Mutational analysis of the Escherichia coli K-12 TolA N-terminal region and characterization of its TolQ-interacting domain by genetic suppression

Abstract

The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. They form two complexes in the cell envelope. Transmembrane domains of TolQ, TolR, and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. The N-terminal transmembrane domain of TolA anchors the protein to the cytoplasmic membrane and interacts with TolQ and TolR. Extensive mutagenesis of the N-terminal part of TolA was carried out to characterize the residues involved in such processes. Mutations affecting the function of TolA resulted in a lack or an alteration in TolA-TolQ or TolR-TolA interactions but did not affect the formation of TolQ-TolR complexes. Our results confirmed the importance of residues serine 18 and histidine 22, which are part of an SHLS motif highly conserved in the TolA and the related TonB proteins from different organisms. Genetic suppression experiments were performed to restore the functional activity of some tolA mutants. The suppressor mutations all affected the first transmembrane helix of TolQ. These results confirmed the essential role of the transmembrane domain of TolA in triggering interactions with TolQ and TolR.

FIG. 1

(A) Mutations isolated in the N-terminal domain of TolA. The upper line represents the sequence of TolA from E. coli. The substitutions generated in this study are indicated in lowercase letters (no mutant phenotype) or capital letters (tol phenotype). (B) Alignment of TolA sequences from E. coli, Haemophilus influenzae, and Pseudomonas aeruginosa. (C) Multiple alignment of TolA and TonB sequences (SwissProt release 35.0, GenBank release 104).

FIG. 2

Subcellular localization of TolA and Pal. Strain JC188 was grown to mid-exponential phase. Inner membranes (IM) and outer membrane (OM) fractions were separated on a sucrose gradient as described in Materials and Methods; 35 fractions were recovered, but only the 23 more relevant are shown. The fraction are shown from the bottom (fraction 1) to the top of the gradient. Densities (d) of the inner and outer membrane fractions are indicated at the top. Only immunoblots of Pal (top) and TolA (bottom) are shown.

FIG. 3

Immunoblot analysis of the TolA and TolR complexes in wild-type and tolA mutant strains cross-linked in vivo for 10 min with 1% formaldehyde. JC188Δorf1tolQRA was transformed with pT7-5 derivatives (controls, 1.5 × 10⁸ cells/well except for pT7-1RA [3 × 10⁸ cells/well]) or with pJEL-1QRA plasmids carrying the tolA mutations (A, lanes 5 to 10, 3 × 10⁸ cells/well). Only results obtained with tolA mutations leading to an altered phenotype are presented. The molecular weights (in thousands) of the standards (See-Blue prestained standards; Novex, San Diego, Calif.) are indicated on the right. Immunoblots were revealed by using antibodies raised against TolA (A) or TolR (B).

FIG. 4

Wheel representation of the transmembrane helix of TolA and of the first transmembrane helix of TolQ. Helices were built by using ideal α-helix parameters (3.6 residues/turn).

FIG. 5

Immunoblot analysis of the TolA and TolR complexes in tolA mutants affected at residue 18 or 22 and in the double tolA tolQ strains. pJEL250 carrying the tolQRA genes was used to transform JC188Δ1QRA. Immunoblots were revealed by using antibodies raised against TolA (A) or TolR (B).