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Oxygenic photosynthetic organisms use Photosystem II (PSII) to oxidize water and reduce plastoquinone. Here, we review the mechanisms by which PSII is assembled and turned over in the model green alga Chlamydomonas reinhardtii. This species has been used to make key discoveries in PSII research due to its metabolic flexibility and amenability to genetic approaches. PSII subunits originate from both nuclear and chloroplastic gene products in Chlamydomonas. Nuclear-encoded PSII subunits are transported into the chloroplast and chloroplast-encoded PSII subunits are translated by a coordinated mechanism. Active PSII dimers are built from discrete reaction center complexes in a process facilitated by assembly factors. The phosphorylation of core subunits affects supercomplex formation and localization within the thylakoid network. Proteolysis primarily targets the D1 subunit, which when replaced, allows PSII to be reactivated and completes a repair cycle. While PSII has been extensively studied using Chlamydomonas as a model species, important questions remain about its assembly and repair which are presented here.
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Genetic approaches to uncover gene products involved in iron-sulfur protein maturation.
Iron-sulfur (FeS) clusters are one of the most ubiquitous and versatile prosthetic groups exploited by nature. FeS clusters aid in conducting redox reactions, carbon activation, and environmental sensing. This chapter presents an overview of the genetic approaches that have been useful for identifying and characterizing bacterial factors involved in FeS protein assembly. Traditional genetic screens that assess viability or conditional auxotrophies, and bioinformatic approaches have identified the majority of the described genes utilized for FeS protein assembly. Herein, we expand upon this list of genetic methods by detailing the use of transposon-sequencing (TnSeq) to identify gene products that are necessary for the proper function of metabolic pathways that require FeS enzymes. TnSeq utilizes the power of genomics and massively parallel DNA sequencing to allow researchers to quantify the necessity of individual gene products for a specific growth condition. This allows for the identification of gene products or gene networks that have a role in a given metabolic process but are not essential for the process. An advantage of this approach is that it allows researchers to identify mutants that have partial phenotypes that are often missed using traditional plate-based selections. Applying TnSeq to address questions of FeS protein maturation will result in a more comprehensive understanding of genetic interactions and factors utilized in FeS biogenesis and FeS protein assembly.
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- Award ID(s):
- 1750624
- PAR ID:
- 10318967
- Date Published:
- Journal Name:
- Methods in molecular biology
- ISSN:
- 1940-6029
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1007/978-1-0716-1605-5_3
