INVESTIGATION OF CYCLIC DINUCLEOTIDE HOMEOSTASIS AND THE HYDROLYSIS OF THEIR LINEAR INTERMEDIATES IN BACTERIA

Abstract

The synthesis of cyclic dinucleotides as signals is one strategy bacteria use to sense and adjust to environmental changes. Cyclases synthesize the cyclic dinucleotide, while phosphodiesterases cleave it to yield a linear diribonucleotide, which is recycled into monoribonucleotides by other enzymes. For many bacteria, cyclic di-GMP (c-di-GMP) regulates the transition from a unicellular motile state to a multicellular sessile community. However, c-di-GMP signaling has been less intensively studied in Gram-positive organisms. Bacillus subtilis is a model for the study of bacterial differentiation, yet how c-di-GMP functions in this organism is not fully understood. This work began with construction of a fluorescent reporter to measure c-di-GMP abundance in B. subtilis, which showed that c-di-GMP levels are strikingly different among differentiated subpopulations. These data highlight how single-cell approaches can be used to analyze metabolic trends within bacterial populations and demonstrate that for some bacteria, c-di-GMP levels are adjusted heterogeneously across bulk populations. The enzymes Orn, NrnA, NrnB, and NrnC have been proposed to act as general 3’-5’ exoribonucleases that preferentially process ‘short’ oligoribonucleotides. Intriguingly, Orn also performs a crucial role in c-di-GMP homeostasis by processing the pGpG generated from c-di-GMP production. To discover the molecular basis for Orn’s ability to ‘select’ short RNAs, and to elucidate the relationship between Orn and the diribonucleotide pGpG, we combined structural, biochemical, and in vivo analyses of RNA cleavage. These data reveal that Orn is not a general exoribonuclease of short RNA oligoribonucleotides, as previously believed, but instead acts as a dedicated ‘diribonucleotidase’. Our studies indicate RNA degradation as a step-wise process with a dedicated enzyme for the clearance of diribonucleotides, which affect cellular physiology and viability. Examination of the roles of NrnA and NrnB is underway. We conducted an initial study to determine if NrnA and NrnB are redundant proteins, as has been proposed, and if they might also act as ‘diribonucleotidases’. These data show that they exhibit different substrate preferences and that they may have unique cellular functions. Therefore this work changes the perception of the role(s) Orn plays and that a re-evaluation of ‘short’ RNases is needed.