System Specificity of the TpsB Transporters of Coexpressed Two-Partner Secretion Systems of Neisseria meningitidis

Abstract

The two-partner secretion (TPS) systems of Gram-negative bacteria consist of a large secreted exoprotein (TpsA) and a transporter protein (TpsB) located in the outer membrane. TpsA targets TpsB for transport across the membrane via its ϳ30-kDa TPS domain located at its N terminus, and this domain is also the minimal secretory unit. Neisseria meningitidis genomes encode up to five TpsAs and two TpsBs. Sequence alignments of TPS domains suggested that these are organized into three systems, while there are two TpsBs, which raised questions on their system specificity. We show here that the TpsB2 transporter of Neisseria meningitidis is able to secrete all types of TPS domains encoded in N. meningitidis and the related species Neisseria lactamica but not domains of Haemophilus influenzae and Pseudomonas aeruginosa. In contrast, the TpsB1 transporter seemed to be specific for its cognate N. meningitidis system and did not secrete the TPS domains of other meningococcal systems. However, TpsB1 did secrete the TPS2b domain of N. lactamica, which is related to the meningococcal TPS2 domains. Apparently, the secretion depends on specific sequences within the TPS domain rather than the overall TPS domain structure.

The Polypeptide Transport-associated (POTRA) Domains of TpsB Transporters Determine the System Specificity of Two-partner Secretion Systems

“…SDS-PAGE and Western Blotting-All procedures were carried out as described earlier (19,26). Briefly, N. meningitidis HB-1 cultures were grown for 4 -6 h to an optical density at 600 nm (A 600 ) of ϳ2.5-3.5 in the presence or absence of 0.1 or 0.25 mM IPTG.…”

“…The constructs of wild-type tpsB2 and tpsB1 ORFs in combination with truncated tpsA1 or tpsA2 ORFs have been described earlier (26). Mutated ORFs were first subcloned downstream of the truncated tpsA ORF in pPU1000 (tpsA1a), pPU1200 (tpsA2a), or pPU1300 (tpsA2b) (19) using the EcoRI and BamHI restriction sites inserted in the primers.…”

“…1C and Table 2). We focused primarily on the TpsB2 protein because this protein showed a relaxed specificity in the secretion of minimal TPS constructs that consist of the signal peptide and TPS domain of one of the TpsAs of the neisserial TPS systems (26).…”

“…1C). The ORFs encoding these mutated TpsB proteins were cloned in a neisserial expression vector downstream of a truncated tpsA2a gene under the control of a lac promoter (26). This truncated tpsA2a includes the TPS domain and represents the minimal TpsA protein that is still secreted.…”

“…The system 3 TpsA is encoded on a genetic island that lacks a gene encoding a dedicated TpsB. Our previous study showed that the TPS domain of TpsA3 is efficiently secreted by TpsB2 (26). In fact, the TpsB2 showed a relaxed system specificity and was able to recognize and secrete TPS domains of N. meningitidis and Neisseria lactamica.…”