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Journal of Bacteriology, June 2008, p. 4210-4217, Vol. 190, No. 12
0021-9193/08/$08.00+0     doi:10.1128/JB.00061-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Transcriptional Regulation of the Nitrile Hydratase Gene Cluster in Pseudomonas chlororaphis B23{triangledown}

Toshihide Sakashita,{dagger} Yoshiteru Hashimoto,{dagger} Ken-Ichi Oinuma, and Michihiko Kobayashi*

Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan

Received 13 January 2008/ Accepted 2 April 2008

An enormous amount of nitrile hydratase (NHase) is inducibly produced by Pseudomonas chlororaphis B23 after addition of methacrylamide as the sole nitrogen source to a medium. The expression pattern of the P. chlororaphis B23 NHase gene cluster in response to addition of methacrylamide to the medium was investigated. Recently, we reported that the NHase gene cluster comprises seven genes (oxdA, amiA, nhpA, nhpB, nhpC, nhpS, and acsA). Sequence analysis of the 1.5-kb region upstream of the oxdA gene revealed the presence of a 936-bp open reading frame (designated nhpR), which should encode a protein with a molecular mass of 35,098. The deduced amino acid sequence of the nhpR product showed similarity to the sequences of transcriptional regulators belonging to the XylS/AraC family. Although the transcription of the eight genes (nhpR, oxdA, amiA, nhpABC, nhpS, and acsA) in the NHase gene cluster was induced significantly in the P. chlororaphis B23 wild-type strain after addition of methacrylamide to the medium, transcription of these genes in the nhpR disruptant was not induced, demonstrating that nhpR codes for a positive transcriptional regulator in the NHase gene cluster. A reverse transcription-PCR experiment revealed that five genes (oxdA, amiA, nhpA, nhpB, and nhpC) are cotranscribed, as are two other genes (nhpS and acsA). The transcription start sites for nhpR, oxdA, nhpA, and nhpS were mapped by primer extension analysis, and putative –12 and –24 {sigma}54-type promoter binding sites were identified. NhpR was found to be the first transcriptional regulator of NHase belonging to the XylS/AraC family.

* Corresponding author. Mailing address: Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan. Phone: (81)-29-853-4679. Fax: (81)-29-853-4605. E-mail: sec-mmb{at}agbi.tsukuba.ac.jp

{triangledown} Published ahead of print on 11 April 2008.

{dagger} T.S. and Y.H. contributed equally to this work.

Journal of Bacteriology, June 2008, p. 4210-4217, Vol. 190, No. 12
0021-9193/08/$08.00+0     doi:10.1128/JB.00061-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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