title: Identification of bacterial guanylate cyclases.
abstract: The ability of bacteria to use cGMP as a second messenger has been controversial for decades. Recently, nucleotide cyclases from Rhodospirillum centenum, GcyA, and Xanthomonas campestris, GuaX, have been shown to possess guanylate cyclase activities. Enzymatic activities of these guanylate cyclases measured in vitro were low, which makes interpretation of the assays ambiguous. Protein sequence analysis at present is insufficient to distinguish between bacterial adenylate and guanylate cyclases, both of which belong to nucleotide cyclases of type III. We developed a simple method for discriminating between guanylate and adenylate cyclase activities in a physiologically relevant bacterial system. The method relies on the use of a mutant cAMP receptor protein, CRPG , constructed here. While wild-type CRP is activated exclusively by cAMP, CRPG can be activated by either cAMP or cGMP. Using CRP- and CRPG -dependent lacZ expression in two E. coli strains, we verified that R. centenum GcyA and X. campestris GuaX have primarily guanylate cyclase activities. Among two other bacterial nucleotide cyclases tested, one, GuaA from Azospillrillum sp. B510, proved to have guanylate cyclase activity, while the other one, Bradyrhizobium japonicum CyaA, turned out to function as an adenylate cyclase. The results obtained with this reporter system were in excellent agreement with direct measurements of cyclic nucleotides secreted by E. coli expressing nucleotide cyclase genes. The simple genetic screen developed here is expected to facilitate identification of bacterial guanylate cyclases and engineering of guanylate cyclases with desired properties.
authors: Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, 82071.
authors: Min-Hyung
authors: MH
authors: Ryu
authors: Hwan
authors: H
authors: Youn
authors: In-Hye
authors: IH
authors: Kang
authors: Mark
authors: M
authors: Gomelsky
mesh_terms: D000262:Adenylyl Cyclases / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D006152:Cyclic GMP / Q000737:chemistry; D057075:Enzyme Assays; D006162:Guanylate Cyclase / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase; D006152:Cyclic GMP
keywords: Escherichia coli; cAMP; cGMP; nucleotide cyclase; second messenger; signal transduction
references: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
references: 10829079
references: J Mol Biol. 2000 Dec 15;304(5):847-59
references: 11124031
references: EMBO J. 2002 Jul 15;21(14):3672-80
references: 12110580
references: Biochemistry. 2002 Oct 1;41(39):11857-67
references: 12269830
references: Proc Natl Acad Sci U S A. 1970 May;66(1):104-10
references: 4320461
references: Proc Natl Acad Sci U S A. 1971 Nov;68(11):2794-8
references: 4330942
references: Biochem Biophys Res Commun. 1974 Jan;56(1):119-26
references: 4362936
references: Gene. 1988 Oct 15;70(1):191-7
references: 2853689
references: J Mol Biol. 1989 Jun 20;207(4):783-96
references: 2547972
references: Nucleic Acids Res. 1994 Aug 11;22(15):2894-901
references: 8065899
references: J Biol Chem. 2006 Jan 13;281(2):1119-27
references: 16260780
references: J Biol Chem. 2007 Feb 9;282(6):3632-9
references: 17158453
references: J Bacteriol. 2008 Jul;190(13):4532-40
references: 18456811
references: Proc Natl Acad Sci U S A. 2009 Apr 28;106(17):6927-32
references: 19359484
references: J Mol Microbiol Biotechnol. 2009;17(1):1-11
references: 19033675
references: Mol Cell Biochem. 2010 Jan;334(1-2):215-9
references: 19943185
references: J Biol Chem. 2010 Dec 31;285(53):41501-8
references: 21030591
references: Mol Microbiol. 2011 Feb;79(3):600-15
references: 21214648
references: Mol Microbiol. 2011 Feb;79(3):562-5
references: 21255104
references: J Bacteriol. 2011 Sep;193(18):4859-68
references: 21764924
references: EMBO J. 2013 Sep 11;32(18):2430-8
references: 23881098
references: EMBO J. 2013 Sep 11;32(18):2421-3
references: 23963313
references: Acta Crystallogr D Biol Crystallogr. 2014 Jun;70(Pt 6):1726-42
references: 24914983
references: Proc Natl Acad Sci U S A. 2014 Jul 15;111(28):10167-72
references: 24982160