Chloramphenicol-resistant Mutants Research Articles

New chloramphenicol resistance locus in Bacillus subtilis.

A spontaneously occurring, noninducible, chloramphenicol-resistant mutant of Bacillus subtilis 168 has a mutation (cam-2) which maps in the ribosomal protein region of the chromosome near dal. Its presence does not confer dependence on chloramphenicol. Ribosomes of the cam-2 strain remained sensitive to chloramphenicol in in vitro protein synthesis. No chloramphenicol acetyltransferase activity could be detected.

Chloramphenicol resistance in an isolate of Phytophthora infestans

Treatment of wild-type P. infestans with chloramphenicol induced the mitochondria to lose their christae, whereas there was no change with a chloramphenicol-resistant mutant. Resistance appeared to be due to reduced uptake and there was no evidence of antibiotic degradation.

Nuclear suppressors of mitochondrial chloramphenicol resistance in Baker's yeast: their use for the isolation of novel mutants

Strains that are genotypically sensitive to chloramphenicol and also contain one of the nuclear suppressors of mitochondrial chloramphenicol resistance (Waxman et al. 1979) were constructed. A manganese mutagenesis on such a strain produced chloramphenicol resistant mutants, most of which resulted from mutations in nuclear genes. These mutants may be either dominant or recessive, and they probably do not code for membrane proteins. The few mitochondrial mutants fall into several classes, but all result from mutations in the 21S rRNA gene. The suppressor allele effectively prevents the appearance of the most common group of mitochondrial mutants (those that map at cap1), and thereby enhances the selection of novel mutants in the region.

Isolation and genetic characterization of streptomycin and chloramphenicol resistant mutants in the mossPhyscomitrella patens

Mutant strains of the mossPhyscomitrella patens that were resistant to the antibiotics streptomycin and chloramphenicol were isolated following UV irradiation. Mutants resistant to streptomycin at 1.7×10−4M showed both dominant and recessive Mendelian and inheritance patterns. Mutants resistant to streptomycin at 1.7 ¢ 10−4M and to chloramphenicol at 3.1×10−4M were, with one exception, cytoplasmically inherited.

Genomic rearrangements in a mouse cell line containing integrated SV40 DNA

In the SV40-transformed mouse embryo fibroblast cell line SVT2/S, genomic rearrangements involving the SV40 DNA and flanking host sequences were identified by Southern blot hybridization using viral DNA as probe. No rearrangements of SV40 DNA integrated into nonpermissive mouse cells have been previously described. The standard arrangement found in the majority of subclones was mapped with 20 restriction enzymes, 10 of which cleave sites within the SV40 DNA. A single copy of a defective integrated viral genome is present, in which the late region is missing from about nucleotide 200 clockwise to about nucleotide 1750. The rest of the viral genome including the origin of replication and T antigen binding region is present and colinear with SV40 DNA, except for an internal repeat of about 1750 bp located between nucleotides 2750 and 4500. Rearrangements were found in 4 out of 20 random subclones of the parental SVT2/S cell line and 3 of the 4 continued to rearrange. The thioguanine-resistant cell line 281-1-4, derived from SVT2/S, remained stable on subculture but a chloramphenicol-resistant mutant, 107-6-4, derived from 281-1-4, was highly unstable. In 107-6-4, unique rearrangements were found in 6 of 31 subclones of a population that had undergone about 25 doublings from a single-cell isolate. The high rate of rearrangement and the sporadic expression of rearrangement potential are characteristic of the transposable controlling elements discovered by McClintock.

NEW ANTIBIOTIC RESISTANCE LOCI IN THE RIBOSOMAL REGION OF YEAST MITOCHONDRIAL DNA

A large number of mitochondrial antibiotic-resistant mutants have been isolated following mutagenesis with manganese. These include several different phenotypic classes of mutants, as distinguished by cross-resistance patterns, that have been found to be allelic at cap1 or ery1; some have been found to be heteroallelic.--Seven chloramphenicol-resistant mutants have been identified that are nonallelic by recombination tests with the three loci (cap1, spi1 and ery1) previously identified in the ribosomal region. Four of these are allelic with each other and define a new locus, cap3; two others are allelic and define another new locus, cap2; the seventh maps at yet a different locus, cap4. One new spiramycin-resistant mutant has been identified that defines still another new locus, spi2. A variety of genetic techniques have been used to map these loci within the ribosomal region of the mitochondrial genome.-Manganese has been shown to be effective in inducing the mutation from omega(-) to omega(n) in many mutants that experience a simultaneous mutation at the closely linked cap1 locus. The omega(n) mutation has also been described in the cap4 mutant, and this locus has been shown to be more closely linked to omega than cap1 is to omega.

Mitochondrial mutagenesis in Saccharomyces cerevisiae I. Ultraviolet radiation

UV efficiently induces mutations in mitDNA, conferring resistance to erythromycin. Mitochondrial chloramphenicol-resistant mutants are probably also induced by UV, but almost 90% of mutants with such phenotype are nonmitochodrial; therefore it is impossible to estimate accurately the frequencies of the induced presumptive mitochondrial cap r mutations.

Suppression of mitochondrially-determined resistance to chloramphenicol and paromomycin by nuclear genes in Saccharomyces cerevisiae

Phenotypic "revertants" of a drug resistant strain of Saccharomyces cerevisiae were induced by mutgenesis with manganese. Several of these drug sensitive mutants have been shown to result from mutations in the nuclear genome that cause phenotypic modification (suppression) of the mitochondrially-determined drug resistant genotype. Four mutants carrying a single recessive nuclear gene capable of modifying mitochondrial chloramphenicol resistance are described; these may be assigned to three complementation groups. Chloramphenicol resistant mutants mapping at five separate mitochondrial loci are described. At least two of the nuclear genes cause modification of mitochondrial chloramphenicol resistance determined by mutations at three of these loci, but the other two loci are apparently non-suppressible by these nuclear alleles. This indicates that these modifiers do not act by causing a general decrease in cellular or mitochondrial permeability to the drug. A single dominant nuclear modifier of mitochondrial paromomycin resistance has been identified. It is non-allelic to and does not interact with the genes modifying mitochondrial chloramphenicol resistance.

Chloramphenicol resistance in Streptococcus pneumoniae: enzymatic acetylation and possible plasmid linkage.

Clinical isolates of Streptococcus pneumoniae resistant to chloramphenicol were observed in France for the first time in 1973. During a 4-year survey, these strains were found to represent 6% of a total of 564 isolates of S. pneumoniae in a general hospital and to belong to 13 different serotypes. One such strain, referred to as BM 6001, was shown to inactivate chloramphenicol, and the process was found to be inducible. The inactivated products were demonstrated to be O-acetoxy esters of chloramphenicol. The synthesis of an inducible chloramphenicol acetyltransferase was shown to be responsible for the inactivation of the drug. The resistant strain was able to transfer the chloramphenicol marker by transformation to competent strains of pneumococci at a frequency of 1% of that observed for control chromosomal markers. The loss of resistance was enhanced by ethidium bromide treatment, but no chloramphenicol-resistant mutant was isolated by mutagenesis of a "cured" clone or naturally susceptible isolates. All attempts to isolate plasmid deoxyribonucleic acid as covalently closed circular molecules from strain BM 6001 have been unsuccessful, but epidemiological evidence and the fact that the genes specifying chloramphenicol acetyltransferase synthesis are usually located on plasmids suggest that this marker may be plasmid-borne in S. pneumoniae.

Genetic damage during thymidylate starvation in Saccharomyces cerevisiae

Thymidylate starvation in a yeast mutant auxotrophic for dTMP caused cell death and the induction of mutations in the mitochondrial genome. After 24 h of starvation almost all surviving cells were respiratory deficient petites. In addition, shorter episodes of dTMP starvation induced chloramphenicol and erythromycin resistant mutants, indicating the occurrence of mitochondrial point mutations. Suboptimal concentrations of exogenous thymidylate were also found to induce petites and a decline in cell viability and the magnitude of these effects was acutely dependent upon the dTMP concentration. Cesium chloride gradient analysis of DNA from cells undergoing thymineless incubation revealed a progressive loss of mitochondrial DNA, and a decrease in the molecular weight of nuclear DNA.

The isolation and genetic characterization of extrachromosomal chloramphenicol and oligomycin-resistant mutants from the petite-negative yeast Kluyveromyces lactis.

Spontaneous mutants of the petite-negative yeast Kluyveromyces lactis, resistant to the antibiotics chloramphenicol and oligomycin, were isolated and genetically characterized. Three chloramphenicol-resistant mutants showed non-Mendelian inheritance when crossed to sensitive parents. Of 5 oligomycin-resistant strains studied, three exhibited resistance due to the presence of an extrachromosomal mutation. The resistance of the other two deriving from a nuclear and recessive mutation. When two factor crosses in trans configuration were performed between two of the chloramphenicol and the five oligomycin-resistant mutants a polarity in recombination was observed with a predominance of sensitive (OSCS) over resistant (ORCR) reciprocal recombinants. Allelism tests carried out among the oligomycin-resistant mutants indicated the presence of at least two distinct extrachromosomal regions responsible for the resistance.

Extranuclear recombination in Aspergillus nidulans: Closely-linked multiple chloramphenicol- and oligomycin-resistance loci

A nuclear, chloramphenicol-sensitive mutant cas-1 has been isolated which is cross sensitive to a number of drugs, including oligomycin and cycloheximide. Approximately one-third of the chloramphenicol-resistant mutants isolated from mutagenized conidia of this strain were found to be extranuclear, and exhibited a variety of phenotypes. One of these mutants, designated (camB51), was slow growing on drug-free medium and recombined at low frequency with the previously described mutant (camA112) (Gunatilleke et al., 1975). The majority of extranuclear oligomycin-resistant mutants isolated from cas-1 were indistinguishable from (oliA1) (Rowlands and Turner, 1973). Two mutants, (oliB322) and (oliB332), with similar but not identical phenotypes to (oli A1), recombined with the latter at low frequency but not with each other, thus representing a new class of extranuclear mutants.

Studies on the development of chloramphenicol resistance in Salmonella typhimurium

Chloramphenicol therapy of acute and chronic Salmonella typhimurium infection in mice did not lead to the development of chloramphenicol resistant mutants of this organism. However, chloramphenicol resistant organisms were readily produced in vitro. Transfer of chloramphenicol resistance from a donor strain of Escherichia coli X12 to a suitable recipient strain of S typhimurium 5235 occurred readily in the intestine of 15 out of 20 conventional mice, following oral administration of large doses of these strains supplemented by chloramphenicol therapy. When a similar system was used in untreated animals, only a small number of chloramphenicol resistant S typhimurium were isolated from three out of 18 mice. The virulence of chloramphenicol resistant S typhimurium produced in vitro and in vivo was similar to that of the sensitive parent strain.

Cytoplasmic inheritance of chloramphenicol resistance in mouse tissue culture cells.

A chloramphenicol-resistant mutant, isolated from mouse A9 cells, was enucleated and fused with a nucleated chloramphenicol-sensitive mouse cell line. Resultant fusion products, cytoplasmic hybrids (or "cybrids"), were selected as resistant to chloramphenicol, and had the nuclear markers and chromosome complement of the chloramphenicol-sensitive parent. These cybrids appeared at the high frequency of 2-8 per 10(4) cells plated. Neither parent produced any colonies when plated under identical selective conditions. Fusion between enucleated chloramphenicol-sensitive cell fragments and the chloramphenicol-sensitive cell produced no resistant colonies, suggesting that chloramphenicol resistance is not due to an increase in the ratio of cytoplasm to nucleus. Furthermore, fusions between resistant and sensitive nucleated cells produced resistant hybrids at a frequency 100 times less than that of resistant cybrids. Thus, these stable chloramphenicol-resistant cybrids result from the fusion of a chloramphenicol-resistant cytoplasm with a chloramphenicol-sensitive cell. It is proposed, therefore, that chloramphenicol resistance is a cytoplasmically inherited characteristic in this mouse cell line.

Mitochondrial antibiotic resistance in yeast: Ribosomal mutants resistant to chloramphenicol, erythromycin and spiramycin

1. Mutants of Saccharomyces cerevisiae, resistant to chloramphenicol, erythromycin and/or spiramycin as a result of a mutation in mtDNA have been characterized biochemically. 2. Three erythromycin-resistant mutants, mapping at two distinct loci on a linkage map of mitochondrial genes, have been examined. Mutants 354 and 514, highly resistant in vivo, possess altered mitochondrial ribosomes. Besides being genetically distinct, these two mutants can be distinguished on the basis of their response to lincomycin and in their quantitative response to erythromycin, carbomycin and spiramycin. Weakly resistant mutant 553 is also a ribosomal one. 3. Chloramphenicol-resistant mutants 321 and 323 also possess altered mitochondrial ribosomes. 4. Acrylamide gel electrophoresis of proteins from the 50-S mitochondrial ribosomal subunit of the wild type and several of the mutants has failed to reveal differences in composition. The possibility that resistance is determined by ribosomal RNA is discussed.

TWO CYTOPLASMICALLY INHERITED CHLORAMPHENICOL RESISTANCE LOCI IN YEAST (SACCHAROMYCES CEREVISIAE)

Cytoplasmically inherited chloramphenicol resistant mutants were tested for allelism and found to be non-allelic. The loci appear linked but it has not yet been possible to obtain a good estimate of the amount of linkage.

Occurrence of Chloramphenicol-acetylating Enzymes in Various Gram-negative Bacilli

The occurrence of a chloramphenicol-acetylating enzyme, similar to that found in Escherichia coli, carrying an R factor was investigated in various gram-negative bacilli. The acetylated products of chloramphenicol were identified by chromatography and quantitatively assayed after benzene extraction. The investigated strains were of the Salmonella-Arizona group, the Klebsiella-Aerobacter group, Serratia marcescens, the Proteus group, and Pseudomonas aeruginosa, most of which were isolated from 1947 to 1957. Both chloramphenicol-sensitive and -resistant strains were included, but none of them was able to transfer chloramphenicol resistance by conjugation. In the Proteus group, a significant level of a chloramphenicol-acetylating enzyme was found in most strains, whether they were sensitive or resistant to chloramphenicol; the resistant strains showed higher levels of the enzyme. Some chloramphenicol-sensitive strains lacked this enzyme. Only the sensitive strains containing the enzyme could easily produce chloramphenicol-resistant mutants with higher enzyme activity. Thus, the chloramphenicol resistance of this group can be reasonably explained on the basis of the chloramphenicol-acetylating enzyme. All of the Pseudomonas aeruginosa strains were resistant to chloramphenicol, and most strains showed low levels of the enzyme (which, however, did not appear sufficient to explain their resistance). All of the strains of the other groups (except one strain of Enterobacter cloacae) lacked the enzyme, although most strains of the Klebsiella-Aerobacter group and of S. marcescens were resistant to chloramphenicol. With respect to the origin of the resistance gene of the R factor, it is noteworthy that the strains of Proteus mirabilis isolated in 1947 possessed this enzyme before the discovery of chloramphenicol.

NEW MODE OF GENETIC TRANSFER IN STREPTOCOCCUS FAECALIS VAR. LIQUEFACIENS.

Raycroft, Ruth E. (The Pennsylvania State University, University Park), and L. N. Zimmerman. New mode of genetic transfer in Streptococcus faecalis var. liquefaciens. J. Bacteriol. 87:799-801. 1964.-When chloramphenicol-resistant mutants are grown in mixed culture with the sensitive wild type of Streptococcus faecalis var. liquefaciens 31, a transfer of chloramphenicol resistance occurs. (The marker is stable through at least five transfers.) The transfer mechanism is not inhibited by deoxyribonuclease, nor is it phage-mediated; genetic transfer is apparently dependent upon cell contact. Although kinetic studies have yet to be completed, preliminary data indicate a high efficiency (2.2 per donor cell in some experiments) of transmission. Two levels of drug resistance, 7 and 50 mug, were transferred; in each case, recipient cells expressed the donor's degree of resistance.

Induction of mutations inSerratia marcescens by a proteosynthesis block

The formation of colour mutations ofSerratia marcescens by the action of chloramphenicol was studied. Pink variants were the commonest; the proportion of white variants was much smaller. Almost 100% mutations were formed in a two-day culture containing 100 μg. chloramphenicol/ml. Comparative experiments showed that the change in pigment formation was hereditary, i.e. that actual mutation, and not selection of chloramphenicol-resistant mutants, occurred. Mutation occurred both in strain 151 and strain HY. The resultant mutations remained constant throughout ten passages on normal nutrient medium. The minimum chloramphenicol concentration which produced an increase in the mutation frequency was 5 μg./ml. The combined effect of X-ray irradiation and chloramphenicol treatment somewhat stimulated the increase in the frequency of mutation as compared with cells which were only irradiated. The increase in the frequency of mutation was much slower on solid medium containing chloramphenicol.

Effect of chloramphenicol on protein and nucleic acid synthesis by Shigella flexneri.

SUMMARY: Resting organisms of Shigella flexneri 3 were able to synthesize protein and nucleic acids in a chemically defined medium. Protein synthesis was markedly inhibited while ribonucleic acid (RNA) synthesis was stimulated by low concentrations of chloramphenicol; deoxyribonucleic acid synthesis was inhibited slightly. Neither glycine, l-phenylalanine nor l-tyrosine annulled the action of the antibiotic. Inhibition of protein synthesis and stimulation of RNA synthesis in resting organisms of a chloramphenicol-resistant mutant of S. flexneri 3 was observed only in the presence of high concentrations of chloramphenicol. The results presented in this paper show that chloramphenicol suppresses the growth of S. flexneri 3 by interfering with the synthesis of protein. The stimulation of RNA synthesis suggests that chloramphenicol may exert its inhibitory action by promoting the formation of RNA with altered biological activity.