Identification of bacterial guanylate cyclases. Issue 5 (9th February 2015)

• Record Type: Journal Article
• Authors: Ryu, Min‐Hyung; Youn, Hwan; Kang, In‐Hye; Gomelsky, Mark
• Is Part Of: Proteins. Volume 83:Issue 5(2015)
• Page Start: 799
• Page End: 804
• Publication Date: 2015-02-09
• Subjects: Proteins -- Periodicals; Proteins -- Periodicals; 572.6
• DOI: 10.1002/prot.24769
• Languages: English
• ISSNs: 0887-3585

Abstract

<abstract abstract-type="main"> <title>ABSTRACT</title> <p>The ability of bacteria to use cGMP as a second messenger has been controversial for decades. Recently, nucleotide cyclases from <italic>Rhodospirillum centenum</italic>, GcyA, and <italic>Xanthomonas campestris</italic>, GuaX, have been shown to possess guanylate cyclase activities. Enzymatic activities of these guanylate cyclases measured <italic>in vitro</italic> 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, CRP<sub>G</sub>, constructed here. While wild‐type CRP is activated exclusively by cAMP, CRP<sub>G</sub> can be activated by either cAMP or cGMP. Using CRP‐ and CRP<sub>G</sub>‐dependent <italic>lacZ</italic> expression in two <italic>E. coli</italic> strains, we verified that <italic>R. centenum</italic> GcyA and <italic>X. campestris</italic> GuaX have primarily guanylate cyclase activities. Among two other bacterial nucleotide cyclases tested, one, GuaA from <italic>Azospillrillum</italic> sp. B510, proved to have guanylate cyclase activity, while the other one, <italic>Bradyrhizobium japonicum</italic> 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 <italic>E. coli</italic> 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. Proteins 2015; 83:799–804. © 2015 Wiley Periodicals, Inc.</p> </abstract>