Evolution of Function of Bacterial Nucleoid-Associated Proteins

Louisiana State University, Baton Rouge LA

Investigators

Abstract

This award supports research focused on understanding the role of important proteins in the structure and function of a bacterial cell. The size of the DNA requires that it be compacted to fit the dimensions of the cell, while still allowing its accessibility to cellular proteins. The bacterial genome is associated with a number of different DNA-binding proteins, including proteins known as HU. Aside from being involved in the organization in DNA, HU proteins function in regulating DNA-dependent processes, such as replication, repair and gene expression and are therefore critical for normal cellular function. In this project, the focus will be on the HU variant encoded by Mycobacterium smegmatis. Mycobacteria encode an unusual two-domain HU protein. What is intriguing about two-domain HU proteins (typically referred to as Hlp, for Histone Like Protein) is that they appear to be encoded only by bacterial species that are particularly adept at resisting environmental stress. For mycobacteria, Hlp has, for example, been reported to be more abundant during dormancy. The goal of this research is to define the functional role of M. smegmatis Hlp, with a specific focus on the role of the lysine-rich extension. Understanding functional roles of mycobacterial Hlp will illuminate a unique aspect of mycobacterial physiology as well as the specific demands of other bacterial species that encode two-domain HU homologs. A combination of DNA-binding assays designed to determine the role of Hlp in DNA compaction are planned, as well as assays aimed at determining the ability of Hlp to protect DNA from damage. M. smegmatis also encodes a protein named Ku, which is expected to function in repair of DNA double strand breaks; Ku shares with Hlp key features of the lysine-rich extension. This shared sequence feature will be exploited to determine the role of the lysine-rich domain in DNA end-joining by both proteins and to ascertain their roles in double strand break repair. A final aim is centered on determining conditions under which Hlp is more abundant and on investigating consequences of Hlp deletion on global gene expression. This line of inquiry is expected to define conditions under which excess Hlp is required and the roles of Hlp in DNA organization, DNA protection, and DNA double strand break repair. This project will integrate research and education by offering the opportunity for both graduate and undergraduate students to acquire research experience and proficiency in techniques associated with analysis of protein-protein and protein-nucleic acid interactions. The involvement of minority students will be emphasized, and students at all levels will be expected to disseminate their research at national meetings.