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Mutational Analysis of the Myxococcus xanthus 4499 Promoter Region Reveals Shared and Unique Properties in Comparison with Other C-Signal-Dependent Promoters

<[ERROR]zaff;1[ERROR]>Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824

Received 8 January 2004/ Accepted 11 March 2004

The bacterium Myxococcus xanthus undergoes multicellular development during times of nutritional stress and uses extracellular signals to coordinate cell behavior. C-signal affects gene expression late in development, including that of 4499, an operon identified by insertion of Tn5 lac into the M. xanthus chromosome. The 4499 promoter region has several sequences in common with those found previously to be important for expression of other C-signal-dependent promoters. To determine if these sequences are important for 4499 promoter activity, the effects of mutations on expression of a downstream reporter gene were tested in M. xanthus. Although the promoter resembles those recognized by Escherichia coli ⁵⁴, mutational analysis implied that a ⁷⁰-type factor likely recognizes the promoter. A 7-bp sequence known as a C box and a 5-bp element located 6 bp upstream of the C box have been shown to be important for expression of other C-signal-dependent promoters. The 4499 promoter region has C boxes centered at –33 and –55 bp, with 5-bp elements located 7 and 8 bp upstream, respectively. A multiple-base-pair mutation in any of these sequences reduced 4499 promoter activity more than twofold. Single base-pair mutations in the C box centered at –33 bp yielded a different pattern of effects on expression than similar mutations in other C boxes, indicating that each functions somewhat differently. An element from about –81 to –77 bp exerted a twofold positive effect on expression but did not appear to be responsible for the C-signal dependence of the 4499 promoter. Mutations in sigD and sigE, which are genes that encode factors, reduced expression from the 4499 promoter. The results provide further insight into the regulation of C-signal-dependent genes, demonstrating both shared and unique properties among the promoter regions so far examined.

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