Display of green fluorescent protein on Escherichia coli cell surface

Shi, Huidong; Su, Wei

doi:10.1016/s0141-0229(00)00281-7

Cited by 70 publications

(66 citation statements)

References 36 publications

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“…Clearly, a signal peptidase motif is missing, allowing PepI or any PepI fusion protein to escape from secP-mediated cleavage. Moreover, the sec machinery appears to be able to translocate GFP fusion proteins to the bacterial surface, as has been demonstrated, e.g., for OmpA-GFP (33) and ChoD-GFP (8) fusion proteins. Certainly, more experimental work is needed for full understanding of the PepImediated bacteriocin immunity; however, the data presented here provide an excellent basis for further studies of this unique phenomenon.…”

Section: Discussionmentioning

confidence: 67%

Localization and Functional Analysis of PepI, the Immunity Peptide of Pep5-Producing Staphylococcus epidermidis Strain 5

Schneider

Pag

Sahl

2004

Appl Environ Microbiol

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Pep5 is a cationic pore-forming lantibiotic produced by Staphylococcus epidermidis strain 5. The producer strain protects itself from the lethal action of its own bacteriocin through the 69-amino-acid immunity peptide PepI. The N-terminal segment of PepI contains a 20-amino-acid stretch of apolar residues, whereas the C terminus is very hydrophilic, with a net positive charge. We used green fluorescent protein (GFP)-PepI fusions to obtain information on its localization in vivo. PepI was found to occur outside the cytoplasm and to accumulate at the membrane-cell wall interface. The extracellular localization appeared essential for conferring immunity. We analyzed the functional role of the specific segments by constructing various mutant peptides, which were also fused to GFP. When the hydrophobic N-terminal segment of PepI was disrupted by introducing charged amino acids, the export of PepI was blocked and clones expressing such mutant peptides were Pep5 sensitive. When PepI was successively shortened at the C terminus, in contrast, its export properties remained unchanged whereas its ability to confer immunity was gradually reduced. The results show that the N-terminal part is required for the transport of PepI and that the C-terminal part is important for conferring the immunity phenotype. A concept based on target shielding is proposed for the PepI immunity mechanism.Lantibiotics, a subgroup of bacteriocins from gram-positive bacteria, are polycyclic peptides containing modified amino acids: in particular, the thioether amino acids lanthionine and 3-methyllanthionine and the dehydroamino acids 2,3-didehydroalanine and 2,3-didehydrobutyrine. Lantibiotics are ribosomally synthesized as precursor peptides, consisting of a leader sequence and a propeptide part (31). The precursor peptides are converted into the mature peptide by posttranslational modification followed by processing and export from the producing cell (for a review, see reference 27). The cationic lantibiotic Pep5 is produced by Staphylococcus epidermidis strain 5 (28) and is classified along with nisin, subtilin, and epidermin as a pore-forming type-A lantibiotic (27). The biosynthetic gene cluster of Pep5 contains the structural gene pepA as well as the genes for posttranslational modification (pepB and pepC), processing (pepP), transport (pepT), and immunity (pepI) (18).Bacteriocin production generally requires a self-protection mechanism for the producer strain (1, 29). Various immunity concepts have been elaborated by the different groups of bacteria; e.g., the immunity proteins of the channel-forming colicins (e.g., colicin A, E1, and B) reside in the cytoplasmic membrane and protect the producer by forming a stoichiometric complex with the respective colicin (10, 32, 36). For the lantibiotic bacteriocins of gram-positive bacteria generally two different mechanisms, which in some cases may even complement each other, are found: the small immunity peptides collectively defined as LanI and the ABC (ATP-binding cassette) transporters LanFEG. Th...

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Section: Discussionmentioning

confidence: 67%

Localization and Functional Analysis of PepI, the Immunity Peptide of Pep5-Producing Staphylococcus epidermidis Strain 5

Schneider

Pag

Sahl

2004

Appl Environ Microbiol

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“…Two different anchoring motifs were employed for the targeting of OPH and GFP onto the cell surface in order to minimize direct competition for the same translocation machinery. To date, only a few anchoring motifs, which include Lpp-OmpA from E. coli and INP from P. syringae, have been shown to target GFP to the cell surface (22,35). Here, the Lpp-OmpA chimera, comprised of a localization domain (Lpp) and a transmembrane domain (OmpA), was employed as an anchoring motif, and a pUC18-based vector, pLOG18, was used to target Lpp-OmpA-GFP to the cell surface of E. coli.…”

Section: Resultsmentioning

confidence: 99%

Development of an Autofluorescent Whole-Cell Biocatalyst by Displaying Dual Functional Moieties on Escherichia coli Cell Surfaces and Construction of a Coculture with Organophosphate-Mineralizing Activity

Yang

Zhu

Yang

et al. 2008

Appl Environ Microbiol

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“…Cell fractionation was carried out firstly for the recombinant strain Pse-w-MT and the original strain Psew using a method based on an ultrasonic differential solubilization of cell compartments with 2% Triton X-200 (v/ v) and a standard sucrose gradient-centrifugation procedure to verify surface localization of fusion protein inaK-N/MT on the recipient strain Pse-w (Shi and Su, 2001). Western blot analysis was used to verify the surface localization of the fusion protein inaK-N/MT-1 on the strain Pse-w-MT cells.…”

Section: Bacterialmentioning

confidence: 99%

Isolation and engineering of plant growth promoting rhizobacteria <i>Pseudomonas aeruginosa</i> for enhanced cadmium bioremediation

Huang¹,

Liu²,

et al. 2016

J. Gen. Appl. Microbiol.

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Although many bacteria are tolerant to heavy metals and play important roles in the immobilization of heavy metals, they cannot always be dependably reproduced under field conditions. In this work, a cadmium (Cd)-resistant bacterium was isolated from a Cd-contaminated oil field and identified as Pseudomonas aeruginosa (Pse-w). We then determined various plant growth promoting features such as the solubilization of phosphate, and the production of indole-3-acetic acid and siderophores. Lastly, we engineered the strain Psew-MT by targeting metallothioneins to the cell surface of Pse-w to immobilize Cd 2+ and promote plant growth. Our data revealed that Pse-w exhibited high levels of resistance to Cd 2+ (4 mM) and showed various plant growth promoting features. The engineered strain Pse-w-MT was found to adsorb Cd 2+ mainly via extracellular deposition, and had an enhanced ability for immobilizing Cd 2+ ions from the external media. Furthermore, the inoculation of Cd-polluted soil with Pse-w-MT significantly elevated the shoot and root biomass and leaf chlorophyll content. Similarly, plants inoculated with Psew-MT demonstrated markedly lower Cd 2+ accumulation in the root and shoot system. It was concluded that plant growth promoting rhizobacteria with a high Cd 2+ tolerance was an ideal candidate to be engineered for bioremediation and plant growth promotion against Cd-induced stress.

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Display of green fluorescent protein on Escherichia coli cell surface

Cited by 70 publications

References 36 publications

Localization and Functional Analysis of PepI, the Immunity Peptide of Pep5-Producing Staphylococcus epidermidis Strain 5

Localization and Functional Analysis of PepI, the Immunity Peptide of Pep5-Producing Staphylococcus epidermidis Strain 5

Development of an Autofluorescent Whole-Cell Biocatalyst by Displaying Dual Functional Moieties on Escherichia coli Cell Surfaces and Construction of a Coculture with Organophosphate-Mineralizing Activity

Isolation and engineering of plant growth promoting rhizobacteria <i>Pseudomonas aeruginosa</i> for enhanced cadmium bioremediation

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