Abstract
Streptomyces coelicolor and Mycobacterium bovis are high G+C gram-positive members of the phylum Actinobacteria. M. bovis is a member of the M. tuberculosis species complex and M. bovis, BCG is the attenuated vaccine strain used as a model organism as it can be manipulated in containment Category 2 laboratories. When cells are exposed to different environments/stresses they need to adapt their physiology and biochemistry for their effective survival. A key mechanism of adaptation is the ability to quickly alter the molecular composition of the cell through the regulation of gene expression both at transcriptional and translational level. While transcriptional regulation of gene expression has been studied extensively, little information regarding translational regulation in bacteria is available. To begin to study translational regulation in Streptomyces and Mycobacterium, a genome-wide approach was adapted to study the extent of translational regulation when cells are exposed to heat shock. As an initial attempt the protocol to isolate polysomes from both S. coelicolor and M. bovis (BCG) using classical sucrose density gradient under normal and heat shock conditions was optimized. Using DNA microarrays and RNA sequencing, the relative distribution of mRNA in the polysome fractions were analysed and compared it to the total transcriptome of the cell. Major heat shock responsive genes such as dnaK, grpE, groEL, groES, lon, hspR and dnaJ were significantly differentially expressed or were found to be translationally/transcriptionally potentiated (significantly differentially expressed genes in both transcription and translation) during heat shock in S. coelicolor, and in M. bovis (BCG) a smaller number of genesincluding SerX coding for tRNA biosynthesis and an ArsR repressor anti-toxin coding gene were shown to be translationally regulated. The study was further extended in S. coelicolor to investigate the use of a method based on the purification of affinity tagged ribosomes as a way to isolate actively translated mRNA. Selected ribosomal proteins were successfully tagged using strep-tag and the presence of tagged ribosomal proteins in cell lysates was checked using blotting techniques.