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Escherichia colisensitivity to tetracycline involves transport and accumulation of the antibiotic within the cell by two different uptake systems: an initial rapid uptake, which occurs over the initial 6 min of contact of the cell with tetracycline, and a slower uptake system, which continues indefinitely and whose rate of uptake is 1/10 that of the rapid system. Only the slow uptake system is blocked by inhibitors of energy-driven systems; it appears to be particularly dependent upon energy from oxidative phosphorylation. Although both uptake systems lead to accumulation of intracellular tetracycline and contribute to the cell's sensitivity, the rapid uptake system appears to be the more important. While these studies confirm active transport of tetracycline into the cell, they demonstrate that a critical uptake system which appears insensitive to metabolic inhibitors occurs initially.

In the presence of suboptimal inducing amounts of dimethylsulfoxide or hexamethylenebisacetamide, a major proportion of uncommitted murine erythroleukemia (MEL) cells was found to be precommitted or primed for commitment, which was demonstrated by their rapid commitment to terminal differentiation when recultured for short periods of time (three to six hours) with fresh inducer. These same cells did not commit if left in the original inducer-containing media or if replated in fresh media without inducer. The two inducers could be interchanged in the priming and postpriming period without affecting the commitment event. However, hemin, an agent that induces hemoglobin synthesis without commitment, showed no ability to enhance commitment of a primed cell population. The rapid commitment of primed cells was inhibited by 12-0-tetradecanoylphorbol-13-acetate and cordycepin but not by cycloheximide. The latter finding indicated that this rapid inducer-dependent commitment event required new RNA synthesis but not new protein synthesis. The precommitment state was lost within six hours of the growth of cells in the absence of inducer but could be sustained longer if cells were incubated in cycloheximide. These studies characterize a precommitment state not previously described and one that appears during chemically induced differentiation in the absence of metabolic inhibitors. The stabilization of these precommitted cells by cycloheximide suggests that the reversibility of precommitment involves new protein synthesis. These findings show that MEL cells proceed to terminal differentiation by accumulating unstable products that must be maintained by the inducer until the final commitment event. © 1987 by Grune & Stratton, Inc.

The plasmid pUC18 contains a lacZ a-complementation gene that codes for a small peptide that can complement the AM15 mutation of the Escherichia colilacZ (β-galacto-sidase) gene, converting bacteria carrying that mutated gene from the lac Z-to the lacZ+phenotype. This plasmid was used in experiments designed to study mutagenesis by human neutrophils. E colicarrying pUC18 were incubated with neutrophils under conditions in which little ingestion of the bacteria took place; the plasmid was then isolated and transformed into an E colistrain (BOZO) that carries the lacZAM15 mutation. Of these transformants, 11 of 205,000 were lac-, suggesting that in these 11, α-comple-mentation had been lost through a mutation. No lac-colonies were detected among several hundred thousand BOZO transformed with plasmid isolated from incubations in which phagocytosis could take place, nor from incubations from which neutrophils were omitted. Despite the lac-phenotype of these 11 transformants, plasmids reiso-lated from nine of them showed normal a-complementing ability when transformed into fresh BOZO. These findings indicated that in these nine, the mutations were located in the chromosomes of the transformed BOZO. It thus appears that on exposure to activated neutrophils, a plasmid may acquire a lesion (?mutation) that can somehow be transferred to the genome of a recipient microorganism, resulting in repair of the damaged plasmid accompanied by mutation of the recipient's chromosome. Restriction mapping of the DNA from four of these nine chromosomal mutants suggested that the mutations did not represent major insertions or deletions in the portion of the bacterial chromosome corresponding to the pUC18 lac operon insert, nor in the remainder of the lacZΔM1 5 gene. These results confirm previous work showing that exposure to activated neutrophils can induce mutations in biological systems, and provides an experimental model in which the mechanism of neutrophil-mediated mutagenesis may be examined.1988 by Grune & Stratton, Inc. 0006-4971/88/7102-0028$3.00/0

ABSTRACTThe contribution of regulatory genes to fluoroquinolone resistance was studied with clinical Escherichia colistrains bearing mutations in gyrAand parCand with different levels of fluoroquinolone resistance. Expression of marAand soxSwas evaluated by Northern blot analysis of isolates that demonstrated increased organic solvent tolerance, a phenotype that has been linked to overexpression of marA,soxS, and rob. Among 25 cyclohexane-tolerant strains detected by a screen for increased organic solvent tolerance (M. Oethinger, W. V. Kern, J. D. Goldman, and S. B. Levy, J. Antimicrob. Chemother. 41:111–114, 1998), we found 5 Mar mutants and 4 Sox mutants. A further Mar mutant was detected among 11 fluoroquinolone-resistant, cyclohexane-susceptible E. colistrains used as controls. Comparison of the marORsequences of clinical Mar mutants with that of E. coliK-12 (GenBank accession no. M96235) revealed point mutations in marRin all mutants which correlated with loss of repressor function as detected in a marO::lacZtranscriptional assay. We found four other amino acid changes in MarR that did not lead to loss of function. Two of these changes, present in 20 of the 35 sequenced marORfragments, identified a variant genotype of marOR. Isolates with the samegyrAand parCmutations showed increased fluoroquinolone resistance when the mutations were accompanied by overexpression of marAor soxS. These data support the hypothesis that high-level fluoroquinolone resistance involves mutations at several chromosomal loci, comprising structural and regulatory genes.

Penicillin has been used to purify large quantities of minicells.

Salmonella enteritidis14C-endotoxin was recovered predominantly from the nuclear and mitochondrial subcellular fractions of livers and spleens of mice and rats, 3.5 hr and 3 days after intravenous administration. Of the recovered radioactivity, 10 to 20% was present in the liver mitochondrial fraction as high-molecular-weight, biologically active material, suggesting the presence of intact endotoxin. Autoradiographic studies demonstrated nuclear and cytoplasmic labeling in the liver and at least nuclear label in spleen cells. The resistance of rats, as compared to mice, to the induction of amyloidosis does not appear to be based on a difference in subcellular localization of endotoxin within the reticuloendothelial system.

Levy, StuartB. (Institut du Radium, Paris, France). Isologous interference with ultraviolet and X-ray irradiated bacteriophage T2. J. Bacteriol. 87:1330–1338. 1964.—Qualitative and quantitative analysis of the interference capacity of an irradiated T2bacteriophage was made with ultraviolet and X-ray irradiation. Two different effects were found to explain the total interference picture in the ultraviolet-irradiated system: exclusion and depression. Exclusion is the absolute inhibition of infectious phage growth in the bacterial host. Depression is the diminution of burst size in instances where the infectious phage has not been excluded. Both effects were seen when the infectious phage was added after the addition of ultraviolet-irradiated phage. Doses between 1,600 and 2,200 ergs/mm2(survivals, ca. 10−7) showed the greatest exclusion effect (70%). Exclusion was lost between 6,500 and 7,500 ergs/mm2. The depression effect was highest (90%) at lower doses (survivals, ca. 10−6), falling off as the dose range went above 1,600 ergs/mm2or survivals of 10−7. Depression was lost at 3,000 ergs/mm2. X-ray irradiation (both direct and indirect) to survivals less than 10−2showed no interference capacity in the phage irradiated. Indirect X-ray irradiation to survivals between 5 and 10% showed 50% exclusion, but no depression.

Both fi+and fi−type R factors function as intact replicative units when segregated into Escherichia coliminicells. Hybridization studies demonstrate that at least 95% of the deoxyribonucleic acid (DNA) in R+minicells is episomal in origin. About half of the DNA can be extracted in a closed circular form and about 75% of the DNA is membrane associated. DNA, ribonucleic acid, and protein synthesis proceeds in R+minicells in contrast to R−minicell controls. The system offers a unique opportunity to study a relatively small replicative unit in a native cell environment and a simple means of isolating large quantities of episomal DNA.

ABSTRACTA carbapenem-resistant clinical isolate of Escherichia coli, which lacked OmpF and OmpC porins, carried a marRmutation and expressed a functional yedS, a normally nontranslated gene. MarR and YedS are described here as having effects on the ability of this strain to resist carbapenems. Additionally, expression of YedS was regulated by the small RNA MicF in a MarA-dependent way. These findings illustrate how broadly bacteria can mutate within a selective clinical setting, in this case, resistance to carbapenems, by altering three porin genes and one regulatory gene.