Functional reconstitution of the E . coli Tat system by subunits obtained from a thermophilic bacterium

The twin arginine transport (Tat) system transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plants. In Eschericha coli, the Tat translocase consists of the integral membrane proteins TatA, TatB and TatC. In this study we have tested the ability of tat genes from the hyperthermophilic bacterium, Aquifex aeolicus, to compensate for the absence of the cognate E. coli tat genes, both individually and in various combinations. Here, we show that the A. aeolicus TatA1, A2 can functionally reconstitute the absence of E. coli TatABC, since Tat translocase activity is observed when these proteins are expressed in cells lacking all the known Tat genes. Introduction The Twin Arginine Transport (Tat) pathway is widespread among prokaryotes and plant thylakoids. In prokaryotes, it serves to export folded or even oligomeric proteins across the inner membrane (1). Invariably, all of its substrate proteins possess the characteristic N-terminal signal peptides that harbor the name giving “twin arginine” motif, S/TRRxFxK. The sizes of substrates, some up to 70 Å in diameter (1), pose the Tat export machinery with the remarkable challenge of opening large pores in membranes that otherwise need to be ion-impermeable. The actual mechanism in doing so is not yet understood. The Tat system does not hydrolyze ATP; instead the whole process appears to be driven by the protonmotive force (2, 3). Since the Tat machinery is absent in humans, from a medical view point understanding its mechanistic features could allow specific targeting of some important pathogenic bacteria (4). Tat has been shown to be absolutely essential for virulence in Pseudomonas (5), Mycobacterium (6), Yersinia (7), and other pathogenic bacteria. Much of our current knowledge about the Tat machinery has come from studying the model Gram-negative bacterium, Escherichia coli. In E. coli, three membrane components of this machinery are currently known, TatA, TatB and TatC (8, 9). The organism also encodes TatE, which was identified as a paralogue of TatA (9). TatB and TatC purify together as a stable complex containing multiple copies of each of them, in equimolar amounts (10). This TatBC complex is addressed as the substrate recognition unit of the Tat machinery based on in vitro biochemical studies (11), which indicate TatC as the primary dock site for twin arginine substrates which are later transferred to TatB. In functional terms, the Tat translocon additionally requires a pore-forming unit. Complexes made of multiple copies of TatA are proposed to form the actual pore within the machinery (11-13). Recently Gohlke et al. (1) obtained single particle electron microscopic images of purified TatA. 3D models of the images show that variable copy numbers of TatA lead to variable diameters of the complexes. Based on this model, they argue that the poreforming unit of the Tat machinery presumably requires this feature, in order to match their pore Functional reconstitution of E.coli Tat system 47 size to the substrate size, thereby restricting any excess pore diameter for ion leakage. Together with other evidence based on purification and cross-linking studies (14-17), a model for Tat targeting and transport has been put forth (8, 9), which has later been reviewed in detail (4). This model envisions dynamical assembly and disassembly of Tat components in the membrane. Accordingly, substrate binding to TatC would constitute the first step in activation of the Tat machinery. This event would then promote the pore-forming unit to adhere to the substrate recognition TatBC unit. When these two units would come together, TatB would mediate the transfer of the substrate from TatC to TatA. At this point, the pore-forming unit would transiently open its pore that channels the substrate to the periplasmic side where the mature substrate is released. Immediately, the pore would get sealed off and the two units would fall apart. In contrast to the E. coli type Tat machinery, the vast majority of the Gram-positive bacteria and some archaea lack a TatB homologue in their genome. Nevertheless, such a minimal Tat machinery consisting of only TatA and TatC as present in Bacillus subtilis was shown to mediate effective translocation (18). There are very few studies so far that address the molecular basis for the difference in component composition between the two types. One study employed random mutagenesis to understand the individual abilities of TatA and TatC to substitute for TatB in the E. coli Tat machinery (19). Surprisingly, all of the mutations that enable Tat activity in absence of TatB were located in tatA. Specifically, they all encoded certain single amino acid substitutions at the amino-terminus of TatA. Not a single activating mutation was found in the tatC gene. Based on the results, the authors suggested that TatA and not TatC represent a bifunctional component that in addition to its own function can also fulfill that of TatB. They extended their interpretation also to TatA of the minimal Tat system that naturally lacks TatB. Sequence and phylogenetic analysis also classifies TatB to be a distant relative of TatA (20). Comparison of the two-component and three-component Tat systems has the potential to yield extensive insight into the role of subunits. For this purpose we transferred tat genes of the minimal Aquifex aeolicus Tat system into E. coli strains, which are deletion mutants for one or more tat genes. In addition, Tat translocase from a hyperthermophile such as A. aeolicus can be used as model system in structural studies for the relatively less stable Tat translocase from other organisms. A. aeolicus is one of the most ancient eubacteria known to date and is distanced from E. coli by approximately four and half billion years. It has two homologues for tatA and one for tatC (20). Surprisingly, our results indicate that A. aeolicus TatA homologues are not only functional in E. coli, but can together complement the entire E. coli Tat system. Materials and Methods Bacterial strains, plasmids and growth conditions: The E. coli strains used in this study are listed in the Table 1. Two types of plasmids differing in their origin of replication and antibiotic resistance that permit mutual co-existence were made for complementation studies. The first type was provided with a col E1 origin and an ampicillin-resistance gene. This type includes pAStatA1,2 that carries in tandem the paralogous aq_64b and aq_64c genes (GenBank) of A. aeolicus and pNWtatA (already available in the lab) that carries the E. coli tatA gene. The second type was provided with a p15 origin and a chloramphenicol-resistance gene and includes pAStat3 that carries the aq_1267 gene of A. aeolicus. All of the tat genes mentioned are under the control of the tac promoter. All of the cell types used were routinely grown aerobically in Luria-Bertani (LB) medium at 37C with appropriate antibiotics, ampicillin (50g/mL) or chloramphenicol (34g/mL). Table 1: Bacterial strains used in this work Strains Relevant information on genotype References MC4100 (wild type) F'araD139Δ(argF-lac)U169 rpsL150 relAI flb5301 ptsF25 rbsR (21) ΔtatA/E MC4100ΔtatAΔtatE (22) ΔtatB MC4100ΔtatB (22) ΔtatC MC4100ΔtatC::ΩSpec (23) DADE MC4100 ΔtatAΔtatBΔtatCΔtatE (9) SDS-sensitivity assay: Cells for SDS-sensitivity assay were inoculated onto LB plate containing 2% SDS and appropriate antibiotics. The plates were incubated at 37C overnight and subsequently examined for growth. Anaerobic growth assay: Strictly anaerobic culturing techniques were employed throughout the growth assay. Basal medium (24) consisted of the following ingredients: (g l): NaHCO3, 2.5; NH4Cl, 1.5; NaH2PO4, 0.6; KCl, 0.1; NaWO4.2H2O, 0.00025; 10 ml l trace element solution (medium 141, DSMZ) and 2% glycerol together with appropriate antibiotics were filter sterilized and degassed. 10 ml serum bottles (Sigma-Aldrich, Germany) were sealed by a butyl rubber stopper in a glove box filled with N2/CO2 (90:10) and then autoclaved. In the glove box, aliquots of the media (8 ml) were dispensed into the sterile serum bottles and inoculated with appropriate culture strains. These cells were grown for 24 hrs at 37C and 175 rpm and their growths were measured in terms of optical density (OD) using spectrometry at 600nm wavelength. The ratios of the OD values of different cell types to that of MC4100 wild type cells were represented in form of a bar diagram. Functional reconstitution of E.coli Tat system 49 Table 1. SDS sensitivity assay to study complementation of Tat activity in E. coli by A. aeolicus Tat subunits. Results A. aeolicus TatA homologues in E. coli Earlier work with the A. aeolicus TatA homologues (14) showed that they mostly localize in the membrane and complement Tat activity in a ∆TatA strain of E. coli. We also examined complementation by either one or both of the A. aeolicus TatA homologues also in E. coli strain DADE, lacking all of the E. coli Tat components. Two approaches were employed in the present study – the SDS sensitivity assay and the anaerobic growth assay. It has been shown previously (25, 26) that E. coli strains lacking an active Tat system are exquisitely sensitive to the presence of SDS. Growth on SDS constitutes thus a qualitative indication for Tat functionality (14, 27) in a given cell type. SDS sensitivity of different E. coli strains expressing A. aeolicus Tat homologues are listed in Table 1. Obviously, the two A. aeolicus TatA homologues in combination complemented the Tat activity in E. coli DADE, whereas A. aeolicus TatC was few times (2 out of 5 times) able to complement E. coli ΔtatB, but not E. coli ΔtatC. In the latter case, it is possible that certain mutations in either E. coli tatA or A. aeolicus tatC may substitute for the absence of tatB. But investigation into occurrences of such mutations is yet to be carried out. In a second approach, we examined anaerobic growth rates of the cell types used in the previous experiment. Results obtained with TatA homologues (Figure1) coincided with the results of the previous assay, confirming the complementation of Tat activity in E. coli DADE by the combined expression of A. aeolicus TatA1 and TatA2. E. coli strain A. aeolicus Tat subunit expressed Growth status