Analysis of the Outer Membrane Protein Assembly in Escherichia coli Using an Assembly-Defective Mutant of the Maltoporin LamB

Abstract

The outer membrane (OM) of Gram-negative bacteria acts as a first line of defense against stresses such as toxic molecules in the external environment and antibiotics. With the slow development of antibiotics and the selective pressure against bacteria, antibiotic-resistant strains of bacteria are emerging relatively fast. Outer membrane proteins (OMPs) play a major role in regulating OM defenses. Yet, OM assembly and the pathways that regulate its formation and maintenance are poorly understood. The Bam complex folds and inserts OMPs into the OM in organisms such as Escherichia coli. Previous work has shown that the Bam complex recognizes unfolded OMP substrates by a C-terminal ß-signal sequence. Preliminary work in the Silhavy lab has identified an assembly defective ß-signal mutant, lamB\(_{G439D}\), in the maltoporin LamB, that is suppressed with the deletion of degP, a gene encoding for a periplasmic protease, at 30°C. Interestingly, at 37ºC, the deletion of degP is lethal in this background, suggesting that unfolded proteins accumulate in the periplasm and create a toxic backup. This temperature sensitivity has allowed us to select for suppressors that are able to overcome the toxic accumulation of unfolded OMPs. Furthermore, we have utilized dextrin uptake as a readout on MacConkey indicator media to detect LamB assembly in these suppressors. Using this approach, we have isolated twenty-three suppressors of lamBG439D and have identified degS\(_{A323E}\) as one suppressor mutation that will provide understanding of the interactions taking part in the OMP assembly pathway.