An Investigation of the Function of the Periplasmic Domain of BamA, an Essential Protein in Gram-Negative Bacteria

Castagneri, Danielle Andrea

Undergraduate Honors Thesis

An Investigation of the Function of the Periplasmic Domain of BamA, an Essential Protein in Gram-Negative Bacteria Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/8049g549t

Abstract

Gram-negative bacteria make up the majority of dangerous health-care associated infections (HAIs), and are becoming increasingly resistant to standard antibiotics. The Beta Barrel Assembly Machine (BAM) is a protein complex in Gram-negative bacteria composed of BamA, an essential member of the Omp85 family, and four associated lipoproteins B-E. The BAM is present in all Gram-negative bacteria and has a pivotal role in outer membrane protein (OMP) insertion, making it an attractive target for study and the development of new antibiotic agents. This investigation probes the importance of conserved residues in BamA, the central component of the BAM complex, and suggests the hypothesis that it may form higher order assemblies important for outer membrane biogenesis. The ConSurf bioinformatics server was used to identify highly evolutionarily conserved surface amino acids that could have putative functional importance. Tryptophan scanning and complementation assays were carried out to characterize potentially functionally important residues directed by this bioinformatics analysis. In addition, single cysteine residues were introduced in the periplasmic domain of BamA and used in crosslinking experiments in an attempt to trap and identify interacting partners. One tryptophan mutant, R36W, failed to complement, suggesting mechanistic importance. Strong crosslinking was observed in cysteine mutants T53C and R160C, supporting a model where BAM organizes into higher order assemblies to mediate folding and insertion of beta-barrel outer membrane proteins.

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