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Optogenetic control of Bacillus subtilis gene expression.

ABSTRACT: The Gram-positive bacterium Bacillus subtilis exhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest into B. subtilis. Following an initial non-functional design, we optimize expression of pathway genes, enhance PCB production via a translational fusion of the biosynthetic enzymes, engineer a strong chimeric output promoter, and increase dynamic range with a miniaturized photosensor kinase. Our final design exhibits over 70-fold activation and rapid response dynamics, making it well-suited to studying a wide range of gene regulatory processes. In addition, the synthetic biology methods we develop to port this pathway should make B. subtilis easier to engineer in the future.

SUBMITTER: Castillo-Hair SM

PROVIDER: S-EPMC6629627 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Optogenetic control of Bacillus subtilis gene expression.

Castillo-Hair Sebastian M SM Baerman Elliot A EA Fujita Masaya M Igoshin Oleg A OA Tabor Jeffrey J JJ

Nature communications 20190715 1

The Gram-positive bacterium Bacillus subtilis exhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest into B. sub ...[more]

PMID: 31308373

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Project description:Previous characterization of Bacillus subtilis hemN, encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation, indicated the presence of a second hemN-like gene (B. Hippler, G. Homuth, T. Hoffmann, C. Hungerer, W. Schumann, and D. Jahn, J. Bacteriol. 179:7181-7185, 1997). The corresponding hemZ gene was found to be split into the two potential open reading frames yhaV and yhaW by a sequencing error of the genome sequencing project. The hemZ gene, encoding a 501-amino-acid protein with a calculated molecular mass of 57,533 Da, complemented a Salmonella typhimurium hemF hemN double mutant under aerobic and anaerobic growth conditions. A B. subtilis hemZ mutant accumulated coproporphyrinogen III under anaerobic growth conditions. A hemN hemZ double mutant exhibited normal aerobic and anaerobic growth, indicating the presence of a third alternative oxygen-independent enzymatic system for coproporphyrinogen III oxidation. The hemY gene, encoding oxygen-dependent protoporphyrinogen IX oxidase with coproporphyrinogen III oxidase side activity, did not significantly contribute to this newly identified system. Growth behavior of hemY mutants revealed the presence of an oxygen-independent protoporphyrinogen IX oxidase in B. subtilis. A monocistronic hemZ mRNA, starting 31 bp upstream of the translational start codon, was detected. Reporter gene fusions of hemZ and hemN demonstrated a fivefold anaerobic induction of both genes under nitrate ammonifying growth conditions. No anaerobic induction was observed for fermentatively growing B. subtilis. The B. subtilis redox regulatory systems encoded by resDE, fnr, and ywiD were indispensable for the observed transcriptional induction. A redox regulation cascade proceeding from an unknown sensor via resDE, through fnr and ywiD to hemN/hemZ, is suggested for the observed coregulation of heme biosynthesis and the anaerobic respiratory energy metabolism. Finally, only hemZ was found to be fivefold induced by the presence of H(2)O(2), indicating further coregulation of heme biosynthesis with the formation of the tetrapyrrole enzyme catalase.

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Optogenetic control of Bacillus subtilis gene expression.

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Optogenetic control of Bacillus subtilis gene expression.

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