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Author ORCID Identifier

https://orcid.org/0000-0002-8101-5604

Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Microbiology

Year Degree Awarded

2019

Month Degree Awarded

May

First Advisor

Yasu S. Morita

Second Advisor

Michele M. Klingbeil

Third Advisor

Sloan Siegrist

Fourth Advisor

Peter Chien

Subject Categories

Bacteriology | Biochemistry | Microbial Physiology | Molecular Genetics

Abstract

The integrity of the multilaminate cell envelope surrounding mycobacteria is critical for survival and pathogenesis. The prevalence of phosphatidylinositol mannosides in the cell envelope suggests an important role in the mycobacterial life cycle. Indeed, deletion of the pimE gene (ΔpimE) encoding the first committed step in phosphatidylinositol hexamannoside biosynthesis in Mycobacterium smegmatis results in the formation of smaller colonies than wild-type colonies on Middlebrook 7H10 agar. To further investigate potential contributors to cell-envelope mannan biosynthesis while taking advantage of this colony morphology defect, we isolated spontaneous suppressor mutants of ΔpimE that reverted to wildtype colony size. Of 22 suppressor mutants, six accumulated significantly shorter lipomannan or lipoarabinomannan. Genome sequencing of these mutants revealed mutations in genes involved in the lipomannan/lipoarabinomannan biosynthesis, such as those encoding the arabinosyltransferase EmbC and the mannosyltransferase MptA. Furthermore, we identified three mutants carrying a mutation in a previously uncharacterized gene, MSMEG_5785, that we designated lmeA. Complementation of these suppressor mutants with lmeA restored the original ΔpimEphenotypes, and deletion of lmeA in wildtype M. smegmatis resulted in smaller lipomannan as observed in the suppressor mutants. LmeA carries a predicted N-terminal signal peptide, and density gradient fractionation and detergent extractability experiments indicated that LmeA localizes to the cell envelope. Using a lipid ELISA assay, we found that LmeA binds to plasma membrane phospholipids such as phosphatidylethanolamine and phosphatidylinositol. LmeA is widespread throughout the Corynebacteriales; therefore, we concluded that LmeA is an evolutionarily conserved cell-envelope protein critical for controlling the mannan chain length of lipomannan/lipoarabinomannan.

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