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

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.

Fig 1. Growth phenotype of C. glutamicum ATCC13032 wild-type, ΔtatAC, ΔtatA/E, and ΔtatB mutants.

Cells were inoculated to an OD₆₀₀ of 0.5 in 750 μl BHIS medium and cultivated in 48-well FlowerPlates in a BioLector system for 48 h at 30°C, 1100 rpm under constant 85% relative humidity. Growth of the respective C. glutamicum strains, as indicated in the figure, was monitored as backscattered light (620 nm; signal gain factor 20) in 15 min intervals. The growth curves show one representative experiment of three independent biological replicates. Standard deviations are given for 10 selected time points. a.u.: arbitrary units.

Fig 2. The growth defect of the C. glutamicum ΔtatB mutant is reversed by expressing the C. glutamicum TatB in trans from plasmid pEC-TatB^CG.

Cells were inoculated to an OD₆₀₀ of 0.5 in 750 μl BHIS medium containing 10 μM IPTG and cultivated in 48-well FlowerPlates in a BioLector system for 48 h at 30°C, 1100 rpm under constant 85% relative humidity. Growth of the respective C. glutamicum strains, as indicated in the figure, was monitored as backscattered light (620 nm; signal gain factor 20) in 15 min intervals. The growth curves show one representative experiment of three independent biological replicates. Standard deviations are given for 10 selected time points. a.u.: arbitrary units.

Fig 3. Analysis of PhoD^CG-GFP secretion in C. glutamicum wild-type and tat mutant strains.

Cultures of C. glutamicum strains expressing the Tat-dependent PhoD^CG-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 pCGPhoD^CG-GFP (lanes 3 and 4), and the pCGPhoD^CG-GFP-containing C. glutamicum mutant strains ΔtatAC (lanes 5 and 6), ΔtatB (lanes 7 and 8), and ΔtatA/E (lanes 9 and 10). p: PhoD^CG-GFP precursor; asterisk: cytosolic degradation product; m: mature-sized GFP protein.

Fig 4. The correct membrane assembly of the physiologically important Tat substrate QcrA is required for unimpaired growth of C. glutamicum.

Cells were inoculated to an OD₆₀₀ of 0.5 in 750 μl BHIS medium containing 100 μM IPTG and cultivated in 48-well FlowerPlates in a BioLector system for 48 h at 30°C, 1100 rpm under constant 85% relative humidity. Growth of the respective C. glutamicum strains, as indicated in the figure, was monitored as backscattered light (620 nm; signal gain factor 20) in 15 min intervals. The growth curves show one representative experiment of three independent biological replicates. Standard deviations are given for 10 selected time points. a.u.: arbitrary units.

Fig 5. The growth defect of a C. glutamicum ΔqcrA ΔtatB double mutant cannot be complemented in trans by plasmid-encoded QcrA.

Cells were inoculated to an OD₆₀₀ of 0.5 in 750 μl BHIS medium containing 100 μM IPTG and cultivated in 48-well FlowerPlates in a BioLector system for 48 h at 30°C, 1100 rpm under constant 85% relative humidity. Growth of the respective C. glutamicum strains, as indicated in the figure, was monitored as backscattered light (620 nm; signal gain factor 20) in 15 min intervals. The growth curves show one representative experiment of three independent biological replicates. Standard deviations are given for 10 selected time points. a.u.: arbitrary units.

Fig 6. SDS sensitivity of E. coli tat mutant strains expressing the C. glutamicum TatB protein from plasmid pHSG-TatB^CG.

MC4100 (wild-type) containing the empty vector pHSG575 (black squares), BØD (ΔtatB) containing pHSG575 (green circles), BØD containing pHSG-TatB^EC (pink triangles), BØD containing pHSG-TatB^CG (purple asterisks), JARV15 (ΔtatA/E) containing pHSG575 (blue diamonds), and JARV15 containing pHSG-TatB^CG (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.