Conserved residues of the putative L6 loop of Escherichia coli BamA play a critical role in the assembly of β-barrel outer membrane proteins, including that of BamA itself

Abstract

Many members of the Omp85 family of proteins form essential β-barrel outer membrane protein (OMP) biogenesis machinery in Gram-negative bacteria, chloroplasts, and mitochondria. In Escherichia coli, BamA, a member of the Omp85 family, folds into an outer membrane-embedded β-barrel domain and a soluble periplasmic polypeptide-transport-associated (POTRA) domain. Although the high-resolution structures of only the BamA POTRA domain of E. coli are available, the crystal structure of FhaC, an Omp85 family member and a component of the two-partner secretion system in Bordetella pertussis, suggests that the BamA β-barrel likely folds into a 16-stranded β-barrel. The FhaC β-barrel is occluded by an N-terminal α-helix and a large β-barrel loop, L6, which carries residues that are highly conserved among the Omp85 family members. Deletion of L6 in FhaC did not affect its biogenesis but abolished its secretion function. In this study, we tested the hypothesis that the conserved residues of the putative L6 loop, which presumably folds back into the lumen of the BamA β-barrel like the FhaC counterpart, play an important role in OMP and/or BamA biogenesis. The conserved (641)RGF(643) residues of L6 were either deleted or replaced with alanine in various permutations. Phenotypic and biochemical characterization of various BamA L6 mutants revealed that the conserved RGF residues are critical for OMP biogenesis. Moreover, three BamA L6 alterations, ΔRGF, AAA, and AGA, produced a conditional lethal phenotype, concomitant with severely reduced BamA levels and folding defects. Thus, the conserved (641)RGF(643) residues of the BamA L6 loop are important for BamA folding and biogenesis.

Fig 1

Amino acid sequence alignment of the putative loop 6 region of the BamA β-barrel. The conserved VRGF motif is shaded. Greek letters represent different classes of the phylum Proteobacteria. The gain-of-function alterations in BamA that reverse the growth and OMP assembly defects of a strain simultaneously lacking BamB and BamE are indicated by arrows (29). Residues altered by site-directed mutagenesis in this study are marked by asterisks.

Fig 2

Growth phenotypes of strains expressing wild-type BamA (WT; sector 1) or various mutant BamA proteins (sectors 2 to 6) on LBA or glycerol minimal agar plates. Plates were incubated for 24 h (LBA) or 36 h (M63 minimal medium) at 30°C and 37°C. BamA was expressed from the pZS21 plasmid replicon.

Fig 3

Antibiotic sensitivities of strains expressing wild-type (WT) or mutant BamA proteins. Antibiotic sensitivities were measured from overnight cultures grown in glycerol minimal medium at 30°C. Zones of inhibition from two independent cultures were measured after 24 h of incubation at 30°C. Paper disks (6.5 mm in diameter) were either presoaked with rifampin (5 μg/ml) or soaked with 10 μl of vancomycin (75 μg/ml). Error bars show standard deviations.

Fig 4

Assessment of OMP and BamA levels and BamA's folding status. (A) OMPs were visualized from purified envelopes by Coomassie blue staining of an SDS (urea)-polyacrylamide gel. Envelopes were purified from overnight-grown cultures in glycerol minimal liquid medium at 30°C. Each lane contained 5 μg of proteins. (B) BamA levels were determined by Western blots from purified envelopes. Membrane blots were probed with antibodies specific to BamA and AcrA, with the latter serving as a gel loading control. BamA levels were determined relative to AcrA and then normalized to the wild-type (WT) value of 1. (C) BamA's folding status was determined by assessing its heat modifiability. Purified envelopes were analyzed as for panel B except that prior to SDS-PAGE analysis, SDS-solubilized samples were either left at room temperature (25°C) or heated (100°C). BamA was detected by Western blot analysis. BamA_D and BamA_F are denatured and folded forms of BamA.

Fig 5

Growth phenotypes of strains expressing wild-type (WT) BamA (sectors 1 and 5) or various mutant BamA proteins (sectors 2 to 4 and 6 to 8) on LBA or glycerol minimal agar plates. Plates were incubated for 24 h (LBA) or 36 h (M63 minimal medium) at 30°C and 37°C. BamA was expressed from the pZS21 plasmid replicon.

Fig 6

Antibiotic sensitivities of strains expressing wild-type (WT) or mutant BamA proteins. The assays were carried out as described in the Fig. 3 legend.

Fig 7

Assessment of OMP (A) and BamA (B) levels and BamA's folding status (C). These assays were carried out as described in the Fig. 4 legend. BamA_D and BamA_F are denatured and folded forms of BamA. Please note that the migration of folded forms of BamA in multiple bands is caused by subtle changes in the pH of the running SDS-PAGE buffer.