Abstract

For single-celled organisms, the cell envelope is the thin barrier between life and death, between the self and the environment. Their survival depends on the integrity and maintenance of this envelope. The cell envelope of Gram-negative organisms is tripartite, consisting of a phospholipid inner membrane, an aqueous periplasmic space containing the peptidoglycan sacculus, and an asymmetric outer membrane bearing phospholipids on the inner leaflet and lipopolysaccharides (LPS) on the outer leaflet. Surface-exposed, highly immunoreactive, and essential to nearly all Gram-negative bacteria, LPS has been extensively studied for its roles in pathogenesis and immunobiology and as a potential therapeutic target for novel antibiotics. Despite advances in these areas, another aspect of LPS biology – its contribution to overall Gram-negative bacterial physiology – remains unclear. Although LPS has been implicated in various aspects of physiology, the primary essential contribution(s) of LPS to Gram-negative physiology are not known. This dissertation identifies factors – both direct and indirect – which render LPS essential to Gram-negative physiology. LPS has been implicated in both cell envelope rigidity and the coordination of cell division and separation. In Chapter 2, I use a combination of chemical and genetic approaches to test which of these roles – if either – is responsible for LPS essentiality in Escherichia coli and Klebsiella pnuemoniae. Further, I test the hypothesis that asynchronous biogenesis of the inner and outer membranes during LPS depletion is an additional factor contributing to LPS essentiality. I demonstrate that, although LPS is critical for cell envelope integrity, preserving cell envelope integrity is not sufficient to preserve culturability of LPS-depleted cells. Using a candidate gene screen to identify mutants with enhanced survival during LPS depletion, I demonstrate that deletion of factors which remove peptidoglycan crosslinks, which is predicted to increase cell wall rigidity, reduces susceptibility to the LPS synthesis inhibitor CHIR-090. However, these mutations are not sufficient to allow survival in the absence of LPS. Altogether, these data are consistent with a model in which cell envelope rigidity is one – but not the sole – factor responsible for LPS essentiality. In the same candidate gene screen, I found that mutations expected to enhance phospholipid retention in the outer leaflet of the outer membrane reduce susceptibility to CHIR-090. This finding is consistent with work in Acinetobacter baumannii demonstrating that biogenesis of the outer membrane in the absence of LPS is rate-limiting for growth. Further, mutations expected to reduce global lipid synthesis, thus reducing inner membrane synthesis to better match reduced outer membrane synthesis during LPS depletion, similarly reduce susceptibility. This data is consistent with known mechanisms of CHIR-090 resistance, which are thought to exhibit the same effect on membrane biogenesis rates. Although LPS has been implicated in cell division since the 1970s, the nature of that connection remains unclear. In this dissertation, I demonstrate that general enhancement of cell division is not sufficient to enhance survival of cells during LPS depletion. Instead, only the gain-of-function allele ftsA* reduced susceptibility to CHIR-090. Although the precise nature of this connection remains unclear, these results implicate the cell division protein FtsA in LPS essentiality by an unknown mechanism. Additionally, I identified two spontaneous mutants – a 6-bp deletion in marR, a regulator of multiple antibiotic resistance, and a transposition of an IS element into the promoter of the gene encoding Lon protease – that substantially reduce susceptibility to CHIR-090 despite exhibiting a decrease in LPS content of approximately 60% relative to untreated cells. Both mutations seem to result in accumulation of the positive regulator of multiple antibiotic resistance MarA. Overexpression of marA is sufficient to enhance survival during LPS depletion by both lpxC tCRISPRi and CHIR-090 treatment. These data argue against a strictly efflux model of resistance and suggest an as-yet undescribed mechanism of increased tolerance to LPS depletion. As a whole, this dissertation provides insight into the essential contributions of LPS to Gram-negative physiology, specifically through cell envelope rigidity, membrane homeostasis, and cell division. Further work in this area will provide a clearer understanding of Gram-negative physiology and, ultimately, a more thorough understanding of how to treat Gram-negative infections.

Committee Chair

Petra Levin

Committee Members

David Rosen; Enrique Rojas; Jennifer Philips; Michael Caparon; Ram Dixit

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Plant & Microbial Biosciences)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

8-8-2025

Language

English (en)

Author's ORCID

https://orcid.org/0000-0002-5356-4198

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Available for download on Saturday, August 07, 2027

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