Translocator Proteins in the Two-partner Secretion Family Have Multiple Domains

Neeraj K Surana,Amy Z Buscher,Gail G Hardy,Susan Grass,Thomas Kehl-Fie,Joseph W St Geme

doi:10.1074/jbc.m600036200

Abstract

The two-partner secretion pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane translocator protein. Previous work has demonstrated that the TpsB protein forms a beta-barrel structure with pore forming activity and facilitates translocation of the TpsA protein across the outer membrane. In this study, we characterized the functional domains of the Haemophilus influenzae HMW1B protein, a TpsB protein that interacts with the H. influenzae HMW1 adhesin. Using c-Myc epitope tag insertions and cysteine substitution mutagenesis, we discovered that HMW1B contains an N-terminal surface-localized domain, an internal periplasmic domain, and a C-terminal membrane anchor. Functional and biochemical analysis of the c-Myc epitope tag insertions and a series of HMW1B deletion constructs demonstrated that the periplasmic domain is required for secretion of HMW1 and that the C-terminal membrane anchor (HMW1B-(234-545)) is capable of oligomerization and pore formation. Similar to our observations with HMW1B, examination of a Bordetella pertussis TpsB protein called FhaC revealed that the C terminus of FhaC (FhaC-(232-585)) is capable of pore formation. We speculate that all TpsB proteins have a modular structure, with a periplasmic domain that interacts with the cognate TpsA protein and with pore forming activity contained within the C terminus.

Highlights

The two-partner secretion (TPS)5 pathway in Gram-negative bacteria is characterized by an exoprotein and a cognate outer membrane translocator protein [1]
If the recombinant protein was present in the outer membrane at wild type levels and the c-Myc epitope was detectable by dot immunoblot analysis of outer membranes but was not detectable by flow cytometry, we concluded that the c-Myc epitope was on the periplasmic side of the outer membrane
These studies demonstrated that HMW1B has an extracellular N terminus, a large internal periplasmic domain, and a C-terminal ␤-barrel with 10 ␤-strands

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Culture Conditions—Escherichia coli DH5␣ (Invitrogen) and E. coli BL21(DE3) [15] are laboratory strains that have been described previously. The plasmids pHAT::FhaC31–585 and pHAT::FhaC232–585 encode the Hia signal sequence, the HAT epitope, and the indicated FhaC residues To construct these plasmids, chromosomal DNA from B. pertussis strain Tohama I was used as template to amplify the coding sequence for the relevant FhaC residues, engineering a SalI site at the 5Ј end and a BamHI site at the 3Ј end. Samples were resuspended in 10 mM HEPES (pH 7.4) supplemented with Complete Mini Protease Inhibitor mixture tablets (Roche Applied Science), and outer membrane proteins were prepared on the basis of Sarkosyl insolubility [23]. The soluble outer membrane fraction was incubated at 25 °C for 30 min with Talon affinity resin (Clontech) pre-equilibrated with 1% Elugent, 20 mM HEPES (pH 8.0), 150 mM NaCl. The Talon resin was washed three times, resuspended in Laemmli buffer, and boiled for 5 min. Data were analyzed using the Student’s t test to determine statistical differences between groups, requiring a probability level of 0.05 for significance

RESULTS

Pro only

DISCUSSION