Organization, expression, and function of Caulobacter crescentus genes needed for assembly and function of the flagellar
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O R I GI N A L P A P E R
D. A. Mullin á N. Ohta á A. H. Mullin á A. Newton
Organization, expression, and function of Caulobacter crescentus genes needed for assembly and function of the ¯agellar hook
Received: 9 August 2000 / Accepted: 21 November 2000 / Published online: 21 February 2001 Ó Springer-Verlag 2001
Abstract This paper reports on the organization, expression, and function of the divergently transcribed ¯bG and ¯aN operons in the hook gene cluster of Caulobacter crescentus. The transcription initiation site of ¯bG was determined previously, and in this work the transcription map was completed by locating the 3¢ end of the mRNA using nuclease S1 protection assays. A previous genetic study had suggested that the ¯bG operon is comprised of four genes; however, the nucleotide sequence revealed three tandemly arranged ORFs that correspond to 5¢¯bG, ¯bH, and ¯gE. FlbG is similar to FliK proteins which are required for termination of hook synthesis, FlbH is similar to FlgD proteins which are essential scaolding proteins that cap the hook during its assembly, and FlgE corresponds to the hook structural protein. The divergently transcribed ¯aN gene codes for a hook associated protein I homolog based on its inferred amino acid sequence similarity to FlgK proteins. Based on the amino acid sequence similarities and phenotypes of mutants, ¯bG, ¯bH, and ¯aN have been renamed ¯iK, ¯gD, and ¯gK. FlgD, FlgE, and FlgK proteins, with apparent molecular masses of 23, 68, and 41 kDa, respectively, were expressed from plasmids in a cell-free coupled transcription-translation system, and a protein corresponding to FliK was identi®ed as part of a 190-kDa FliK-LacZ fusion protein. We present evidence showing that, in addition to its role in termination of hook synthesis, FliK is also required for initiation of hook assembly. Key words Caulobacter ¯agellum á Flagellar hook á Hook assembly á ¯iK operon á ¯gK Communicated by K. Isono D. A. Mullin (&) á A. H. Mullin Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118, USA E-mail: [email protected] Fax: +1-504-865-6785 N. Ohta á A. Newton Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
Introduction During the cell cycle, ¯agellum biosynthesis in Caulobacter crescentus is subject to complex regulation at the levels of gene expression and protein subunit assembly (Gober and Marques 1995; Wu and Newton 1997). Although the structure of the C. crescentus ¯agellum is very similar to that described in Salmonella typhimurium and Escherichia coli (Aizawa 1996; Macnab 1996), the organization of the gene regulatory hierarchy diers in several signi®cant respects (Wu and Newton 1997). Epistasis tests and studies of gene expression during the cell cycle have allowed most of the C. crescentus ¯agellar genes to be placed into one of four classes which are organized into a regulatory hierarchy. The class I-IV genes are characterized by their sequence of periodic expression during the course of the cell cycle and the observation th
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