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PubMed1977

Aminocyclitol.

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Humans↗
PubMed2001

Phenotypic switching of antibiotic resistance circumvents permanent costs in Staphylococcus aureus.

Bacterial antibiotic resistance is often associated with a fitness cost in the absence of the antibiotic [1,2]. We have examined a resistance mechanism in Staphylococcus aureus that negates these costs. Exposure to gentamicin both in vitro and in vivo has been reported to result in the emergence of a gentamicin-resistant small colony variant (SCV)[3-8]. We show that the emergence of SCVs following exposure to gentamicin results from a rapid switch and that bacteria exposed to cycles of gentamicin followed by antibiotic-free medium repeatedly switched between a resistant SCV and a sensitive parental phenotype (revertants). The fitness of revertants relative to S. aureus with stable gentamicin resistance was greater in drug-free media, which suggests that S. aureus has evolved an inducible and reversible resistance mechanism that circumvents a permanent cost to fitness.

Adaptation, Physiological↗
PubMed1977

The in vitro drug resistance of Salmonella dublin isolated from cattle.

The resistance of Salmonella dublin to eight antibacterial drugs was assessed under diagnostic laboratory conditions. Two techniques were used consecutively and the differences in the results are described. Strains of S. dublin isolated from either calves, adult cattle or abortion material showed no differences in resistance for either of the techniques, though differences between the two techniques were found.

Animals↗
PubMed1987

Inhibition of colonization of the chicken alimentary tract with Salmonella typhimurium gram-negative facultatively anaerobic bacteria.

Oral administration of strains of food poisoning salmonellas to day-old chickens produced a profound inhibition in the subsequent colonization of the caeca by a strain of Salmonella typhimurium given one day later. Closely related genera were unable to produce a similar inhibition. The inhibition was not the result of bacteriophages produced by the first strain. Neither was it the result of an immunological response by the host induced by the first strain. In additional experiments in day-old chickens, inhibition of an Escherichia coli Nalr strain and of a Citrobacter sp. Nalr strain was produced by the antibiotic-sensitive forms of the homologous strains while strains from other genera did not produce any inhibition. When an avirulent mutant of S. typhimurium was used for pre-treatment a statistically significant reduction in the excretion of the super-infecting S. typhimurium Nalr strain over several weeks was produced. A genus specific inhibition was reproduced in vitro by mixed culture experiments. Live cultures were necessary for in vitro inhibition. Killed cells or a culture supernatant produced no inhibition.

Animals↗
PubMed1987

Analysis of penicillinase-producing Neisseria gonorrhoeae isolates in Madrid (Spain) from 1983-85.

Between April 1983 and December 1985, 576 strains of Neisseria gonorrhoeae were isolated in our laboratory from patients attending Sexually Transmitted Diseases (STD) clinics. Of these, 61 (10.6%) were penicillinase-producing. Studies on these strains by plasmid analysis, auxotyping and serogrouping showed that the predominant type strains harboured the Asian resistance plasmid, were prototrophic, and were of serogroup W II/W III. About half of the strains, both of the African and Asian type, harboured the transfer plasmid. Strains of serogroup W II/W III were less sensitive to tetracycline and cefoxitin than serogroup W I strains.

Cefoxitin↗
PubMed1995

Mutations at positions 13 and/or 914 in Escherichia coli 16S ribosomal RNA interfere with the initiation of protein synthesis.

Mutations at positions 13 (U-->A) and/or 914 (A-->U) of Escherichia coli 16S rRNA severely affect cell growth and protein synthesis, when expressed in vivo in a vector encoding an rrn operon under control of an inducible promoter. In vitro assays using extension inhibition indicate that the mutations interfere with the formation of the 30S translational initiation complex, which can account for their effect on cell growth. The two mutations destabilize an adjacent pseudoknot helix in which bases 17-19 pair to bases 916-918. This was shown by the increased binding of an oligodeoxyribonucleotide probe complementary to one strand of the pseudoknot helix, and by the increased reactivity to kethoxal of base G917 within this helix. These observations suggest that this pseudoknot helix participates in the formation of the 30S translational initiation complex.

Adenine↗
PubMed2005

The relationship between aminoglycosides' RNA binding proclivity and their antiplasmid effect on an IncB plasmid.

Bacteria routinely become resistant to antibiotics through the uptake of plasmids that encode resistance-mediating proteins. Such plasmid-based resistance is seen extensively in clinical settings and has been documented for a wide variety of bacterial infections from both Gram-positive and Gram-negative organisms. We recently reported that a small molecule could be used to mimic a natural process of plasmid elimination, called plasmid incompatibility, and that the addition of this compound causes elimination of an IncB plasmid from E. coli and a subsequent resensitization to antibiotics [DeNap, Thomas, Musk, and Hergenrother (2004) J. Am. Chem. Soc. 126, 15402-15404]. Described herein is a further substantiation and validation of the notion that plasmid incompatibility can be mimicked with small molecules that bind to important RNA targets controlling plasmid replication. In this study, the dissociation constant and stoichiometry of RNA binding are determined for 12 aminoglycosides with stem-loop I (SLI) of the IncB replication machinery. Importantly, it is found that compounds that do not bind to this RNA replication control element fail to induce plasmid loss in vivo, whereas those that do bind to the RNA typically cause measurable plasmid loss. These results highlight the potential for targeting key RNA regions for induction of plasmid loss and the subsequent resensitization of bacteria to antibiotics.

Aminoglycosides↗
PubMed1993

The 3'-keto-diol equilibrium of trospectomycin sulfate bulk drug and freeze-dried formulation: solid-state carbon-13 cross-polarization magic angle spinning (CP/MAS) and high-resolution carbon-13 nuclear magnetic resonance (NMR) spectroscopy studies.

Understanding how moisture interacts with a drug or formulation is a critical component of product development. This study demonstrates how water affects the 3'-gem-diol<==>3'-keto equilibrium in trospectomycin sulfate bulk drug and freeze-dried formulation, as probed by solid-state carbon-13 cross-polarization magic angle spinning (CP/MAS) and high-resolution nuclear magnetic resonance (NMR) spectroscopy. Drying the bulk drug or formulation to low water levels dehydrates trospectomycin sulfate from the diol to the keto form. Carbon-13 CP/MAS NMR spectroscopy measures the keto drug concentration in solid samples directly. The bulk drug, which contains approximately 16% water, is more than 90% in the 3'-diol form. Oven drying to < 3% water converts approximately 75% of the drug to the 3'-keto form. The drug is formulated as a freeze-dried, sterile powder that can contain up to 12% water depending on the freeze-drying conditions. These studies show that the 3'-keto concentration rises uniformly (up to 75%) with decreasing residual water in the freeze-dried cake. The keto-diol equilibrium was also studied in solution by high-resolution carbon-13 NMR experiments, and it was found that raising the temperature or using dimethyl sulfoxide (DMSO) as a solvent also dehydrates the drug. For example, in aqueous solution at 25 degrees C, nearly all (> 95%) of the drug is in the 3'-diol form. After equilibration at 60 degrees C, however, the 3'-keto content increases to 7%, and in d6-DMSO solvent at 25 degrees C the drug is mostly (60%) in the 3'-keto form.

Carbon Isotopes↗
PubMed1981

Escherichia coli ribosomal protein S8 feedback regulates part of spc operon.

In Escherichia coli the genes coding for the 52 ribosomal proteins (r-proteins) are organized into a number of transcription units located at various regions on the bacterial genome. The expression of r-protein genes is balanced so that individual r-protein synthesis rates change coordinately in response to changing environmental conditions, and significant amounts of free r-proteins do not exist in the cellular pool. We have suggested a model for the balanced regulation of r-protein gene expression, namely that r-protein synthesis and ribosome assembly are coupled so that r-proteins not incorporated into ribosomes prevent the further translation of r-protein mRNA by a feedback regulatory mechanism. The model was tested in vitro by examining the effect of purified r-proteins on DNA directed r-protein synthesis, and in vivo by examining the effect of overproduction of certain r-proteins on the synthesis rates of other r-proteins. In vitro experiments have revealed that some r-proteins (L1, L4, L10, S4 and S8) can selectively inhibit the synthesis of r-proteins whose genes are in the same operon as their own, and that this specific feedback regulation occurs at the level of translation rather than at the level of transcription of mRNA. Regulatory roles for L1, S4 and L4 have also been established by in vivo experiments. We have studied further the feedback regulatory properties of S8 in vivo and in vitro, and report here that the protein regulates a part of the spc operon.

Bacterial Proteins↗
PubMed2003

Direct measurement of the transfer rate of chloroplast DNA into the nucleus.

Gene transfer from the chloroplast to the nucleus has occurred over evolutionary time. Functional gene establishment in the nucleus is rare, but DNA transfer without functionality is presumably more frequent. Here, we measured directly the transfer rate of chloroplast DNA (cpDNA) into the nucleus of tobacco plants (Nicotiana tabacum). To visualize this process, a nucleus-specific neomycin phosphotransferase gene (neoSTLS2) was integrated into the chloroplast genome, and the transfer of cpDNA to the nucleus was detected by screening for kanamycin-resistant seedlings in progeny. A screen for kanamycin-resistant seedlings was conducted with about 250,000 progeny produced by fertilization of wild-type females with pollen from plants containing cp-neoSTLS2. Sixteen plants of independent origin were identified and their progenies showed stable inheritance of neoSTLS2, characteristic of nuclear genes. Thus, we provide a quantitative estimate of one transposition event in about 16,000 pollen grains for the frequency of transfer of cpDNA to the nucleus. In addition to its evident role in organellar evolution, transposition of cpDNA to the nucleus in tobacco occurs at a rate that must have significant consequences for existing nuclear genes.

Biological Transport↗
PubMed2001

Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step.

We have developed technologies that simplify genomic library construction and screening, substantially reducing both the time and the cost associated with traditional library screening methods and facilitating the generation of gene-targeting constructs. By taking advantage of homologous recombination in Escherichia coli, we were able to use as little as 80 bp of total sequence homology to screen for a specific gene from a genomic library in plasmid or phage form. This method, called recombination cloning (REC), takes only a few days instead of the several weeks required for traditional plaque-lift methods. In addition, because every clone in the mouse genomic library we have constructed has a negative selection marker adjacent to the genomic insert, REC screening can generate gene-targeting vectors in one step, from library screening to finished construct. Conditional targeting constructs can be generated easily with minimal additional manipulation.

Animals↗