Date of Degree

2-2017

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Cheng, Haiping

Committee Members

Edward Jarroll

Renuka Sankaran

David Calhoun

Jonathan Dworkin

Subject Categories

Bacteriology | Cell and Developmental Biology | Cell Biology | Laboratory and Basic Science Research | Microbial Physiology | Molecular Genetics

Keywords

S. meliloti, Medicago sativa, alfalfa, ExoR, microbe interaction, invasion switch

Abstract

The free-living Gram-negative soil bacterium, Sinorhizobium meliloti, must switch into its host-invading form in order to infect the root hairs of the host plant, alfalfa (Medicago sativa), and establish a nitrogen-fixing symbiosis. The activation of the switch is believed to occur inside the infection chamber that is formed by curling of the root hairs. It is not fully understood what signals in the environment of the root hairs trigger and infection chamber S. meliloti to switch into a host-invading form since these signals were not extensively examined until now. This switch can be observed directly, due to the inverse relationship between flagella and succinoglycan production seen in S. meliloti. The free-living S. meliloti synthesize flagella but not succinoglycan, while the host-invading cells produce succinoglycan but not flagella. The ExoR-ExoS/ChvI (RSI) Invasion Switch, is controlled mainly through the amount of the periplasmic ExoR protein. The ExoR protein functions as the repressor of the ExoS in the ExoS/ChvI two-component system, so that the reduction of ExoR leads to the activation of succinoglycan production, suppression of flagella production, and expression of hundreds of genes regulated by the ExoS/ChvI system. The RSI Invasion Switch also has an auto-regulatory mechanism to ensure that switch is seemingly turned on in the presence of high and persistent host signals. The presence of auto-regulation of the exoR gene expression by its own protein and the original search being focused on the ExoS, instead of recently, discovered ExoR has contributed to the delay in identifying signals sensed by the RSI Invasion Switch. In this study, a new screening system was devised to search for potential host signals that influence the RSI Invasion Switch. A loss of function exoR mutant carrying the plasmid with the exoR gene expressed from a weak promoter was exposed to potential signals, including chemicals and differing growth conditions. In the absence of host signals, the ExoR expressed from a weak promoter is sufficient to suppress the succinoglycan and flagella phenotypes of the exoR mutant. The presence of true host signals would lead to a reduction of ExoR that could no longer suppress the loss of function exoR mutation, reverting to the mutant phenotype. Altogether, this allowed us to identify a set of conditions that affected the state of the RSI Invasion Switch by turning it on or off. These conditions were further examined for their abilities to inversely change the expression of both flagellum and succinoglycan biosynthesis genes, along with their abilities to change the levels of periplasmic ExoR. With the discovery of close homologs of the RSI Invasion Switch in 47 other plant and animal pathogens, our findings of potential host-invasion signals could greatly enhance the effort to control these pathogens, in addition to improving nitrogen-fixing symbiosis.

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