In partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Biology

In the

School of Biological Sciences

Alexander Klementiev

Will defend his dissertation

Probing Bacterial Biofilm Physiology using Electrochemistry and Mass Spectrometry Techniques

20, April, 2023

11 AM

Price Gilbert Library 4222

 Thesis Advisor:

Marvin Whiteley, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Committee Members:

Sam Brown, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Stephen Diggle, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

William Ratcliff, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Neha Garg, Ph.D.

School of Chemistry and Biochemistry

Georgia Institute of Technology

ABSTRACT:

During infection, bacteria form complex, spatially-organized communities that involve physical and chemical interactions. These interactions are key to community function, allowing bacteria to evade host defenses and persist despite an often robust immune response. While imaging technologies have allowed assessment of the spatial organization of these communities, we know little about the chemical environment. In this thesis, I leverage electrochemical and mass spectrometry techniques to study the chemical environment surrounding bacterial biofilms at the micron scale. Using electrochemical methods, including scanning electrochemical microscopy, I discovered that biofilms of the opportunistic pathogen Pseudomonas aeruginosa actively deplete oxygen immediately adjacent to the biofilms, forming stable oxygen gradients that extend over 100 microns from the surface of the biofilm. These oxygen gradients persist even upon exposure to high levels of antibiotics. While electrochemical methods allow for the targeted quantification of specific molecules, untargeted mass spectrometry approaches capture the global chemical profile. Using mass spectrometry, we study the interactions between the oral pathogen Aggregatibacter actinomycetemcomitans and the oral commensal Streptococcus gordonii, which are etiological agents of periodontitis. Among the thousands of molecules detected, we focus on understanding the role of glutathione produced by A. actinomycetemcomitans and the benefit it provides to S. gordonii. Together, these tools provide complementary methods to eavesdrop on the chemical interactions which shape bacterial infections.