scholarly journals Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

2021 ◽  
Author(s):  
Devon A. Stork ◽  
Georgia R. Squyres ◽  
Erkin Kuru ◽  
Katarzyna A. Gromek ◽  
Jonathan Rittichier ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Genetic Code ◽  
Cell Biology ◽  
Stop Codon ◽  
Positive Bacterium ◽  
E Coli ◽  
Binding Interface ◽  
Genetic Code Expansion ◽  
And Control

AbstractBacillus subtilis is a model Gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, we demonstrate broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

scholarly journals Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Devon A. Stork ◽  
Georgia R. Squyres ◽  
Erkin Kuru ◽  
Katarzyna A. Gromek ◽  
Jonathan Rittichier ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Genetic Code ◽  
Cell Biology ◽  
Stop Codon ◽  
Positive Bacterium ◽  
E Coli ◽  
Binding Interface ◽  
Genetic Code Expansion ◽  
And Control

AbstractBacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

scholarly journals Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells

2018 ◽  
Author(s):  
Birthe Meineke ◽  
Johannes Heimgärtner ◽  
Lorenzo Lafranchi ◽  
Simon J Elsässer
Keyword(s):  
Genetic Code ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Human Gut ◽  
Methanosarcina Mazei ◽  
Genetic Code Expansion

ABSTRACTGenetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

scholarly journals Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Naomi Shimokawa-Chiba ◽  
Claudia Müller ◽  
Keigo Fujiwara ◽  
Bertrand Beckert ◽  
Koreaki Ito ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stop Codon ◽  
Release Factor ◽  
Positive Bacterium ◽  
Gram Positive ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Dead End ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus

AbstractRescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

scholarly journals ResQ, a release factor-dependent ribosome rescue factor in the Gram-positive bacterium Bacillus subtilis

2019 ◽  
Author(s):  
Naomi Shimokawa-Chiba ◽  
Claudia Müller ◽  
Keigo Fujiwara ◽  
Bertrand Beckert ◽  
Koreaki Ito ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stop Codon ◽  
Release Factor ◽  
Active Conformation ◽  
Positive Bacterium ◽  
Gram Positive ◽  
E Coli ◽  
Dead End ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus

SummaryRescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named ResQ. Genetic analysis shows that B. subtilis requires the function of either trans-translation or ResQ for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-EM characterization demonstrates that ResQ binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although ResQ is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

scholarly journals Generation of photocaged nanobodies for in vivo applications using genetic code expansion and computationally guided protein engineering

2021 ◽  
Author(s):  
Jack M O'Shea ◽  
Angeliki Goutou ◽  
Cyrus Sethna ◽  
Christopher W Wood ◽  
Sebastian Greiss
Keyword(s):  
Genetic Code ◽  
Protein Interactions ◽  
Protein Function ◽  
Binding Interface ◽  
Cellular Environment ◽  
Computational Alanine Scanning ◽  
Genetic Code Expansion ◽  
Target Binding ◽  
Dynamics Simulations

Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular-dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies. These variants exhibit photo-activatable binding triggered by illumination with 365nm light. We demonstrate that the photocaged nanobodies we have created are highly robust and function in a complex cellular environment. We apply them to control subcellular protein localisation in the nematode worm C. elegans. Our approach provides a rare example of computationally designed proteins being directly applied in living animals and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

scholarly journals Isolation of RNase H Genes That Are Essential for Growth of Bacillus subtilis 168

1999 ◽  
Vol 181 (7) ◽  
pp. 2118-2123 ◽  
Author(s):  
Mitsuhiro Itaya ◽  
Akira Omori ◽  
Shigenori Kanaya ◽  
Robert J. Crouch ◽  
Teruo Tanaka ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rnase H ◽  
Positive Bacterium ◽  
E Coli ◽  
Cellular Processes ◽  
Dna Library ◽  
Bacillus Subtilis 168 ◽  
Genes Encoding

ABSTRACT Two genes encoding functional RNase H (EC 3.1.26.4 ) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilisRNase HIII. The B. subtilis genome has anrnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB andrnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both thernhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the singlernhC homologue in the small genome ofMycoplasma species.

scholarly journals Generation of Recombinant Mammalian Selenoproteins through Genetic Code Expansion with Photocaged Selenocysteine

2019 ◽  
Author(s):  
Jennifer C. Peeler ◽  
Rachel E. Kelemen ◽  
Masahiro Abo ◽  
Laura C. Edinger ◽  
Jingjia Chen ◽  
...  
Keyword(s):  
Genetic Code ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Biological Evaluation ◽  
Trna Synthetase ◽  
Secis Element ◽  
E Coli ◽  
Domains Of Life ◽  
Translation Mechanism ◽  
Genetic Code Expansion

ABSTRACTSelenoproteins contain the amino acid selenocysteine and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a non-canonical translation mechanism requiring suppression of the UGA stop codon, and a selenocysteine insertion sequence (SECIS) element in the 3’ untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element, and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged selenocysteine (DMNB-Sec) at the UAG amber stop codon. Recombinantly expressed selenoproteins can be photoactivated in living cells with spatial and temporal control. Using this approach, the native selenoprotein methionine-R-sulfoxide reductase 1 is generated and activated in mammalian cells. The ability to site-specifically introduce selenocysteine directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.

Context effects of genetic code expansion by stop codon suppression

2018 ◽  
Vol 46 ◽  
pp. 146-155 ◽  
Author(s):  
Yonatan Chemla ◽  
Eden Ozer ◽  
Itay Algov ◽  
Lital Alfonta
Keyword(s):  
Genetic Code ◽  
Context Effects ◽  
Stop Codon ◽  
Genetic Code Expansion

scholarly journals Preventive antimicrobial action and tissue architecture ameliorations of Bacillus subtilis in challenged broilers

2021 ◽  
Vol 14 (2) ◽  
pp. 523-536
Author(s):  
Essam S. Soliman ◽  
Rania T. Hamad ◽  
Mona S. Abdallah
Keyword(s):  
Bacillus Subtilis ◽  
Broiler Chickens ◽  
Trichophyton Mentagrophytes ◽  
Antimicrobial Activities ◽  
Lymphoid Hyperplasia ◽  
Bursa Of Fabricius ◽  
Tissue Architecture ◽  
E Coli

Background and Aim: Probiotics improve intestinal balance through bacterial antagonism and competitive exclusion. This study aimed to investigate the in vitro antimicrobial activity, as well as the in vivo preventive, immunological, productive, and histopathological modifications produced by probiotic Bacillus subtilis. Materials and Methods: The in vitro antimicrobial activities of B. subtilis (5×106 CFU/g; 0.5, 1.0*, 1.5, and 2.0 g/L) were tested against Escherichia coli O157: H7, Salmonella Typhimurium, Candida albicans, and Trichophyton mentagrophytes after exposure times of 0.25, 0.5, 1, and 2 h using minimal inhibitory concentration procedures. A total of 320 1-day-old female Ross broiler chickens were divided into five groups. Four out of the five groups were supplemented with 0.5, 1.0*, 1.5, and 2.0 g/L probiotic B. subtilis from the age of 1 day old. Supplemented 14-day-old broiler chickens were challenged with only E. coli O157: H7 (4.5×1012 CFU/mL) and S. Typhimurium (1.2×107 CFU/mL). A total of 2461 samples (256 microbial-probiotic mixtures, 315 sera, 315 duodenal swabs, and 1575 organs) were collected. Results: The in vitro results revealed highly significant (p<0.001) killing rates at all-time points in 2.0 g/L B. subtilis: 99.9%, 90.0%, 95.6%, and 98.8% against E. coli, S. Typhimurium, C. albicans, and T. mentagrophytes, respectively. Broilers supplemented with 1.5 and 2.0 g/L B. subtilis revealed highly significant increases (p<0.01) in body weights, weight gains, carcass weights, edible organs' weights, immune organs' weights, biochemical profile, and immunoglobulin concentrations, as well as highly significant declines (p<0.01) in total bacterial, Enterobacteriaceae, and Salmonella counts. Histopathological photomicrographs revealed pronounced improvements and near-normal pictures of the livers and hearts of broilers with lymphoid hyperplasia in the bursa of Fabricius, thymus, and spleen after supplementation with 2.0 g/L B. subtilis. Conclusion: The studies revealed that 1.5-2.0 g of probiotic B. subtilis at a concentration of 5×106 CFU/g/L water was able to improve performance, enhance immunity, and tissue architecture, and produce direct antimicrobial actions.

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