title: Engineering of Bacillus subtilis strains to allow rapid characterization of heterologous diguanylate cyclases and phosphodiesterases.
abstract: Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis.
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, USA Biochemistry Graduate Program, Indiana University, Bloomington, Indiana, USA.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Gao
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, USA.
authors.forename: Xiao
authors.initials: X
authors.lastname: Dong
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, USA Biochemistry Graduate Program, Indiana University, Bloomington, Indiana, USA.
authors.forename: Sundharraman
authors.initials: S
authors.lastname: Subramanian
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, USA.
authors.forename: Paige M
authors.initials: PM
authors.lastname: Matthews
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, USA.
authors.forename: Caleb A
authors.initials: CA
authors.lastname: Cooper
authors.affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Kearns
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, USA Biochemistry Graduate Program, Indiana University, Bloomington, Indiana, USA cedann@indiana.edu.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Dann
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development; D016360:Clostridioides difficile / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005453:Fluorescence; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D010727:Phosphoric Diester Hydrolases / Q000235:genetics* / Q000378:metabolism; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics* / Q000378:metabolism; D058928:Riboswitch / Q000235:genetics; D015398:Signal Transduction; D019076:Transgenes; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D058928:Riboswitch; C062025:bis(3',5')-cyclic diguanylic acid; D010727:Phosphoric Diester Hydrolases; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP
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