Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria

Meißner, Daniel; Vollstedt, Annah; Dijl, Jan Maarten van; Freudl, Roland

doi:10.1007/s00253-007-0934-8

Cited by 71 publications

(74 citation statements)

References 49 publications

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“…GFP was fused to the N terminus of YhcS to preserve the proper topology and avoid the secretion of GFP. GFP is not normally functional when translocated to the extracellular side of the membrane in bacteria (23,41).…”

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confidence: 99%

Functional Characterization and Localization of a Bacillus subtilis Sortase and Its Substrate and Use of This Sortase System To Covalently Anchor a Heterologous Protein to the B. subtilis Cell Wall for Surface Display

Liew¹,

Wang²,

Wong³

2011

Journal of Bacteriology

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Sortases catalyze the covalent anchoring of proteins to the cell surface on Gram-positive bacteria. Bioinformatic analysis suggests the presence of structural genes encoding sortases and their substrates in the Bacillus subtilis genome. In this study, a ␤-lactamase reporter was fused to the cell wall anchoring domain from a putative sortase substrate, YhcR. Covalent anchoring of this fusion protein to the cell wall was confirmed by using the eight-protease-deficient B. subtilis strain WB800 as the host. Inactivation of yhcS abolished the cell wall anchoring reaction. The amounts of fusion protein anchored to the cell wall were proportional to the levels of YhcS. These data demonstrate that YhcS and YhcR are the sortase and sortase substrate, respectively, in B. subtilis. Furthermore, yhcS is not essential for the survival of B. subtilis under the cultivation condition tested. YhcR fusions were distributed helically in the lateral cell wall. Interestingly, when viewed with an epifluorescence microscope, YhcS also appeared to form short helical arcs. This is the first report to illustrate such distribution of sortases in a rod-shaped bacterium. Models for the spatial distribution of both the sortase and its substrate are discussed. The amount of the reporters displayed on the surface was unambiguously quantified via a unique strategy. Under optimal conditions with the overproduction of YhcS, 47,300 YhcR fusions could be displayed per cell. Displayed reporters were biologically functional and surface accessible. Characterization of the sortase-substrate system allowed the successful development of a YhcR-based covalent surface display system. This system may have various biotechnological applications.

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confidence: 99%

Functional Characterization and Localization of a Bacillus subtilis Sortase and Its Substrate and Use of This Sortase System To Covalently Anchor a Heterologous Protein to the B. subtilis Cell Wall for Surface Display

Liew¹,

Wang²,

Wong³

2011

Journal of Bacteriology

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“…Recent metabolic engineering studies have shown that C. glutamicum is also capable of producing a variety of other commercially interesting compounds, e.g., other L-amino acids (4), D-amino acids (5), organic acids such as succinate (6)(7)(8)(9), diamines such as cadaverine (10,11) or putrescine (12), biofuels such as ethanol or isobutanol (13)(14)(15), and proteins (16)(17)(18).…”

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confidence: 99%

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

Klaffl¹,

Brocker²,

Kalinowski³

et al. 2013

Journal of Bacteriology

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c DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase, led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2, and IolR. Determination of the phosphoenolpyruvate carboxykinase activity of the ⌬ramA, ⌬gntR1 ⌬gntR2, and ⌬iolR deletion mutants indicated that RamA represses pck during growth on glucose about 2-fold, whereas GntR1, GntR2, and IolR activate pck expression about 2-fold irrespective of whether glucose or acetate served as the carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in a divergent orientation with respect to a iol gene cluster, encoding proteins involved in myoinositol uptake and degradation. Comparative DNA microarray analysis of the ⌬iolR mutant and the parental wild-type strain revealed strongly (>100-fold) elevated mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT1, and iolR. IolR therefore is presumably subject to negative autoregulation. A consensus DNA binding motif (5=-KGWCHTRACA-3=) which corresponds well to those of other GntR-type regulators of the HutC family was identified. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism.

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“…We also showed that pro-TG and pro-PG could be produced using the Tat pathway in C. glutamicum (20,21). More recently, Tat pathway-dependent secretion of GFP has been shown to be far more efficient in C. glutamicum than in two other gram-positive bacteria, B. subtilis and Staphylococcus carnosus (28), indicating that C. glutamicum could be a useful host for the production of heterologous proteins.…”

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confidence: 80%

TatABC Overexpression Improves Corynebacterium glutamicum Tat-Dependent Protein Secretion

Kikuchi

Itaya

Date

et al. 2009

Appl Environ Microbiol

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The twin-arginine translocation (Tat) pathway in Corynebacterium glutamicum has been described previously. The minimal functional Tat system in C. glutamicum required TatA and TatC but did not require TatB, although this component was required for maximal efficiency of Tat-dependent secretion. We previously demonstrated that Chryseobacterium proteolyticum pro-protein glutaminase (pro-PG) and Streptomyces mobaraensis pro-transglutaminase (pro-TG) could be secreted via the Tat pathway in C. glutamicum. Here we report that the amounts of pro-PG secreted were more than threefold larger when TatC or TatAC was overexpressed, and there was a further threefold increase when TatABC was overexpressed. These results show that the amount of TatC protein is the first bottleneck and the amount of TatB protein is the second bottleneck in Tat-dependent protein secretion in C. glutamicum. In addition, the amount of pro-TG that accumulated via the Tat pathway when TatABC was overexpressed with the TorA signal peptide in C. glutamicum was larger than the amount that accumulated via the Sec pathway. We concluded that TatABC overexpression improves Tat-dependent pro-PG and pro-TG secretion in C. glutamicum.Protein deamidation, which hydrolyzes the amido groups on glutamine or asparagine residues in proteins, is of great interest to the food industry because it has potential applications for improving the usefulness of a variety of food proteins (11, 41). A new protein glutaminase (PG), catalyzing the deamidation of proteins, was discovered in culture supernatants of Chryseobacterium proteolyticum (46, 47). However, the amount of PG produced by C. proteolyticum was too small for industrial applications. Streptomyces mobaraensis transglutaminase (TG) has been used in the food industry (50) to bind meat and fish and in gelled food products such as jelly, yogurt, and cheese. Moreover, this enzyme has great potential for use in the manufacture of materials found in cosmetics, thermostable microcapsules, and carriers of immobilized enzymes. Hence, there is a need to develop a more efficient system for production of TG.Extracellular protein production has several advantages over a cytoplasmic system. In many cases, the N-terminal amino acid residue of the secreted protein is identical to that of the natural protein, because the signal peptide is completely removed by a specific signal peptidase during secretion (44). In addition, due to disulfide bond formation, the correct protein conformation is more likely to occur when the enzyme is secreted, because the extracellular environment is more oxidative than the cytoplasmic environment (26). Finally, purification of a secreted protein is likely to be easier, as there are fewer contaminating proteins in the culture medium than in the cytoplasm.Most proteins secreted by bacteria are translocated across the cytoplasmic membrane by the Sec pathway, which acts on unfolded proteins using the energy provided by ATP hydrolysis (4, 33). Recently, the novel twin-arginine translocation (Tat) pathway wa...

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Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria

Cited by 71 publications

References 49 publications

Functional Characterization and Localization of a Bacillus subtilis Sortase and Its Substrate and Use of This Sortase System To Covalently Anchor a Heterologous Protein to the B. subtilis Cell Wall for Surface Display

Functional Characterization and Localization of a Bacillus subtilis Sortase and Its Substrate and Use of This Sortase System To Covalently Anchor a Heterologous Protein to the B. subtilis Cell Wall for Surface Display

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

TatABC Overexpression Improves Corynebacterium glutamicum Tat-Dependent Protein Secretion

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