A Junction between Differentiating Bacterial Cells 公开

Abstract

A Junction between Differentiating Bacterial Cells
By Jeffrey Meisner

When nutrient availability is insufficient to sustain growth, Bacillus subtilis can form a metabolically dormant and environmentally resistant cell type called an endospore. Endospore formation involves the differentiation of two adjacent daughter cells, the mother cell and forespore. The differentiation of these two cells involves an ordered series of morphological changes governed by parallel and interlocked transcriptional programs. The transition from the early to late transcription programs is thought to be controlled by a junction composed of the eight SpoIIIA proteins in the mother cell and SpoIIQ in the forespore. Based on remote homology between SpoIIIAH and the YscJ family of ring-forming proteins, we hypothesized that SpoIIIAH and SpoIIQ form a channel through which the mother cell and forespore communicate. In support of this hypothesis, we demonstrated that the extracellular domains of SpoIIIAH and SpoIIQ are accessible to modification by an enzyme produced in the forespore. To begin to understand molecular basis for the assembly of the putative channel, we examined the interaction of the purified extracellular domains of these proteins. We demonstrated that the putative ring-forming YscJ domain of SpoIIIAH recognizes the degenerate LytM domain of SpoIIQ. By analogy with YscJ proteins, we hypothesized that putative SpoIIIAH channel serves as a structural scaffold for the assembly of a specialized export apparatus consisting of the other seven SpoIIIA proteins. Consistent with this hypothesis, we showed that SpoIIIAA is homologous to the superfamily of ATP hydrolases that provide the energy for various types of protein secretion systems. We also analyzed the predicted membrane topology of SpoIIIAB, SpoIIIAC, SpoIIIAD, and SpoIIIAE, and identified important residues in each. Together, these data support a new model for understanding the mechanisms that control the differentiation of the mother cell and forespore during endospore formation.

Table of Contents

TABLE OF CONTENTS

Chapter 1. General Introduction...1

Chapter 2. A channel connecting the mother cell and forespore...25

Chapter 3. A LytM domain dictates the localization of proteins to the mother cell-forespore interface...62
Chapter 4. SpoIIIAA is a secretion superfamily ATPase...100

Chapter 5. Bioinformatics analysis and site-directed mutagenesis of SpoIIIAB, SpoIIIAC, SpoIIIAD, and SpoIIIAE...125

Chapter 6. General Discussion...154

LIST OF FIGURES AND TABLES

Chapter 1

Figure 1. Morphological changes during sporulation...13
Figure 2. Regulatory network controlling the decision to initiate sporulation...14
Figure 3. Parallel and interlocked transcriptional programs governing the differentiation of the forespore and mother cell...15

Chapter 2

Figure 1. Similarity between SpoIIIAH and YscJ/FliF protein family...42
Figure 2. Compartmentalized biotinylation assay...43
Figure 3. Western blot analysis of BirA accumulation...44
Figure 4. Forespore-specific biotinylation of C-terminal BAP-tagged SpoIIIAH...45
Figure 5. Compartmentalization of SpoIIIAH-BAP production and BirA activity...46
Figure 6. Forespore-specific biotinylation of SpoIIIAH-BAP in a sigG mutant...47
Figure 7. Forespore-specific biotinylation of C-terminal BAP-tagged SpoIIQ...48
Figure 8. Degradation of biotinyl-SpoIIIAH-BAP after engulfment...49
Table 1. Bacterial strains...50
Table 2. Plasmids...52
Table 3. Oligonucleotide primers...53
Table 4. Sporulation efficiencies of strains carrying BAP-tagged alleles...54

Chapter 3

Figure 1. Cartoon representation of the SpoIIIAH-SpoIIQ complex...79
Figure 2. Sequence alignment of SpoIIQ and S. aureus LytM (1QWY) based on HHpred...81
Figure 3. Biochemical analysis of the SpoIIIAH-SpoIIQ complex...82
Figure 4. Gel filtration chromatography of the interaction of and SpoIIQ43-283 and truncated SpoIIIAH proteins...84
Figure 5. Gel filtration chromatography of the interaction of and SpoIIIAH25-218 and truncated SpoIIQ proteins...85
Figure 6. Gel filtration chromatography of the interaction of and SpoIIIAG51-229 with SpoIIIAH25-218, SpoIIQ43-283, or the SpoIIIAH25-218 - SpoIIQ43-283 complex...87
Figure 7. HHpred sequence alignment of SpoIIIAG and YscJ-FliF family (PF01514)...89
Table 1. Oligonucleotide primers...90
Table 2. Plasmids...91
Table 3. Bacterial strains...92
Table 4. Thermodynamic parameters determined by ITC for the interaction of truncated SpoIIIAH proteins (syringe) and SpoIIQ43-283 (sample cell)...93
Table 5. Thermodynamic parameters determined by ITC for the interaction of truncated SpoIIQ proteins (syringe) and SpoIIIAH25-218 (sample cell)...94

Chapter 4

Figure 1. Sequence alignment of SpoIIIAA and archaeal secretion superfamily ATPase GspE (Archaeoglobus fulgidus)...112
Figure 2. Multiple sequence alignment of N2-C1 sub-domains from SpoIIIAA orthologs...113
Figure 3. Subdomain composition of GspE, PilT, VirB11, and SpoIIIAA...114
Figure 4. Comparative structural model of the SpoIIIAA N2-C1 subdomains using afGspE as the template...115
Figure 5. Effects of spoIIIAA mutations on σ^G activity...116
Table 1. Oligonucleotide primers...117
Table 2. Plasmids...118
Table 3. Bacterial strains...119
Table 4. Complementation of spoIIIAA deletion by wild-type and mutant alleles...120
Table 5. Complementation of spoIIIAA deletion by orthologous alleles...121

Chapter 5

Figure 1. Multiple sequence alignment of SpoIIIAB orthologs...135
Figure 2. Multiple sequence alignment of SpoIIIAC orthologs...136
Figure 3. Multiple sequence alignment of SpoIIIAD orthologs...137
Figure 4. Multiple sequence alignment of SpoIIIAE orthologs...138
Table 1. Oligonucleotide primers...140
Table 2. Plasmids...143
Table 3. Bacterial strains...145
Table 4. Predicted transmembrane segments of SpoIIIAB, SpoIIIAC, SpoIIIAD, and SpoIIIAE...147
Table 5. Complementation of spoIIIAB deletion by mutant spoIIIAB alleles...148
Table 6. Complementation of spoIIIAC-spoIIIAD deletion by mutant spoIIIAC-spoIIIAD alleles...149
Table 7. Complementation of spoIIIAE deletion by mutant spoIIIAE alleles...150

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Microbiology
关键词	sporulation cellular differentiation Bacillus subtilis intercellular junction
Committee Chair / Thesis Advisor	Moran Jr., Charles, Emory University
Committee Members	Shafer, William M, Emory University Rather, Phil N, Emory University Churchward, Gordon, Emory University Scott, June R, Emory University

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