The Dps (DNA-Binding Protein from Starved Cells) proteins are a class of stress-specific proteins with a major role in protecting DNA during the stationary phase of bacterial growth, through direct physical binding as well as ferroxidation. These proteins are characteristically dodecameric in nature. Mycobacterium smegmatis, which is the model organism used in this study has two Dps homologues- MsDps1 and MsDps2. MsDps1, that has previously been studied, is exceptional in having trimeric as well as dodecameric states in vitro. This work focuses on the functional domains of MsDps1, with respect to its oligomerisation and DNA binding property, the identification of a new Dps homologue MsDps2, the in vitro characterization of MsDps2 and elucidation of a possible function of the protein in the physiology of Mycobacterium smegmatis.
The Thesis is organized as shown below:
Chapter 1: The literature on the bacterial stationary phase physiology and the role of Dps has been reviewed in this chapter. It gives a brief introduction of the background of the present study including the stationary phase response of bacteria and the significance of studying bacteria under stress as apart from ideal conditions of growth, which has been the conventional approach until recently. The advantages of using Mycobacterium smegmatis as a model system, and its starvation-induced stationary phase are also discussed. An introduction to the Dps proteins as a family of proteins branched off from ferritins and nucleoid proteins is explained. A brief summary of the ferritin and nucleoid proteins is given. Similarities connecting Dps to both these protein families is described. The review of earlier work done in our laboratory on the mycobacterial MsDps1 protein is also presented.
Chapter 2: involves the study of the solution properties of the protein including its ability to oligomerize in vitro. The MsDps1 protein exists in two forms, a trimer and a dodecamer. The
trimer form is a unique feature of the M.smegmatis homologue. Dps proteins from other sources are characteristically dodecameric. Earlier studies have shown that the trimeric form of the protein can perform ferroxidation while the dodecamer can bind to DNA. The dodecamer can also perform ferroxidation and accumulate the oxidized iron in its negatively charged core. In this chapter, we show that the trimeric form is extremely stable, under various conditions of pHs. The protein, when over expressed in M.smegmatis, also shows the presence of the trimer, thus ruling out the effect of heterologous expression of the protein in E.coli. We further report here, the ideal conditions for dodecamerisation of the protein from trimer to dodecamer, which binds to DNA. The dodecamer once formed is also highly stable and does not revert back to the trimeric form. The structural stability of the dodecamer is expected, as it is the fully functional form of the protein that physically protects the DNA from stress. However, the high stability of the trimeric form and its precise conversion into a stable dodecamer is intriguing. It is interesting to study the functional significance in vivo of the oligomerisation process in MsDps1. In addition, we looked at the effect of over expression of the protein on the overall phenotype of Mycobacterium smegmatis, as evidenced by the colony morphology and find no visible alteration, when compared with the wild type.
Chapter 3: deals with a more detailed structural analysis of the MsDps1 protein. The role of N and C termini of the protein in maintaining a stable oligomeric structure is studied by making an N-terminal deletion mutant of the protein which is found to be unable to form a dodecamer in solution. On the other hand, MsDps1 with a 16 amino acid C-terminal deletion, MsDpsΔC16, is able to form stable oligomeric structures, when the N-terminal is intact. A previous deletion reported from our laboratory with 26 amino acids deleted from the C-terminal tail, called MsDpsΔC26 showed inability to form stable oligomeric structures in vitro. Putting together all the above results, a model for the interaction of the N and C-terminal tails of the protein in maintaining a stable dodecamer is presented. A demarcation of the C-terminal tail of MsDps1 into regions determining the oligomeric stability and DNA binding was also inferred. The MsDpsΔC16 protein, does not bind to DNA although it forms a stable dodecamer. A further deletion of 10 amino acids, as seen in a previously made construct, MsDpsΔC26 disrupts both the DNA binding as well as the oligomeric stability of the protein.
Chapter 4: describes the discovery of a new homolog of the Dps protein in M.smegmatis. It was named as MsDps2. Bio-informatics analysis carried out on the complete genome data of Mycobacterium smegmatis yielded a second homologue of Dps in addition to the one already present and characterized. Interestingly, out of the 300 homogues of Dps found in bacteria, only 195 are present as single copies in a bacterium. The rest exist as more than one homologue in the same bacterial genome. The basic characterization of this new Dps homologue and its confirmation as a Dps family member is the focus of this chapter.
Chapter 5: deals with the possible functions of the new protein MsDps2. Electron micrography shows that the purified protein forms stable nucleoprotein-like complexes. Over expression of the MsDps2 proteins presents no difference in the colony morphology when compared with the wild-type. Western analysis shows that the MsDps2 protein is not expressed under normal conditions tested for growth. MsDps1, on the other hand shows expression under conditions of starvation and osmotic stress, as has been established previously in the laboratory. Hence, it can be inferred that the new protein MsDps2 does not perform the same function as MsDps1. However, the in vivo function of this protein remains an important question to be addressed. The appearance of in vitro nucleoid structures involving this protein under the electron microscope, suggests a possible role for this protein in the formation and stabilization of the mycobacterial nucleoid. Indeed extensive evidence for the same exists for the E.coli protein.
Chapter 6: describes the results obtained from the sequence comparison of MsDps2 with other Dps proteins listed in the TIGR database. ClustalW sequence analysis, followed by the construction of a phylogenetic tree using the MEGA software, suggests that the mycobacterial Dps proteins fall into two separate groups, represented by the MsDps1 and MsDps2 homologues from Mycobacterium smegmatis.
Chapter 7 Summary and Conclusions: A summary of the work presented in the thesis is given followed by the appendix sections. Appendix 1 includes list and maps of plasmids used. Appendix 2 details the theoretical DNA and protein sequences of the recombinant clones generated in the study and theoretical physical and chemical properties of the proteins studied, as
calculated with the Expasy Protparam software. Appendix 3 includes raw data obtained from the bio-informatic analysis of MsDps2, obtained using ClustalW analysis.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/918 |
| Date | 02 1900 |
| Creators | Saraswathi, Ramachandran |
| Contributors | Chatterji, Dipankar |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Relation | G22935 |
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