The third TatA subunit TatAc of Bacillus subtilis can form active twin - 1 arginine translocases with the TatCd and TatCy subunits 2 3 Carmine

23 Two independent twin-arginine translocases (Tat) for protein secretion were previously 24 identified in the Gram-positive bacterium Bacillus subtilis. These consist of the TatAd-TatCd 25 and TatAy-TatCy subunits. The function of a third TatA subunit named TatAc was unknown. 26 Here we show that TatAc can form active protein translocases with TatCd and TatCy. 27 28 29 Protein transport from the cytoplasm to different bacterial compartments or the external milieu is 30 facilitated by dedicated molecular machines (6). Among these protein translocases, the twin31 arginine translocases (Tat) stand out, because they permit the passage of tightly folded proteins 32 across the cytoplasmic membrane. The proteins translocated by Tat are synthesized with signal 33 peptides that contain a well-conserved twin-arginine (RR) motif for specific targeting to a 34 membrane-embedded Tat translocase (13, 17, 23). The Tat translocases of Gram-negative 35 bacteria, such as Escherichia coli, are composed of three subunits named TatA, TatB and TatC 36 (5, 18). The formation of an active protein-conducting channel is believed to require the 37 formation of a supercomplex composed of a TatABC heterotrimeric complex and homo38 oligomeric TatA complexes (1, 8). In contrast, most Gram-positive bacteria possess minimized 39 Tat translocases that contain only TatA and TatC subunits. Nevertheless, various studies indicate 40 that these TatAC translocases employ a mechanism similar to that of the TatABC translocases of 41 Gram-negative bacteria (10, 17). 42 The Gram-positive bacterium Bacillus subtilis is a well-known ‘cell factory’ for secretory protein 43 production (20, 21). In this organism two Tat translocases are known to operate in parallel. The 44 TatAdCd translocase consists of the TatAd and TatCd subunits and the TatAyCy translocase 45 on N ovem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom consists of the TatAy and TatCy subunits (11, 12, 15). While the TatAdCd translocase is 46 produced mainly under conditions of phosphate starvation (12, 14, 15), the TatAyCy translocase 47 is expressed under all tested conditions (12, 14). Interestingly, B. subtilis produces a third TatA 48 subunit named TatAc (12). The function of TatAc has remained enigmatic due to the fact that no 49 phenotype was so far detectable for tatAc mutant B. subtilis cells (11, 12, 20). Therefore, the 50 present studies were aimed at determining whether TatAc can actually form active translocases 51 in combination with TatCd or TatCy. This possibility was tested by expressing the respective tat 52 genes in E. coli, because the activity and assembly of Bacillus Tat translocases can be assayed 53 more readily in this organism than in B. subtilis (2). For this purpose, the tatAc gene was 54 amplified from the B. subtilis genome (GenBank/EMBL/DDBJ accession number AL009126) 55 and cloned into plasmid pBAD24, resulting in pBAD-Ac. Next, the tatCd and tatCy genes were 56 PCR-amplified such that the respective proteins contain a C-terminal strepII-tag. The amplified 57 tatCd-strepII and tatCy-strepII genes were cloned into pBAD-Ac, resulting in pBAD-AcCd58 Strep and pBAD-AcCy-Strep, respectively. These vectors were subsequently used to transform 59 E. coli ΔtatABCDE cells, which lack all E. coli tat genes. Next, the resulting strains were tested 60 for their ability to transport the previously identified E. coli Tat substrates TorA, AmiA and 61 AmiC (3, 4, 9). To monitor TorA export to the periplasm, E. coli cells were grown anaerobically 62 until mid-exponential growth phase, and these cells were then subjected to sub-cellular 63 fractionation as previously described (16). The periplasmic, cytoplasmic, and membrane 64 fractions thus obtained were separated on a 10% native polyacrylamide gel that was 65 subsequently assayed for Trimethylamine N-oxide (TMAO) reductase activity as described 66 previously (4, 19). The results in Figure 1 show that the ΔtatABCDE cells producing TatAc plus 67 TatCd, or TatAc plus TatCy were capable of transporting active TorA to the periplasm. In 68 on N ovem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom contrast, ΔtatABCDE cells expressing only tatAc, tatCy or tatCd were not able to export active 69 TorA to this subcellular location. This showed for the first time that TatAc was able to form 70 active translocases in combination with TatCd or TatCy. To further investigate the activity of 71 these translocases, we tested the export of AmiA and AmiC, which are both required for cell wall 72 biosynthesis in E. coli (3, 9). Cells that do not export these molecules to the periplasm grow in 73 long chains, as is observed for the E. coli ΔtatABCDE strain (Fig. 2, A and B (9)). As shown by 74 phase contrast microscopy, the bacteria producing TatAc plus TatCd, or TatAc plus TatCy 75 showed the wild-type phenotype although some slightly longer chains were still detectable (Fig. 76 2, C and D). This is indicative of active export of AmiA and/or AmiC to the periplasm, providing 77 further support for the idea that active TatAcCd and TatAcCy complexes can be formed in E. 78 coli. 79 To demonstrate the formation of TatAcCd and TatAcCy complexes, a blue native (BN) PAGE 80 analysis was performed. For this purpose, membranes were isolated from cells expressing TatCd81 strepII, TatCy-strepII, TatAc-TatCd-strepII, or TatAc-TatCy-strepII. In addition, cells producing 82 TatAc-strepII from plasmid pBAD24 were included in the analyses. Upon solubilization in 2% 83 digitonin, membrane proteins were separated by BN PAGE, followed by immunoblotting with 84 antibodies against the strep-II tag. As shown in Figure 3, TatCd-strepII and TatCy-strepII alone 85 formed bands of ~66 kDa. In addition, TatCd-strepII formed a minor band of ~100 kDa. TatAc86 strepII expressed by itself formed a small homogeneous complex of ~100 kDa. Importantly, 87 when TatAc (non-tagged) was co-expressed with either TatCd-strepII or TatCy-strepII, bands of 88 ~230 kDa or ~200 kDa respectively were observed. This showed that TatAc does indeed form 89 membrane-embedded complexes with TatCd and TatCy. 90 on N ovem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom In conclusion, our present studies document for the first time that the hitherto enigmatic third 91 TatA subunit of B. subtilis known as TatAc can engage in the formation of active TatAC type 92 translocases. The results also show that TatAc has no particular preference for partnering with 93 TatCd or TatCy. It is noteworthy that the identified TatAcCd and TatAcCy complexes appear to 94 be homogeneous and relatively small in size (~230 to 200 kDa). Interestingly, previous studies in 95 B. subtilis have shown that the co-expression of TatAc and TatCd or TatAc and TatCy does not 96 facilitate export of the known Tat substrates PhoD and YwbN (7). Furthermore, a recent tiling 97 array analysis across 104 conditions has shown that tatAc is expressed under most conditions 98 (14). These previous observations together with our present findings suggest that TatAcCd and 99 TatAcCy translocases could be involved in the specific export of as yet unidentified Tat 100 substrates in B. subtilis. However, it has to be noted here that our present observations on TatAc 101 function were made upon heterologous expression in E. coli, and that it remains to be assessed 102 whether TatAc fulfils the same functions in B. subtilis. 103 104 Acknowledgements 105 C.G.M., J.B., C.R. and J.M.v.D. were supported through the CEU projects PITN-GA-2008106 215524 and 244093, and the transnational SysMO projects BACELL SysMO 1 and 2 through the 107 Research Council for Earth and Life Sciences of the Netherlands Organization for Scientific 108 Research. 109 110 on N ovem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom Reference list 111 112 1. Baglieri, J., Beck, D., Vasisht, N., Smith, C., Robinson, C. 2011. Structure of the TatA 113 paralog, TatE, suggests a structurally homogeneous form of Tat protein translocase that 114 transports folded proteins of differing diameter. J. Biol. Chem. 287(10):7335-44. 115 116 2. Barnett, J.P., van der Ploeg, R., Eijlander, R.T., Nenninger, A., Mendel, S., 117 Rozeboom, R., Kuipers, O.P., van Dijl, J.M., Robinson, C. 2009. The twin-arginine 118 translocation (Tat) systems from Bacillus subtilis display a conserved mode of complex 119 organization and similar substrate recognition requirements. FEBS J. 276(1):232-43. 120 121 3. Bernhardt, T. G., de Boer, P. A. 2003. The Escherichia coli amidase AmiC is a 122 periplasmic septal ring component exported via the twin-arginine transport pathway. Mol. 123 Microbiol. 48:1171–1182 124 125 4. Bolhuis, A., Mathers, J.E., Thomas, J.D., Barrett, C.M.L., and Robinson, C. 2001. 126 TatB and TatC Form a Functional and Structural Unit of the Twin-arginine Translocase 127 from Escherichia coli. Journal of Biological Chemistry 276:20213-20219 128 129 5. Bogsch, E. G., Sargent, F., Stanley, N. R., Berks, B. C., Robinson, C., Palmer, T. 13