Mistranslation and genetic variability: the effect of streptomycin

Streptomycin is an aminoglycoside antibiotic that acts at the level of protein synthesis. Exposure to sublethal concentrations of this antibiotic increased significantly the number of Arg+ mutants derived from an Escherichia coli argE3 (ochre) rpsL31 (streptomycin-resistant) strain. The vast majority of these mutants appeared on selective minimal medium plates with streptomycin (200 micro g/ml) during stationary phase, after 6-10 days incubation at 37 degrees C. Derivative mutD5 or mutL or mutS mutants, carrying a faulty varepsilon subunit of DNA polymerase or a defective mismatch DNA-repair protein, respectively, also showed higher numbers of Arg+ mutants on selective medium with streptomycin than on medium without streptomycin. Interestingly, with these DNA-repair mutants about 50% of the Arg+ mutants generated in the presence of streptomycin appeared during the first 5 days of incubation. These observations suggest that the activities of these fidelity-repair proteins prevent in the parental strain the early appearance of the supernumerary Arg+ mutants on the selective medium with streptomycin. The appearance of Arg+ mutants on the plates with streptomycin was not significantly altered by recA, rpoS or dps mutations. A high percentage of the Arg+ mutants arising in the presence of streptomycin were streptomycin-dependent for growth without arginine (Arg+ St-D). These types of mutants displayed a Ram (for ribosomal ambiguity) phenotype, manifested by increased misreading, assayed by in vitro and in vivo experiments and by leakiness on several selective minimal media. Genetic data indicated that these mutants carry a mutation located at about 74 min of the E.coli map that relieves the high translational fidelity conferred by the rpsL mutation. These studies suggest that the growth-limiting conditions of the assay system used, as well as the presence of streptomycin, which causes an increased production of altered proteins, favours the appearance and growth of compensatory Arg+ mutants.     

Genetic study of plasmid-associated high-frequency mutations to streptomycin resistance in Escherichia coli]

Escherichia coli CTR1(RT1)RHfm1) carrying two H-factors and having unusually high frequency of mutation to high level streptomycin resistance is studied. The high frequency of mutation (about 10(-6) to streptomycin resistance is connected with the presence of R factor RHfm1, controlling the resistance to chloramphenicol and low level streptomacin resistance, but not with RT1, controlling the resistance to tetracycline. Spontaneous or ethidium bromide-induced loss of RHfm1 is accompanied by a decrease of the mutation frequency to 10(-9). RHfm1 is efficiently transmissible to other strains at 28 degrees C. The acquisition of RHfm1 by strains of E. coli K-12 ans S. typhimurium LT2 was followed by a 1000--10000-fold increase of the frequejcy of mutation to streptomycin resistance. Some streptomycin resistant mutants were isolated, and chromosome location of the mutations was demonstrated. The streptomycin resistant mutants were unable to transmit high level of resistance to streptomycin with R factor, but only low level one. The loss of RHfm1 by streptomycin resistant mutants was accompanied by the return to the streptomycin sensitivity of the initial R- strans (E. coli K-12 mutants) or by a decrease of the streptomycin resistance to the level, only 2-fold higher than that of R- wild type (E. coli CTR1 mutant). Thus, the mutantions had practically no effect on streptomycin resistance of R- strains, but could lead to high resistance phenotypes in the presence of RHfm1. The mutant loci in all three studied strains were found to be closely linked to the locus "fus" on the genetic map of E. coli.     

Genetic antagonism and hypermutability in Mycobacterium smegmatis

Multidrug-resistant strains of Mycobacterium tuberculosis are a serious and continuing human health problem. Such strains may contain as many as four or five different mutations, and M. tuberculosis strains that are resistant to both streptomycin and rifampin contain mutations in the rpsL and rpoB genes, respectively. Coexisting mutations of this kind in Escherichia coli have been shown to interact negatively (S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 72:2084-2087, 1975; S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 74:1157-1161, 1977). We investigated this possibility in Mycobacterium smegmatis by analyzing the frequency and nature of spontaneous mutants that are resistant to either streptomycin or rifampin or to both antibiotics. Mutants resistant to streptomycin were isolated from characterized rifampin-resistant mutants of M. smegmatis under selection either for one or for both antibiotics. Similarly, mutants resistant to rifampin were isolated from streptomycin-resistant strains. The second antibiotic resistance mutation occurred at a lower frequency in both cases. Surprisingly, in both cases a very high rate of reversion of the initial antibiotic resistance allele was detected when single antibiotic selection was used; the majority of strains resistant to only one antibiotic were isolated by this process. Determinations of rates of mutation to antibiotic resistance in M. smegmatis showed that the frequencies were enhanced up to 10(4)-fold during stationary phase. If such behavior is also typical of slow-growing pathogenic mycobacteria, these studies suggest that the generation of multiply drug-resistant strains by successive mutations may be a more complex genetic phenomenon than suspected.     

Media dependence of translational mutant phenotype

Abstract We have measured the growth rates of some ribosomal mutants of Escherichia coli in different growth media. The mutants are a streptomycin resistant (SmR) mutation in rpsL; a partially streptomycin dependent (SmP) mutation in rpsL; a ribosome ambiguity mutant (ram) in rpsD; a ram mutant in rpsE as well as a mutant defective in tRNA modification, mia A. The data show that the growth rates of all mutants are less inhibited in poor media than they are in rich ones. The translation rates and nonsense suppression levels for each mutant are not significantly different in rich and poor media, which shows that the ribosomal mutant phenotypes are maintained under different growth conditions. These results suggest that the degree of growth inhibition for mutants with altered translation machinery is dependent on the growth conditions. In addition, the data suggest that bacteria are able to physiologically compensate for the loss of growth efficiency in such mutants, particularly, under poor growth conditions.     

Relationship between mutant amidases of Pseudomonas aeruginosa and hydroxyurea as an inhibitor.

Summary Hydroxyurea inhibited growth of Pseudomonas aeruginosa strain AI 3 on media containing either acetanilide (N-phenyl acetamide) or acetamide as sole carbon sources. Mutants resistant to hydroxyurea inhibition of growth on acetanilide (OUCH strains) and acetamide (AmOUCH strains) displayed altered growth properties on various amide media compared with the parent strain AI 3. AI 3 amidase, which catalyses the initial step in the metabolism of acetanilide and acetamide, was inhibited by hydroxyurea in a time-dependent reaction that was slowly reversible at pH 7.2 Compared with AI 3 amidase, amidases from the OUCH mutants were much less sensitive to inhibition by hydroxyurea and showed altered substrate specificities and pH/activity profiles; amidases from the AmOUCH mutants were more sensitive to hydroxyurea inhibition but showed increased activity towards acetamide. Association of resistance to hydroxyurea inhibition with a mutation in the amidase structural gene of strain OUCH 4 was confirmed by transduction.     

通过获得链霉素抗性基因突变株筛选小诺霉素高产菌株

通过链霉素对小诺霉素产生菌 (Micromonospora purpura) 49 1 2 #菌株孢子致死浓度的测定 ,采用诱变剂EMS 3种不同诱变剂量对菌株的孢子进行诱变处理 ,诱变处理的孢子涂布在含链霉素致死浓度的改良高氏平板上 ,获得大量的链霉素抗性基因突变株 ,然后从链霉素抗性基因突变株进一步筛选小诺霉素高产菌株 ,获得小诺霉素菌株 49 1 2 3菌株。在摇瓶条件下 ,其产小诺霉素生物活性单位比出发菌株 49 1 2 #的摇瓶发酵单位提高了 40 %以上。小诺霉素的组分比由出发菌株的C2b∶C1a的 5∶5提高到 8∶2。C2b有效组分提高了 30 %;链霉素抗性基因突变与小诺霉素发酵单位突变之间 ,小诺霉素正突变率达到 40 %,负突变率达 2 6%,正突变大于负突变     

Induction and characterization of streptomycin-resistant mutants in Capsicum praetermissum

Streptomycin-resistant mutants were isolated from mutagenised cotyledon explants of Capsicum praetermissum Heiser & Smith. The explants were mutagenised with N-ethyl-N-nitrosourea, which resulted in a high frequency of streptomycin-resistant mutants (18.0%) and a low frequency of chlorophyll-deficient (albino) mutants (8.0%). Complete streptomycin-resistant plantlets were obtained after rooting of the regenerated green shoots on rooting medium containing 1.0 mg L-1 IAA and 500 mg L-1 streptomycin sulphate. Leaf-segment assay of these plantlets revealed that they were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, and spectinomycin. Reciprocal crosses between streptomycin-resistant and -sensitive plants showed a non-Mendelian transmission of resistance by female parents.     

Effects of streptomycin on diploid tobacco callus cultures and the isolation of resistant mutants

Summary Diploid callus cultures from four genetic lines of tobacco (Nicotiana tabacum) were grown in the presence of 0–2 mg/ml streptomycin sulfate. These genetic lines are closely related with regard to nuclear genes, but differ in their respective cytoplasmic genomes. In calli from all these four lines, the formation of green color was inhibited at 0.5 mg/ml streptomycin. Small differences were observed between the four lines in their respective growth on medium containing streptomycin. Streptomycin resistant mutants were isolated from cultures at high drug concentrations, using as criteria for isolation either the ability of the callus to grow green, or a better rate of growth as indicated by the size of white callus.     

Effect of cycloheximide on development of methotrexate resistance of Chinese hamster ovary cells treated with inhibitors of DNA synthesis.

We examined the effects of 18 h of incubation of Chinese hamster ovary (CHO K1) cells with cycloheximide, hydroxyurea, and aphidicolin. Treatment of cells with cycloheximide alone at a concentration adequate to inhibit DNA synthesis to less than 10% of control was significantly less cytotoxic and clastogenic than treatment with hydroxyurea or aphidicolin, did not induce unbalanced cellular growth, and had no effect on the frequency of resistant cells in methotrexate selections compared with control cells. When combined with hydroxyurea or aphidicolin and compared with the effects of either drug alone, cycloheximide blocked the induction of unbalanced growth during drug treatment, reduced the frequency of chromosomal aberrations in recovering cell populations, and decreased cell killing. In addition, the increased frequency of methotrexate-resistant cells observed after treatment with hydroxyurea or aphidicolin was eliminated when cycloheximide was present during drug treatment.     

A simple procedure for the isolation of streptomycin resistant plants in Solanaceae

A system has been developed for rapid selection of streptomycin resistant mutants, as adventitious shoots arising from explants of several Solanaceous species. Efficient mutagenesis was achieved by incubating shoot culture-derived leaf strips with 1 or 5 mM nitroso-methylurea, for 90 or 120 min. In Nicotiana tabacum and Lycopersicon peruvianum these treatments resulted in white or variegated adventitious shoots from up to 3.5% of explants placed on medium promoting shoot regeneration. Chlorophyll deficiencies were only observed very rarely in Solanum nigrum. Streptomycin resistant shoots were obtained from leaf explants placed on medium containing 500 mg l^-1 streptomycin sulphate, under which conditions explants are bleached and adventitious shoot development suppressed. Green adventitious s shoots appeared at a frequency dependent both on the mutagenic treatment and on the species. The best response was with S. nigrum where >70% of the explants produced streptomycin resistant shoots, most of which retained their resistance on subsequent testing. Maternal inheritance of streptomycin resistance has been confirmed for several N. tabacum and S. nigrum mutants, and there is also evidence for paternal transmission in the latter species. The procedure has been successfully extended to other species, including N. sylvestris and N. plumbaginifolia, and also to obtain spectinomycin resistant mutants.Communicated by R. Hagemann     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci.

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

Improvement of antibiotic titers from Streptomyces bacteria by interactive continuous selection

We have applied a technique of interactive continuous selection (ICS) to the isolation of streptomycin-resistant mutants of the streptomycin-producing organism, Streptomyces griseus. A series of mutants, each with a different colonial morphology and expressing successively greater resistance to streptomycin, was isolated during the course of selection. Takeover of the mutants has been correlated with changes in on-line estimates of streptomycin concentration such that these estimates may be used as a real-time measure of the genetic state of the cell population. When grown in the medium employed for ICS, mutants expressed increased antibiotic production titers; the best mutant produced 10 to 20 times more streptomycin than the parent strain. Absolute improvements in the maximum specific growth rate and intrinsic resistance to streptomycin did not account for the observed growth advantage of all mutants. Rather, each mutant exhibited relative increases in specific growth rate at increasing concentrations of streptomycin. (c) 1996 John Wiley & Sons, Inc.     

The induction of supersuppressor mutants of Bacillus subtilis by ethyl methanesulphonate and the posttreatment modification of mutation yield

Summary Supersuppressor mutants have been induced in a strain of Bacillus subtilis with the chemical mutagen ethyl methanesulphonate. The yield of mutants recovered is dependent on the degree of supplementation of the selective plating medium with minute quantities of either nutrient broth or the previously required growth supplements. The optimal quantities of these medial additives have been established and the superiority of nutrient broth described. This broth effect has been shown to be due to components of the nutrient broth other than the previously required growth substances.This stimulatory effect of nutrient broth on mutation yield is completed after approximately 2 hours incubation on solid medium. Conversely, absence of broth during the first two hours of incubation on solid medium leads to a time-dependent, irreversible decline in mutation frequency even when incubation is continued upon selective agar with added broth. In liquid media, mutation frequency decline takes place in a manner similar to that observed on solid media, but the stimulatory effects of broth upon mutation fixation and mutant recovery are no longer evident. A decline in mutation frequency occurs in liquid of any composition, but addition of leucine and uracil reduces its degree. It is suggested that the additional phenomena detected in liquid media are due to a liquid holding recovery, although the removal of residual, as yet unreacted, mutagen or mutagenic intermediates into liquid cannot be ruled out.     

Cytochrome oxidase deficiency protects Escherichia coli from cell death but not from filamentation due to thymine deficiency or DNA polymerase inactivation

Temperature-sensitive DNA polymerase mutants (dnaE) are protected from cell death on incubation at nonpermissive temperature by mutation in the cydA gene controlling cytochrome bd oxidase. Protection is observed in complex (Luria-Bertani [LB]) medium but not on minimal medium. The cydA mutation protects a thymine-deficient strain from death in the absence of thymine on LB but not on minimal medium. Both dnaE and Deltathy mutants filament under nonpermissive conditions. Filamentation per se is not the cause of cell death, because the dnaE cydA double mutant forms long filaments after 24 h of incubation in LB medium at nonpermissive temperature. These filaments have multiply dispersed nucleoids and produce colonies on return to permissive conditions. The protective effect of a deficiency of cydA at high temperature is itself suppressed by overexpression of cytochrome bo3, indicating that the phenomenon is related to energy metabolism rather than to a specific effect of the cydA protein. We propose that filamentation and cell death resulting from thymine deprivation or slowing of DNA synthesis are not sequential events but occur in response to the same or a similar signal which is modulated in complex medium by cytochrome bd oxidase. The events which follow inhibition of replication fork progression due to either polymerase inactivation, thymine deprivation, or hydroxyurea inhibition differ in detail from those following actual DNA damage.     

Spontaneous Auxotrophic and Pigmented Mutants Occurring at High Frequency in Bacillus pumilus NRRL B-3275

Broth cultures of Bacillus pumilus NRRL B-3275 (BpB1) grown at 25, 30, or 37 C contain 1 to 2% spontaneous auxotrophic mutants in both the exponential and stationary phases of growth. Of 70 such mutants isolated from cultures grown at 37 C, approximately two-thirds reverted at such a high frequency as to preclude their study. Of the remaining 22 mutants, 18 required a single amino acid, 1 required adenine, and 1 required uracil. Two of the auxotrophs each required two unrelated amino acids resulting from two independent mutations. All of the mutations reverted spontaneously. Enhanced reversion of approximately one-third of the mutations was obtained with nitrosoguanidine, ethyl methane sulfonate, or diethyl sulfate, or with more than one of these mutagens. The reversion of one mutation was enhanced by 2-aminopurine. The reversion of the remaining mutations was not enhanced by the above mutagens, nor by mutagens known to induce (and revert) frameshift mutations in other bacterial systems. Nine of 10 mutants examined did not show a selective growth advantage over the parents. All but three of the mutations could be linked by PBS1 transduction to one of the previously described auxotrophic markers in strain BpB1. No evidence was obtained for clustering of the mutations on the BpB1 genome. Six of the mutations conferred a requirement for serine. One linked by transduction to trp-2, three linked to argA1, and two (ser-2, -3) linked to argO1. Pigmented mutants (containing a carotenoid-like pigment), which occur spontaneously in BpB1 cultures at a frequency on the order of 1 to 5 mutants per 10(4) cells, link by transduction to ser-2, -3. Spontaneous mutants of strain BpB1 resistant to rifampin, streptomycin, erythromycin, 5-fluorouracil, or 5-methyltryptophan occur at a frequency similar to that of strains of B. pumilus which do not exhibit a high rate of spontaneous mutation to auxotrophy. It is suggested that certain sites or regions of the BpB1 genome exhibit a high rate of spontaneous mutation.     

Adaptation of Spirogyra insignis (Chlorophyta) to an extreme natural environment (sulphureous waters) through preselective mutations

Adaptation of Spirogyra insignis (Chlorophyceae) to growth and survival in an extreme natural environment (sulphureous waters from La Hedionda Spa, S. Spain) was analysed by using an experimental model. Photosynthesis and growth of the alga were inhibited when it was cultured in La Hedionda Spa waters (LHW), but after further incubation for several weeks, the culture survived due to the growth of a variant that was resistant to LHW. A Luria-Delbruck fluctuation analysis was carried out to distinguish between resistant filaments arising from rare spontaneous mutations and resistant filaments arising from other mechanisms of adaptation. It was demonstrated that the resistant filaments arose randomly by rare spontaneous mutations before the addition of LHW (preselective mutations). The rate of spontaneous mutation from sensitivity to resistance was 2.7 x 10(-7) mutants per cell division. Since LHW(resistant) mutants have a diminished growth rate, they are maintained in nonsulphureous natural waters as the result of a balance between new resistants arising from spontaneous mutation and resistants eliminated by natural selection. Thus, recurrence of rare spontaneous preselective mutations ensures the survival of the alga in sulphureous waters.     

Study on the rapid, secondary-growth mutants of Staphylococcus epidermidis (author's transl)

According to colony type, growth rate and development of secondary growth on the proteose-peptone No. 3 mannitol salt agar (PMS) and the nutrient agar (NA) media, Staphylococcus epidermidis may be classified into three groups. Group I includes strains which develop smooth colonies on both media. Group II consists of those which show rapid propagation of entire clones and develop secondary growth on the PMS medium, but grow only smooth colonies on the NA medium, and which may be called reversible mutants. Group III includes thoes which show secondary growth on both PMS and NA as well as other media, which may be called irreversible mutants. One percent proteose-peptone No. 3 and 5--7% NaCl are the essential ingredients for the induction of mutation, and mannitol can enhance it. Except the high sensitivity of the reversible mutants of human origin, the three groups of chicken origin showed similar drug susceptibility to biosynthesis inhibitors of protein and cell wall. On the HI medium, chloramphenicol inhibited secondary growth of irreversible mutants at 25.0 microgram/ml minimal antimutagenesis concentration (MAC), whereas streptomycin, penicillin, erythromycin and oxytetracycline did not at all. The irreversible mutants had higher resistance to biosynthesis inhibitors of DNA or RNA, e.g. mitomycin C (MMC), novobiocin (NOV) and rifampicin (RIF), than the other two groups. On the HI medium, MMC at the MAC of 0.16 microgram/ml, NA at 25.0 microgram/ml and NOV at 2.5 microgram/ml inhibited the secondary growth of irreversible mutants, but RIF did not. To the irreversible mutants, the MIC and MAC of NA on the PMS medium were both higher than those on the HI medium. The MACs of MMC and NOV on the PMS medium were also higher than those on the HI medium, but their geometric mean MIC remained almost unchanged on both media. Because the MACs of MMC (0.31 microgram/ml) and NA (100.0 microgram/ml) to the reversible mutants on the PMS medium were much similar to those of the irreversible mutants, it suggests that both groups had the similar mutation mechanism.     

Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium

Most chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium, the nucleotide substitution AAA42 --> AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eighty-one independent lineages of the low-fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be > or = 10-7 per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin-sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single-nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.     

Chloroplast genes in Chlamydomonas affecting organelle ribosomes. Genetic and biochemical analysis of analysis of antibiotic-resistant mutants at several gene loci.

Six chloroplast gene mutants of Chlamydomonas reinhardtii resistant to spectinomycin, erythromycin, or streptomycin have been assessed for antibiotic resistance of their chloroplast ribosomes. Four of these mutations clearly confer high levels of antibiotic resistance on the chloroplast ribosomes both in vivo. Although one mutant resistant to streptomycin and one resistant to spectinomycin have chloroplast ribosomes as sensitive to antibiotics as those of wild type in vivo, these mutations can be shown to alter the wildtype sensitivity of chloroplast ribosomes in polynucleotide-directed amino acid incorporation in vitro. Genetic analysis of these six chloroplast mutants and three similar mutants (Sager, 1972), two of which have been shown to affect chloroplast ribosomes (Mets and Bogorad, 1972; Schlanger and Sager, 1974), indicates that in Chlamydomonas at least three chloroplast gene loci can affect streptomycin resistance of chloroplast ribosomes and that two can affect erythromycin resistance. The three spectinomycin-resistant mutants examined appear to be alleles at a single chloroplast gene locus, but may represent mutations at two different sites within the same gene. Unlike wild type, the streptomycin and spectinomycin resistant mutants which have chloroplast ribosomes sensitive to antibiotics in vivo, grow well in the presence of antibiotic by respiring exogenously supplied acetate as a carbon source, and have normal levels of cytochrome oxidase activity and cyanide-sensitive respiration. We conclude that mitochondrial protein synthesis in these mutants is resistant to these antibiotics, whereas in wild type it is sensitive. To explain the behavior of these two chloroplast gene mutants as well as other one-step mutants which are resistant both photosynthetically and when respiring acetate in the dark, we have postulated that a mutation in a single chloroplast gene may result in alteration of both chloroplast and mitochondrial ribosomes. Mitochondrial resistance would appear to be the minimal necessary condition for survival of all such mutants, and antibiotic-resistant chloroplast ribosomes would be necessary for survival only under photosynthetic conditions.