The extreme C‐terminus is required for secretion of both the native polygalacturonase (PehA) and PehA‐Bla hybrid proteins in Erwinia carotovora subsp. carotovora

Abstract

A set of gene fusions was constructed between the pehA gene encoding the secreted endopolygalacturonase (PehA) and the bla gene coding for a normally periplasmic beta-lactamase (Bla). The resulting hybrid proteins were specifically and actively routed out of the cells via the Out-terminal branch of the general secretory pathway (GSP) in Erwinia carotovora subsp. carotovora (Ecc), provided that no more than the last two amino acids (aa) of the PehA domain were excluded from the fusion. However, both PehA-Bla hybrid proteins and PehA variants lacking at least four aa from the C-terminus of the PehA accumulated in the periplasm. Also, overexpression of the gene fusions prevented extracellular targeting of the hybrid proteins. Site-directed mutagenesis of the codons -4 and -3 (encoding Asn-373 and Val-374, respectively) from the end of the pehA gene and analysis of the protein products suggested that the Val-374 was important both for the structure and secretion of PehA, while the Asn-373 proved to be insignificant. We conclude that: (i) the GSP of Ecc is capable of secreting heterologous proteins; (ii) as the PehA protein can accommodate C-terminal extensions, secretion can occur with no part of the proposed targeting signal lying within the C-terminal extremity of a secreted molecule; and (iii) residues within the C-terminus of PehA play a role in secretion, possibly through stabilization of a structure needed for proper exposition of the proposed targeting motif.

Influence of Deletions within Domain II of Exotoxin A on Its Extracellular Secretion from Pseudomonas aeruginosa

“…In the case of P. aeruginosa PE, residues 60 to 120 appeared to be sufficient for directing ␤-lactamase secretion (26). However, in the case of E. carotovora polygalacturonase (PehA), no more than the last two amino acids of the PehA could be excluded from a PehA-Bla hybrid without blocking secretion (29,30). Passenger proteins might contain structures incompatible with efficient secretion by the type II machineries.…”

Protein secretion and the pathogenesis of bacterial infections

“…Studies on many different type II substrates have identified a large variety of domains and amino acids that are required for secretion (Hamood et al 1989;Kornacker and Pugsley 1990;He et al 1991b;Wong and Buckley 1991;Py et al 1993;Palomaki and Saarilahti 1995;Sauvonnet et al 1995;Lu and Lory 1996). For example, two spatially separated domains of pullulanase (PulA) seem required for substrate recognition (Sauvonnet and Pugsley 1996).…”

On the path to uncover the bacterial type II secretion system

“…It was also suggested that P. aeruginosa exotoxin A (ToxA) contains two separate secretion signals [132], while alteration of another region also affects secretion efficiency [133]. Finally, the polygalacturonase PehA of E. carotovora was found to contain three separate domains involved in T2SS targeting [134,135]. As reported above, the secretion signal may be composed of residues from different locations in the linear polypeptide chain, which are brought together into a conformational patch during protein folding [136].…”