Selection of PSII-Deficient Mutants of Synechocystis sp. PCC 6803 Created by Targeted in vitro Random Mutagenesis of psbAII Gene Based on Nitrofurantoin Resistance

AbstractWe describe the selection of Anticalins against a common tumour surface antigen, human Hsp70, using functional display on liveEscherichia colicells as fusion with a truncated EspP autotransporter. While found intracellularly in normal cells, Hsp70 is frequently exposed in a membrane-bound state on the surface of tumour cells and, even more pronounced, in metastases or after radiochemotherapy. Employing a recombinant Hsp70 fragment comprising residues 383-548 as the target, Anticalins were selected from a naïve bacterial library. The Anticalin with the highest affinity (KD=13 nm), as determined towards recombinant full-length Hsp70 by real-time surface plasmon resonance analysis, was improved toKD=510 pmby doped random mutagenesis and another cycle ofE. colisurface display, followed by rational combination of mutations. This Anticalin, which recognises a linear peptide epitope located in the interdomain linker of Hsp70, was demonstrated to specifically bind Hsp70 in its membrane-associated form in immunofluorescence microscopy and via flow cytometry using the FaDu cell line, which is positive for surface Hsp70. The radiolabelled and PASylated Anticalin revealed specific tumour accumulation in xenograft mice using positron emission tomography (PET) imaging. Furthermore, after enzymatic coupling to the protein toxin gelonin, the Anticalin showed potent cytotoxicity on FaDu cellsin vitro.

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Fluorescence quenching of the phycobilisome terminal emitter LCM from the cyanobacterium Synechocystis sp. PCC 6803 detected in vivo and in vitro

Journal of Photochemistry and Photobiology B Biology ◽

10.1016/j.jphotobiol.2013.05.014 ◽

2013 ◽

Vol 125 ◽

pp. 137-145 ◽

Cited By ~ 31

Author(s):

Igor N. Stadnichuk ◽

Mikhail F. Yanyushin ◽

Gábor Bernát ◽

Dmitry V. Zlenko ◽

Pavel M. Krasilnikov ◽

...

Keyword(s):

Fluorescence Quenching ◽

Synechocystis Sp ◽

Pcc 6803

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Perception and transduction of low-temperature signals to induce desaturation of fatty acids

Biochemical Society Transactions ◽

10.1042/bst0280628 ◽

2000 ◽

Vol 28 (6) ◽

pp. 628-630 ◽

Cited By ~ 32

Author(s):

I. Suzuki ◽

D. A. Los ◽

N. Murata

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Low Temperature ◽

Membrane Lipids ◽

Response Regulator ◽

Random Mutagenesis ◽

Fatty Acid Desaturases ◽

Histidine Kinases ◽

Synechocystis Sp ◽

Pcc 6803

When cells of the cyanobacterium Synechocystis sp. PCC 6803 are exposed to a low temperature, genes for fatty acid desaturases are expressed with resultant increases in the degree of unsaturation of fatty acids in membrane lipids. However, the sensor and transducers of low-temperature signals had not yet been identified. In order to identify these components we applied to the cyanobacterium Synechocystis sp. PCC 6803 the systematic disruption of all 43 putative genes for histidine kinases and random mutagenesis of the whole genome in conjunction with screening by the transcriptional activity of the promoter of the desB gene for the ω 3 desaturase. This allowed us to identify two histidine kinases and a response regulator as components of the perception and transduction of low-temperature signals for the expression of genes for fatty acid desaturases.

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Targeting of an Expressed Insect Selective Neurotoxin by its Recombinant Baculovirus: Pharmacokinetic and Pharmacodynamic Aspects

10.32747/1995.7571354.bard ◽

1995 ◽

Author(s):

Eliahu Zlotkin ◽

Shizuo G. Kamita ◽

Nor Chejanovsky ◽

S. Maeda

Keyword(s):

Random Mutagenesis ◽

Recombinant Baculovirus ◽

Silkworm Larvae ◽

In Vitro System ◽

Large Numbers ◽

Target Sites ◽

Local Supply ◽

Insect Cns ◽

Selection Of

Objectives: 1) Clarification of the mode of potentiation of an expressed insect selective neurotoxin (AaIT) by its recombinant baculovirus. 2) In vitro formation and/or modification of neuroactive polypeptides for the design of new improved recombinant baculoviruses. Results: 1) A combined utilization of bioassays, LM-cytochemistry, the highly resolutive EM immunogold and electrical recording from the CNS of baculovirus and AaIT - expressing – recombinant baculovirus infected larvae it has been shown that the recombinant virus potentiates the effect of the toxin. Potentiation is achieved through its continuous expression in the infected tracheal epithelia thus providing a: a) Local supply of freshly produced toxin in the vicinity of its traget sites; b) Translocation of the expressed toxin to the insect CNS. The latter exposes the recombinant toxin to new, critical, target sites which are inaccessible through the natural route of scorpion envenomation. 2) Subjecting a recombinant AaIT toxin to a newly designed system of random mutagenesis results in large numbers of new AaIT genes with amino acid substitutions. The new or modified toxin genes were inserted into a linear BmNPV expressed in silkworm cell culture and assayed on blowfly and silkworm larvae. Thus a system for mass formation and screening of neuroactive agents was developed. Contribution to agriculture: 1) Demonstration of the insecticidal mechanism, capacity and utility of the combination of neuroactive polypeptides and recombinant pathogens. 2) Development of a simple in vitro system for the formation and selection of new neuroactive polypeptides.

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Random Mutagenesis Targeted to the psbAII Gene of Synechocystis sp. PCC 6803 to Identify Functionally Important Residues in the D1 Protein of the Photosystem II Reaction Center

Plant and Cell Physiology ◽

10.1093/pcp/pcf066 ◽

2002 ◽

Vol 43 (5) ◽

pp. 540-548 ◽

Cited By ~ 4

Author(s):

Akihiro Yamasato ◽

Tomoe Kamada ◽

Kimiyuki Satoh

Keyword(s):

Photosystem Ii ◽

Reaction Center ◽

Random Mutagenesis ◽

D1 Protein ◽

Synechocystis Sp ◽

Pcc 6803

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Four Novel Genes Required for Optimal Photoautotrophic Growth of the Cyanobacterium Synechocystis sp. Strain PCC 6803 Identified by In Vitro Transposon Mutagenesis

Journal of Bacteriology ◽

10.1128/jb.186.3.875-879.2004 ◽

2004 ◽

Vol 186 (3) ◽

pp. 875-879 ◽

Cited By ~ 13

Author(s):

Shulu Zhang ◽

Susan M. Laborde ◽

Laurie K. Frankel ◽

Terry M. Bricker

Keyword(s):

Coat Protein ◽

Malic Enzyme ◽

Transposon Mutagenesis ◽

Hypothetical Proteins ◽

Photoautotrophic Growth ◽

First Report ◽

In Vitro Transposon Mutagenesis ◽

Synechocystis Sp ◽

Pcc 6803

ABSTRACT Four novel Synechocystis sp. strain PCC 6803 genes (sll1495, sll0804, slr1306, and slr1125) which encode hypothetical proteins were determined by transposon mutagenesis to be required for optimal photoautotrophic growth. Mutations were also recovered in ccmK4, a carboxysome coat protein homologue, and me, the decarboxylating NADP+-dependent malic enzyme. This is the first report that these known genes are required for optimal photoautotrophy.

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