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The third TatA subunit TatAc of Bacillus subtilis can form active twin-1
arginine translocases with the TatCd and TatCy subunits 2
3
Carmine G. Monteferrante1#, Jacopo Baglieri2#, Colin Robinson2 and 4
Jan Maarten van Dijl1*. 5
6
1Department of Medical Microbiology, University of Groningen and University Medical Center 7
Groningen, Hanzeplein 1, P.O. box 30001, 9700 RB Groningen, the Netherlands, 8
2School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom. 9
#These authors contributed equally to this work 10
11
12
13
14
* To whom correspondence should be addressed: Department of Medical Microbiology, 15
University Medical Center Groningen, Hanzeplein 1, P.O. box 30001, 9700 RB Groningen, the 16
Netherlands, Telephone: +31-50-3615187, Fax: +31-50-3619105, E-mail: 17
19
20
Running title: The TatAcCd and TatAcCy translocases of Bacillus 21
Keywords: Bacillus subtilis / Escherichia coli / TatAc / TatCd / TatCy 22
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01108-12 AEM Accepts, published online ahead of print on 27 April 2012
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Abstract 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 twin-31
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 homo-38
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
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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-AcCd-58
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
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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 TatCd-81
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. TatAc-86
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
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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-2008-106
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
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Reference list 111
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FIGURE 1. B. subtilis TatAcCd and TatAcCy facilitate TorA export in E. coli 214
Cells of E. coli ΔtatABCDE were subjected to subcellular fractionation. Proteins in the obtained 215
periplasmic (P), membrane (M) and cytoplasmic (C) fractions were separated by native PAGE 216
and the gels were subsequently analyzed for TMAO reductase (TorA) activity. Strains used in 217
this analysis were E. coli MC4100 (WT), E. coli ΔtatABCDE (Δtat), or E. coli ΔtatABCDE 218
expressing: B. subtilis TatAc from plasmid pBAD-Ac-Strep (Δtat + TatAc), B. subtilis TatCd 219
from plasmid pBAD-Cd-Strep (Δtat + TatCd), B. subtilis TatCy from plasmid pBAD-Cy-Strep 220
(Δtat + TatCy), B. subtilis TatAcCd from plasmid pBAD-AcCd-Strep (Δtat + TatAcCd), or B. 221
subtilis TatAcCy from plasmid pBAD-AcCy-Strep (Δtat + TatAcCy). The position of active full-222
length TorA is indicated. TorA* indicates a faster migrating form of TorA (22). 223
224
FIGURE 2. B. subtilis TatAcCd and TatAcCy facilitate AmiA and AmiC export in E. coli 225
The export of AmiA and AmiC in E. coli was assayed indirectly by assessing the chain length of 226
exponentially growing cells. A, E. coli MC4100 (WT); B, E. coli ΔtatABCDE forms long chains 227
due to the mislocalization of AmiA and AmiC (9); C, E. coli ΔtatABCDE producing TatAcCd 228
from plasmid pBAD-AcCd-Strep; D, E. coli ΔtatABCDE producing TatAcCy from plasmid 229
pBAD-AcCy-Strep. As evidenced by the significantly reduced chain length, the export of AmiA 230
and AmiC in E. coli ΔtatABCDE is at least partially restored by the production of TatAcCd or 231
TatAcCy. 232
233
FIGURE 3. TatAcCd and TatAcCy form discrete complexes 234
To investigate complex formation, TatCd-strepII, TatCy-strepII, TatAc-strepII, TatAc-TatCd-235
strepII or TatAc-TatCy-strepII were expressed in E. coli ΔtatABCDE. Next, membranes from the 236
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respective cells were isolated. Membrane proteins were then solubilised in 2% digitonin and 237
separated by blue-native PAGE. The gels were immunoblotted using strepII-specific antibodies 238
and a secondary anti-mouse IgG horseradish peroxidise conjugate. The EZ-ECL detection kit 239
was used to visualize bound antibodies. The mobility of molecular mass markers (left panel) and 240
strepII-tagged proteins and complexes is indicated (kDa). TatCd* indicates a TatCd complex 241
with higher molecular weight. 242
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