Open AccessFunctions of the BamBCDE Lipoproteins Revealed by Bypass Mutations in BamA
Author(s) -
Elizabeth M. Hart,
Thomas J. Silhavy
Publication year - 2020
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.00401-20
Subject(s) - bama , biology , bacterial outer membrane , function (biology) , folding (dsp implementation) , protein folding , mutation , mutant , membrane protein , barrel (horology) , microbiology and biotechnology , biochemistry , membrane , escherichia coli , gene , materials science , engineering , electrical engineering , composite material
The heteropentomeric β-barrel assembly machine (BAM complex) is responsible for folding and inserting a diverse array of β-barrel outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria. The BAM complex contains two essential proteins, the β-barrel OMP BamA and a lipoprotein BamD, whereas the auxiliary lipoproteins BamBCE are individually nonessential. Here, we identify and characterize three bamA mutations, the E-to-K change at position 470 ( bamA E470K ), the A-to-P change at position 496 ( bamA A496P ), and the A-to-S change at position 499 ( bamA A499S ), that suppress the otherwise lethal Δ bamD , Δ bamB Δ bamC Δ bamE , and Δ bamC Δ bamD Δ bamE mutations. The viability of cells lacking different combinations of BAM complex lipoproteins provides the opportunity to examine the role of the individual proteins in OMP assembly. Results show that, in wild-type cells, BamBCE share a redundant function; at least one of these lipoproteins must be present to allow BamD to coordinate productively with BamA. Besides BamA regulation, BamD shares an additional essential function that is redundant with a second function of BamB. Remarkably, bamA E470K suppresses both, allowing the construction of a BAM complex composed solely of BamA E470K that is able to assemble OMPs in the absence of BamBCDE. This work demonstrates that the BAM complex lipoproteins do not participate in the catalytic folding of OMP substrates but rather function to increase the efficiency of the assembly process by coordinating and regulating the assembly of diverse OMP substrates. IMPORTANCE The folding and insertion of β-barrel outer membrane proteins (OMPs) are conserved processes in mitochondria, chloroplasts, and Gram-negative bacteria. In Gram-negative bacteria, OMPs are assembled into the outer membrane (OM) by the heteropentomeric β-barrel assembly machine (BAM complex). In this study, we probe the function of the individual BAM proteins and how they coordinate assembly of a diverse family of OMPs. Furthermore, we identify a gain-of-function bamA mutant capable of assembling OMPs independently of all four other BAM proteins. This work advances our understanding of OMP assembly and sheds light on how this process is distinct in Gram-negative bacteria.