Summary: Halophilic archaea thrive in environments with salt concentrations approaching saturation. However, little is known about the way in which these organisms stabilize their secreted proteins in such ‘hostile’ conditions. Here, we present data suggesting that the utilization of protein translocation pathways for protein secretion by the Halobacteriaceae differs significantly from that of non‐haloarchaea, and most probably represents an adaptation to the high‐salt environment. Although most proteins are secreted via the general secretion (Sec) machinery, the twin‐arginine translocation (Tat) pathway is mainly used for the secretion of redox proteins and is distinct from the Sec pathway, in that it allows cytoplasmic folding of secreted proteins. tatfind (developed in this study) was used for systematic whole‐genome analysis of Halobacterium sp. NRC‐1 and several other prokaryotes to identify putative Tat substrates. Our analyses revealed that the vast majority of haloarchaeal secreted proteins were predicted substrates of the Tat pathway. Strikingly, most of these putative Tat substrates were non‐redox proteins, the homologues of which in non‐haloarchaea were identified as putative Sec substrates. We confirmed experimentally that the secretion of one such putative Tat substrate depended on the twin‐arginine motif in its signal sequence. This extensive utilization of the Tat pathway in haloarchaea suggests an evolutionary adaptation to high‐salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion.

The biosynthesis of pyoverdines –

In Escherichia coli, the Tat system does not translocate Tat signal sequence fused PhoA (RR-PhoA), as it requires disulfide formation for folding. Here we show that such a RR-PhoA construct can be efficiently targeted to the Tat translocon, but the transport is not completed. RR-PhoA is detectable in a 580-kDa TatBC-containing complex, which is the first substrate-bound TatBC complex detected in a bacterial system so far. A second TatBC complex near 440 kDa comprises most of the TatB and TatC but is devoid of RR-PhoA. The targeting of PhoA to the Tat translocon depends on the twin-arginine motif and results in severe growth defects. This physiological effect is likely to be due to proton leakage at the cytoplasmic membrane. The results point to mechanistic incompatibilities of the Tat system with unfolded proteins such as RR-PhoA. There does not exist an intrinsic quality control at the TatBC complex itself, although correct folding is inevitable for Tat-dependent translocation.