Cyclic di-GMP metabolism in Shewanella oneidensis MR-1 [electronic resource]

Shewanella oneidensis MR-1 is a gram-negative, facultative gamma-proteobacterium with the ability to utilize a wide assortment of different electron acceptors for growth, including iron(III), manganese(III) and (IV), nitrate, nitrite, thiosulfate, sulfite, trimethylamine N-oxide (TMAO), fumarate, uranium(VI), dimethyl sulfoxide (DMSO) and elemental sulfur. This metabolic versatility and ability to reduce metals has made S. oneidensis the subject of research in the fields of bioremediation and microbial biofuels. Many of these applications require the formation of biofilms by this microbe. Microbial biofilms are surface-associated communities of microorganisms, and are ubiquitous and profoundly impact the environment and human health. Recently, the bacterial signaling molecule cyclic di-GMP (c-di-GMP) was found to affect many physiological and metabolic functions in biofilm formation, as well as in cell cycle progression, expression of virulence factors and flagellar genes, production of exopolysaccharides, control of flagellar movement, quorum sensing, and the stress response. The environmental and cellular factors controlling c-di-GMP signaling are numerous and diverse, but it is not well understood how these factors modulate c-di-GMP levels and metabolism as well as control the target responses. Diguanylate cyclases containing a 'GGDEF' amino acid motif and c-di-GMP-specific phosphodiesterases characterized by an 'EAL' amino acid motif are known to alter intracellular c-di-GMP concentrations. Many of these enzymes also contain sensor domains such as the Per-Arnt-Sim (PAS) domain, which is known to perceive changes in redox potential, oxygen, other small molecular ligands, or light, as well as to facilitate protein-protein interactions. Biofilm formation in Shewanella oneidensis MR-1 is known to be controlled by c-di-GMP; however, the c-di-GMP signaling network in this microorganism has not been explored until now. Here, I present the results of genetic and biochemical analyses of one GGDEF domain protein and three PAS-GGDEF-EAL domain proteins present in this microorganism, and describe hitherto unknown downstream targets of c-di-GMP signaling. First, the GGDEF domain protein MxdA, which is required for formation of three-dimensional biofilms in Shewanella oneidensis MR-1, was previously hypothesized, based on genetic data, to act as a diguanylate cyclase (DGC). I demonstrate here that MxdA does not exhibit diguanylate cyclase activity in vitro; however, the protein controls the cellular level of c-di-GMP in S. oneidensis indirectly. Second, I characterized the PAS-GGDEF-EAL domain protein SO0341, here named BgdA, from Shewanella oneidensis MR-1. A bgdA deletion mutant exhibited a lower growth rate in minimal media than did the wild type strain. This phenotype was rescued by external addition of the branched-chain amino acids isoleucine, leucine and valine. Genetic evidence indicates that BgdA activates expression of two ilvE isozymes, which catalyze the final step in the biosynthetic pathways of these amino acids. In in vitro enzyme activity assays, BgdA demonstrated both diguanylate cyclase (DGC) and c-di-GMP-specific phosphodiesterase (PDE) activity. However, mutations in the EAL and GGDEF domains that effectively abolished the respective PDE and DGC activities did not affect S. oneidensis MR-1 growth or change ilvM expression levels, indicating that these activities were not necessary for the regulation of ilvE transcription. These results collectively suggest that BgdA acts as a bifunctional enzyme in vivo, with one role involving the regulation of branched-chain amino acid biosynthesis and the other, yet to be determined, affecting c-di-GMP metabolism. Third, I present genetic and biochemical analyses of the PAS-GGDEF-EAL domain protein SO0437, renamed SarP, from Shewanella oneidensis MR-1. A sarP deletion mutant exhibited decreased swimming motility and increased biofilm formation under medium-rich growth conditions. Genetic evidence indicated that during biofilm growth, SarP acts as a regulator of numerous genes involved in sulfate uptake and assimilation. Addition of sulfate to sarP deletion mutants enhanced the motility phenotype. In vitro enzyme activity assays with purified SarP indicated that the protein exhibited PDE but no DGC activity. A mutation in the EAL domain effectively abolishing the PDE activity was constructed, and the corresponding mutant strain showed that the regulation of sulfate metabolism by SarP required its PDE activity. Finally, I conducted genetic and biochemical analyses of the NIT-HAMP-PAS-GGDEF-EAL domain protein SO0141 from Shewanella oneidensis MR-1. Mutants defective in SO0141 exhibit reduced swimming motility under anaerobic conditions. Genetic evidence indicates that SO0141 functions as a regulator of genes involved in molybdate transport and molybdopterin biosynthesis, and that this regulation is dependent on the PDE activity of SO0141.

Keywords: Chemical Engineering
Advisor: Spormann Alfred M. primary advisor. Thesis advisor
Khosla Chaitan
Swartz James R. advisor. Thesis advisor