Biochemical studies with bi-resistant mutants (ethambutol plus streptomycin and isoniazid plus streptomycin) ofMycobacterium smegmatis ATCC 607

Biochemical characteristics of bi-resistant mutants (resistant to ethambutol plus streptomycin or isoniazid plus streptomycin) of mycobacteria isolated by replica plating fromMycobacterium smegmatis ATCC were compared with those of the drug-susceptible strains. Reduced incorporation of [¹⁴C]uracil, [³H]lysine and [¹⁴C]acetate into RNA, protein and phospholipids respectively was seen in the resistant mutants. Total phosphorlipids were enhanced in ethambutol plus streptomycin resistant mutant and decreased in isoniazid plus streptomycin resistant mutant. There were similar changes in levels of individual phospholipids. The resistant mutants revealed an accumulation of phospholipids in the cell wall, and a marked decrease of phospholipids in the cell membrane in comparison to the susceptible strain. Several qualitative alterations in the polypeptide profile (with respect to number and molecular weight) of the crude protein extract and of different subcellular compartments were seen in the resistant mutants.     

The effects of antibiotics on hemolytic behavior of red cells

Human blood was sheared between rotating polyethylene disks and plasma hemoglobin measured at intervals to produce kinetic hemolysis curves (KHC), plotted as free hemoglobin concentration vs time. The KHC produced by blood samples incubated in the presence of penicillin, streptomycin, gentamicin, and amikacin lie always below those for control samples, indicating a reduction in hemolysis; this reduction was greater as the drug concentration was increased. Explanations in terms of alterations in red cell structure were sought by several characterization tests of amikacin-loaded blood samples. Drug-localization studies demonstrated that significant fractions of the total dosage were associated with the red-cell membrane. Resistive pulse spectroscopy was used to show how amikacin affected cell size, deformability, and osmotic fragility; results were sensitive to storage age of the blood. In all cases, the effect of shearing was to reduce cell size, deformability, and osmotic fragility. Mechanisms for hemolytic protection by drugs are proposed.     

Alterations in the number of rRNA operons within the Bacillus subtilis genome

Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.     

The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors

Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed. DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (VioR) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids. Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions. Several useful cloning vectors were constructed. They are able to maintain large (greater than 30 kb) DNA inserts, with stable inheritance at a low copy number (1-2 per chromosome) and without structural rearrangements, in Streptomyces hosts. The vectors have a broad host range in the genus Streptomyces. One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli.     

Survival of orally administered isolated intestinalLactobacillus acidophilus in different parts of gastrointestinal tract of mice

A strain ofLactobacillus acidophilus (Strain HF) was isolated from human faeces. A chloramphenicol resistant strain (HFCm) and a strain (HFCmSm) restant to both chloramphenicol and streptomycin were developed from the isolated strain (HF). All the three strains showed similarin vitro susceptibility against host defence factors like gastric acid, bile salts and volatile as well as non-volatile fatty acids.In vivo tests were done by feeding these strains to mice. When the resistant strains were orally administered along with the antibiotic(s) they were stable up to 72 h     

Aminoglycoside-3″-adenyltransferase confers resistance to spectinomycin and streptomycin in Nicotiana tabacum

The bacterial gene aad A encodes the enzyme aminoglycoside-3-adenyltransferase that confers resistance to spectinomycin and streptomycin in Escherichia coli. Chimeric genes have been constructed for expression in plants, and were introduced into Nicotiana tabacum by Agrobacterium binary transformation vectors. Spectinomycin or streptomycin in selective concentrations prevent greening of N. tabacum calli. Transgenic clones, however, formed green calli on selective media containing spectinomycin, streptomycin, or both drugs. Resistance was inherited as a dominant Mendelian trait in the seed progeny. Resistance conferred by the chimeric aad A gene can be used as a color marker similar to the resistance conferred by the streptomycin phosphotransferase gene to streptomycin.     

Streptomycin retards the phenotypic maturation of chick myogenic cells

Summary As part of an effort to optimize conditions required for the complete maturation of muscle cells in vitro, we have investigated the effects of the antibiotics penicillin, streptomycin, and Fungizone (amphotericin B) on the development of cultured chick embryo skeletal muscle. It is shown that even low dosages of streptomycin, but not penicillin or Fungizone, retard protein synthesis and accumulation in these cultures. Myosin accumulation was also reduced and the appearance of striations in fused cells was delayed in myotubes formed in medium containing streptomycin. Additional data suggest that this overall retardation of myogenesis is due to the influence of streptomycin on maturing myotubes rather than early proliferation and cell fusion. These results are discussed with regard to recent efforts to promote the full maturation of muscle cells grown in culture. This research was supported by National Institutes of Health Grant NS 155882 and a Task Force on Drug Development Research Contract from The Muscular Dystrophy Association.     

Nitrous acid mutagenesis of duplex DNA as a three-component system.

Purified native Hemophilus influenzae DNA is relatively insusceptible to nitrous acid (NA) mutagenesis in vitro, but is readily mutated following denaturation. NA mutagenicity for duplex DNA is significantly increased in the presence of various alcohols, glycols, phenols or primary amines. Phenol-extracted DNA contains dissociable contaminants of low molecular weight that enhance NA mutagenesis. Enhancement of NA mutagenesis by phenol and by spermine is due to the formation of unstable molecular species. We propose that reactive organic nitroso compounds are formed which then serve as delivery vehicles to promote mutagenicity of native DNA, perhaps via transnitrosation reactions. Similar reactions probably occur in vivo to promote NA-induced base substitution (but not frameshift) mutations in Salmonella typhimurium and in Escherichia coli. The possible significance of these observations to carcinogenesis is discussed.     

Survival and transfer in the human gut of poorly mobilizable (pBR322) and of transferable plasmids from the same carrier E. coli

We examined the survival of a host Escherichia coli K-12 bacterium containing two transferable plasmids (pLM2, pSL222-4) and one poorly mobilizable plasmid (pBR322), and the transfer of these three plasmids to endogenous bacteria in the human intestinal tract. The survival of this plasmid-carrying host organism in four human volunteers was 3.5 to 6 days at recovery rates of 10^?1 to 10^?4. This finding was similar to our previous survival data on the same organism bearing a single plasmid. The K-12 strain appeared to be under a strong selective disadvantage in the human gut, since, even when bearing a tetracycline-resistant plasmid, its titer did not increase despite the administration of tetracycline. Studies of transferability showed that, while the transfer-depressed incFII plasmid pSL222-4 transferred at a frequency of 10^?1 in culture, its transfer in the human gut was much less frequent. The number of new recipients per donor cell ingested was about 10^?5, which included new recipients arising by multiplication. The recovery of pSL222-4 transcipients was enhanced by the administration of tetracycline on day 6. Neither the transfer-repressed, broad host range incP plasmid pLM2, nor the plasmid pBR322, could be detected in any endogenous host bacteria. Using the transfer and mobilization frequencies obtained in culture and the number of new recipients of pSL222-4 in the intestinal tract, we estimated that any in vivo mobilization of pBR322 to a new recipient could not occur at a frequency higher than 10^?12.     

Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III

We have cloned in Escherichia coli and sequenced a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The resistance gene was located by analysis of the initiation and termination codons in an open reading frame (ORF) of 792 bp. The deduced gene product, a 3'5'-aminoglycoside phosphotransferase of type III, has an Mr of 29,200. Comparison of its amino acid sequence with those of type I (Oka et al., 1981) and type II (Beck et al., 1982) 3' phosphotransferase, from transposable elements Tn903 and Tn5, respectively, indicated a statistically significant structural relationship between these enzymes from phylogenetically remote bacterial genera. The degree of homology observed indicate that phosphotransferase type III and type I genes have diverged from a common ancestor and that the phosphotransferase type II gene has emerged more recently from the type I evolutionary pathway.