Two genes (arg-3 and arp-6) marked by arginine auxotrophy and a gene (sac-2) marked by resistance to serum killing were found to be linked by cotransformation. Evidence derived from the linkage of these markers to spc (spectinomycin resistance) suggests that sac-2 may be genetically distinct from a sac gene previously described.
Step-wise intrinsic resistance to ampicillin in Neisseria gonorrhoeae was analyzed genetically by DNA-medi-ated transformation experiments. A first-step ampicillin-resistant (Amprl) mutant and a second-step ampicillinresistant (Ampr2) mutant generated during sequential selection were used in these studies.
ITH the introduction of drug resistances into Pneumococcus by DNA trans-W f ormation (HOTCHKISS 1951), it became clear that heritable traits not presumably connected to the classic capsular antigens (AVERY, MACLEOD and MCCARTY 1944; AUSTRIAN and MACLEOD 1949) could also be carried by DNA. Certain practical and conceptual developments were derived from this finding. Selective resistance markers allowed quantitative estimation of the number of cells converted, the quantitative improvement of transformation recipes, and the determination of the degree of independence of various marker pairs. Independence of penicillin and streptomycin resistance markers from capsule-type transforming agents was interpreted as indicating the existence of differently marked regions of DNA (considered then to be separate molecules) within the total DNA of a single cell line (HOTCHKISS 1951). The specificity of resistancetransforming units for each kind of drug, and the correspondence of each unit quantitatively to the spontaneous mutational steps of cumulative penicillin resistance, indicated the close parallelism of DNA-carried factors and the units of mutational history present in the cell lines.The original data on penircillin-resistant Pneumococci were briefly reported in 1951 and summarized thereafter at various times. Other demonstrations of transferable drug resistance were soon reported (ALEXANDER and LEIDY 1953) and served, as have later developments in bacterial, and eventually bacteriophage, genetics to demonstrate that DNA can bear a wide variety of traits besides surface antigens. Meanwhile, modifications in the Pneumococcal strains, markers, and techniques used have made it unfeasible to reproduce precisely the conditions of the early experiments. The present article reports a reexamination in the light of modern techniques, of the system which played a considerable part in fashioning our early views on the molecular basis of genetics. In it we describe four identifiable units of penicillin resistance in currently available material, their transfer by DNA to recipient cells, their cooperative phenotypic effects in multiply marked cells, and the indications of certain linkages exhibited by some of them.
Several different systems for the conversion of sorbitol to hexose have been described in microorganisms. In the pseudomonads, sorbitol may be oxidized to fructose (Shaw, 1956) or to sorbose (Sebek and Randles, 1952). The latter investigators concluded that the conversion of sorbitol to fructose or sorbose did not involve phosphorylation since carbon dioxide was not liberated from bicarbonate buffer upon incubabation of whole cells, adenosine triphosphate, and hexitol. Cell-free preparations of Acetobacter suboxydans have been shown to dissimilate sorbitol by three different pathways. In addition to a dehydrogenase, presumably a flavoprotein which catalyzes the oxidation of sorbitol to hexose (Widmer et al., 1956; Arcus and Edson, 1956) two alternate enzyme systems found in the soluble portions of the cells have been described (Cummins et al., 1957a). Separation of these systems revealed the existence of a DPN3and a TPN-linked sorbitol dehydrogenase (Cummins et al., 1957b). The product of the DPN-linked oxidation was found to be fructose whereas the latter formed sorbose. Studies by Wolff and Kaplan (1956a) demonstrated that D-sorbitol-6-phosphate is dehydrogenated by cell-free extracts of Escherichia coli strain B. The phosphorylated route was indicated as the major pathway of sorbitol dissimilation since the specific activity of extracts of sorbitol-grown cells was approximately 60-fold greater for sorbitol-6-phosphate as compared to sorbitol. The results presented in this paper describe the isolation, purification, and properties of a DPN-linked enzyme from Lactobacillus casei 1 This investigation was supported by a grantin-aid from the Gustavus and Louise Pfeiffer Research Foundation. 2 Lederle Medical Student Research Fellow. 3 The following abbreviations are used: diphosphopyridine nucleotide, oxidized and reduced, DPN and DPNH; triphosphopyridine nucleotide. TPN; tris(hydroxymethyl)aminomethane, Tris. which catalyzes the oxidation of D-sorbitol-6phosphate to fructose-6-phosphate. MATERIALS AND METHODS Organism and culture conditions. L. casei strain ATCC 4646 was employed in this study. Stock
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