Mutation of a Cytoplasmic Gene in Chlamydomonas Alters Chloroplast Ribosome Function

Abstract

A mutation, car, determining resistance to several macrolide antibiotics, including carbomycin, has been identified in the alga Chlamydomonas as cytoplasmic, and mapped in the known cytoplasmic linkage group close to genes determining resistance to other antibiotics, including streptomycin, erythromycin, and spectinomycin. The A serious gap in our understanding of cellular organization concerns the precise functional role of cytoplasmic genes (1). All cytoplasmic genes so far examined in the alga Chlamydomonas reinhardi show the same pattern of maternal inheritance and map within the same linkage group, long postulated to be located in chloroplast DNA and to code for numerous chloroplast proteins (1-4). Gillham et al. (5) have reported that chloroplast ribosomes from some cytoplasmic mutants of Chlamydomonas were 66 S rather than 70 S, and Mets and Bogorad (6) found that chloroplast ribosomes from a cytoplasmic erythromycin-resistant mutant were impaired in their erythromycin-binding ability. This paper reports the correlation between a mutation to resistance to the macrolide antibiotic carbomycin, that maps in the cytoplasmic linkage group and an alteration in chloroplast ribosome function, attributed to a change in a ribosomal protein.We compare the effects of antibiotics on the proteinsynthesizing activity of 70S chloroplast ribosomes from wildtype and antibiotic-resistant mutant strains. For this purpose it was necessary to develop an in vitro protein-synthesizing system that used chloroplast ribosomes. This paper reports the first demonstration of in vitro incorporating activity of chloroplast ribosomes from Chlamydomonas. We show that high concentrations of Mg++ and spermidine activate 70S chloroplast ribosomes, and low concentrations activate 80S cytoplasmic ribosomes. Both in S-30 extracts and in fractionated systems, the 70S ribosomes of the resistant mutant were resistant to carbomycin, whereas the ribosomes from the wildtype strain were sensitive.

Localization of Five Antibiotic Resistances at the Subunit Level in Chloroplast Ribosomes of Chlamydomonas

“…Wild-Type strain 21 gr, media, and growing conditions have been previously described (7). Non-Mendelian pnutant strains-each resistant to one of the antibiotics streptomycin, spectinomycin, carbomycin, and cleocin-were selected after streptomycin mutagenesis (14) of wild-type strain 21 gr by growth on antibiotic-containing agar.…”

“…The pattern of subunit localization, mirroring that found in bacterial ribosomes (8) as well as the low levels of cross-resistance between mutants, provide strong, albeit indirect, evidence that each of the mutant genes responsible for resistance codes for a chloroplast ribosomal protein. Several lines of evidence 1715 that non-Mendelian mutations to drug resistance in Chlamydomonas induce alterations in chloroplast ribosomes have been previously reported (7,(9)(10)(11)(12)(13).…”

“…Mutations conferring resistance to streptomycin (sm2), neamine (nea), spectinomycin (spc), carbomycin (car), and cleocin (cle) each show non-Mendelian inheritance and map in a single linkage group (1,6,7). Results to be reported here provide further evidence that this linkage group is located in chloroplast DNA; 70S ribosomes and reassociated subunits from wild-type and five resistant mutant strains each carrying one of these mutant genes were examined for their antibiotic resistance in a poly(U)-directed phenylalanine incorporating system.…”

Can a non-Mendelian Mutation Affect Both Chloroplast and Mitochondrial Ribosomes?

“…Evidence already exists that both nuclear and UP mutations to antibiotic resistance affect chloroplast ribosomes in C. reinhardtii (9,15,22,42,43). Our hypothesis that certain ribosomal proteins of the chloroplast and mitochondria share genetic determinants may explain the high incidence of one-step mutations to antibiotic resistance which grow equally well phototrophically, mixotrophically, or heterotrophically (44).…”

Binding of dihydrostreptomycin to ribosomes and ribosomal subunits from streptomycin‐resistant mutants of Chlamydomonas reinhardi

“…In this connection a comparative study of the ribosomes from organelles and cytoplasm is of great importance. For the investigation of chloroplast ribosomes and chloroplastcytoplasmic interactions the unicellular green alga Chlamydomonas reinhardi is widely used [3], and mutants with altered 70 S ribosomes have been decribed [4,5].…”