Redox Control and Mechanism of Spx-activated Transcription in Bacillus subtilis By Cierra A. Birch A Dissertation Presented to the Division of Environmental and Biomolecular Systems at Oregon Health and Science University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology School of Medicine Oregon Health and Science University July 2017 Table of Contents List of tables………………………………………………………………………………………………...............vi List of figures………………………………………………………………………………………………….........vi Acknowledgements……………………...………………………………………………………………….....viii Abstract ………………………………………………………………………………………………………...….…ix Chapter 1: Introduction…………………………………………………………………………………............................…1 Introduction to Bacillus subtilis and bacterial stress response…………………………..……..1 ROS production and oxidative stress response………………………….………..............................2 RNA polymerase and transcriptional control……………………………………………………….....7 RNA polymerase (RNAP): structure and assembly………………………………………….........7 Overview of bacterial transcription…………………………………………....................................10 Recruitment-based activated-transcription…………………………………………....................11 Activated-transcription by RNAP appropriation………...………………………………...........13 B. subtilis SigB-dependent general stress response.………………………………….…………...16 Redox control of oxidative stress response.………………………………………………….……....17 Introduction to Spx...................................................................................................................................19 B. subtilis Spx, a global transcriptional regulator…………………………………………..........19 Spx in other bacteria………………………………………………………………………………………....22 Control of Spx………………………………………………………………………………………………......23 Major research aims…………………………………………........................................................................30 Chapter 2, Part I: Exploring the role of the RNAP α subunit in Spx-activated transcription..............32 Introduction…………………………………………........................................................................................32 Results…………………………………………...................................................................................................37 Alignment of B. subtilis αCTD with those of other classes of bacteria.........................37 Complementation approach to uncover the role of putatively essential ii αCTD residues in Spx-activated transcription.......................................................................39 Effect on disulfide stress sensitivity of ectopically expressed rpoA mutant alleles. …………………………………….................................................................................42 Effect of ectopically expressed rpoA alleles on Spx-activated transcription in vivo. …………………………....................................................................................44 Salt stress uncovers defects in Spx-activated expression in cells expressing rpoAR261A, rpoAE255A and rpoAE298A mutant alleles……………....…..46 Evidence the rpoAE255A does not function in αCTD-σA interaction during Spx-activated transcription...........................................................................................................50 Purification of active RNAP from strains bearing ectopically expressed rpoA-His6 and mutant derivatives…..........................................................................................51 The rpoAE255A, rpoAE298A and rpoAR261A mutations confer defects in Spx interaction with RNAP and/or α subunit..................................................................53 rpoAE255A and rpoAE298A mutations reduce Spx-enhanced affinity of RNAP for promoter DNA…........................................................................................................59 Discussion…...............................................................................................................................................61 Chapter 2, Part II: Mutational analysis of possible σA-interacting Spx amino acid residues............65 Introduction...............................................................................................................................................65 Possible multi-subunit contact between Spx and multiple subunits of RNAP.......65 Structural determinants of Spx activity..................................................................................66 Spx amino acid residues K43, D40, and Q77 may function in Spx-RNAP interaction...........................................................................................................................................69 Results..........................................................................................................................................................73 Differential structural requirements of Spx activity among members of the Spx regulon...................................................................................................................................................73 sigAR362A affects control of Phyperspank- expressed proteins………...…………………73 iii Discussion...................................................................................................................................................77 Materials and methods (Part I).........................................................................................................76 Materials and methods (Part II).......................................................................................................76 Chapter 3: Redox control and Spx-dependent regulation of thiol homeostasis genes ........91 Introduction...............................................................................................................................................91 Thiol stress and redox buffering................................................................................…………...91 Functions of bacillithiol (BSH) .....................................................................................................93 Role of bacillithiol in other bacteria...........................................................................................97 Biosynthesis of BSH...........................................................................................................................98 Genomic organization of B. subtilis BSH biosynthetic genes.........................................101 B. subtilis ypjD operon organization is conserved in B. anthracis...............................102 Cis-acting elements of Spx-activated promoters...............................................................103 Architecture of the bsh operon promoters...........................................................................105 Results........................................................................................................................................................109 Effects of PypjD truncations on Spx-dependent activation..........................................109 Differential redox requirements of Spx activity among the Spx regulon……....…111 Differential redox requirements of B. subtilis Spx during activation of B. subtilis ypjD and B. anthracis ypjD (BA1554)………………......................................113 Effects of ypjD promoter mutations on Spx-dependent activation...........................117 Discussion..................................................................................................................................................121 Speculations on the functional significance of the ypjD operon organization….124 Materials and methods.......................................................................................................................109 Chapter 4: Summaries, future directions, and final remarks..........................................................132 Exploring the role of the RNAP α subunit in Spx-activated transcription.............132 Mutational analysis of possible σA-interacting Spx amino acid residues...............133 iv Redox control and Spx-dependent regulation of thiol homeostasis genes…........135 Final remarks..........................................................................................................................................138 v Tables Table 2.1 B. subtilis strains and plasmids used in Chapter 2: Part I……………….……...86 Table 2.2 Oligonucleotides used in Chapter 2: Part I………………………..…...……….….....87 Table 2.3 B. subtilis strains and plasmids used in Chapter 2: Part II…….…..………......89 Table 2.4 Oligonucleotides used in Chapter 2: Part II………………………………………......90 Table 3.1 B. subtilis strains and plasmids used in Chapter 3..……………….………….…130 Table 3.2 Oligonucleotides used in Chapter 3…………………………………………..………..131 Figures Figure 1.1 ROS production....................................................................................................................4 Figure 1.2 ROS and cysteine thiol oxidation..................................................................................6 Figure 1.3 RNAP holoenzyme subunits............................................................................................9 Figure 1.4 Recruitment-based transcriptional activation…………………………....…….…12 Figure 1.5 Pre-recruitment-based transcriptional activation….........................................15 Figure 1.6 Spx functions as a transcriptional activator and repressor...........................20 Figure 1.7 ClpXP-dependent proteolytic control of