suggest that M. xanthus uses a series of enhancer binding proteins (EBPs) for the coordinated expression of developmental genes. Mutations in the EBP genes nla6 and nla28 have pronounced effects on developmental gene expression starting at 1 hour post-starvation. Since the nla6 and nla28 mutants form fruiting bodies but fail to sporulate normally, we speculated that the Nla6 and Nla28 proteins are primarily involved in the activation of sporulation genes. Using a combination of DNA microarray analysis and bioinfomatics, we identified eleven putative developmental promoter targets for Nla6 and ten for Nla28. Gel shift assays were performed using the purified DNA binding domains of Nla6 (Nla6 DBD) and Nla28 (Nla28 DBD) and fragments of potential developmental promoter targets. We found that Nla28 DBD was capable of binding to all 10 promoter fragments, and when we scanned these fragments we found similar tandem repeat sequences. Subsequent in vitro promoter binding studies support the idea that these tandem repeats are bona fide Nla28 binding sites. Additional developmental promoter targets for Nla28 have been uncovered by searching the M. xanthus genome sequence for Nla28 binding sites. Nla6 DBD bound to all developmental promoter fragments tested so far, and we have uncovered similar tandem repeat binding sequences within these promoter fragments. We are now in the process of confirming that the tandem repeats are Nla6 binding sites. Putative Nla6 and Nla28 target genes have been inactivated and all of these mutations produce phenotypes similar to those produced by the nla6 and nla28 mutations themselves; strains carrying target gene mutations are capable of forming fruiting bodies, but they show strong sporulation defects. These findings support our proposal that Nla6 and Nla28 are key regulators of M. xanthus sporulation genes.