Abstract 1355 Oxygen sensing diguanylate cyclase proteins control metal homeostasis and stress tolerance in the Gram-negative bacteria

Bacteria utilize globin coupled sensor (GCS) proteins to sense and respond to oxygen and other diatomic gaseous ligands. A subgroup of GCS proteins contains a diguanylate cyclase (DGC) output domain which is linked to the sensory domain through a variable middle domain. DGC proteins synthesize the second messenger cyclic-di-GMP (c-di-GMP), which influences motility, biofilm formation, and virulence in bacteria. DGC containing GCS proteins have been studied in various organisms, such as Pectobacterium carotovorum, an important plant pathogen. P. carotovorum (Pcc) contains one GCS protein, PccDgcO. PccDgcO synthesizes c-di-GMP upon oxygen binding and regulates motility, virulence, and biofilm formation under aerobic conditions. Another DGC containing GCS protein, EcDgcO, is found in the Gram-negative model organism Escherichia coli, and is a homolog of PccDgcO. EcDgcO is co-transcribed and forms a complex in vivo with an O2-sensing c-di-GMP phosphodiesterase (PDE), EcPdeO. The EcDgcO-PdeO complex is a unique c-di-GMP metabolic complex, in which both the DGC and PDE are active, and respond to the same signals. This work seeks to understand the cellular processes and pathways regulated by PccDgcO and the EcDgcO-PdeO complex, using methods such as, RNA sequencing and differential expression analysis, ICP-MS, physiological assays, mass spectrometry, and bioinformatic tools, among others. Using deletion strains of the respective bacterial species, this work investigates the roles these proteins play in their respective organisms, compares similarities and differences in the functions controlled in each species, and aims to use these proteins as models to understand local vs. global c-di-GMP signaling. Results show that both GCS proteins affect Mn tolerance in their respective organisms, and that in E. coli the ΔdgcO-pdeO strain shows a decreased tolerance to H2O2 under high Mn stress, while WT and ΔdgcO Pcc show differences in adaptation to changes in oxygen levels. These results, among others, reveal that GCS proteins with DGC output domains control a broad range of phenotypes relating to stress tolerance, motility, biofilm formation, and central metabolism, and highlight the broad role c-di-GMP plays in bacteria. This work was supported by NSF CHE1352040 (EW), NSF CHE2003350 (EW), and Frasch Foundation Grant 824-H17 (EW).