Treick , R. W. (Indiana University, Bloomington), and W. A. Konetzka . Physiological state of Escherichia coli and the inhibition of deoxyribonucleic acid synthesis by phenethyl alcohol. J. Bacteriol. 88: 1580–1584. 1964.—The effects of concentration of phenethyl alcohol (PEA) and the physiological state of the cells on inhibition of macromolecular synthesis in Escherichia coli were investigated. Deoxyribonucleic acid (DNA) synthesis by cells of E. coli from the maximum stationary phase is completely inhibited by 0.32% (v/v) PEA immediately upon addition of the inhibitor, although there is a net increase in the synthesis of ribonucleic acid (RNA) and protein. However, DNA synthesis in cells from the exponential phase is inhibited only after an increase which corresponds to 1.4 to 1.6 times the amount of DNA present at the time of PEA addition. In a randomly dividing culture, this increment of DNA synthesis presumably represents completion of the DNA replication cycle initiated at the time of PEA addition. By programming the addition and removal of PEA, DNA synthesis can be made to proceed in stepwise increments corresponding to doublings of the DNA. The data indicate that the DNA being replicated at the time of PEA addition completes the replication cycle and, although there is net synthesis of RNA and protein, no initiation of a second cycle of DNA replication occurs until the removal of the inhibitor.

A detailed characterization of the lysine biosynthetic pathway in plants is yet to be completed. It is, however, assumed that the diaminopimelic acid pathway exists in the plant kingdom, as commonly described forEscherichia coli.Modification and refinement of lytic complementation, a technique previously utilized in bacterial systems, facilitated the isolation of a functional Diaminopimelate Dehydrogenase gene from aGlycine max nuclear gene library. The isolated gene codes for the enzyme meso-diaminopimelate dehydrogenase. The coding capacity for the enzyme was originally contained on a 6.6kb fragment in a Charon 4a soybean gene bank. Subcloning of the 6.6kb fragment resulted in the recombinant plasmid pMW75. Subsequent subcloning resulted in a 4.05 kb fragment contained in pLW14. One region of homology was observed upon hybridization to EcoR1 digested soybean DNA. Homologous sequences were also observed in Triticum DNA.Meso-diaminopimelate dehydrogenase activity was demonstrated inGlycine max embryos. Maximum enzymatic activity of the cloned enzyme was observed at a pH of 8.0. The enzyme encoded by the soybean gene has an apparent molecular weight of 67 000.

The earliest report of the cultivation in vitro of pathogenic leptospires is that of Inada et al. (1916). From 1916 until the present, various media, both liquid and semisolid, have been developed and used for research and diagnostic purposes. Examples of such media are those of Korthof (1932), Chang (1947), Stuart (1946), and Cox (1955). A common denominator for growth of leptospires which is present in all of the media is some type of body fluid such as serum, whole blood, or ascites fluid. The literature contains few reports on the suc

Plants of Mimulus cardinalis Douglas (Scrophulariaceae) were grown in axenic culture from seed for 28 days on a minimal salts medium supple-mented with L-lysine, L-methionine, L-threonine, L-isoleucine, DL-or L-homoserine or DL-homocysteine, alone or in combinations ranging from 5 to 1000 μM. Abnormal growth was observed at the higher concentrations of all these aminoacidsexcept homocysteine. The lysine inhibition was significantly reduced by methionine, homocysteine or isoleucine. The threonine inhibition was significantly reduced by methionine or homocysteine. A combination of lysine and threonine at 1 MM was lethal. This synergistic effect was prevented when methionine, homoserine or homocysteine were added to the lysine-threonine combination. These results can be explained in terms of end-product control of aspartokinase and homoserine dehydrogenase by lysine and threonine, respectively, in the biosynthetic pathway to these aspartic-acid-derived amino acids.

A modification of the Colowick and Womack procedure for measuring ligand binding by macromolecules is described for drug binding by bacteria. This technique is based on the determination of drug concentration in the dialysate from a bacteria-drug mixture at equilibrium. The dialysis cell of the original procedure was replaced by a Minibeaker (Bio-Rad), which has a larger membrane surface area, and the dialysate was continuously monitored with a spectrophotometer equipped with a flow cell. With this system, only 3 min was required to determine the amount of cetylpyridinium chloride bound by Escherichia coli K-12 strain P678. Possible applications of the technique are discussed.

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65animals were injected into separate areas of the skin with 40 pg of cell walls and 40 pg of protoplasm of homologous organisms. At 48 hours postinjection, protoplasm had caused little reaction a t the site of inoculation, whereas cell walls had produced severe reaction in the 6 animals that had received an initial injection of cell walls. The lesions were red and edematous and varied from 20 x 7 to 38 x 35 mm in diameter. The 3 animals that had received protoplasm as the initial inoculum developed flat lesions varying from 7 x 7 to 13 x 13 mrn in diameter following the second inoculation of cell walls, although the 3 normal animals failed to respond. These results demonstrate that cell walls contain fractions capable of inducing hypersensitivity of the delayed type and of eliciting skin reactions in sensitized animals.Initial lesions produced by mycobacteria injected into the skin of rabbits do not appear until after 4 or 5 days. In this respect these organisms differ from B . tularense and Hemophilus pertussis suspensions which produce visible effects in rabbits within 24 hours( 1 0 ) . Initial and sensitivity reactions produced by fractions of mycobacteria can be differentiated by their time of appearance. Details of these and other studies will be discussed later.Summary. A technic was described for separation of cell wall and internal protoplasm from cells of Mycobacteria. Data are presented which demonstrated that cell walls produced lesions when injected intradermally into rabbits, whereas protoplasm failed to produce these lesions. Cell walls were also shown to be capable of inducing hypersensitivity of the delayed type. Separation of these morphological elements resulted in definite separation of one element possessing certain biological activities from material not possessing these characteristics. Such initial fractionation of the cell should facilitate purification and characterization of substances which elicit typical tissue responses to infection with tubercle bacilli.

Treatment of strains of Escherichia coli with 0.25 % phenethyl alcohol resulted in a reversible inhibition of DNA synthesis and cell division. Comparisons of sedimentation behavior for nucleoids isolated prior to and following phenethyl alcohol exposure indicated a dramatic increase in sedimentation velocity, from 3,220 S to 11,770 S. However, following extended phenethyl alcohol exposure, nucleoid sedimentation velocities began to decline spontaneously and to approach that of nucleoids obtained from untreated cultures. Following removal of phenethyl alcohol, DNA synthesis and cell division resumed and nucleoid sedimentation velocities returned to that of controls. Analysis of nucleoids isolated from phenethyl alcohol treated cultures demonstrated the presence of high levels of cosedimenting membrane proteins, indicating that nucleoids were not released from the membrane even though DNA synthesis had terminated. Results are discussed in relation to previously reported phenethyl alcohol-induced perturbations of various cellular processes.