1887

Abstract

The second messenger, bis-(3′,5′)-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host–pathogen interactions. Bioinformatics analyses predicted that , an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen encodes only a single diguanylate cyclase. One of the unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that expression altered colony morphology, motility and biofilm formation of PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography–mass spectrometry confirmed that expression increased cyclic di-GMP production in PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in expressing could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to growth, .

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2016-09-01
2019-12-13
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