Nucleoid gene regulation in mycobacteria

[摘要] ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis disease, remains one of the leadingcauses of death worldwide. The ongoing tuberculosis epidemic and poor treatment outcomes can beattributed to the emergence of antibiotic resistant mycobacterial organisms as well as persistent M.tuberculosis infections. It is therefore imperative that we improve our understanding of M. tuberculosispathogenesis to develop more effective anti-tuberculosis drugs and treatment strategies. M. tuberculosis isknown to encounter adverse stress conditions such as nutrient starvation, hypoxia, nitrosative stress andlow pH during infection and understanding how this pathogen adapts to its host environment is crucial infinding ways to circumvent tuberculosis disease. Conventional methods of investigating transcriptionalresponses, such as microarrays and ChIP-seq, are limited to the investigation of a single protein. Althoughvaluable information has been obtained through this approach, adaptation to stress is likely mediatedthrough several regulatory elements. Furthermore, new methodologies are required to identify theseproteins. For example, nucleoid-associated proteins (NAPs), a group of global transcriptional regulatorswhich have the unique ability to shape the bacterial chromosome, have been shown to be involved in themycobacterial stress response. However, due to poor sequence homology with other bacterial organismsand conventional methodologies, only seven nucleoid associated proteins have been identified formycobacterial organisms compared to 12 NAPs in Escherichia coli.Nucleoprotein - Mass Spectrometry (NP-MS) makes use of affinity purification of formaldehyde cross-linkedRNA polymerase transcriptional complexes to identify proteins involved in chromosomal structure, andtranscriptional and translational processes using mass spectrometry. This approach successfully identifiedvarious DNA and RNA associated proteins as well as numerous proteins associated with energy, carbon,lipid and amino acid metabolism. These results suggested that NP-MS was effective in isolating not onlyproteins directly involved in transcription and translation but also proteins associated with the RNApolymerase transcriptional complex and DNA. MSMEG_1060, MSMEG_2695, MSMEG_3754,MSMEG_4306 and MSMEG_5512 were identified as possible nucleic acid associated proteins throughcomparison of conserved proteins domains and were subjected to further investigation. Episomalexpression of these proteins as FLAG-tagged fusion proteins in M. smegmatis revealed MSMEG_1060, MSMEG_2695, MSMEG_4306 and MSMEG_5512 to be putative DNA-associated proteins whilst no DNA association was found for MSMEG_3754.NP-MS was applied to investigate differences between DNA-associated proteins in exponential andstationary phase cultures. Investigation into the proteins which make up the RNA polymerase transcriptionalcomplex and its associated proteins in stationary phase M. smegmatis cultures, revealed that NP-MS couldeffectively be used to identify proteins which are required for adaptation to stress in this organism. These included the dormancy response regulator DevR, the ribosome hibernation promoting factor (hpf), heatshock protein HspX and the universal stress proteins MSMEG_3811, MSMEG_3945 and MSMEG_3950.These results demonstrated the ability of the developed NP-MS method to identify the proteins whichmediate DNA structure, and transcriptional and translational changes in M. smegmatis. Furthermore, wepropose that NP-MS can be used to investigate the proteins associated with the RNA polymerase complexin not only mycobacterial species but also other bacterial organisms.

[发布机构] Stellenbosch University