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.     

Escherichia coli ras locus: its involvement in radiation repair

There are several classes of Escherichia coli mutants defective in radiation repair. These include strains defective in pyrimidine dimer excision, in photoreactivation, in recombination, in repair of X-ray damage, and ultraviolet (UV)-conditional mutants which do not divide after UV. Another mutant (ras(-)) has been isolated. The ras(-) has increased UV sensitivity, but only slightly increased X-ray sensitivity (1.5-fold increase). Ability to effect genetic recombination, to reactivate irradiated bacteriophage T1, and to be photoreactivated is normal. UV-induced mutation frequency is greatly increased in the mutant. The ras(-) apparently lacks the ability to repair some UV damage in the bacterial cell but can repair UV damage to bacteriophage DNA. The ras locus is located between lac and purE on the chromosome map.     

The dependence of UV-induced reversions to prototrophy on the streptomycin resistance allele in Escherichia coli

UV-induced mutability to prototrophy depended on the alleles of the strA locus. The yield of UV-induced suppressor mutations was decreased ten-fold in a Str^r strain compared with an isogenic Str^s strain. This decrease was due to the inability of the majority of suppressors to be expressed phenotypically in the Str^r strain in the course of selection. The addition of streptomycin to the selective media raised the number of selected suppressor revertants, by making such expression possible. The probable relation of streptomycin resistance and UV-induced suppressors is discussed.     

A clinical isolate of transposon Tn5 expressing streptomycin resistance in Escherichia coli.

The central region of transposon Tn5 carries three antibiotic resistance markers: neo, ble, and str. The str gene codes for a phosphotransferase that inactivates streptomycin. This activity is phenotypically expressed in several gram-negative bacteria but not in Escherichia coli. We identified a Tn5 variant in E. coli clinical isolates that express streptomycin resistance. This transposon carries a 6-base-pair deletion within the str gene, near the 3' end. The same kind of mutation had been previously obtained experimentally from Tn5.     

Characteristics of copper-induced streptomycin transport in Escherichia coli

Copper dependent uptake of streptomycin by resting E. coli cells was studied. It was shown that copper stimulates the aminoglycoside uptake only when bacteria possess endogenic energy sources. Additional accumulation of positive charged molecules of the antibiotic is accompanied by partial depolarisation of the membrane, its steady state distribution between cells and the medium corresponding to the resulted value of the membrane potential. On the basis of the data obtained it was suggested that under the influence of copper membrane permeability for streptomycin increases.     

Spreading of streptomycin antibiotic resistance gene in Escherichia coli plasmids demonstrated by Southern blot analysis

Plasmids from E. coli strains of 38 donors were transconjugated to common recipient SY663 Escherichia coli K12. The restriction patterns of the isolated plasmids were highly heterogenous. However, the streptomycin (Sm) resistance genes of the plasmids were identical or closely homologous in 29 of the 33 plasmids conferring Sm resistance. These data were based on Southern blot analysis, using the Sm resistance gene (encoding aminoglycoside phosphoryl transferase) as probe cut out from pBP1 plasmid. Our data suggest an extensive spreading of streptomycin resistance gene of this type.     

A mutation in the 530 loop of Escherichia coli 16S ribosomal RNA causes resistance to streptomycin.

Oligonucleotide-directed mutagenesis was used to introduce an A to C transversion at position 523 in the 16S ribosomal RNA gene of Escherichia coli rrnB operon cloned in plasmid pKK3535. E. coli cells transformed with the mutated plasmid were resistant to streptomycin. The mutated ribosomes isolated from these cells were not stimulated by streptomycin to misread the message in a poly(U)-directed assay. They were also restrictive to the stimulation of misreading by other error-promoting related aminoglycoside antibiotics such as neomycin, kanamycin or gentamicin, which do not compete for the streptomycin binding site. The 530 loop where the mutation in the 16S rRNA is located has been mapped at the external surface of the 30S subunit, and is therefore distal from the streptomycin binding site at the subunit interface. Our results support the conclusion that the mutation at position 523 in the 16S rRNA does not interfere with the binding of streptomycin, but prevents the drug from inducing conformational changes in the 530 loop which account for its miscoding effect. Since this effect primarily results from a perturbation of the translational proofreading control, our results also provide evidence that the 530 loop of the 16S rRNA is involved in this accuracy control.     

Role of constitutive and inducible repair in radiation resistance of Escherichia coli

Radiation resistance of Escherichia coil cells depends on how efficiently DNA is recovered after damage, which is determined by the function of constitutive and inducible repair branches. The effects of additional mutations of the key genes of constitutive and inducible repair (recA, lexA, recB, polA, lig, gyr, recE, recO, recR, recJ, recQ, uvrD, helD, recN, and ruv) on radiation resistance were studied in E. coli K-12 strain AB 1157 and highly radiation-resistant isogenic strain Gam(r)444. An optimal balance ensuring a high gamma resistance of the Gam(r)444 radiation-resistant E. coli mutant was due to expression of the key SOS repair genes (recA, lexA, recN, and ruv) and activation of the presynaptic functions of the RecF homologous recombination pathway as a result of a possible mutation of the uvrD gene, which codes for repair helicase II.     

Reversal of the streptomycin injury of Escherichia coli

The number of viable Escherichia coli in a young, actively growing culture is decreased approximately 99.9 per cent by a 30 second exposure to 25 phig. streptomycin/ml. The injury induced by the antibiotic is only potentially lethal, however, and may be reversed by subculture within 5 minutes into fresh culture medium, NH(4)NO(3), NH(4)Cl, (NH(4))(2)HPO(4), NH(4) citrate, and NH(4) tartrate. Subculturing into water, glucose, or MgSO(4) results in a more marked decrease in the number of viable organisms. In KNO(3), NaNO(3), K(2)HPO(4), and Na(2)SO(4) solutions reversal occurs first, followed by a rapid decrease in viability. True reversal of the streptomycin injury takes place, as demonstrated by the rapid rate of recovery to the viable count of the original culture. Development of resistance has been eliminated as the cause of regrowth since the streptomycin sensitivity of recovered cultures remained the same as that of the original culture. The use of water as diluent for viability determinations potentiates the lethal effect of streptomycin activity. Several compounds, at various dilutions, substituted for water as the diluent gave rise to four types of responses, group I, NH(4)NO(3), NH(4)Cl, KNO(3), NaNO(3), Ca(NO(3))(2), showed complete reversal of the streptomycin injury at all levels of the salts tested, from 0.01 to 0.5 M concentrations. Group II, NaCl and K(2)HPO(4) showed complete reversal at 0.03 and 0.1 M. Group III, glucose and urea allowed complete reversal at 0.5 M. Group IV, glycerol and glycerine showed no reversal at 0.5 M concentration. The reversal of the streptomycin injury to young actively growing bacteria is suggested as a tool for studying the pathology of the injury to the cells.