The Universal Stress Proteins of Bacteria

Abstract

Universal stress proteins (USPs) are a widespread and abundant protein family often linked to survival during stress. However, their exact biochemical and cellular roles are incompletely understood. Mycobacterium tuberculosis (Mtb) has 10 USPs, of which Rv1636 appears to be unique in its domain structure and being the only USP conserved in M. leprae. Over-expression of Rv1636 in M. smegmatis indicated that this protein does not share the growth arrest phenotype of another Mtb USP, Rv2623, suggesting distinct roles for the Mtb USPs. Purified Rv1636 was shown to have novel nucleotide binding capabilities when subjected to UV crosslinking. A range of site-directed mutants of Rv1636 were produced, including mutations within a predicted nucleotide binding motif, with the aim of identifying and characterising key residues within the Rv1636 protein. Further putative biochemical activities, including nucleotide triphosphatase, nucyleotidylyation and auto-phosphorylation were also investigated in vitro; however Rv1636 could not be shown to definitively possess these activities, raising the possibility that addition factors may be present in vivo. Bioinformatic analysis of Rv1636 has provided an in-depth look at the protein. The crystal structure of Rv1636 shows a strand swapped dimer conformation that appears to block the predicted nucleotide binding site, providing a possible reason for the low NTPase activity previously observed. Truncated Rv1636 constructs were successfully generated, in which the strand swapped dimer was disrupted, and subjected to biophysical analysis, including analytical ultracentrifugation and size exclusion chromatography combined with multi-angle light scattering. Previous Mtb single-USP mutants are known to have no phenotype under a range of stress conditions. For this reason the P. aeruginosa USP PA3309, which does possess a stress survival phenotype, was also investigated. This provided the opportunity to investigate the role of USPs and their putative nucleotide binding motif in vivo. Site-directed mutants of PA3309 were generated to investigate the role of the nucleotide binding motif in vivo. It remains to be determined if the survival defect observed for ΔPA3309 strain can be complemented with these mutants as the vector system used in these experiments proved unable to integrate into the attB site of the genome. Through the analysis of the USPs from mycobacteria and Pseudomonas, the aim was to elucidate a greater understanding of the role of Rv1636 in Mtb and the role of USPs in general. The bioinformatic and biochemical analyses of USPs, in addition to the site directed mutants generated as a result of this work, will provide a strong foundation for future studies.