Mutational Analysis of the Myxococcus xanthus Ω4400 Promoter Region Provides Insight into Developmental Gene Regulation by C Signaling

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Abstract
Myxococcus xanthus utilizes extracellular signals during development to coordinate cell movement, differentiation, and changes in gene expression. One of these signals, the C signal, regulates the expression of many genes, including Ω4400, a gene identified by an insertion of Tn 5 lac into the chromosome. Expression of Tn 5 lac Ω4400 is reduced in csgA mutant cells, which fail to perform C signaling, and the promoter region has several sequences similar to sequences found in the regulatory regions of other C-signal-dependent genes. One such gene, Ω4403, depends absolutely on the C signal for expression, and its promoter region has been characterized previously by mutational analysis. To determine if the similar sequences within the Ω4400 and Ω4403 regulatory regions function in the same way, deletion analysis and site-directed mutagenesis of the Ω4400 promoter region were performed. A 7-bp sequence centered at −49 bp, termed a C box, is identical in the Ω4400 and Ω4403 promoter regions, yet mutations in the individual base pairs affected expression from the two promoters very differently. Also, a single-base-pair change within a similar 5-bp element, which is centered at −61 bp in both promoter regions, had very different effects on the activities of the two promoters. Further mutational analysis showed that two regions are important for Ω4400 expression; one region, from −63 to −31 bp, is required for Ω4400 expression, and the other, from −86 to −81 bp, exerts a two- to fourfold effect on expression and is at least partially responsible for the C signal dependence of the Ω4400 promoter. Mutations in sigD and sigE , which are genes that encode σ factors, abolished and reduced Ω4400 expression, respectively. Expression of Ω4400 in actB or actC mutants correlated well with the altered levels of C signal produced in these mutants. Our results provide the first detailed analysis of an M. xanthus regulatory region that depends partially on C signaling for expression and indicate that similar DNA sequences in the Ω4400 and Ω4403 promoter regions function differently.