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A procedure has been developed whereby paper chromatograms of agents which induce lambda bacteriophage in Escherichia coli can be developed using bioautographs with a lysogenic test system. Well-defined plaque-forming zones are produced indicating the area on the paper chromatogram where the active inducing material can be located. A mixture of the bacteriophage-inducing antibiotic, mitomycin C, and the noninducing antibiotic, paromomycin, was resolved into its components on paper strips with an ethyl acetate-methanol solvent system. The location of both antibiotics could thus be readily observed. Antibacterial and inducing activities were found to be identical with a crude fermentation solid, NSC-B-158,791. The use of this procedure for resolution of multicomponent inducing activities in antibiotic beers and for characterization of active components which may be potential antitumor antibiotics is indicated.

Resting cells of Escherichia coli strain D302(bioD302) can synthesize 7,8-diaminopelargonic acid from 7-keto-8-aminopelargonic acid. The product of this aminotransferase reaction has been identified by paper chromatography and electrophoresis. Glucose enhances the vitamer yield twofold. Of the 19 amino acids tested as amino donors, only methionine proved to be significantly stimulatory. In cell-free extracts, however, methionine was completely inactive unless both adenosine triphosphate (ATP) and Mg(2+) were present. S-Adenosyl-l-methionine (SAM) was about 10 times more effective than methionine, ATP, and Mg(2+). The optimal conditions for the reaction were determined, and substrate inhibition was found for 7-keto-8-aminopelargonic acid. It has been possible to eliminate certain impurities as amino donors in the commercial preparation of SAM and those that may arise in enzymatic reactions in which SAM is a substrate. The direct participation of SAM in the aminotransferase reaction seems a likely possibility.

A threonine dehydrataseless mutant of Escherichia coli, Crookes strain, was observed to grow on an acetate minimal medium without the usual requirement for isoleucine supplementation. Both the wild-type Crookes strain and a threonine auxotroph metabolized l-glutamate-1-(14)C to l-isoleucine-1-(14)C with no appreciable randomization, suggesting that a pathway for isoleucine formation from glutamate via beta-methylaspartate, beta-methyloxaloacetate, and alpha-ketobutyrate was possible in addition to the pathway from threonine and alpha-ketobutyrate. Crude cell-free extracts formed (14)C-beta-methylaspartate from (14)C-glutamate, and the conversion of beta-methylaspartate to alpha-ketobutyrate was also demonstrated, thus supporting the conclusion that glutamate can serve as a precursor of alpha-ketobutyrate (and isoleucine) without the necessary involvement of threonine as an intermediate.

A pathway for the incorporation of 2-aminopurine into deoxyribonucleic acid (DNA) was studied in cell-free extracts of Escherichia coli. It was demonstrated that the free base can be converted to the deoxynucleoside, and that the deoxynucleotide can be phosphorylated to the di- and triphosphates and then incorporated into the DNA. From a consideration of the individual reactions in crude extracts, it is likely that the rate-limiting step in this pathway is the formation of the deoxynucleotide. Of especial interest is the observation that 2-aminopurine may be viewed as an analogue of either guanine or adenine, depending on which enzymatic step is being considered. On the one hand, it resembles guanine in that it is specifically converted from the mono- to the diphosphate by guanylate kinase and not by adenylate kinase. On the other hand, it replaces adenine rather than guanine in the DNA synthesized with purified DNA polymerases. E. coli DNA polymerase utilizes aminopurine deoxynucleoside triphosphate as a substrate for DNA synthesis much better than does purified phage T5-induced DNA polymerase and is also much less inhibited by this analogue than the T5 enzyme. These experiments in vitro correlate with known differential effects of 2-aminopurine on E. coli and phage in vivo.

A heptose-deficient mutant of Escherichia coli has been isolated and from it a glycolipid, consisting of lipid A and 2-keto-3-deoxyoctonate (KDO), has been extracted with diisobutylketone-acetic acid-water. Based on beta-hydroxymyristic acid, the extractable glycolipid accounts for a major portion of the total lipid A in this mutant. A glycolipid, purified from the lipid extract by a combination of silicic acid and Sephadex LH-60 chromatography, contains glucosamine, phosphate, KDO, acetyl groups, and fatty acids in the following molar ratios: 1:2:2:1.7:5. These components account for over 80% of the lipid by weight. The fatty acid pattern of the glycolipid is typical of lipid A, the major component being beta-hydroxymyristic acid. The lipid also contains an amino sugar which appears to be 4-amino-4-deoxyarabinose. With the use of an ion-exchange paper chromatographic technique, gram-negative bacteria can be rapidly screened for the presence of this glycolipid. The mutant is believed to have a leaky defect in either biosynthesis of heptose or its incorporation into lipopolysaccharide. The lipopolysaccharide from the mutant contains only about a third as much heptose, glucose, and galactose as the parent CR34, a K-12 derivative. Chemical analysis and phage typing suggest that CR34 contains an incomplete core polysaccharide devoid of glucosamine.

The carbon skeleton of glucose is extensively randomized during conversion to cell wall glucosamine by Escherichia coli K-12. Exogenous glucosamine-1-(14)C is selectively oxidized, and isotope incorporation into cellular glucosamine is greatly diluted during assimilation. A mutant unable to grow with N-acetylglucosamine as a carbon and energy source was isolated from E. coli K-12. This mutant was found to be defective in glucosamine-6-phosphate deaminase. Glucosamine-1-(14)C and N-acetylglucosamine-1-(14)C were assimilated during the growth of mutant cultures without degradation or carbon randomization. Assimilated isotopic carbon resided entirely in cell wall glucosamine and muramic acid. Some isotope dilution occurred from biosynthesis, but at high concentrations (0.2 mm) of added N-acetylglucosamine nearly all cellular amino sugar was derived from the exogenous source. Growth of the mutant was inhibited with 1 mmN-acetylglucosamine.

With a stepwise degradation and terminal labeling procedure the 3'-terminal sequence of E. coli 16S ribosomal RNA is shown to be Pyd-A-C-C-U-C-C-U-U-A(OH). It is suggested that this region of the RNA is able to interact with mRNA and that the 3'-terminal U-U-A(OH) is involved in the termination of protein synthesis through base-pairing with terminator codons. The sequence A-C-C-U-C-C could recognize a conserved sequence found in the ribosome binding sites of various coliphage mRNAs; it may thus be involved in the formation of the mRNA.30S subunit complex.

A modified purification is described for an enzyme, from Escherichiacoli B, which polymerizes deoxyribonucleoside-5' diphosphates. Under appropriate conditions, the enzyme will add a single deoxyribonucleotide residue to a deoxyribo-oligonucleotide primer. At all stages, the enzyme activity copurified with the activity which will polymerize adenosine-5' diphosphate (polynucleotide phosphorylase). Studies of heat stability, the effect of various temperatures of reaction and of disc gel electrophoresis failed to provide evidence that the two activities are separable.

A dinucleoside monophosphate was isolated from 5-bromouracil-induced filaments of a thymine auxotroph of Escherichia coli K-12. The dinucleoside monophosphate was fractioned from a [(14)C]5-bromouracil-labeled perchloric acid extract using Dowex-1-formate ion-exchange chromatography. Sephadex chromatography revealed its molecular weight to be 710. Snake venom phosphodiesterase digest of the dinucleoside monophosphate yielded [(14)C]5-bromouridine and adenosine 5'-monophosphate. The presence of [(14)C]5-bromouracil in bacterial ribonucleic acid indicates that ribonucleic acid, which had incorporated 5-bromouracil, was the probable source of this dinucleoside monophosphate, 5-bromouridylyl-(3' --> 5')-adenosine.

Trileucine is utilized as a source of leucine for growth of strains of Escherichia coli K-12 that are deficient in the oligopeptide transport system (Opp). Trithreonine is toxic to E. coli K-12. Opp- mutants of E. coli K-12 retain complete sensitivity to this tripeptide. Moreover, E. coli W, which is resistant to trithreonine, can utlize this tripeptide as a threonine source and this capability is fully maintained in E. coli W (Opp-). A spontaneous trithreonine-resistant mutant of E. coli K-12 (Opp-) has been isolated that has an impaired growth response to trileucine and is resistant to trithreonine. Trileucine competes with the uptake of trithreonine as measured by its ability to relieve trithreonine toxicity in E. coli K-12. It is concluded that trileucine as well as trithreonine are transported into E. coli K-12 or W by a common uptake system that is distinct from the Opp system. Trimethionine can act as a competitor of trileucine or trithreonine-supported growth and as an antagonist of trithreonine toxicity in Opp- mutants. It is concluded that trimethionine is recognized by the trileucine-trithreonine transport system. Trithreonine, trimethionine, and trileucine are also transported by the Opp system, as they all relieve triornithine toxicity towards E. coli W and compete with tetralysine utilization as lysine source for growth of a lysine auxotroph of this strain.

The 5'-terminal sequence of mRNA from the galactose operon of E. coli has been determined. gal RNA is synthesized in vitro by means of a kinetically controlled purified transcription system and is isolated by a two-step RNA.DNA hybridization scheme. The following sequence of the first 77 nucleotides has been deduced by analysis of oligonucleotides produced by digestion with T(1), pancreatic, and carboxymethylated pancreatic ribonucleases: ppA-U-A-C-C-A-U-A-A-G-C-C-U-A-A-U-G-G-A-G-C-G-A-A-U-U-A-U-G-A-G-A-G-U-U-C-U-G-G-U-U-A-C-C-G-G-U-G-G-U-A-G-C-G-G-U-U-A-C-A-U-U-G-G-A-A-G-U-C-A-U-A-C-C-U-G-U. Residues 27-77 correspond to the amino terminal 17 amino acids of UDPgalactose 4-epimerase (EC 5.1.3.2), the protein specified by the promoter-proximal structural gene of the operon. A self-complementary sequence occurs near the 5' terminus; 12 of 15 nucleotides between residues 4 and 18 are symmetrically located about position 11. The sequence of residues 22-33 closely resembles part of the lactose operator sequence reported previously [Gilbert, W. & Maxam, A. (1973) Proc. Nat. Acad. Sci. USA 70, 3581-3584].

Wild-type strains of Escherichia coli K-12 accumulate toxic concentrations of methylglyoxal when grown in medium containing adenosine 3',5'-monophosphate and either d-xylose, l-arabinose, or d-glucose-6-phosphate, independent of the presence of other carbon sources. Mutations at a locus called cxm specifically block methylglyoxal formation from xylose in the presence of adenosine 3',5'-monophosphate. Accumulation in medium containing xylose, studied in some detail, is dependent on the ability to utilize xylose and is associated with an increased rate of xylose utilization without changes in levels of xylose isomerase. These results suggest that adenosine 3',5'-monophosphate results in induction of excessively high levels of an early rate-limiting step in xylose metabolism. This step may be the transport of xylose into the cell. The resulting excessive rates of xylose catabolism could stimulate methylglyoxal formation by overburdening late steps in glycolysis.

An enzyme that liberates uracil from single-stranded and double-stranded DNA containing deaminated cytosine residues and from deoxycytidylate-deoxyuridylate copolymers in the absence of Mg(++) has been purified 30-fold from cell extracts of E. coli. The enzyme does not release uracil from deoxyuridine, dUMP, uridine, or RNA, nor does it liberate the normally occurring pyrimidine bases, cytosine and thymine, from DNA. The enzymatic cleavage of N-glycosidic bonds in DNA occurs without concomitant cleavage of phosphodiester bonds, resulting in the formation of free uracil and DNA strands of unaltered chain length that contain apyrimidinic sites as reaction products. The enzyme may be active in DNA repair, converting deaminated dCMP residues to an easily repairable form.