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Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

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Cyclic GMP controls Rhodospirillum centenum cyst development.

Jeremiah N Marden¹, Qian Dong, Sugata Roychowdhury

¹Indiana University, Bloomington, IN 47405, USA University of California at Berkeley, Berkeley, CA 94720, USA.

Molecular Microbiology

|January 11, 2011

Summary

This study reveals that cyclic guanosine monophosphate (cGMP) regulates development in the bacterium Rhodospirillum centenum. A guanylyl cyclase enzyme is essential for cGMP synthesis and cyst formation in this prokaryote.

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Area of Science:

Microbiology
Molecular Biology
Biochemistry

Background:

Guanylyl cyclases are typically found in eukaryotes, while adenylyl cyclases are widespread.
The role of cyclic guanosine monophosphate (cGMP) in prokaryotic systems is largely unexplored.

Purpose of the Study:

To investigate the presence and function of guanylyl cyclase in prokaryotes.
To determine the role of cGMP in bacterial development, specifically in Rhodospirillum centenum cyst formation.

Main Methods:

Genetic deletion of guanylyl cyclase in R. centenum.
Purification and in vitro enzymatic assays of the R. centenum guanylyl cyclase.
Analysis of a cyclic AMP (cAMP) receptor protein (CRP) homologue's interaction with cGMP using isothermal calorimetry (ITC) and differential scanning fluorimetry (DSF).

Main Results:

Rhodospirillum centenum secretes cGMP during cyst development.
A guanylyl cyclase deletion mutant exhibited defects in cGMP synthesis and cyst formation.
The purified R. centenum guanylyl cyclase synthesized cGMP from GTP in vitro.
A CRP homologue linked to the cyclase was stabilized by cGMP, not cAMP, and its deletion impaired cyst development.

Conclusions:

cGMP plays a critical role in regulating prokaryotic development.
This finding has implications for understanding bacterial interactions with plants, as similar cyclases are found in plant-associated bacteria.
Azospirillum brasilense also synthesizes cGMP during cyst induction, suggesting a conserved mechanism.