High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes

Abstract

Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100 -250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This ⌬nd4 mutation causes a misassembly of complex I, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex I. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex I, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex I.green alga ͉ mitochondrial DNA mutagenesis ͉ telomere ͉ complex I assembly ͉ respiratory-deficient mutant

Investigating the dynamics of recombinant protein secretion from a microalgal host

“…Currently this alga has the most well developed molecular toolkit of any eukaryotic 90 microalgae, and transformation of nuclear, chloroplast, and mitochondrial genomes is 91 possible (Bateman and Purton 2000; Kindle 1990; Remacle et al 2006). Chloroplast based 92 recombinant protein (RP) expression in this organism has been shown to achieve titres up 93 to 21% total soluble protein (TSP) (Surzycki et al 2009).…”

Inactivation of genes coding for mitochondrial Nd7 and Nd9 complex I subunits in Chlamydomonas reinhardtii. Impact of complex I loss on respiration and energetic metabolism

“…This provides an advantage to screen for complex I mutants, as they display robust growth in the light but slow growth under heterotrophic conditions . Several mutations in four of the complex I subunits (ND1, 4, 5, and 6) encoded by the mitochondrial genome have been described so far in Chlamydomonas Cardol et al, 2002;Larosa et al, 2012;Remacle et al, 2001;Remacle et al, 2006). Several complex I-deficient mutants (called amc for "assembly of mitochondrial complex I) of nuclear origin have also been isolated by using a mutagenesis approach (Barbieri et al, 2011).…”

Respiratory-deficient mutants of the unicellular green alga Chlamydomonas: A review

“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 16 [17], (c) [68], (d) [69], (e) [70], (f) [71], (g) [72], (h) [73], (i) [74], (j) [75], (k) [76], (l) [77], (m) [22], (n) [29], (o) [78], (p) [58], (q) [79], (r)[34] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 17 ((a) [48], (b) [45], (c) [47], (d) [43], (e) [44], (f) [39], (g) …”

“…More recently, mitochondrial transformation was set up with the aim of performing site-directed mutagenesis of the respiratory subunits encoded by the mitochondrial genome (e.g., [43], [44]). Therefore C. reinhardtii is the only photosynthetic organism where reverse genetics is possible in mitochondria.…”

“…In addition, mitochondrial transformation by biolistic device allowed obtaining mutants for the nd4 gene (Δnd4, L157P ND4) [43,44]. The basis for the isolation of such transformants relies on the fact that, as stated before, complex I mutants are able to grow in the dark, albeit slower than the wild-type strain.…”