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A tamB homolog is involved in maintenance of cell envelope integrity and stress resistance of Deinococcus radiodurans

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ABSTRACT

The translocation and assembly module (TAM) in bacteria consists of TamA and TamB that form a complex to control the transport and secretion of outer membrane proteins. Herein, we demonstrated that the DR_1462-DR_1461-DR_1460 gene loci on chromosome 1 of Deinococcus radiodurans, which lacks tamA homologs, is a tamB homolog (DR_146T) with two tamB motifs and a DUF490 motif. Mutation of DR_146T resulted in cell envelope peeling and a decrease in resistance to shear stress and osmotic pressure, as well as an increase in oxidative stress resistance, consistent with the phenotype of a surface layer (S-layer) protein SlpA (DR_2577) mutant, demonstrating the involvement of DR_146T in maintenance of cell envelope integrity. The 123 kDa SlpA was absent and only its fragments were present in the cell envelope of DR_146T mutant, suggesting that DR_146T might be involved in maintenance of the S-layer. A mutant lacking the DUF490 motif displayed only a slight alteration in phenotype compared with the wild type, suggesting DUF490 is less important than tamB motif for the function of DR_146T. These findings enhance our understanding of the properties of the multilayered envelope in extremophilic D. radiodurans, as well as the diversity and functions of TAMs in bacteria.

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Survival of D. radiodurans wild type and mutant strains exposed to H2O2 treatment.The ∆DR_146T and ∆DR_2577 showed higher resistance to H2O2 and increased Mn/Fe ratio than D. radiodurans wild type. (a) Comparison of the sensitivity of wild-type (DRWT), ∆DR_146T and ∆DR_2577 strains under H2O2 treatment. Cells (107 CFU ml−1) were dripped onto TGY plates following H2O2 treatment for 30 min and dilution with sterile phosphate buffer (0.1 M, pH 7.4). Different dilutions of cell cultures are indicated in the figure. (b) Survival assays of D. radiodurans wild type and ∆DR_146T strains. Cells were suspended in phosphate buffer (107 CFU ml−1), treated with H2O2 for 30 min, and the survival fraction was measured by counting bacterial colonies of treated compared with the untreated samples (0 mM H2O2). (c) ICP-MS analysis of the relative metal ion (Fe, Zn, Cu, Mn) content in wild type and mutant strains. (d) Ratio of Mn and Fe ion content in wild type and mutant strains. Experiments were independently performed three times. P-values indicate the significance compared with wild type cells.
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f6: Survival of D. radiodurans wild type and mutant strains exposed to H2O2 treatment.The ∆DR_146T and ∆DR_2577 showed higher resistance to H2O2 and increased Mn/Fe ratio than D. radiodurans wild type. (a) Comparison of the sensitivity of wild-type (DRWT), ∆DR_146T and ∆DR_2577 strains under H2O2 treatment. Cells (107 CFU ml−1) were dripped onto TGY plates following H2O2 treatment for 30 min and dilution with sterile phosphate buffer (0.1 M, pH 7.4). Different dilutions of cell cultures are indicated in the figure. (b) Survival assays of D. radiodurans wild type and ∆DR_146T strains. Cells were suspended in phosphate buffer (107 CFU ml−1), treated with H2O2 for 30 min, and the survival fraction was measured by counting bacterial colonies of treated compared with the untreated samples (0 mM H2O2). (c) ICP-MS analysis of the relative metal ion (Fe, Zn, Cu, Mn) content in wild type and mutant strains. (d) Ratio of Mn and Fe ion content in wild type and mutant strains. Experiments were independently performed three times. P-values indicate the significance compared with wild type cells.

Mentions: The resistance of ∆DR_146T and the SlpA mutant (∆DR_2577) to oxidative stress was much higher than that of wild type cells (Fig. 6a and b). The ∆DR_146T strain could survive high concentrations of H2O2 (160 mM) without any obvious decline in viability compared with controls (0 mM H2O2), indicating that disruption of DR_146T leads to an increase in resistance to oxidative stress. Previous studies demonstrated that accumulation of manganese ions (Mn2+) and a higher intracellular Mn/Fe ratio contribute to oxidative stress resistance in D. radiodurans through Mn complex-mediated ROS scavenging252634. Thus, we measured the metal ion content in mutant and wild type cells by inductively-coupled plasma-mass spectrometry (ICP-MS) (Fig. 6c and d). The Mn ion content in ∆DR_146T and ∆DR_2577 was almost twice that of wild type cells, while the Fe and Zn ion content were slightly reduced in the mutants. Therefore, the H2O2 resistance of ∆DR_146T might be attributed to the increased Mn/Fe ratio in the mutant cells.


A tamB homolog is involved in maintenance of cell envelope integrity and stress resistance of Deinococcus radiodurans
Survival of D. radiodurans wild type and mutant strains exposed to H2O2 treatment.The ∆DR_146T and ∆DR_2577 showed higher resistance to H2O2 and increased Mn/Fe ratio than D. radiodurans wild type. (a) Comparison of the sensitivity of wild-type (DRWT), ∆DR_146T and ∆DR_2577 strains under H2O2 treatment. Cells (107 CFU ml−1) were dripped onto TGY plates following H2O2 treatment for 30 min and dilution with sterile phosphate buffer (0.1 M, pH 7.4). Different dilutions of cell cultures are indicated in the figure. (b) Survival assays of D. radiodurans wild type and ∆DR_146T strains. Cells were suspended in phosphate buffer (107 CFU ml−1), treated with H2O2 for 30 min, and the survival fraction was measured by counting bacterial colonies of treated compared with the untreated samples (0 mM H2O2). (c) ICP-MS analysis of the relative metal ion (Fe, Zn, Cu, Mn) content in wild type and mutant strains. (d) Ratio of Mn and Fe ion content in wild type and mutant strains. Experiments were independently performed three times. P-values indicate the significance compared with wild type cells.
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f6: Survival of D. radiodurans wild type and mutant strains exposed to H2O2 treatment.The ∆DR_146T and ∆DR_2577 showed higher resistance to H2O2 and increased Mn/Fe ratio than D. radiodurans wild type. (a) Comparison of the sensitivity of wild-type (DRWT), ∆DR_146T and ∆DR_2577 strains under H2O2 treatment. Cells (107 CFU ml−1) were dripped onto TGY plates following H2O2 treatment for 30 min and dilution with sterile phosphate buffer (0.1 M, pH 7.4). Different dilutions of cell cultures are indicated in the figure. (b) Survival assays of D. radiodurans wild type and ∆DR_146T strains. Cells were suspended in phosphate buffer (107 CFU ml−1), treated with H2O2 for 30 min, and the survival fraction was measured by counting bacterial colonies of treated compared with the untreated samples (0 mM H2O2). (c) ICP-MS analysis of the relative metal ion (Fe, Zn, Cu, Mn) content in wild type and mutant strains. (d) Ratio of Mn and Fe ion content in wild type and mutant strains. Experiments were independently performed three times. P-values indicate the significance compared with wild type cells.
Mentions: The resistance of ∆DR_146T and the SlpA mutant (∆DR_2577) to oxidative stress was much higher than that of wild type cells (Fig. 6a and b). The ∆DR_146T strain could survive high concentrations of H2O2 (160 mM) without any obvious decline in viability compared with controls (0 mM H2O2), indicating that disruption of DR_146T leads to an increase in resistance to oxidative stress. Previous studies demonstrated that accumulation of manganese ions (Mn2+) and a higher intracellular Mn/Fe ratio contribute to oxidative stress resistance in D. radiodurans through Mn complex-mediated ROS scavenging252634. Thus, we measured the metal ion content in mutant and wild type cells by inductively-coupled plasma-mass spectrometry (ICP-MS) (Fig. 6c and d). The Mn ion content in ∆DR_146T and ∆DR_2577 was almost twice that of wild type cells, while the Fe and Zn ion content were slightly reduced in the mutants. Therefore, the H2O2 resistance of ∆DR_146T might be attributed to the increased Mn/Fe ratio in the mutant cells.

View Article: PubMed Central - PubMed

ABSTRACT

The translocation and assembly module (TAM) in bacteria consists of TamA and TamB that form a complex to control the transport and secretion of outer membrane proteins. Herein, we demonstrated that the DR_1462-DR_1461-DR_1460 gene loci on chromosome 1 of Deinococcus radiodurans, which lacks tamA homologs, is a tamB homolog (DR_146T) with two tamB motifs and a DUF490 motif. Mutation of DR_146T resulted in cell envelope peeling and a decrease in resistance to shear stress and osmotic pressure, as well as an increase in oxidative stress resistance, consistent with the phenotype of a surface layer (S-layer) protein SlpA (DR_2577) mutant, demonstrating the involvement of DR_146T in maintenance of cell envelope integrity. The 123 kDa SlpA was absent and only its fragments were present in the cell envelope of DR_146T mutant, suggesting that DR_146T might be involved in maintenance of the S-layer. A mutant lacking the DUF490 motif displayed only a slight alteration in phenotype compared with the wild type, suggesting DUF490 is less important than tamB motif for the function of DR_146T. These findings enhance our understanding of the properties of the multilayered envelope in extremophilic D. radiodurans, as well as the diversity and functions of TAMs in bacteria.

No MeSH data available.


Related in: MedlinePlus