An investigation of cell wall lytic enzymes in Streptomyces coelicolor

Abstract

An increasing appreciation for the role of small RNA regulators prompted us to investigate the scope of RNA regulation in the bacterium, Streptomyces coelicolor. Our search revealed an antisense RNA that corresponds to the upstream region of four genes encoding cell wall cleavage enzymes (cell wall hydrolases), and a previously uncharacterized population of transfer RNA (tRNA) cleavage products. Further characterization of the 'tRNAs led to the discovery that S. coelicolor tRNAs are cleaved into 'tRNA halves' in a developmentally regulated fashion. All tRNAs seem to be susceptible to tRNA cleavage, although a bias was detected for tRNAs specifying highly used codons. To date, our work is the sole description of 'tRNA half production in a bacterium, and recent studies suggest that it is a widespread phenomenon among eukaryotic organisms. In a separate line of investigation, we noticed that a previous study had predicted that the genes associated with the antisense RNA are under the control of a riboswitch- a regulatory RNA element that directly controls gene expression in response to specific conditions. Our multifaceted characterization of this system began with the construction and phenotypic analyses of deletion mutant strains for several of the cell wall hydrolase-encoding genes. We demonstrate that S. coelicolor cell wall hydrolases are involved in germination, vegetative growth, and sporulation. Finally, we studied the potential for riboswitch regulation of one of the cell wall hydrolase-encoding genes, rpfA. RpfA is a resuscitation: Qromoting factor protein that is important for the revival of dormant bacteria, including the human pathogen and S. coelicolor relative - Mycobacterium tuberculosis. Our investigation uncovered evidence suggesting that the riboswitch region is involved in the regulation of rpfA, and we identified specific conditions under which it is repressed. This work represents a novel paradigm in the regulation of cell wall hydrolase expression.