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Journal of Bacteriology, November 2004, p. 7112-7122, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7112-7122.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Classification and Strength Measurement of Stationary-Phase Promoters by Use of a Newly Developed Promoter Cloning Vector

Tomohiro Shimada,1,2 Hideki Makinoshima,1 Yoshito Ogawa,1 Takeyoshi Miki,3 Michihisa Maeda,2 and Akira Ishihama1,4*

Nippon Institute for Biological Science, Division of Molecular Biology, Ome, Tokyo,1 Meiji University, Faculty of Agriculture, Kawasaki, Kanagawa,2 Fukuoka Dental College, Department of Biology, Fukuoka,3 Hosei University, Faculty of Engineering and Research Center for Micro-Nano Technology, Koganei, Tokyo, Japan4

Received 21 June 2004/ Accepted 30 July 2004

When an Escherichia coli culture changes from exponential growth to the stationary phase, expression of growth-related genes levels off, while a number of stationary-phase-specific genes are turned on. To gain insight into the growth phase-dependent global regulation of genome transcription, we analyzed the strength and specificity of promoters associated with the stationary-phase genes. For the in vivo assay of promoter activity, 300- to 500-bp DNA fragments upstream from the translation initiation codon were isolated and inserted into a newly constructed doubly fluorescent protein (DFP) vector. The activity of test promoters was determined by measuring the green fluorescent protein (GFP). To avoid the possible influence of plasmid copy number, the level of transcription of reference promoter lacUV5 on the same plasmid was determined by measuring the red fluorescent protein (RFP). Thus, the activities of test promoters could be easily and accurately determined by determining the GFP/RFP ratio. Analysis of the culture time-dependent variation of 100 test promoters indicated that (i) a major group of the stationary-phase promoters are up-regulated only in the presence of RpoS sigma; (ii) the phase-coupled increase in the activity of some promoters takes place even in the absence of RpoS; and (iii) the activity of some promoters increases in the absence of RpoS. This classification was confirmed by testing in vitro transcription by using reconstituted RpoD and RpoS holoenzymes.

* Corresponding author. Mailing address: Nippon Institute for Biological Science, Shinmachi 9-2221, Ome, Tokyo 198-0024, Japan. Phone: (81) 428 33 1071. Fax: (81) 428 33 1072. E-mail: aishiham{at}nibs.or.jp.

Journal of Bacteriology, November 2004, p. 7112-7122, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7112-7122.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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