Abstract
The two-partner secretion (TPS) systems of Gram-negative bacteria secrete large TpsA exoproteins by a dedicated TpsB transporter in the outer membrane. TpsBs contain an N-terminal module located in the periplasm that includes two polypeptide transport-associated (POTRA) domains. These are thought to initiate secretion of a TpsA by binding its N-terminal secretion signal, called the TPS domain. Neisseria meningitidis encodes up to five TpsA proteins that are secreted via only two TpsB transporters: TpsB1 and TpsB2. Of these two, the TpsB2 recognizes the TPS domains of all TpsAs, despite their sequence diversity. By contrast, the TpsB1 shows a limited recognition of a TPS domain that is shared by two TpsAs. The difference in substrate specificity of the TpsBs enabled us to investigate the role of the POTRA domains in the selection of TPS domains. We tested secretion of TPS domains or full-length TpsAs by TpsB mutants with deleted, duplicated, and exchanged POTRA domains. Exchanging the two POTRA domains of a TpsB resulted in a switch in specificity. Furthermore, exchanging a single POTRA domain showed that each of the two domains contributed to the cargo selection. Remarkably, the order of the POTRA domains could be reversed without affecting substrate selection, but this aberrant order did result in an alternatively processed secretion product. Our results suggest that secretion of a TpsA is initiated by engaging both POTRA domains of a TpsB transporter and that these select the cognate TpsAs for secretion.
Highlights
TpsB transporters secrete large exoproteins across the outer membrane of Gram-negative bacteria
polypeptide transport-associated (POTRA) Domain Assignment in TpsB1 and TpsB2—To construct various POTRA mutants of TpsB1 and TpsB2 derived from N. meningitidis strain H44/76, we used the crystal structure of FhaC [8] to build structural models
We have used this difference in secretion specificity to investigate the role of the periplasmic POTRA domains of the TpsB transporters in the recognition and secretion of TpsAs
Summary
TpsB transporters secrete large exoproteins across the outer membrane of Gram-negative bacteria. The two-partner secretion (TPS) systems of Gram-negative bacteria secrete large TpsA exoproteins by a dedicated TpsB transporter in the outer membrane. TpsBs contain an N-terminal module located in the periplasm that includes two polypeptide transport-associated (POTRA) domains These are thought to initiate secretion of a TpsA by binding its N-terminal secretion signal, called the TPS domain. We have used this difference in substrate specificity between TpsB2 and TpsB1 to investigate the function of the POTRA domains in the recognition and selection of TPS domains of secreted TpsA proteins. We found that both POTRA domains are crucial for and define this specificity. The order of the POTRA domains seems irrelevant for secretion
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