Genetic and genomic analysis of RNases in model cyanobacteria
Cyanobacteria are diverse photosynthetic microbes with the ability to convert CO2 into useful products. However, metabolic engineering of cyanobacteria remains challenging because of the limited resources for modifying the expression of endogenous and exogenous biochemical pathways. Fine-tuned control of protein production will be critical to optimize the biological conversion of CO2 into desirable molecules. Messenger RNAs (mRNAs) are labile intermediates that play critical roles in determining the translation rate and steady-state protein concentrations in the cell. The majority of studies on mRNA turnover have focused on the model heterotrophic bacteria Escherichia coli and Bacillus subtilis. These studies have elucidated many RNA modifying and processing enzymes and have highlighted the differences between these Gram-negative and Gram-positive bacteria, respectively. In contrast, much less is known about mRNA turnover in cyanobacteria. We generated a compendium of the major ribonucleases (RNases) and provide an in-depth analysis of RNase III-like enzymes in commonly studied and diverse cyanobacteria. Furthermore, using targeted gene deletion, we genetically dissected the RNases in Synechococcus sp. PCC 7002, one of the fastest growing and industrially attractive cyanobacterial strains. We found that all three cyanobacterial homologs of RNase III and a member of the RNase II/R family are not essential under standard laboratory conditions, while homologs of RNase E/G, RNase J1/J2, PNPase, and a different member of the RNase II/R family appear to be essential for growth. This work will enhance our understanding of native control of gene expression and will facilitate the development of an RNA-based toolkit for metabolic engineering in cyanobacteria.
KeywordsmRNA RNA Ribonuclease Photosynthesis Cyanobacteria Synthetic biology Biofuels Comparative genomics
We thank Dr. Bryant for providing the pSRA81 vector. We thank Dr. Himadri Pakrasi for providing access to the oxygen electrode used in physiological measurements. We thank Kymberleigh Romano, Alex Linz, and Alex Sanchez for their help with preliminary experiments. Funding for this work was provided by the Department of Energy (DE-SC0010329). GCG is the recipient of an NIH Biotechnology Training Fellowship (NIGMS—5 T32 GM08349). JCC and BFP conceived and designed the study. JCC and GCG performed experiments. JCC, GCG, and BFP analyzed and interpreted the data. JCC and BFP wrote the manuscript.
- Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie JM, Gascuel O (2008) Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36(Web Server issue):W465–W469Google Scholar
- Eisenhut M, Georg J, Klahn S, Sakurai I, Mustila H, Zhang P, Hess WR, Aro EM (2012) The antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 prevents premature expression of the flv4-2 operon upon shift in inorganic carbon supply. J Biol Chem 287(40):33153–33162PubMedCentralCrossRefPubMedGoogle Scholar
- Kaberdin VR, Miczak A, Jakobsen JS, Lin-Chao S, McDowall KJ, von Gabain A (1998) The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly. Proc Natl Acad Sci USA 95(20):11637–11642PubMedCentralCrossRefPubMedGoogle Scholar
- Murphy RC, Gasparich GE, Bryant DA, Porter RD (1990) Nucleotide sequence and further characterization of the Synechococcus sp. strain PCC 7002 recA gene: complementation of a cyanobacterial recA mutation by the Escherichia coli recA gene. J Bacteriol 172(2):967–976Google Scholar
- Shih PM, Wu D, Latifi A, Axen SD, Fewer DP, Talla E, Calteau A, Cai F, de Marsac NT, Rippka R, Herdman M, Sivonen K, Coursin T, Laurent T, Goodwin L, Nolan M, Davenport KW, Han CS, Rubin EM, Eisen JA, Woyke T, Gugger M, Kerfeld CA (2013) Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. Proc Natl Acad Sci USA 110(3):1053–1058PubMedCentralCrossRefPubMedGoogle Scholar
- Stevens SEJ, Patterson COP, Meyers J (1973) The production of hydrogen peroxide by blue-green algae: a survey. J Phycol 9:427–430Google Scholar