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1 INRS-Institut Armand-Frappier, Université du Québec, 531 blvd des Prairies, Ville de Laval, Québec, Canada H7V 1B7
2 Centre d'étude et de Valorization de la Diversité Microbienne, Département de biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada J1K 2R1
Correspondence
Rolf Morosoli
rolf.morosoli{at}inrs-iaf.uquebec.ca
The availability of the complete genome sequence of Streptomyces coelicolor A3(2) has allowed the prediction of the Tat-exported proteins of this Gram-positive bacterium. To predict secreted proteins that potentially use the Tat pathway for their secretion, the TATscan program was developed. This program identified 129 putative Tat substrates. To test the validity of these predictions, nine signal sequences, including three which were not identified by existing prediction programs, were selected and fused to the structural xlnC gene in place of its native signal sequence. Xylanase C (XlnC) is a cofactorless enzyme which is secreted in an active form exclusively through the Tat-dependent pathway by Streptomyces lividans. Among the nine chosen signal sequences, seven were shown to be Tat-dependent, one was Sec-dependent and one was probably not a signal sequence. The seven Tat-dependent signal sequences comprised two lipoprotein signal sequences and three sequences not predicted by previous programs. Pulse–chase experiments showed that the precursor-processing rate in the seven transformants was generally slower than wild-type XlnC, indicating that these signal peptides were not equivalent in secretion. This suggested that there might be some incompatibility between the signal peptide and the reporter protein fused to it. To test this possibility, the signal peptides were fused to a cofactorless chitosanase (SCO0677), a Tat-dependent protein validated in this work but structurally different from XlnC. With some fluctuations, similar results were obtained with this enzyme, indicating that the type of folding of the reporter protein had little effect on the Tat secretion process.
A complete set of 129 candidate signal sequences is available as supplementary data with the online version of this paper.
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