Characterisation of the AsmA-like protein family in Escherichia coli

Holyfield, Rachel B ORCID: 0009-0003-9387-0733 (2025). Characterisation of the AsmA-like protein family in Escherichia coli. University of Birmingham. Ph.D.

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Abstract

Understanding the mechanisms of cell envelope biogenesis in Gram-negative bacteria could reveal novel therapeutic targets to combat rising antimicrobial resistance. Despite decades of research, the fundamental process of phospholipid transport from the inner membrane to the outer membrane of the Gram-negative cell envelope has long remained an elusive process. Recently, the AsmA-like superfamily have emerged as likely contenders of this intermembrane phospholipid transport. With six AsmA-like members in E. coli (YhdP, TamB, YdbH, YhjG, AsmA and YicH) and mostly unknown structures and functions for each, many questions have now emerged about their exact role in the cell and their potential mechanisms of action. Here, we present the phenotypic profiling of the asmA-like superfamily, supporting recent evidence of an essential and redundant function in outer membrane biogenesis between yhdP and tamB, identifying a novel gain-of-fitness phenotype and providing evidence of a genetic interaction between yhdP and asmA. We also describe trials to express and purify these proteins, discovering that they are often prone to breakdown and aggregation. Nevertheless, we then present low-resolution cryo-EM structures of YhjG and YicH, the first experimental structures of any of the full-length AsmA-like proteins. We show the first evidence of phospholipid transport via YdbH, YhjG and YicH using an in vitro FRET-based phospholipid transport assay and reveal a spontaneous mechanism of phospholipid entry through coarse-grained molecular dynamics simulations. This investigation sheds light on the role of AsmA-like proteins in phospholipid transport and lays the groundwork for future structural and mechanistic studies of both this protein family and other phospholipid transporter proteins.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):