Membrane-Protein Diffusion in E. coli: A Random Walk in a Heterogeneous Landscape

The function of cvpA, a bacterial gene predicted to encode an inner membrane protein, is largely unknown. Early studies in E. coli linked cvpA to Colicin V secretion and recent work revealed that it is required for robust intestinal colonization by diverse enteric pathogens. In enterohemorrhagic E. coli (EHEC), cvpA is required for resistance to the bile salt deoxycholate (DOC). Here, we carried out genome-scale transposon-insertion mutagenesis and spontaneous suppressor analysis to uncover cvpA’s genetic interactions and identify common pathways that rescue the sensitivity of a ΔcvpA EHEC mutant to DOC. These screens demonstrated that mutations predicted to activate the σE-mediated extracytoplasmic stress response bypass the ΔcvpA mutant’s susceptibility to DOC. Consistent with this idea, we found that deletions in rseA and msbB and direct overexpression of rpoE restored DOC resistance to the ΔcvpA mutant. Analysis of the distribution of CvpA homologs revealed that this inner membrane protein is conserved across diverse bacterial phyla, in both enteric and non-enteric bacteria that are not exposed to bile. Together, our findings suggest that CvpA plays a role in cell envelope homeostasis in response to DOC and similar stress stimuli in diverse bacterial species. IMPORTANCE Several enteric pathogens, including Enterohemorrhagic E. coli (EHEC), require CvpA to robustly colonize the intestine. This inner membrane protein is also important for secretion of a colicin and EHEC resistance to the bile salt deoxycholate (DOC), but its function is unknown. Genetic analyses carried out here showed that activation of the σE-mediated extracytoplasmic stress response restored the resistance of a cvpA mutant to DOC, suggesting that CvpA plays a role in cell envelope homeostasis. The conservation of CvpA across diverse bacterial phyla suggests that this membrane protein facilitates cell envelope homeostasis in response to varied cell envelope perturbations.

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Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2109169119 ◽

2021 ◽

Vol 119 (1) ◽

pp. e2109169119

Author(s):

Kristen A. Gaffney ◽

Ruiqiong Guo ◽

Michael D. Bridges ◽

Shaima Muhammednazaar ◽

Daoyang Chen ◽

...

Keyword(s):

Membrane Protein ◽

Lipid Bilayer ◽

Lipid Bilayers ◽

Limited Proteolysis ◽

Water Soluble ◽

Disordered Proteins ◽

Resonance Spectroscopy ◽

Denatured State ◽

E Coli ◽

State Ensemble

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron–electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli’s lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

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