In partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Biology

In the

School of Biological Sciences

Siu Lung (Michael) Ng

Will defend his dissertation

Evolution of Bacterial Type VI Secretion System Regulation and Defense 

18th August 2022

2 p.m.

Price Gilbert Library 4222 or https://gatech.zoom.us/j/7036781501

 Thesis Advisor:

Brian Hammer, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Committee Members:

Marvin Whiteley, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

William Ratcliff, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Frank Stewart, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Department of Microbiology and Immunology

Montana State University

Peter Yunker, Ph.D.

School of Physics

Georgia Institute of Technology

ABSTRACT:

Bacteria in nature live in communities with other organisms where resources and space are typically lacking. To compete, bacteria have evolved strategies to increase fitness by eliminating unwanted competitors with secreted goods such as traditional, diffusible antibiotics. A more recently described mechanism of antagonism is a contact-dependent ”nanoharpoon”, the type VI secretion system (T6), which kills neighboring ”target” cells by directly injecting lethal toxins.

The broadly distributed T6 weapon plays an important role in competition and the pathogenicity of many Gram-negative bacteria including Vibrio cholerae, with prior studies demonstrating a role for the V. cholerae T6 in host colonization and infection. The majority of our understanding of this apparatus in V. cholerae is based on studies with isolates that contribute to disease. The current regulatory model of T6 control describes a requirement of one of the two transcription factors: QstR, which is induced by external chemical cues, or TfoY, which is activated by intracellular signals. By contrast, our knowledge of the T6 of V. cholerae strains from non-human sources is more limited. So too, the structure of the T6 apparatus, the toxic effector proteins delivered by it, and their corresponding immunity proteins have garnered much attention. Yet we know little about how microbes may protect

themselves against T6 attack.

In my dissertation, I describe two major contributions to the field. First, I determined that QstR and TfoY are either dispensable or only play a minor role in V. cholerae strains isolated from nonhuman sources, which instead display constitutive T6 activity in laboratory conditions. To begin to determine the mechanism responsible for regulatory differences between human and nonhuman strains, I successfully mapped the promoter region of the major T6 gene cluster and identified a single-nucleotide polymorphism (SNP) in the DNA sequence upstream of the promoter that affects the regulation of T6. Human-derived, QstR-regulated strains encode a guanine (G) at this position, while environmental strains carry a thymine (T), with varying contributions of TfoY. These results are consistent with a ”pathoadaptive” theory that V. cholerae dampens the T6 regulation during infection and displays a constitutive T6 activity in natural environments. Second, I participated in the discovery that target cells have two major mechanisms to resist T6 attacks. Using experimental evolution, I help uncover that Escherichia coli becomes less susceptible to T6 antagonism following several hundred generations of repeated T6-mediated competition. We identified three genes that contribute to the T6 resistance: apaH disruption, a specific yjeP missense mutation, and yejM mutations that result in C-terminus disruptions. The T6 resistance is greater when E. coli carries both the yjeP and yejM mutations. These mutations however are pleiotropic, reducing growth rate and causing other collateral effects, supporting a hypothesis that evolution of T6 resistance in natural communities is likely constrained by fitness effects. This dissertation provides insight into how T6 aggression and resistance are two facets of an evolutionary arms race: with killer cells evolving strategies for antagonism that provide selective pressure favoring target cells with T6 resistance.