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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

SDS sensitivity of E. coli tat mutant strains expressing the C. glutamicum TatB protein from plasmid pHSG-TatBCG.MC4100 (wild-type) containing the empty vector pHSG575 (black squares), BØD (ΔtatB) containing pHSG575 (green circles), BØD containing pHSG-TatBEC (pink triangles), BØD containing pHSG-TatBCG (purple asterisks), JARV15 (ΔtatA/E) containing pHSG575 (blue diamonds), and JARV15 containing pHSG-TatBCG (red open pentagons) were analyzed for SDS sensitivity using the assay described by Ize et al. [40]. The cells were grown for 3 h in LB medium containing 100 μM IPTG in the presence of various SDS concentrations as outlined in the Material and Methods section. 100% survival is defined as the optical density of each strain after 3 h growth in LB medium without SDS. The experiments were performed in triplicate using biologically independent replicates and the respective standard deviations are indicated.
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pone.0123413.g006: SDS sensitivity of E. coli tat mutant strains expressing the C. glutamicum TatB protein from plasmid pHSG-TatBCG.MC4100 (wild-type) containing the empty vector pHSG575 (black squares), BØD (ΔtatB) containing pHSG575 (green circles), BØD containing pHSG-TatBEC (pink triangles), BØD containing pHSG-TatBCG (purple asterisks), JARV15 (ΔtatA/E) containing pHSG575 (blue diamonds), and JARV15 containing pHSG-TatBCG (red open pentagons) were analyzed for SDS sensitivity using the assay described by Ize et al. [40]. The cells were grown for 3 h in LB medium containing 100 μM IPTG in the presence of various SDS concentrations as outlined in the Material and Methods section. 100% survival is defined as the optical density of each strain after 3 h growth in LB medium without SDS. The experiments were performed in triplicate using biologically independent replicates and the respective standard deviations are indicated.

Mentions: E. coli tat mutants possess pleiotropic cell envelope defects that are caused by the mislocalization of two Tat-dependent periplasmic amidases (AmiA and AmiC) that are involved in cell wall turnover [40]. Due to this fact, the E. coli tat mutants are sensitive to the presence of sodium dodecyl sulfate (SDS), unless they are complemented with a tat gene that allows for the formation of a functionally active Tat translocase [41]. To investigate whether the C. glutamicum TatB can indeed perform the specialized functions associated with the E. coli TatB, we tested whether the C. glutamicum TatB can complement the E. coli ΔtatB mutant strain BØD [17]. Following the protocol established by Ize et al. [40], the respective cells were grown in the presence of various SDS concentrations and the % survival of each strain was determined for the SDS concentrations tested (Fig 6). In contrast to the MC4100 wild-type strain (black squares), the uncomplemented ΔtatB mutant (green circles) is highly sensitive to SDS. As expected, SDS resistance of BØD could be restored by complementation with plasmid-encoded E. coli TatB (pink triangles). Likewise, restoration of SDS resistance was observed when the C. glutamicum TatB was expressed in BØD (purple asterisks), clearly demonstrating that the heterologous TatB can fulfill the dedicated function of the E. coli TatB protein. In contrast, expression of the C. glutamicum TatB from plasmid pHSG-TatBCG in the ΔtatA/E mutant JARV15 [11] did not result in the formation of a functional Tat translocase, since the corresponding strain (red open pentagons) showed the same SDS sensitivity as the uncomplemented ΔtatA/E mutant (blue diamonds). These results strongly suggest that the C. glutamicum TatB protein in fact is a bona fide TatB protein that is functionally equivalent to the TatB proteins present in TatABC-type Tat translocases.


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

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

SDS sensitivity of E. coli tat mutant strains expressing the C. glutamicum TatB protein from plasmid pHSG-TatBCG.MC4100 (wild-type) containing the empty vector pHSG575 (black squares), BØD (ΔtatB) containing pHSG575 (green circles), BØD containing pHSG-TatBEC (pink triangles), BØD containing pHSG-TatBCG (purple asterisks), JARV15 (ΔtatA/E) containing pHSG575 (blue diamonds), and JARV15 containing pHSG-TatBCG (red open pentagons) were analyzed for SDS sensitivity using the assay described by Ize et al. [40]. The cells were grown for 3 h in LB medium containing 100 μM IPTG in the presence of various SDS concentrations as outlined in the Material and Methods section. 100% survival is defined as the optical density of each strain after 3 h growth in LB medium without SDS. The experiments were performed in triplicate using biologically independent replicates and the respective standard deviations are indicated.
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Related In: Results  -  Collection

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

pone.0123413.g006: SDS sensitivity of E. coli tat mutant strains expressing the C. glutamicum TatB protein from plasmid pHSG-TatBCG.MC4100 (wild-type) containing the empty vector pHSG575 (black squares), BØD (ΔtatB) containing pHSG575 (green circles), BØD containing pHSG-TatBEC (pink triangles), BØD containing pHSG-TatBCG (purple asterisks), JARV15 (ΔtatA/E) containing pHSG575 (blue diamonds), and JARV15 containing pHSG-TatBCG (red open pentagons) were analyzed for SDS sensitivity using the assay described by Ize et al. [40]. The cells were grown for 3 h in LB medium containing 100 μM IPTG in the presence of various SDS concentrations as outlined in the Material and Methods section. 100% survival is defined as the optical density of each strain after 3 h growth in LB medium without SDS. The experiments were performed in triplicate using biologically independent replicates and the respective standard deviations are indicated.
Mentions: E. coli tat mutants possess pleiotropic cell envelope defects that are caused by the mislocalization of two Tat-dependent periplasmic amidases (AmiA and AmiC) that are involved in cell wall turnover [40]. Due to this fact, the E. coli tat mutants are sensitive to the presence of sodium dodecyl sulfate (SDS), unless they are complemented with a tat gene that allows for the formation of a functionally active Tat translocase [41]. To investigate whether the C. glutamicum TatB can indeed perform the specialized functions associated with the E. coli TatB, we tested whether the C. glutamicum TatB can complement the E. coli ΔtatB mutant strain BØD [17]. Following the protocol established by Ize et al. [40], the respective cells were grown in the presence of various SDS concentrations and the % survival of each strain was determined for the SDS concentrations tested (Fig 6). In contrast to the MC4100 wild-type strain (black squares), the uncomplemented ΔtatB mutant (green circles) is highly sensitive to SDS. As expected, SDS resistance of BØD could be restored by complementation with plasmid-encoded E. coli TatB (pink triangles). Likewise, restoration of SDS resistance was observed when the C. glutamicum TatB was expressed in BØD (purple asterisks), clearly demonstrating that the heterologous TatB can fulfill the dedicated function of the E. coli TatB protein. In contrast, expression of the C. glutamicum TatB from plasmid pHSG-TatBCG in the ΔtatA/E mutant JARV15 [11] did not result in the formation of a functional Tat translocase, since the corresponding strain (red open pentagons) showed the same SDS sensitivity as the uncomplemented ΔtatA/E mutant (blue diamonds). These results strongly suggest that the C. glutamicum TatB protein in fact is a bona fide TatB protein that is functionally equivalent to the TatB proteins present in TatABC-type Tat translocases.

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