UC Berkeley

Abstract

Regulation of Biomineralization by an HtrA Protease in Magnetotactic Bacteria

by

Patrick Jeffrey Browne

Doctor of Philosophy in Microbiology

University of California, Berkeley

Professor Arash Komeili, Chair

Magnetotactic bacteria are a unique group of bacteria characterized by their ability to manufacture magnetic crystals for the purpose of aligning with geo-magnetic fields. They offer a unique opportunity for merging the study of bacterial cellular biology, classic molecular genetics, biochemistry, and evolution, both on the small scale of individual proteins, and a larger system of proteins with a defined complex function. This dissertation will first look at the genetics and evolution of magnetotactic bacteria as a whole before focusing on MamE, an HtrA protease conserved throughout magnetotactic bacteria. HtrA proteases are ubiquitous throughout all domains of life and serves several different functions. This dissertation will show that MamE is a multifunctional protein and will also highlight other magnetosome related genes that either regulate MamE’s activity or are targets of proteolysis.

The first chapter of this dissertation is a to-be-submitted review article and introduces the genetics of magnetotactic bacteria (MTB), a paraphyletic group of gram-negative bacteria that use around a hundred genes, located in a defined gene island, to create magnetosomes, membrane-bound compartments containing a magnetic iron crystal. By organizing magnetosomes into a chain, cells are able to align with geo-magnetic fields. It will discuss the most well-conserved genes whose general functions are known. It also describes the interesting evolutionary history of the magnetosome gene island (MAI) which despite being an island seemingly capable of horizontal gene transfer, does not contain other hallmarks of recent HGT and closely matches the phylogenetic tree.

The second chapter, a published primary research article (Hershey et al., PLOS Biology 2016, and) is focused on MamO, a protein that also appears to also be an HtrA protease similar to MamE. However, closer examination shows that is contains no protease activity and is instead a metal-binding protein most likely involved in crystal nucleation. It is also shown to not only be a target of MamE proteolysis but also regulate MamE’s activity. Finally, it contains an analysis of MamOs throughout MTB and shows that they are not actually a single protein family. Instead, four separate lineages of MTB have all acquired inactive proteases in a convergent manner.

The third chapter, a published primary research article (Hershey et al., Journal of Biological Chemistry 2016) is the first to focus primarily on MamE. Unlike MamO, this chapter shows that MamE is a bonafide protease and identifies several targets of MamE, including MamE itself, MamO, and MamP. It also interrogates the kinetics of MamE’s protease activity and shows that MamE processes its targets in a regulated manner.

The fourth chapter, to be submitted as a primary research article, also investigates MamE and its targets, this time beginning with a proteomic approach. Cells carrying an inactive form of MamE have deformed smaller crystals. Using a novel method of cell lysis and magnetosome separation, wild-type and mutant magnetosome are compared. This method uncovered a few potential additional targets of MamE proteolysis, one of which was confirmed in vitro. This protein, MamD was also further investigated and determined to possibly function as a biomineralization inhibitor. This chapter also discusses the proteome of the magnetosome as a whole, defining proteins enriched in the magnetosome

Finally, the fifth chapter contains unpublished material related to MamE and ends with concluding thoughts and perspectives. It includes a preliminary investigation of how magnetosome proteins reach the magnetosome, a poorly understood topic, but one that MamE also plays a role in. It will also identify one further potential target of MamE proteolysis, MamT, and investigates how the magnetochrome domain, shared by MamE, MamP, and MamT is crucial to the function of all three proteins but not in identical ways. Finally, it will include thoughts on ways to move forward in the field of magnetosome genetics and molecular biology.