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Development in Caulobacter Crescentus (Dlfferentiation/Phosphorylation/Cell Cyde) SHUI PING WANG, PREM L

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Development in Caulobacter Crescentus (Dlfferentiation/Phosphorylation/Cell Cyde) SHUI PING WANG, PREM L Proc. Nati. Acad. Sci. USA Vol. 90, pp. 630-634, January 1993 Developmental Biology A histidine protein kinase is involved in polar organelle development in Caulobacter crescentus (dlfferentiation/phosphorylation/cell cyde) SHUI PING WANG, PREM L. SHARMA*, PATRICIA V. SCHOENLEINt, AND BERT ELYt Department of Biological Sciences, University of South Carolina, Columbia, SC 29208 Communicated by Richard M. Losick, September 3, 1992 ABSTRACT Mutations having pleiotropic effects on polar the flagellin proteins, are nonchemotactic, and have a re- organelle development (pod) in Caulobacter crescentus have duced ability to release assembled flagallar filaments prior to been identified and shown to occur in at least 13 genes scattered stalk development (1, 2). Also, pleC mutants are resistant to throughout the genome. Mutations at each locus affect a unique polar bacteriophage, produce a paralyzed flagellum, and are combination of polar traits, suggesting that complex interac- stalkless in the presence of excess phosphate (3-5). In tions occur among these genes. The DNA sequence of one of contrast, pleA mutants are resistant to polar bacteriophage these genes, pkeC, indites that it is homologous to members and do not synthesize a flagellum (3-5). It has been suggested of the family of histidine protein kinase genes. Members ofthis that these genes encode regulatory elements that control the family include the sensor components of the bacterial two- expression of genes involved in polar development (1, 2, 5). component regulatory systems. Furthermore, in vitro experi- To provide evidence that these genes encode regulatory ments demonstrated that the PleC protein was capable of elements, we have cloned, sequenced, and analyzed one of autophosphorylation. These results suggest that the PieC pro- these genes, pleC.§ Comparison of the derived amino acid tein (and perhaps the proteins encoded by the other pod genes sequence of the PleC protein to known sequences suggests as well) regulates the expes of genes involved in polar that it is a histidine-specific protein kinase similar to the organelle development through the phosphorylation of key sensor proteins involved in two-component regulatory sys- regulatory proteins. The use of a phosphorelay system cued to tems. The kinase activity of the PleC protein was demon- internal changes in the cell would provide a mechanis for strated by showing that the C-terminal portion of the protein coordinating major changes in gene expression with the com- was capable of autophosphorylation. Thus, we propose that pletion of specific cell cycle events. the PleC protein is involved in a phosphorelay system that functions to regulate polar organelle development. To iden- tify additional genes involved in this regulatory system, we Differentiation in Caulobacter crescentus is observed as an isolated a collection of mutants with altered polar organelle ordered series of events at the poles of the cell. Each cell development. division results in two cell types, a stalked cell like the parent and a motile swarmer cell. The two new poles formed at cell division lack polar organelles, while the older poles contain MATERIALS AND METHODS polar organelles produced during previous rounds of differ- Bacterial Strains and Growth Conditions. The bacterial entiation. After cell division, DNA replication is initiated strains used in this study are all derivatives of the C. immediately in the stalked progeny cell, followed by the crescentus wild-type strain CB15. Growth conditions and assembly of a set of polar organelles including a flagellum, genetic techniques have been described (6-8). Bacterial cells pili, and phage receptors at the newly created pole. The were observed by phase-contrast light microscopy, in the resulting predivisional cell has a different set of organelles at light microscope after staining (9), or by electron microscopy each pole, the original stalk at one end and the flagellum, pili, (10). Resistance to bacteriophage 4Cbk was determined by and phage receptors at the other. spotting dilutions of a phage lysate on lawns of bacteria in After a period of motility, the swarmer cell releases the PYE soft agar (11). flagellum into the culture medium, and the remainder of the Molecular Techniques. Recombinant DNA experiments polar organelles are replaced by a stalk. An exception is the were performed by using techniques described by Sambrook holdfast material, which is found at the base ofthe flagellum. et al. (12). Single-stranded DNA for sequencing was obtained The holdfast material is retained and ends up at the tip ofthe from derivatives of the Bluescript plasmids, and sequencing stalk. Consequently, the holdfast material serves as a con- reactions were performed with Sequenase according to the venient landmark to demonstrate that the stalk is formed at directions of the manufacturer (United States Biochemical). the flagellar pole. The resulting stalked pole is terminally Western analyses and pulsed-field gel electrophoresis were differentiated and remains unchanged during subsequent performed as described (2, 13). cell-division cycles. Thus, newly formed poles undergo two Autophosphorylation Assay. Escherichia coli cells contain- series of differentiation events. First, as part of a predivi- ing the lacZ: .pleC fusion were grown at 370C to an optical sional cell, they assemble a flagellum, pili, and phage recep- density at 600 nm of0.6, isopropyl j3-D-thiogalactopyranoside tors. Subsequently, as a differentiated pole on a swarmer cell, (IPTG) was added at a final concentration of 1 mM, and the they lose the first set of organelles and replace them with a stalk. Abbreviation: IPTG, isopropyl j-D-thiogalactopyranoside. Several mutations that have multiple effects on differenti- *Present address: Dana-Farber Cancer Institute and Department of ation of the polar organelles have been described previously. Biological Chemistry and Molecular Pharmacology, Harvard Med- For instance,flbT mutants make bibbed stalks, overproduce ical School, Boston, MA 02115. tPresent address: Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M91449). 630 Downloaded by guest on September 29, 2021 AILDke'velopmental Biology: Wang et aL Proc. Nati. Acad. Sci. USA 90 (1993) 631 culture was incubated for an additional 2.5 hr. The resulting at position 588 is the most likely start codon for thepleCgene, cells were collected by centrifugation, washed with phos- and it is preceded by a ribosome-binding site. The stop codon phate-saline buffer (14), and resuspended in phosphate saline for the pleC gene is located at position 3114, generating a buffer in 2.5% of their original volume. After storage at protein with 842 amino acid residues (Mr 88,915). The region -700C overnight, the cells were disrupted by two passages upstream of the pleC gene contained the 3' end of a gene through a French pressure cell at 20,000 psi (1 psi = 6.89 kPa), transcribed in the same direction as pleC and separated from and the unbroken cells and cell debris were removed by pleC by 167 base pairs (bp). The third coding region was 67 centrifugation at 1900 x g. The cellular proteins were sepa- bp downstream of the pleC gene and could encode another rated by SDS/polyacrylamide gel electrophoresis and puri- protein of 326 amino acid residues. fied as described by Hager and Burgess (15). The PleC fusion To address the role of the pleC gene, the deduced amino proteins were renatured and phosphorylated essentially as acid sequence derived from the pleC gene was used in a described by Jin et al. (16). To test the stability of the homology search of the GenBank data base. The results phosphorylated fusion proteins, the acrylamide gels contain- indicated that the PleC protein shares a high level of homol- ing the phosphorylated proteins were treated with either 0.5 ogy in the C-terminal region with the products ofthe histidine M NaOH for 2 hr at room temperature or with 5% trichlo- protein kinase gene family. Proteins in this gene family are roacetic acid for 2 hr at 680C. The treated gels were neutral- capable of autophosphorylation at a conserved histidine (16, ized with 0.1 M Tris (pH 7.5), rinsed with 18). PleC contains an appropriate histidine within a highly water, and exposed conserved sequence centered at amino acid 610 (Fig. 2). The to the Kodak x-ray film. proteins with histidine protein kinase regions most homolo- gous to PleC were the family of sensor proteins from two RESULTS component regulatory systems, such as Bordatella pertussis BvgS (19) and Escherichia coli RcsC (18) (Fig. 2). The sensor pkC Gene Encodes a Histidine Protein Kinase. A cosmid proteins are often transmembrane proteins responsible for clone (pLSG7) containing the pleC gene was identified by receiving a signal from either outside or inside a cell. Once the complementation ofSC618 (pleC147) (17). Additional exper- sensor receives the signal, it is autophosphorylated by its iments demonstrated that a subclone of pLSG7, pSCC25, histidine protein kinase region and then transfers the phos- containing a 4.4-kilobase (kb) Xho I-Xho I fragment com- phate moiety to another protein called the response
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