Regulation of a muralytic resuscitation-promoting factor in Streptomyces coelicolor

Abstract

To survive inhospitable conditions, bacteria can form fortified stress-resistant cells that are unable to grow and have little to no detectable metabolic activity. Upon detecting improved environmental conditions, these cells can reactivate metabolism and resume growth. A common aspect of establishing and exiting dormancy involves the extensive remodeling of the bacterial cell wall. In the actinobacteria, the ‘resuscitation-promoting factors’ (Rpfs) orchestrate the exit from dormancy through their muralytic activities. The expression and activity of muralytic enzymes are tightly regulated. Excessive activity could weaken the cell wall, leading to lysis, while insufficient activity, in the case of the Rpfs, could compromise resuscitation from a dormant state. rpf genes are regulated at the level of transcription initiation; however, these genes are likely also regulated post-transcriptionally and post-translationally. For example, in Streptomyces coelicolor, rpfA is associated with several regulatory RNAs: a riboswitch motif is located within the 5′ untranslated region, while a small RNA-encoding gene is located immediately downstream of the rpfA stop codon. In this work, we probed multi-level rpfA regulation in S. coelicolor, placing a particular emphasis on RNA-based control mechanisms acting post-transcriptionally. We found that rpfA was regulated at many levels: transcription initiation, transcript elongation and protein stability. We also discovered that rpfA served as a regulatory hub, receiving input from three second messengers with known functions in dormancy and/or resuscitation. During the course of this work, we also uncovered a novel riboswitch regulatory mechanism and identified previously uncharacterized activity determinants for this riboswitch class. Finally, we showed that the downstream-encoded small RNA impacted the expression of genes involved in microaerobic metabolism and, through its predicted regulation of rpfA expression, may coordinate cell wall remodeling and dormancy/resuscitation with the hypoxic response. This work dramatically expands our understanding of rpf gene regulation and actinobacterial resuscitation.