Regulation and Function of the Swarming Inhibitor disA in Proteus mirabilis Open Access
Barker, Bree Ann Szostek (2013)
Abstract
Proteus mirabilis is a Gram negative bacterium known for its form of multicellular surface motility, termed swarming. After encountering a solid surface P. mirabilis differentiates from a vegetative rod into an enlongated, hyperflagellated swarmer cell. Swarmer cells interact to migrate away from the central inoculum. After migration the cells undergo a process of consolidation where swarmer cells return to their vegetative form to prepare for another onset of swarming. While a great deal is known about the regulation and initiation of differentiation and migration, little is known about how the cell undergoes consolidation. Our lab previously identified a decarboxylase inhibitor of swarming, disA, which completely abolishes swarming when overexpressed. Homology to aromatic amino acid decarboxylases and an increase in disA transcription over the swarm cycle led us to hypothesize DisA may produce a consolidation signal, particularly phenethylamine. We have further characterized the disA locus by identifying the transcriptional start site as a guanine residue seventy basepairs upstream of the open reading frame. Site-directed mutagenesis also identified an extended -10 promoter element. Further work identified mutation of umoB, the P. mirabilis homologue of igaA, increased disA expression and demonstrated the upregulation is mediated by the Rcs phosphorelay. Additionally, exogenous addition of phenethylamine decreased disA expression. We have also demonstrated overexpressing disA is able to inhibit motility in other Gram negative organisms known to swarm, but does not affect organisms with flhDC independent motility. Significant work has been performed to identify the product and substrate of DisA. Metabolomic analysis of disA mutant and overexpressing strains suggests DisA may be involved in regulating swarming by modifying the levels of central metabolic intermediates. Supplementation of media with 10mM succinate, fumarate or malate, three intermediates of the tricarboxylic acid cycle (TCA), increased swarming motility of a disA mutant strain under anaerobic conditions. Tested TCA cycle intermediates had no effect on motility of the disA mutant during aerobic growth. Wild-type motility was also unaffected by all tested metabolites under aerobic and anaerobic growth. These data suggest DisA may function to regulate swarming by altering the levels of central metabolites in the cell, particularly fumarate and aspartate.
Table of Contents
Abstract Acknowledgements
Table of Contents
List of Tables and Figures
Chapter 1: Introduction........................................................................................................................................................................1
Chapter 2: Regulation of gene expression during swarmer cell differentiation in Proteus mirabilis....................................................................54
Chapter 3: Expression of the DisA amino acid decarboxylase from Proteus mirabilis inhibits motility and class 2 flagellar gene expression in
Escherichia coli.................................................................................................................................................................................93
Chapter 4: Regulation of the swarming inhibitor disA in Proteus mirabilis...................................................................................................121
Chapter 5: Supplementary Data..........................................................................................................................................................154
Chapter 6: Discussion.......................................................................................................................................................................178
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