Functional Display of Active Bovine Adrenodoxin on the Surface of E. coli by Chemical Incorporation of the [2Fe–2S] Cluster

Jose, Joachim; Bernhardt, Rita; Hannemann, Frank

doi:10.1002/1439-7633(20010903)2:9<695::aid-cbic695>3.0.co;2-s

Cited by 54 publications

(74 citation statements)

References 32 publications

(52 reference statements)

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“…This shows that under native conditions the AIDA autotransporter is preferentially expressed on the bacterial surface as a monomer, is also able to form dimers, and does not form larger oligomers. The ability of the AIDA translocator to form dimers in vivo is also supported by the surface presentation of functional bovine adrenodoxin and of sorbitol dehydrogenase, enzymes that are active only as dimers (15)(16)(17).…”

Section: Discussionmentioning

confidence: 99%

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Arrangement of the Translocator of the Autotransporter Adhesin Involved in Diffuse Adherence on the Bacterial Surface

et al. 2005

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Autotransporters of gram-negative bacteria are single-peptide secretion systems that consist of a functional N-terminal ␣-domain ("passenger") fused to a C-terminal ␤-domain ("translocator"). How passenger proteins are translocated through the outer membrane has not been resolved, and at present essentially three different models are discussed. In the widely accepted "hairpin model" the passenger proteins are translocated through a channel formed by the ␤-barrel of the translocator that is integrated in the outer membrane. This model has been challenged by a recent proposal for a general autotransporter model suggesting that there is a hexameric translocation pore that is generated by the oligomerization of six ␤-domains. A third model suggests that conserved Omp85 participates in autotransporter integration and passenger protein translocation. To examine these models, in this study we investigated the presence of putative oligomeric structures of the translocator of the autotransporter adhesin involved in diffuse adherence (AIDA) in vivo by cross-linking techniques. Furthermore, the capacity of isolated AIDA fusion proteins to form oligomers was studied in vitro by several complementary analytical techniques, such as analytical gel filtration, electron microscopy, immunogold labeling, and cross-linking of recombinant autotransporter proteins in which different passenger proteins were fused to the AIDA translocator. Our results show that the AIDA translocator is mostly present as a monomer. Only a fraction of the AIDA autotransporter was found to form dimers on the bacterial surface and in solution. Higher-order structures, such as hexamers, were not detected either in vivo or in vitro and can therefore be excluded as functional moieties for the AIDA autotransporter.

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Section: Discussionmentioning

confidence: 99%

“…After cleavage the AIDA-I adhesin remains noncovalently associated with the bacterial surface. In AIDA and other autotransporter proteins the authentic passenger can be replaced by heterologous antigens which are functionally expressed on the bacterial surface (7,15,16,23,24,38).…”

mentioning

confidence: 99%

Arrangement of the Translocator of the Autotransporter Adhesin Involved in Diffuse Adherence on the Bacterial Surface

et al. 2005

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“…The seminal work on secretion has also demonstrated the potential biotechnological exploitation of the autotransporter secretion pathway, namely, by displaying heterologous proteins on the bacterial surface (453). The possible applications of this autodisplay system are numerous and include (i) exposure of antigenic determinants for vaccine development (142,267,316), (ii) expression of peptide libraries for epitope mapping or antibody specificity test (258), (iii) display of receptor or ligand for binding assays or purification (499,500), (iv) functional domain analyses of a heterologous protein (59,499,500), and (v) bioconversion by expressing enzymatic activity on the bacterial surface (229,230,276). In conclusion, further understanding of the autotransporter secretion pathway and the functions of the passenger domains will facilitate a richer view of the mechanisms and evolution of bacterial pathogenesis and provide important practical applications for the medical and biotechnological communities.…”

Section: Future Directionsmentioning

confidence: 99%

Type V Protein Secretion Pathway: the Autotransporter Story

Henderson

Navarro-Garcı́a

Desvaux

et al. 2004

Microbiol Mol Biol Rev

713

795

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Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have developed several systems for protein secretion. The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins. Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria. Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis. However, most of the autotransporter proteins remain to be characterized. In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function

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“…With an aim to develop a novel system for the display of enzymes, we examined the E. coli outer membrane protein FadL as a potential anchoring motif. Several strategies have been developed to fuse target proteins to the anchoring motif: N-terminal fusion, sandwich fusion, and Cterminal fusion (11,16,26,32). Among them, we employed a C-terminal deletion-fusion strategy, as it allows display of relatively large proteins of up to 60 kDa (21).…”

Section: Discussionmentioning

confidence: 93%

“…Activity and stability of cell surface-displayed lipase. To determine the optimal conditions of cell surface-displayed lipase, reactions were carried out at various temperatures ranging from 16 . Cell surface-displayed lipase showed the maximum activity at 50°C.…”

Section: Resultsmentioning

confidence: 99%

Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis

Lee

Choi²,

Park³

et al. 2004

Appl Environ Microbiol

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We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50°C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70°C, and retained over 90% of the full activity after incubation at 50°C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.

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Functional Display of Active Bovine Adrenodoxin on the Surface of E. coli by Chemical Incorporation of the [2Fe–2S] Cluster

Cited by 54 publications

References 32 publications

Arrangement of the Translocator of the Autotransporter Adhesin Involved in Diffuse Adherence on the Bacterial Surface

Arrangement of the Translocator of the Autotransporter Adhesin Involved in Diffuse Adherence on the Bacterial Surface

Type V Protein Secretion Pathway: the Autotransporter Story

Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis

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