CHAPTER 1 Introduction to [Fe-S] cluster biosynthesis: the requirement of a controlled expression system in A. vinelandii. Iron-sulfur [Fe-S] clusters are one of nature’s simplest and most functionally versatile co-factors (Beinert et al. 1997; Fontecave 2006). Proteins that contain [Fe-S] clusters are often referred to as [Fe-S] proteins and they participate in essential biological processes such as photosynthesis, respiration and nitrogen fixation. The functions of [Fe- S] clusters can include electron transfer, catalysis, environmental sensing or structural integrity. The simplest and most common types of [Fe-S] clusters are [2Fe-2S]- and [4Fe-4S] clusters that are usually attached to their protein partners via cysteine ligands. The biosynthesis of [Fe-S] clusters and assembly into target proteins occurs in a controlled manner involving a number of biosynthetic proteins, some of which are highly conserved (with regard to their basic features, functions and primary sequences) in both prokaryotes and eukaryotes. Proteins involved in [Fe-S] cluster biosynthesis were first identified in the Gram-negative, nitrogen-fixing bacterium, Azotobacter vinelandii (Zheng et al. 1993). Early genetic studies identified two major gene regions involved in [Fe-S] cluster biosynthesis: the nitrogen-fixation- (nif)-specific biosynthetic genes (Zheng et al. 1993) and the ‘housekeeping’ iron sulfur cluster (isc) biosynthetic genes (Zheng et al. 1998). The nif-specific genes encode proteins involved in the assembly of [Fe-S] clusters necessary for biological nitrogen fixation and are only expressed under diazotrophic conditions. The isc operon, encodes proteins required for the biosynthesis and maturation of [Fe-S] proteins necessary for general ‘housekeeping’ cellular activities like respiration and carbon assimilation. The Nif and Isc systems share two common core functions involving cysteine desulfurases (NifS and IscS), which act as sulfur donors, and scaffold proteins (NifU and IscU), which act as sulfur and iron acceptors. The Isc system however, has additional accessory proteins, such as molecular chaperones (HscB and HscA), a ferredoxin (Fdx) and another potential scaffold/iron binding protein (IscA) whose function in the [Fe-S] cluster biosynthetic pathway remains unclear and is the subject of intense research. 1 Our current understanding of specific in vivo interactions that take place during [Fe-S] cluster biosynthesis is predominantly derived from in vitro data involving purified proteins which are often combined at concentrations that are unlikely to represent physiological quantities (reviewed in Johnson et al. 2005). Furthermore, the involvement of chaperone proteins with ATPase activity suggest that although IscS/NifS and IscU/NifU alone may be the minimum requirements for cluster formation and transfer in vitro, the process may prove quite different in vivo. Genetic studies to address some of these issues and to confirm the bona fide in vivo role of various components of the isc cluster in [Fe-S] assembly has begun in a number of laboratories. Saccharomyces cerevisiae and Arabidopsis thaliana, which contain homologs to all the A. vinelandii Isc components, serve as excellent genetic models for studying [Fe-S] cluster biosynthesis in eukaryotes (reviewed in Barras et al 2005 and Balk and Lobreaux 2005). Discoveries in these areas continue to have a major impact on our current understanding of the [Fe-S] cluster biosynthetic pathway. Genetic work conducted in prokaryotes has predominantly focused on the isc region from Escherichia coli. Key findings to date include the following: (i) plasmid-directed over-expression of the entire isc operon increases the yield of over-expressed recombinant [Fe-S] proteins that contain a correctly assembled [Fe-S] cluster (Nakamura et al. 1999); (ii) inactivation of iscS ,iscU, iscA, hscBA and fdx individually results in growth defects and a marked decrease in the maturation of a variety of [Fe-S] proteins (Takahashi and Nakamura 1999; Schwartz et al. 2000; Tokumoto and Takahashi 2001); (iii) E. coli contains a second [Fe-S] cluster biosynthetic region, known as Suf, that seems to serve as an alternate housekeeping [Fe-S] cluster system under conditions of oxidative stress (Takahashi and Tokumoto 2002; Outten et al. 2004); (iv) IscS activity is also necessary for the mobilization of sulfur in the biosynthetic pathways of other essential cell components such as biotin, thiamin, thiolated tRNA and Mo cofactor maturation (Lauhon and Kambampati 2000; Skovran and Downs 2000; Leimkuhler and Rajagopalan 2001; Mueller et al. 2001). Chapter 2 provides a comprehensive review, published in the Annual Reviews of Biochemistry, that summarizes our current knowledge of [Fe-S] cluster biosynthesis. 2 In A.vinelandii, unlike E.coli, it has not been possible to isolate strains with individual deletions in iscS, iscU, iscA, hscB, hscA or fdx (Zheng et al. 1998). This has led to the assumption that most, if not all, of these “housekeeping genes” provide the only machinery available to the cell for performing essential [Fe-S] cluster biosynthetic functions. The A. vinelandii genome does not contain a suf operon counterpart, therefore using this bacterium as our model system offers us an advantage in attempting to study the detailed roles of the isc proteins since we can conduct mutational analyses on the isc operon without the complication of a second “housekeeping” region. It also offers us the opportunity to investigate the basis of the apparent target specificity between the Isc and Nif systems, which unlike the E. coli Isc and Suf sytems, do not seem capable of performing redundant functions. In order to conduct an extensive functional analysis of the essential isc operon of A. vinelandii, a non-plasmid based genetic system was developed to control the expression of the isc genes, providing the opportunity to investigate the physiological effects resulting from the depletion of an individual isc gene product under different growth conditions. This system essentially involves strains which contain two copies of the isc operon, the endogenous copy which is controlled by its normal promoter and a second copy controlled by an inducible promoter. The effect of deletions or mutations placed in genes within the first, endogenous isc copy can be monitored by repression of gene transcription from the second intact isc copy. Development of this controlled expression system in A. vinelandii was made possible by the discovery of a promoter within the sucrose metabolic region of the A. vinelandii genome that can be fused to any number of target proteins and whose expression can be controlled by the presence or absence of sucrose in the growth media. The development and the application of this controlled expression system in A. vinelandii has been the main focus of my work. A full description of the construction of this genetic system and how it was used to identify new potential roles for certain Isc components is presented in Chapter 3 as a manuscript submitted for publication in the Journal of Bacteriology. This system was also used to further our understanding of 3 potential overlapping functions between the Nif and Isc systems of A. vinelandii. In Chapter 4, experiments which I performed in collaboration with Dr. Patricia Dos Santos, are described in two parts: Part 1 presents experimental work that was published in Biochemical Society Transactions showing that under normal laboratory growth conditions, the Isc and Nif systems are not functionally equivalent and therefore incapable of performing overlapping functions. Part 2 describes more recent experimental work showing conditions under which overlapping functions between the two systems can be forced. This data provides new clues as to what features of each system determines target specificity. The development of an arabinose-based protein over-expression system in A. vinelandii by Dr. Dos Santos has allowed substantial progress to be made in this area, and experiments in Part 2 are part of a manuscript in preparation of which I am second author. In Appendix 1, evidence is presented which highlights a potential use of this system for the purification of physiologically produced, apo-forms of [Fe-S] proteins. A full list of all the plasmids and strains constructed for the purpose of this study are described in Appendices 2 and 3., respectively. Finally, Chapter 5 summarizes the main findings of this work and how it contributes to the field of [Fe-S] cluster biogenesis. An attempt is made to evaluate some of the key observations regarding IscA and HscBA and to propose new hypotheses for a specific role in the biosynthetic process. Future experimental work is proposed, emphasizing the value of a genetic approach in identifying possible interactions between members of the Isc machinery. 4 CHAPTER 2 Literature Review: Structure, Function and Formation of Biological Iron-Sulfur Clusters Published in: Annual Reviews of Biochemistry 74:247-81 (2005) Deborah Johnson1, Dennis R. Dean1, Archer D. Smith2, Michael K. Johnson2 This manuscript describes the background and recent literature on the three known [Fe-S] cluster biosynthetic machineries (Nif, Isc and Suf systems) which are involved in the formation of biological [Fe-S] clusters and the maturation of [Fe-S] proteins. Emphasis is placed on the similarities and specificities of each system based on genetic, physiological, biochemical and structural data that has accumulated since the discovery of the Nif system from Azotobacter