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Journal of Bacteriology, May 2004, p. 2946-2955, Vol. 186, No. 10
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.10.2946-2955.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Functional Dissection of the XpsN (GspC) Protein of the Xanthomonas campestris pv. campestris Type II Secretion Machinery

Hsien-Min Lee,^1, Juine-Ruey Chen,^1, Hai-Lun Lee,^1, Wei-Ming Leu,² Ling-Yun Chen,³ and Nien-Tai Hu^1*

Institute of Biochemistry,¹ Institute of Biotechnology, National Chung Hsing University,² Institute of Biochemistry, Chung Shan Medical University, Taichung, Taiwan, Republic of China³

Received 26 August 2003/ Accepted 29 January 2004

Type II secretion machinery is composed of 12 to 15 proteins for translocating extracellular proteins across the outer membrane. XpsL, XpsM, and XpsN are components of such machinery in the plant pathogen Xanthomonas campestris pv. campestris. All are bitopic cytoplasmic-membrane proteins, each with a large C-terminal periplasmic domain. They have been demonstrated to form a dissociable ternary complex. By analyzing the C-terminally truncated XpsN and PhoA fusions, we discovered that truncation of the C-terminal 103 residues produced a functional protein, albeit present below detectable levels. Furthermore, just the first 46 residues, encompassing the membrane-spanning sequence (residues 10 to 32), are sufficient to keep XpsL and XpsM at normal abundance. XpsN46(His₆), synthesized in Escherichia coli, is able to associate in a membrane-mixing experiment with the XpsL-XpsM complex preassembled in X. campestris pv. campestris. The XpsN N-terminal 46 residues are apparently sufficient not only for maintaining XpsL and XpsM at normal levels but also for their stable association. The membrane-spanning sequence of XpsN was not replaceable by that of TetA. However, coimmunoprecipitation with XpsL and XpsM was observed for XpsN97::PhoA, but not XpsN46::PhoA. Only XpsN97::PhoA is dominant negative. Single alanine substitutions for three charged residues within the region between residues 47 and 97 made the protein nonfunctional. In addition, the R78A mutant XpsN protein was pulled down by XpsL-XpsM(His₆) immobilized on an Ni-nitrilotriacetic acid column to a lesser extent than the wild-type XpsN. Therefore, in addition to the N-terminal 46 residues, the region between residues 47 and 97 of XpsN probably also plays an important role in interaction with XpsL-XpsM.

Present address: Institute of Medical Biotechnology, Chungtai Institute of Health Sciences and Technology, Taichung, Taiwan 406, Republic of China.

Present address: Institute of Molecular Biology, Academia Sinica R.O.C., Taipei, Taiwan 115, Republic of China.

Present address: Liver Research Unit, Chang Gung Memorial Hospital, Taoyuan, Taiwan 333, Republic of China.

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