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.

Keywords: Escherichia coli; cAMP; cGMP; nucleotide cyclase; second messenger; signal transduction.

Figure 1

DNA-binding of CRP and CRP_G at various concentrations of cAMP and cGMP. In vitro DNA binding was measured by fluorescence anisotropy.

Figure 2

The CRP/CRP_G reporter system for detecting adenylate and guanylate cyclase activities. A, The BL21 [DE3] cya crp strains expressing wild-type CRP from pRKcrp (A) or CRP_G from pRKcrp_G (G) were grown with or without the addition of cAMP or cGMP. The strain expressing CRP developed blue color only in the presence of cAMP whereas the strain expressing CRP_G developed blue color in the presence of either cAMP or cGMP. B, Heterologous nucleotide cyclases were introduced into the BL21 [DE3] cya crp mutants expressing CRP or CRP_G. The CyaB1 (catalytic domain) and CyaA (full-length) produced blue color in both strains, while GcyA, GuaA and GuaX produced blue colonies only in the presence of CRP_G. The medium contained 50 μM IPTG. C, Analysis of secreted cNMPs from the E. coli strains expressing nucleotide cyclases. The same strains as in panel B were used. Cyclic nucleotide levels were measured by ELISA using cAMP- and cGMP-specific antibodies. Average data and standard deviations from three independent experiments are shown.