Identification of an asymmetrically localized sensor histidine kinase responsible for temporally and spatially regulated transcription

Science

Volumn 274, Issue 5287, 1996, Pages 597-601

  Wingrove, James A ^a   Gober, James W ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

HISTIDINE; PROTEIN KINASE; TRANSCRIPTION FACTOR;

ARTICLE; BACTERIAL GENETICS; CAULOBACTER CRESCENTUS; CELL DIVISION; CONTROLLED STUDY; ENZYME PHOSPHORYLATION; FLAGELLUM; GENE EXPRESSION REGULATION; GENETIC TRANSCRIPTION; NONHUMAN; PRIORITY JOURNAL; PROMOTER REGION; SENSOR; SIGNAL TRANSDUCTION; TRANSCRIPTION INITIATION; TRANSCRIPTION REGULATION;

BACTERIAL PROTEINS; CAULOBACTER CRESCENTUS; CELL DIVISION; DNA-BINDING PROTEINS; GENE EXPRESSION REGULATION, BACTERIAL; GENES, BACTERIAL; MUTATION; PHOSPHORYLATION; PROMOTER REGIONS (GENETICS); PROTEIN KINASES; RECOMBINANT FUSION PROTEINS; TRANSCRIPTION FACTORS; TRANSCRIPTION, GENETIC;

BACTERIA (MICROORGANISMS); CAULOBACTER VIBRIOIDES;

EID: 0029823740     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5287.597     Document Type: Article

References (40)

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17. D52E/S14OF was generated, overexpressed, and purified as described (5).
18. D52E/S14OF was generated, overexpressed, and purified as described (5).
19. O52E/S140F were done as described above.
20. 32P-FlbE were incubated in either 0.2 N HCl or 1.0 N NaOH for 45 min at 55°C.
21. 32P-FlbE were incubated in either 0.2 N HCl or 1.0 N NaOH for 45 min at 55°C.
22. 32P-FlbE were incubated in either 0.2 N HCl or 1.0 N NaOH for 45 min at 55°C.
23. To create a flbE deletion mutant (UC3003), we integrated a pJBZ-sacB vector [J. A Wingrove, unpublished data; H. P. Schweizer, Mol. Microbiol. 6, 1195 (1992)] containing a 490-base pair (bp) internal fragment of the flbE gene into the C. crescentus genome by homologous recombination. Integrants were selected for resistance to kanamycin and judged nonmotile by microscopy. The disrupted strain was subcultured three successive times onto PYE plates containing 5% sucrose. Genomic DNA was isolated from sucrose-tolerant, nonmotile, kanamycin-sensitive colonies, digested with Eco Rl, and then probed by Southern (DNA) hybridization with a 1150-bp Eco Rl fragment containing the entire flbE gene. The flbE deletion strain (UC3003) was assayed for motility on PYE swarmer plates containing 0.3% agar. Complementation of the UC3003 phenotype could be accomplished by introducing a 796-bp Sal l-Eco Rl fragment containing flbE on the plasmid pMR4, indicating that the deletion affected only flbE and not other genes within the fliF operon
24. To create a flbE deletion mutant (UC3003), we integrated a pJBZ-sacB vector [J. A Wingrove, unpublished data; H. P. Schweizer, Mol. Microbiol. 6, 1195 (1992)] containing a 490-base pair (bp) internal fragment of the flbE gene into the C. crescentus genome by homologous recombination. Integrants were selected for resistance to kanamycin and judged nonmotile by microscopy. The disrupted strain was subcultured three successive times onto PYE plates containing 5% sucrose. Genomic DNA was isolated from sucrose-tolerant, nonmotile, kanamycin-sensitive colonies, digested with Eco Rl, and then probed by Southern (DNA) hybridization with a 1150-bp Eco Rl fragment containing the entire flbE gene. The flbE deletion strain (UC3003) was assayed for motility on PYE swarmer plates containing 0.3% agar. Complementation of the UC3003 phenotype could be accomplished by introducing a 796-bp Sal l-Eco Rl fragment containing flbE on the plasmid pMR4, indicating that the deletion affected only flbE and not other genes within the fliF operon
25. 35S-label (ICN, lrvine, CA), followed by the addition of cold methionine. Cells were allowed to divide, and subsequent progeny cells were re-isolated by Ludox density centrifugation. Proteins were immunoprecipitated, subjected to SDS-PAGE, and visualized by fluorography.
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27. J. A. Wingrove and J. W. Gober, unpublished data.
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30. Immunofluorescence microscopy was performed on strains containing either FlbE-M2, MCP-M2, or β-Gal expressed from the fliF promoter, essentially as described [J. R. Maddock and L. Shapiro, Science 259, 1717 (1993)]. Fluorescent membrane labeling was accomplished by adding DiA (Molecular Probes, OR) to a concentration of 20 μg/ml during the primary antibody incubation step.
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33. The FlbE-LacZ protein fusion was constructed by creating an in-frame fusion at codon 54 of flbE and codon 8 of lacZ. The fusion contains the flbE upstream sequences through the putative input domain. The flbE sequence ends at the start of the glutamine-rich linker (Fig. 1). The lacZ portion of the fusion contains sequences up to amino acid residue 652 and is enzymatically inactive.
34. pJS 14 is a multicopy plasmid with broad host range and Is derived from pBBRIMCS [M. E. Kovach, R. W. Phillips, P. H. Elzer. R. M. I. Roop, K. M. Peterson, Biotechniques 16, 800 (1994)].
35. K. C. Quon, G T. Marczynski, L. Shapiro, Cell 84, 83 (1996)
36. J. A. Wingrove and J. W. Gober, data not shown.
37. Bacterial strains, plasmids, and growth conditions. Caulobacter crescentus strain NA1000 was used as a wild-type, synchronizable motile strain. SC1048 is a mutant strain with a Tn5 insertion in the fliP gene (13). LS1298 is a mutant strain containing a disruption in the fliF gene [U. Jenal and L Shapiro, EMBO J. 15, 2393 (1996)]. Construction of the fliF-lacZ, flbG-lacZ, fljL-lacZ, and fljK-lacZ transcription fusions are described elsewhere (3, 4). Unless otherwise noted, strains were grown either in PYE media [J. S Poindexter, Bacteriol. Rev. 28. 231 (1964) ] or minimal M2-glucose media [I. Contreras, L. Shapiro, S. Henry, J. Bacteriol. 135, 1130 (1978)] at 31°C.
38. Bacterial strains, plasmids, and growth conditions. Caulobacter crescentus strain NA1000 was used as a wild-type, synchronizable motile strain. SC1048 is a mutant strain with a Tn5 insertion in the fliP gene (13). LS1298 is a mutant strain containing a disruption in the fliF gene [U. Jenal and L Shapiro, EMBO J. 15, 2393 (1996)]. Construction of the fliF-lacZ, flbG-lacZ, fljL-lacZ, and fljK-lacZ transcription fusions are described elsewhere (3, 4). Unless otherwise noted, strains were grown either in PYE media [J. S Poindexter, Bacteriol. Rev. 28. 231 (1964) ] or minimal M2-glucose media [I. Contreras, L. Shapiro, S. Henry, J. Bacteriol. 135, 1130 (1978)] at 31°C.
39. Bacterial strains, plasmids, and growth conditions. Caulobacter crescentus strain NA1000 was used as a wild-type, synchronizable motile strain. SC1048 is a mutant strain with a Tn5 insertion in the fliP gene (13). LS1298 is a mutant strain containing a disruption in the fliF gene [U. Jenal and L Shapiro, EMBO J. 15, 2393 (1996)]. Construction of the fliF-lacZ, flbG-lacZ, fljL-lacZ, and fljK-lacZ transcription fusions are described elsewhere (3, 4). Unless otherwise noted, strains were grown either in PYE media [J. S Poindexter, Bacteriol. Rev. 28. 231 (1964) ] or minimal M2-glucose media [I. Contreras, L. Shapiro, S. Henry, J. Bacteriol. 135, 1130 (1978)] at 31°C.
40. We thank U. Jenal for LS1298, M.R.K. Alley for the MCP-M2 construct, and J. Maddock for suggesting the use of DiA. We are also grateful to L. Shapiro, R. Losick, and members of our laboratory for helpful comments on the manuscript. J.A.W. was supported by USPHS predoctoral fellowship GM-07104 and a University of California Dissertation Year Fellowship. This work was supported by Public Health Service grant GM48417 from the National Institutes of Health and Junior Faculty Research Award JFRA-466 from the American Cancer Society (J.W.G.).