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Journal of Bacteriology, February 2004, p. 661-671, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.661-671.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of the Myxococcus xanthus {Omega}4400 Promoter Region Provides Insight into Developmental Gene Regulation by C Signaling

Deborah R. Yoder and Lee Kroos*

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824

Received 2 September 2003/ Accepted 30 October 2003

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 {Omega}4400, a gene identified by an insertion of Tn5 lac into the chromosome. Expression of Tn5 lac {Omega}4400 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, {Omega}4403, 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 {Omega}4400 and {Omega}4403 regulatory regions function in the same way, deletion analysis and site-directed mutagenesis of the {Omega}4400 promoter region were performed. A 7-bp sequence centered at -49 bp, termed a C box, is identical in the {Omega}4400 and {Omega}4403 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 {Omega}4400 expression; one region, from -63 to -31 bp, is required for {Omega}4400 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 {Omega}4400 promoter. Mutations in sigD and sigE, which are genes that encode {sigma} factors, abolished and reduced {Omega}4400 expression, respectively. Expression of {Omega}4400 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 {Omega}4400 and {Omega}4403 promoter regions function differently.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824. Phone: (517) 355-9726. Fax: (517) 353-9334. E-mail: kroos{at}pilot.msu.edu.

Journal of Bacteriology, February 2004, p. 661-671, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.661-671.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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