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Journal of Bacteriology, June 2002, p. 3348-3359, Vol. 184, No. 12
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.12.3348-3359.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of the {Omega}4514 Regulatory Region, a Developmental Promoter of Myxococcus xanthus That Is Transcribed In Vitro by the Major Vegetative RNA Polymerase

Tong Hao, Dvora Biran,,{dagger} Gregory J. Velicer,,{ddagger} and Lee Kroos*

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

Received 11 December 2001/ Accepted 29 March 2002

{Omega}4514 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. DNA upstream of the insertion site was cloned, and the promoter was localized. The promoter resembles vegetative promoters in sequence, and {sigma}A RNA polymerase, the major form of RNA polymerase in growing M. xanthus, initiated transcription from this promoter in vitro. Two complete open reading frames were identified downstream of the promoter and before the {Omega}4514 insertion. The first gene product (ORF1) has a putative helix-turn-helix DNA-binding motif and shows sequence similarity to transcriptional regulators. ORF2 is most similar to subunit A of glutaconate coenzyme A (CoA) transferase, which is involved in glutamate fermentation. Tn5 lac {Omega}4514 is inserted in the third codon of ORF3, which is similar to subunit B of glutaconate CoA-transferase. An orf1 disruption mutant exhibited a mild sporulation defect, whereas neither a disruption of orf2 nor insertion {Omega}4514 in orf3 caused a defect. Based on DNA sequence analysis, the three genes are likely to be cotranscribed with a fourth gene whose product is similar to alcohol dehydrogenases. ORF1 delays and reduces expression of the operon during development, but relief from this negative autoregulation does not fully explain the regulation of the operon, because expression from a small promoter-containing fragment is strongly induced during development of an orf1 mutant. Also, multiple upstream DNA elements are necessary for full developmental expression. These results suggest that transcriptional activation also regulates the operon. {Omega}4514 is the first example of a developmentally regulated M. xanthus operon that is transcribed by the major vegetative RNA polymerase, and its regulation appears to involve both negative autoregulation by ORF1 and positive regulation by one or more transcriptional activators.

* 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.

{dagger} Present address: Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel 69978.

{ddagger} Present address: Department of Evolutionary Biology, Max Planck Institute for Developmental Biology, D-72076 Tübingen, Germany.

Journal of Bacteriology, June 2002, p. 3348-3359, Vol. 184, No. 12
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.12.3348-3359.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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