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A TatABC-type Tat translocase is required for unimpaired aerobic growth of Corynebacterium glutamicum ATCC13032.

Oertel D, Schmitz S, Freudl R - PLoS ONE (2015)

Bottom Line: Furthermore, our results clearly show that TatB, besides TatA and TatC, is strictly required for unimpaired aerobic growth.In addition, TatB was also found to be essential for the secretion of a heterologous Tat-dependent model protein into the C. glutamicum culture supernatant.Together with our finding that expression of the C. glutamicum TatB in an E. coli ΔtatB mutant strain resulted in the formation of an active Tat translocase, our results clearly indicate that a TatABC translocase is used as the physiologically relevant functional unit for Tat-dependent protein translocation in C. glutamicum and, most likely, also in other TatB-containing Actinobacteria.

View Article: PubMed Central - PubMed

Affiliation: Institut für Bio- und Geowissenschaften 1, IBG1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany.

ABSTRACT
The twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Escherichia coli and other Gram-negative bacteria possess a TatABC-type Tat translocase in which each of the three inner membrane proteins TatA, TatB, and TatC performs a mechanistically distinct function. In contrast, low-GC Gram-positive bacteria, such as Bacillus subtilis, use a TatAC-type minimal Tat translocase in which the TatB function is carried out by a bifunctional TatA. In high-GC Gram-positive Actinobacteria, such as Mycobacterium tuberculosis and Corynebacterium glutamicum, tatA, tatB, and tatC genes can be identified, suggesting that these organisms, just like E. coli, might use TatABC-type Tat translocases as well. However, since contrary to this view a previous study has suggested that C. glutamicum might in fact use a TatAC translocase with TatB only playing a minor role, we reexamined the requirement of TatB for Tat-dependent protein translocation in this microorganism. Under aerobic conditions, the misassembly of the Rieske iron-sulfur protein QcrA was identified as a major reason for the severe growth defect of Tat-defective C. glutamicum mutant strains. Furthermore, our results clearly show that TatB, besides TatA and TatC, is strictly required for unimpaired aerobic growth. In addition, TatB was also found to be essential for the secretion of a heterologous Tat-dependent model protein into the C. glutamicum culture supernatant. Together with our finding that expression of the C. glutamicum TatB in an E. coli ΔtatB mutant strain resulted in the formation of an active Tat translocase, our results clearly indicate that a TatABC translocase is used as the physiologically relevant functional unit for Tat-dependent protein translocation in C. glutamicum and, most likely, also in other TatB-containing Actinobacteria.

No MeSH data available.


Related in: MedlinePlus

Analysis of PhoDCG-GFP secretion in C. glutamicum wild-type and tat mutant strains.Cultures of C. glutamicum strains expressing the Tat-dependent PhoDCG-GFP model protein [35] were fractionated into cells (C) and supernatant (S). Samples of the fractions corresponding to an equal number of cells (i.e. an OD600 of 1.0) were subjected to SDS-PAGE and immunoblotting using GFP-specific antibodies. The following strains were analyzed: C. glutamicum wild-type (wt) containing the empty vector pEKEx2 as negative control (lanes 1 and 2), C. glutamicum wild-type (wt) containing plasmid pCGPhoDCG-GFP (lanes 3 and 4), and the pCGPhoDCG-GFP-containing C. glutamicum mutant strains ΔtatAC (lanes 5 and 6), ΔtatB (lanes 7 and 8), and ΔtatA/E (lanes 9 and 10). p: PhoDCG-GFP precursor; asterisk: cytosolic degradation product; m: mature-sized GFP protein.
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pone.0123413.g003: Analysis of PhoDCG-GFP secretion in C. glutamicum wild-type and tat mutant strains.Cultures of C. glutamicum strains expressing the Tat-dependent PhoDCG-GFP model protein [35] were fractionated into cells (C) and supernatant (S). Samples of the fractions corresponding to an equal number of cells (i.e. an OD600 of 1.0) were subjected to SDS-PAGE and immunoblotting using GFP-specific antibodies. The following strains were analyzed: C. glutamicum wild-type (wt) containing the empty vector pEKEx2 as negative control (lanes 1 and 2), C. glutamicum wild-type (wt) containing plasmid pCGPhoDCG-GFP (lanes 3 and 4), and the pCGPhoDCG-GFP-containing C. glutamicum mutant strains ΔtatAC (lanes 5 and 6), ΔtatB (lanes 7 and 8), and ΔtatA/E (lanes 9 and 10). p: PhoDCG-GFP precursor; asterisk: cytosolic degradation product; m: mature-sized GFP protein.

Mentions: Next, Tat-dependent protein translocation was analyzed using a previously described model protein (PhoDCG-GFP [35]) consisting of the signal peptide of the C. glutamicum Tat substrate PhoD (an alkaline phosphatase) fused to green fluorescent protein (GFP). As shown in Fig 3, large amounts of mature GFP are present in the supernatant of C. glutamicum wild-type containing plasmid pCGPhoDCG-GFP (lane 4). Furthermore, hardly any detectable GFP-derived polypeptides were present in the cellular fraction of the same strain (lane 3). In contrast, no secreted GFP protein is found in the supernatant of the ΔtatAC, ΔtatA/E, or ΔtatB mutant strains (lanes 6, 8, 10) and, in the cell fractions of the corresponding strains (lanes 5, 7, 9), an accumulation of PhoDCG-GFP precursor and degradation products of it can be observed. These results clearly indicate that TatA, TatB, and TatC are equally important for the translocation of PhoDCG-GFP across the cytoplasmic membrane of C. glutamicum.


A TatABC-type Tat translocase is required for unimpaired aerobic growth of Corynebacterium glutamicum ATCC13032.

Oertel D, Schmitz S, Freudl R - PLoS ONE (2015)

Analysis of PhoDCG-GFP secretion in C. glutamicum wild-type and tat mutant strains.Cultures of C. glutamicum strains expressing the Tat-dependent PhoDCG-GFP model protein [35] were fractionated into cells (C) and supernatant (S). Samples of the fractions corresponding to an equal number of cells (i.e. an OD600 of 1.0) were subjected to SDS-PAGE and immunoblotting using GFP-specific antibodies. The following strains were analyzed: C. glutamicum wild-type (wt) containing the empty vector pEKEx2 as negative control (lanes 1 and 2), C. glutamicum wild-type (wt) containing plasmid pCGPhoDCG-GFP (lanes 3 and 4), and the pCGPhoDCG-GFP-containing C. glutamicum mutant strains ΔtatAC (lanes 5 and 6), ΔtatB (lanes 7 and 8), and ΔtatA/E (lanes 9 and 10). p: PhoDCG-GFP precursor; asterisk: cytosolic degradation product; m: mature-sized GFP protein.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383559&req=5

pone.0123413.g003: Analysis of PhoDCG-GFP secretion in C. glutamicum wild-type and tat mutant strains.Cultures of C. glutamicum strains expressing the Tat-dependent PhoDCG-GFP model protein [35] were fractionated into cells (C) and supernatant (S). Samples of the fractions corresponding to an equal number of cells (i.e. an OD600 of 1.0) were subjected to SDS-PAGE and immunoblotting using GFP-specific antibodies. The following strains were analyzed: C. glutamicum wild-type (wt) containing the empty vector pEKEx2 as negative control (lanes 1 and 2), C. glutamicum wild-type (wt) containing plasmid pCGPhoDCG-GFP (lanes 3 and 4), and the pCGPhoDCG-GFP-containing C. glutamicum mutant strains ΔtatAC (lanes 5 and 6), ΔtatB (lanes 7 and 8), and ΔtatA/E (lanes 9 and 10). p: PhoDCG-GFP precursor; asterisk: cytosolic degradation product; m: mature-sized GFP protein.
Mentions: Next, Tat-dependent protein translocation was analyzed using a previously described model protein (PhoDCG-GFP [35]) consisting of the signal peptide of the C. glutamicum Tat substrate PhoD (an alkaline phosphatase) fused to green fluorescent protein (GFP). As shown in Fig 3, large amounts of mature GFP are present in the supernatant of C. glutamicum wild-type containing plasmid pCGPhoDCG-GFP (lane 4). Furthermore, hardly any detectable GFP-derived polypeptides were present in the cellular fraction of the same strain (lane 3). In contrast, no secreted GFP protein is found in the supernatant of the ΔtatAC, ΔtatA/E, or ΔtatB mutant strains (lanes 6, 8, 10) and, in the cell fractions of the corresponding strains (lanes 5, 7, 9), an accumulation of PhoDCG-GFP precursor and degradation products of it can be observed. These results clearly indicate that TatA, TatB, and TatC are equally important for the translocation of PhoDCG-GFP across the cytoplasmic membrane of C. glutamicum.

Bottom Line: Furthermore, our results clearly show that TatB, besides TatA and TatC, is strictly required for unimpaired aerobic growth.In addition, TatB was also found to be essential for the secretion of a heterologous Tat-dependent model protein into the C. glutamicum culture supernatant.Together with our finding that expression of the C. glutamicum TatB in an E. coli ΔtatB mutant strain resulted in the formation of an active Tat translocase, our results clearly indicate that a TatABC translocase is used as the physiologically relevant functional unit for Tat-dependent protein translocation in C. glutamicum and, most likely, also in other TatB-containing Actinobacteria.

View Article: PubMed Central - PubMed

Affiliation: Institut für Bio- und Geowissenschaften 1, IBG1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany.

ABSTRACT
The twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Escherichia coli and other Gram-negative bacteria possess a TatABC-type Tat translocase in which each of the three inner membrane proteins TatA, TatB, and TatC performs a mechanistically distinct function. In contrast, low-GC Gram-positive bacteria, such as Bacillus subtilis, use a TatAC-type minimal Tat translocase in which the TatB function is carried out by a bifunctional TatA. In high-GC Gram-positive Actinobacteria, such as Mycobacterium tuberculosis and Corynebacterium glutamicum, tatA, tatB, and tatC genes can be identified, suggesting that these organisms, just like E. coli, might use TatABC-type Tat translocases as well. However, since contrary to this view a previous study has suggested that C. glutamicum might in fact use a TatAC translocase with TatB only playing a minor role, we reexamined the requirement of TatB for Tat-dependent protein translocation in this microorganism. Under aerobic conditions, the misassembly of the Rieske iron-sulfur protein QcrA was identified as a major reason for the severe growth defect of Tat-defective C. glutamicum mutant strains. Furthermore, our results clearly show that TatB, besides TatA and TatC, is strictly required for unimpaired aerobic growth. In addition, TatB was also found to be essential for the secretion of a heterologous Tat-dependent model protein into the C. glutamicum culture supernatant. Together with our finding that expression of the C. glutamicum TatB in an E. coli ΔtatB mutant strain resulted in the formation of an active Tat translocase, our results clearly indicate that a TatABC translocase is used as the physiologically relevant functional unit for Tat-dependent protein translocation in C. glutamicum and, most likely, also in other TatB-containing Actinobacteria.

No MeSH data available.


Related in: MedlinePlus