The Hydrophobic Core of Twin-Arginine Signal Sequences Orchestrates Specific Binding to Tat-Pathway Related Chaperones

Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA) and TorD (specific for trimethylamine N-oxide reductase, TorA). Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function

Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways

Transport of Folded Proteins by the Tat System

The twin-arginine protein translocation (Tat) system has been characterized in bacteria, archaea and the chloroplast thylakoidal membrane. This system is distinct from other protein transport systems with respect to two key features. Firstly, it accepts cargo proteins with an N-terminal signal peptide that carries the canonical twin-arginine motif, which is essential for transport. Second, the Tat system only accepts and translocates fully folded cargo proteins across the respective membrane. Here, we review the core essential features of folded protein transport via the bacterial Tat system, using the three-component TatABC system of Escherichia coli and the two-component TatAC systems of Bacillus subtilis as the main examples. In particular, we address features of twin-arginine signal peptides, the essential Tat components and how they assemble into different complexes, mechanistic features and energetics of Tat-dependent protein translocation, cytoplasmic chaperoning of Tat cargo proteins, and the remarkable proofreading capabilities of the Tat system. In doing so, we present the current state of our understanding of Tat-dependent protein translocation across biological membranes, which may serve as a lead for future investigations

SASP: targeted delivery to Gram-negative pathogens

Background: Gram-negative bacteria are responsible for significant morbidity and mortality worldwide. Multi-drug resistance emergence has rendered many therapies ineffective. New therapies are urgently required to widen treatment options. SASPject technology delivers small acid-soluble spore protein (SASP) genes to target bacteria using modified bacteriophage vectors, resulting in rapid killing. SASP is a unique antibacterial protein that non-specifically binds bacterial DNA and halts DNA replication and gene expression. In this study we present the first data for a Gram-negative targeted SASPject vector (PT3.1) which shows in vitro activity against Escherichia coli (EC) and Pseudomonas aeruginosa (PA). Methods: We evaluated efficacy of SASPject PT3.1 vs. EC (N=5) & PA (N=5) using a microtitre tray fixed duration (3 h) kill method. Log-phase cultures (1x105 cfu/mL) were prepared in supplemented (MgSO4 & CaCl2, 5 mM; glucose, 0.1% w/v) LB broth (LBC) & exposed to 2x108 plaque forming units (pfu)/mL of PT3.1. PT3.1 antimicrobial activity was determined using agar-based culture following incubation (37oC). Additionally, rate of PT3.1 kill was determined using a kill-curve technique; a selection of EC & PA strains from the fixed duration kill study were evaluated & bacterial viable counts determined over 3 h on LBC agar. Results: SASPject PT3.1 elicited good antimicrobial activity vs. EC & PA evaluated in this study; the median reduction in viable counts for PT3.1-treated cultures in the fixed 3 h kill was 99.1%. Kill curve data suggested rapid EC & PA killing; viable counts (log10 cfu/mL range) of PT3.1-treated cultures were 2.36->4.01, 3.04-4.06, and 3.40-4.16 lower than corresponding controls after 1, 2, and 3 h respectively. Conclusions: 1. SASPject PT3.1 demonstrated good antimicrobial activity vs. EC & PA evaluated in this study in a fixed 3 h exposure period 2. PT3.1 was bactericidal (≥3 log10 cfu/mL decline) vs. 3 of 4 isolates (1EC & 2PA) evaluated in time-kill curves after 1 h and against all isolates after 2 h 3. Further evaluations of Gram-negative SASPject phage are warrantedNon peer reviewe