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To utilize autoradiographic colony-sorting techniques (C. R. H. Raetz, Proc. Natl. Acad. Sci. U.S.A. 72:2274-2278, 1975) for the isolation of mutants with unstable enzymes, we report a new desiccation-induced lysis method, compatible with low temperatures. Furthermore, a general, two-step protocol is presented for clonal detection of hydrolytic reactions. The advantages of these critical modifications are demonstrated with the membrane enzymes glycerol 3-phosphate acyltransferase and cytidine 5'-diphosphate-diglyceride hydrolase.

Starvation of a mid-log-phase culture of Escherichia coli B/r for nitrogen, phosphate, or carbon resulted in methylation of a membrane-associated protein of about 43,000 daltons (P-43) in the presence of chloramphenicol and [methyl-3H]methionine. The in vivo methylation reaction occurred with a doubling time of 2 to 5 min and was followed by a slower demethylation process. Addition of the missing nutrient to a starving culture immediately prevented further methylation of P-43. P-43 methylation is not related to the methylated chemotaxis proteins because P-43 is methylated in response to a different spectrum of nutrients and because P-43 is methylated on lysine residues. The characteristics of P-43 are similar to those of a methylated protein previously described in Bacillus subtilis and B. licheniformis (R. W. Bernlohr, A. L. Saha, C. C. Young, B. R. Toth, and K. J. Golden, J. Bacteriol. 170:4113-4118, 1988; K. J. Golden and R. W. Bernlohr, Mol. Gen. Genet. 220:1-7, 1989) and are consistent with the proposal that methylation of this protein functions in nutrient sensing.

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.

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.

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.

Studies on the utilization of leucine peptide amides as a source of leucine for a leucine auxotroph showed that in general compounds with the structure leu-chi amide (where chi is any amide) are utilized as well as the free peptide, but that compounds with the structure chi-leu amide (where chi is not leucine) are used less effectively than the free peptide. Growth and enzymological experiments indicated that the lower capacity of Escherichia coli to utilize amides of the structure chi-leu amide is not a result of poor transport of these compounds, but rather the inability to rapidly liberate leucine from the amide when it is supplied to the cell in the form of a peptide. Competition studies indicated that the peptide amides enter the cell via the oligopeptide permease system.

The stereospecificity of peptide utilization in Escherichia coli K-12 4212, a methionine auxotroph, was investigated using diastereomers of trimethionine and trimethionine methyl ester. Of the eight stereoisomers examined, only l-Met-l-Met-l-Met, l-Met-l-Met-d-Met, and d-Met-l-Met-l-Met and the corresponding methyl esters serve as growth substrates. Triornithine-resistant mutants of strain 4212 were isolated which failed to transport d-Met-l-Met-l-Met. These results provide evidence that an oligopeptide containing a d residue at its amine terminus can enter E. coli by the oligopeptide transport system.

Chemical analyses of the carbohydrate composition of lipopolysaccharides (LPS) from a number of LPS mutants were used to propose a schematic composition for the LPS from Escherichia coli K-12. The formula contains four regions: the first consists of lipid A, ketodeoxyoctonoic acid, and a phosphorous component; the second contains only heptose; the third only glucose; and the fourth additional glucose, galactose, and rhamnose. LPS from E. coli B may have a similar composition but lacks the galactose and rhamnose units. A set of LPS-specific bacteriophages were used for comparing three mutants of Salmonella with a number of LPS mutants of E. coli K-12. The results confirm that there are basic similarities in the first and second regions of the LPS structure; they also support the four region divisions of the LPS formula. Paper chromatography was used for characterization of 32-P-labeled LPS from different strains of E. coli and Salmonella. The Rf values for LPS varied from 0.27 to 0.75 depending on the amounts of carbohydrates in the molecule. LPS from all strains studied was homogenous except for strain D31 which produced two types of LPS. Mild acid hydrolysis of labeled LPS liberated lipid A and two other components with phosphate, one of which was assigned to the first region. It is suggested that paper chromatography can be used in biosynthetic studies concerning regions 2 to 4.

The ability of Escherichia coli K-12 4212 to utilize a variety of oligopeptides as sources of required amino acids was examined. Triornithine-resistant mutants of this strain were oligopeptide permease deficient (Opp-) as judged by their inability to utilize (Lys)3 and (Lys)4 as sources of lysine and their resistance to the toxic tripeptide (Val)3. These same mutants were able to grow when Met-Met-Met, Met-Gly-Met, Met-Gly-Gly, Gly-Met-Gly, Gly-Gly-Met, Gly-Met-Met, Met-Met-Gly, or Leu-Leu-Leu were supplied in place of the requisite amino acid. The system mediating the uptake of these peptides, herein designated Opr I, was not able to transport N-alpha-acetylated peptides, nor the tetrapeptides Met-Gly-Met-Met, Met-Met-Gly-Met, or Met-Met-Met-Gly. Competition experiments indicated that trimethionine and trileucine enter E. coli K-12 via either Opp or Opr I. Analogous results were found using the methionine, leucine-requiring auxotroph E. coli B163. It appears that more than one oligopeptide transport system exists in E. coli and that the system mediating peptide uptake is complex.

The kinetics of radioactive phosphate incorporation into the adenosine and guanosine nucleoside triphosphate termini of bacterial ribonucleic acid (RNA) was studied. Knowledge obtained in a previous investigation of the kinetics of phosphate incorporation into their precursors, adenosine 5'-triphosphate and guanosine 5'-triphosphate, allowed calculation of the average half-lives of these termini, which were found to be approximately 170 s at 21.5 C for both. The ratio between the number of nucleotides incorporated into the interior of RNA chains per second and the number of termini synthesized per second was calculated by several methods and found to be between 4,000 and 8,000. Assuming that the initiation of synthesis of a RNA chain by deoxyribonucleic acid-dependent RNA polymerase always produces a triphosphate termini and that some termini do not have half-lives so short as to not be seen in this study (less than 10 s), this is the apparent average length of the transcriptional unit. The implication of these findings to the genetic organization of transfer RNA genes is discussed.

A procedure has been devised that allows selection of mutants defective in the beta-methylgalactoside transport system (mgl) of Escherichia coli. This procedure utilizes the compound 2R-glyceryl-beta-d-galactopyranoside (glycerylgalactoside), which is known to be transported by only two transport system in E. coli, namely, the lactose and the beta-methylgalactoside transport systems. Mutants lacking glycerol-3-phosphate dehydrogenase (glpD) are sensitive to glycerol. Similarly, mutants lacking uridine diphosphate-galactose-4-epimerase (galE) are sensitive to galactose. Glycerylgalactoside is an inducer of the lactose operon and also a substrate for beta-galactosidase. Thus, a mgl(+)glpD galE lacY strain will not grow in the presence of glycerylgalactoside owing to accumulated glycerol-3-phosphate, galactose-1-phosphate, and uridine diphosphate-galactose. We have constructed such a strain and shown that mgl mutants can be obtained by selecting for those that grow in the presence of glycerylgalactoside.

Escherichia coli was grown in a medium containing [1-(14)C]methionine and [methyl-(3)H]methionine, and the (3)H/(14)C ratio was determined for each of the ribosomal proteins derived from the 70S ribosome. Evidence indicates that six proteins from the 50S subunit were methylated: L7, L9, L11, L12, L18, and L33. Methylation of several other 50S proteins (such as L1, L3, L5, etc.) may also occur. The methylated amino acids in protein L11 have been characterized further and found to be predominately epsilon-trimethyllysine. A small amount of a compound tentatively identified as N(G), N'(G)-dimethylarginine was also detected.

The net accumulation of tetracyclines by Escherichia coli as a function of concentration was shown to be biphasic. At concentrations less than the bacteriostatic levels, the mode of uptake was not azide-sensitive and was considered to be physical adsorption on the cell surface. At concentrations above the minimal inhibitory level, a second, azide-sensitive, uptake component was functional in addition to the surface adsorption process. This second energy-requiring mode was judged to represent penetration of the cytoplasmic membrane by tetracycline molecules to their sites of inhibitory action. Each mode for a given tetracycline and culture is expressed algebraically by a characteristic Freundlich equation. Resistance in E. coli is shown to be a result of diminished transport of antibiotic. However, this resistance was due not to a reduction or loss of a transport mechanism but rather to a requirement for higher antibiotic concentrations before the second mode of uptake could become operative.

We have extended our investigation of nicotinamide deamidation in the stomach of conventional rats. The bacterial species in the pars preventricularis were identified as Flavobacterium peregrinum, Escherichia coli, Streptococcus faecalis, and Lactobacillus acidophilus, listed in order of decreasing deamidase activity. Nicotinamide-7-(14)C ingested into rat stomach was rapidly deamidated to nicotinic acid. These results contribute to the accumulated evidence that microorganisms present in the pars preventricularis of rat stomach are responsible for the deamidation of nicotinamide to nicotinic acid, a known precursor of mammalian pyridine nucleotides.

The specific activities of l-alanine:d-alanine racemase, d-alanine:d-alanine ligase, and the l-alanine, d-glutamic acid, meso-diaminopimelic acid, and d-alanyl-d-alanine adding enzymes were followed during growth of Escherichia coli. The specific activities were nearly independent of the growth phase. d-Alanine:d-alanine ligase was inhibited by d-alanyl-d-alanine, d-cycloserine, glycine, and glycyl-glycine. l-Alanine:d-alanine racemase was found to be sensitive to d-cycloserine, glycine, and glycyl-glycine. The l-alanine adding enzyme was inhibited by glycine and glycyl-glycine.

Infection of Escherichia coli with amber mutants of phage fd, in contrast to infection with wild-type phage, leads to cell death and the proliferation of intracytoplasmic membranes observed in electron micrographs at the poles of the cells. The accumulation of membranes correlates with changes in structural phospholipids, especially a marked increase in the apparent rate of formation and total amount of cardiolipin (from 4 to 20% of total radioactive phospholipids), and a compensating decline in phosphatidylethanolamine.

The synthesis and resolution of the diastereoisomers of d-methionine sulfoxide in high yield is described. Growth of two methionine auxotrophs (strains HfrC and AB1932) on the d-methionine sulfoxides is slower than on l-methionine, and the resultant cells are markedly derepressed for three enzymes of the methionine regulon (cystathionine synthetase, cystathionase, and S-adenosyl-l-methionine synthetase). Strain HfrC grows more rapidly on the sulfoxides and shows less derepression than strain AB1932. Although growth on d-methionine-d-sulfoxide is much slower than on d-methionine-l-sulfoxide (two- to threefold increase in division times), cells grown on d-methionine-l-sulfoxide generally have higher enzyme activities. The sulfoxides of d-methionine appear to provide a useful supplement to obtain methionine-limited growth in Escherichia coli.

Cell-free extracts of Escherichia coli were active in catalyzing the synthesis of a biotin vitamer from 7,8-diaminopelargonic acid. The vitamer was identified as desthiobiotin on the basis of its chromatographic and electrophoretic characteristics and its biotin activities for a variety of microorganisms. The reaction was stimulated five-fold by bicarbonate, suggesting that an "active CO(2)" was incorporated into the carbonyl carbon of desthiobiotin. The enzyme was demonstrable in a wild-type (K-12) and in all biotin mutants of E. coli that were tested, with the exception of a strain which was able to grow on desthiobiotin but not on diaminopelargonic acid. Furthermore, the enzyme was repressible by biotin in all of the strains tested. These results are consistent with the hypothesis that the biosynthesis of desthiobiotin from 7,8-diaminopelargonic acid is an obligatory step in the biosynthetic pathway of biotin in E. coli.

Chloroethylthiamine was found to inhibit an entrapment of thiamine as thiamine monophosphate by blocking thiamine monophosphokinase in the cytoplasm after thiamine was taken up by the cells of Escherichia coli.

Mutants having low levels of polynucleotide phosphorylase activity grow poorly at 45 C. All revertants isolated for their ability to grow better at that temperature also regained higher levels of polynucleotide phosphorylase and the ability to be induced for tryptophanase. Thus, a physiological role is implied for the enzyme polynculeotide phosphorylase.

The growth on pseudouridine of two pyrimidine auxotrophs of Escherichia coli (Bu(-) and W63-86) was markedly enhanced when glycerol replaced glucose as a carbon source or when adenosine 3':5'-cyclic monophosphoric acid was added to medium containing glucose. These results indicated that an enzyme catalyzing a reaction in the pathway of pseudouridine conversion to uracil was sensitive to catabolite repression. The following pathway is proposed for pseudouridine utilization: [Formula: see text] [Formula: see text] Pseudouridylate synthetase was sensitive to catabolite repression in strains Bu(-) and W63-86. In contrast, strains B5RU and W5RU, mutants of Bu(-) and W63-86 which were selected for their ability to grow rapidly on pseudouridine in the presence of glucose, had high levels of pseudouridylate synthetase in the presence of glucose. In the case of B5RU but not W5RU, synthetase activity was greater in cells grown on glycerol or on glucose plus adenosine 3':5-cyclic monophosphoric acid than on glucose.

dl-Selenomethionine is as efficient a methyl donor for nucleic acid methylation in both Bacillus megaterium KM and Escherichia coli WWU as dl-methionine.

Anacker, R. L. (Rocky Mountain Laboratory, Hamilton, Mont.), W. D. Bickel, W. T. Haskins, K. C. Milner, E. Ribi, and J. A. Rudbach. Frequency of occurrence of native hapten among enterobacterial species. J. Bacteriol. 91:1427-1433. 1966.-Smooth cultures of representative Enterobacteriaceae were screened for the presence of native hapten, a substance previously extracted with trichloroacetic acid from the protoplasmic fraction of one strain each of Escherichia coli O111:B4 and O113. Trichloroacetic acid extracts of protoplasmic fractions of the cells were analyzed for chemical composition, for constituent sugars by paper chromatography, for immunochemical relationship to endotoxin purified by gel filtration, for sedimentation behavior, and for pyrogenicity in rabbits and lethal toxicity in chick embryos. Extracts from two of three additional strains of E. coli O113, all five additional strains of E. coli O111:B4, and one strain each of E. coli O26:B6 and O55:B5 were similar to previously described native hapten in chemical composition, sedimentation properties (S(20,w), 3.7 to 5.2), biological potency (usually less than 0.1% that of corresponding endotoxin), and immunochemical relationship to endotoxin. Extracts of one strain each of E. coli O127:B8, Serratia marcescens, Citrobacter freundii, Salmonella enteritidis, and of two lipopolysaccharide-deficient mutants of S. enteritidis differed from typical native hapten. The biosynthetic relationship of native hapten to endotoxin has not yet been revealed.

Cephaloridine and cephalexin had no effect on ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or protein synthesis in Escherichia coli. However, cephalosporin 7/30 [7-(S-benzylthioacetamido)-cephem-3-ylmethyl-N -dimethyldithiocarbamate-4-carboxylic acid] and dimethyldithiocarbamate (DMDT), which occupies the side chain at position 3 in the 7/30 molecule, inhibited protein synthesis (and, to a lesser extent, RNA and DNA syntheses) in E. coli and had an inhibitory effect on the growth of Saccharomyces carlsbergensis. A bioautograph technique showed that two inhibitory spots were obtained with 7/30 but only one such spot with cephaloridine. Release of DMDT onto or in the bacterial cell may be responsible for "unusual" mode of action of cephalosporin 7/30.

A mutant of Escherichia coli resembles its parent in taking up actinomycin after treatment with ethylenediaminetetraacetic acid but differs in that it survives this uptake and excretes actinomycin at an increased rate.

The enzymatic synthesis of 7-oxo-8-aminopelargonic acid (7-KAP) from pimelyl-coenzyme A and l-alanine was demonstrated in cell-free extracts of a biotin mutant of Escherichia coli K-12 which excretes only 7-KAP into the growth medium. This biotin vitamer was identified by its chromatographic and electrophoretic properties. The enzyme (7-KAP synthetase) was repressed when the organism was grown in biotin concentrations greater than 0.2 ng/ml. The parent strain and members of other mutant groups that excrete 7-KAP, in addition to other vitamers, also exhibited synthetase activity. A mutant group that failed to excrete 7-KAP was further sub-divided into three groups, one of which lacked synthetase activity. These results are discussed in relation to a previously proposed scheme for biotin biosynthesis in which the formation of 7-KAP is considered the point of entry for pimelic acid into the biotin pathway.

Galactosyl-mannitol can be transported into cells of Escherichia coli by beta-galactoside permease and can be hydrolyzed rapidly to mannitol and galactose by beta-galactosidase. When a mutant strain lacking enzyme I of the phosphoenolpyruvate phosphotransferase system and constitutive in the lactose system was presented with galactosyl-mannitol in which the mannitol moiety was labeled with (3)H, the liberated mannitol remained inside the cell if the Enzyme II complex of the phosphoenolpyruvate phosphotransferase system for mannitol was uninduced. It is postualted that one of the enzyme II proteins can still catalyze translocation of mannitol across the cell membrane even when phsophorylation is not possible.

The Escherichia coli lon(-) mutants apparently are defective in the ability to recommence cell division after temporary periods of deoxyribonucleic acid (DNA) synthesis inhibition. They are also more susceptible to cell division inhibition by the basic dye, crystal violet (CV), than are lon(+) strains. In enriched broth, the lon(+) strain continued to grow and divide in the presence of CV, but lon(-) cell division was inhibited and filamentous growth resulted. In a supplemented minimal medium containing CV, lon(-) cell division was only temporarily inhibited. There was no detectable specific effect on DNA synthesis, although CV slowed the rate of mass increase in both media. Trichloroacetic acid-insoluble lipid synthesis was preferentially inhibited in both lon(+) and lon(-) strains. In CV-containing enriched broth, diaminopimelic acid incorporation into trichloroacetic acid-insoluble compounds occurred at a rate greater than the rate of mass increase in both lon(+) and lon(-) strains. In a CV-containing supplemented minimal medium, diaminopimelic acid was incorporated to a greater extent by lon(-) cells than by lon(+) cells.

Use of a yeast-lactobacillus differential microbiological assay permitted investigation into the synthesis of biotin vitamers by a variety of bacteria. A major portion of the biotin activity was found extracellularly. The level of total biotin (assayable with yeast) greatly exceeded the level of true biotin (assayed with lactobacillus). Values for intracellular biotin generally showed good agreement between the assays, suggesting the presence of only true biotin within the cells. Bioautographic analysis of the medium after growth of each organism revealed the presence of large amounts of a vitamer which corresponded to dl-desthiobiotin on the basis of Rf value and biological activity. Biotin, when detected at all, was at very low concentrations. Also, an avidin-uncombinable vitamer was synthesized by a majority of the bacteria. Addition of d-biotin to the growth medium prevented completely the synthesis of both vitamers of biotin. d-Biotin-d-sulfoxide had no effect on the synthesis of desthiobiotin or the avidin-uncombinable vitamer. Addition of dl-desthiobiotin did not prevent its own synthesis nor that of the other vitamer. Control of vitamer synthesis is therefore highly specific for d-biotin. The avidin-uncombinable vitamer was produced only at repressed levels in the presence of high concentrations of both d-biotin and dl-desthiobiotin, which suggested that it is not a degradation product of these substances. A possible mechanism for the overproduction of the biosynthetic precursors of biotin is presented.

Moss, C. Wayne (North Carolina State University, Raleigh), and M. L. Speck. Identification of nutritional components in Trypticase responsible for recovery of Escherichia coli injured by freezing. J. Bacteriol. 91:1098-1104. 1966.-Freezing and storage of Escherichia coli at -20 C resulted in nonlethal or "metabolic" injury to a proportion of the surviving population. The injury was manifested as an increased nutritional requirement after freezing. Injured cells could not grow on a minimal agar medium, but could develop on Trypticase Soy Agar. The percentage of injured survivors varied among strains, but was little affected by altering the freezing menstruum. Trypticase was found to be the component in Trypticase Soy Agar responsible for the recovery of injured cells, and contained five closely related peptides that possessed most of the biological activity. Isolation of the peptides was accomplished by Sephadex gel chromatography, paper chromatography, and high-voltage paper electrophoresis. Hydrolysis of the peptides destroyed the ability to restore injured cells.

4-N-hydroxy-cytidine was found to substitute for uridine as a pyrimidine supplement for the growth of Escherichia coli Bu(-). Measurement of the incorporation of 4-N-hydroxy-cytidine-2-(14)C into ribonucleic acid and deoxyribonucleic acid revealed that this compound was converted to cytidine or uridine before utilization. Two pathways for metabolism were considered: (i) the reduction of 4-N-hydroxy-cytidine to cytidine followed by deamination, (ii) the direct hydrolysis of hydroxylamine from 4-N-hydroxy-cytidine to yield uridine. A threefold increase in cytidine (deoxycytidine) deaminase (EC 3.5.4.5) activity, when the cells were grown on 4-N-hydroxy-cytidine, suggested the involvement of this enzyme. More direct proof was obtained by purifying the deaminase 185-fold and finding that it released hydroxylamine from 4-N-hydroxy-cytidine at one-fiftieth the rate at which ammonia was removed from cytidine. This result is consistent with the slower rate of growth of the Bu(-) cells on 4-N-hydroxy-cytidine than cytidine and suggests that the second pathway is the major route for utilization of this compound.

This study is concerned with the isolation and characterization of the enzyme, S-adenosylmethionine:ribosomal ribonucleic acid-adenine (N(6-)) methyl-transferase [rRNA-adenine (N(6)-) methylase] of Escherichia coli strain B, which is responsible for the formation of N(6)-methyladenine moieties in ribosomal ribonucleic acids (rRNA). A 1,500-fold purified preparation of the species-specific methyltransferase methylates a limited number of adenine moieties in heterologous rRNA (Micrococcus lysodeikticus and Bacillus subtilis) and methyl-deficient homologous rRNA. The site recognition mechanism does not require intact 16 or 23S rRNA. The enzyme does not utilize transfer ribonucleic acid as a methyl acceptor nor does it synthesize 2-methyladenine or N(6)-dimethyladenine moieties. Mg(2+), spermine, K(+), and Na(+) increase the reaction rate but not the extent of methylation; elevated concentrations of the cations inhibit markedly. The purified preparations utilize 9-beta-ribosyl-2,6-diaminopurine (DAPR) as a methyl acceptor with the synthesis of 9-beta-ribosyl-6-amino-2-methylaminopurine. A comparison of the two activities demonstrated that one methyltransferase is responsible for the methylation of both DAPR and rRNA. This property provides a sensitive assay procedure unaffected by ribonucleases and independent of any specificity exhibited by rRNA methyl acceptors.

Vender, Joyce (Indiana University, Bloomington), Kunthala Jayaraman, and H. V. Rickenberg. Metabolism of glutamic acid in a mutant of Escherichia coli. J. Bacteriol. 90:1304-1307. 1965.-A mutant strain of Escherichia coli W1485 was selected for its ability to utilize glutamic acid as the sole source of carbon. Growth of the mutant on glutamic acid led to the repression of glutamic acid dehydrogenase formation. The mutant differed from the wild-type strain in that glutamic decarboxylase activity was absent from the mutant under conditions of growth which supported the formation of this enzyme in the parent strain. Evidence is presented which suggests that loss of the decarboxylase activity results in the acquisition of the ability to utilize glutamic acid as sole source of carbon; a pathway of glutamate utilization via transamination is proposed.

The nucleotide pools of certain mucoid, colanic acid-synthesizing strains of Escherichia coli, Salmonella typhimurium, and Aerobacter cloacae were examined, and in all cases the nucleotide sugars uridine-5'-diphosphate glucose (UDPG), uridine-5'-diphosphate galactose (UDPGal), guanosine-5'-diphosphate fucose (GDPF), and uridine-5'-diphosphate glucuronic acid (UDPGA) were detected. It is postulated that these nucleotide sugars are precursors in the synthesis of colanic acid. The levels of these nucleotide sugars and of the enzymes involved in their synthesis were examined in a number of mucoid strains and compared with the levels found in certain strains which were repressed in the synthesis of colanic acid, only becoming mucoid when grown in the presence of p-fluorophenylalanine (PFA). The levels of UDPG and UDPGal and the enzymes involved in their synthesis were substantially the same in both mucoid and repressed types, but the levels of UDPGA and GDPF and of some of the enzymes involved in their synthesis were much higher in mucoid strains. When repressed strains were grown in the presence of PFA, the levels of UDPGA and GDPF approached those found in mucoid strains. The existence of an operon, containing genes coding for certain key enzymes involved in colanic acid synthesis has been suggested.

Some properties of a d-glutamic acid auxotroph of Escherichia coli B were studied. The mutant cells lysed in the absence of d-glutamic acid. Murein synthesis was impaired, accompanied by accumulation of uridine-5'-diphosphate-N-acetyl-muramyl-l-alanine (UDP-MurNac-l-Ala), as was shown by incubation of the mutant cells in a cell wall medium containing l-[(14)C]alanine. After incubation of the parental strain in a cell wall medium containing l-[(14)C]glutamic acid, the acid-precipitable radioactivity was lysozyme degradable to a large extent. Radioactive UDP-MurNac-pentapeptide was isolated from the l-[(14)C]glutamic acid-labeled parental cells. After hydrolysis, the label was exclusively present in glutamic acid, the majority of which had the stereo-isomeric d-configuration. Compared to the parent the mutant incorporated less l-[(14)C]glutamic acid from the wall medium into acid-precipitable material. Lysozyme degraded a smaller percentage of the acid-precipitable material of the mutant than of that of the parent. No radioactive uridine nucleotide precursors could be isolated from the mutant under these conditions. Attempts to identify the enzymatic defect in this mutant were not successful. The activity of UDP-MurNac-l-Ala:d-glutamic acid ligase (ADP; EC 6.3.2.9) (d-glutamic acid adding enzyme) is not affected by the mutation. Possible pathways for d-glutamic acid biosynthesis in E. coli B are discussed.

The shape of Escherichia coli is fixed by the form of the sacculus. This sacculus is a macromolecule made up from the polymer murein. In an investigation of the possible factors determining the shape of the sacculus, we attempted to resolve between two fundamental alternatives. (i) Is the shape of the sacculus automatically fixed by its chemical composition? or (ii) does a special morphogenetic system exist which determines the shape of the sacculus? An analysis of sacculi from cells grown in poor and rich media and harvested at different stages of growth was made. Significant variations in the composition of murein were found, whereas the general shape of the cells remained unchanged. This finding stands opposed to the assumption of a strict correlation between chemistry and shape of the sacculus. The second alternative was investigated by attempting to change artificially the shape of the sacculus by modifying the form of the hypothetical morphogenetic system. Rod-shaped cells were converted into spherical spheroplasts which were subsequently allowed to reform a new spherical sacculus. In chemical composition this spherical sacculus was found to be indistinguishable from the rod-shaped sacculus. This finding is taken as evidence for the existence of a distinct morphogenetic apparatus in the cell wall whose form is reflected by the shape of the sacculus.

The inhibition of Escherichia coli strain B and strain W-11 by 6-methylpurine depended on the formation of 6-methylpurine ribonucleotide by the action of adenine phosphoribosyltransferase (AMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.7). 6-Methylpurine ribonucleotide inhibited the de novo synthesis of purines, presumably via pseudofeedback inhibition of phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14). The same mechanism accounted for its inhibition of adenylosuccinate synthetase [IMP: l-aspartate ligase (GDP), EC 6.3.4.4]. Adenine and 6-methylaminopurine prevented inhibition by competing for the action of adenine phosphoribosyltransferase. In addition, adenine reversed this inhibition by replenishing the AMP to bypass both sites of inhibition. Nonproliferating suspensions of strain B-94, which lacked adenylosuccinate lyase (EC 4.3.2.2), converted exogenous hypoxanthine and aspartate to succinoadenine derivatives which accumulated in the medium. Compounds which inhibited adenylosuccinate synthetase inhibited accumulation of the succinoadenine derivatives. A method was described for the isolation of mutants which potentially possessed an altered adenylosuccinate synthetase.

From Escherichia coli B, mutants were prepared that lacked the enzymes adenosine deaminase, cytidine deaminase, and purine nucleoside phosphorylase. In each case, the mutant lacked enzyme activity for both ribonucleoside and deoxyribonucleoside. Mutants lacking purine nucleoside phosphorylase lost the capacity to cleave the nucleosides of adenine, guanine, and hypoxanthine.

Strain NP 315 of Escherichia coli possesses a thermolabile fructose-1, 6-diphosphate (FDP) aldolase; its growth on carbohydrate substrates is inhibited probably as a consequence of the accumulation of high intracellular levels of FDP. Studies of one class of phenotypic revertants of strain NP 315 which have regained their ability to grow on C(6) substrates at 40 C showed that in these strains the buildup of the inhibitory FDP pool is prevented by additional mutations in enzymes catalyzing the conversion of the substrate offered in the medium to FDP. For example, mutations affecting 6-phosphogluconate dehydrogenase activity (gnd(-)) may be selected in great number without any mutagenesis and enrichment simply by isolating revertants of strain NP 315 able to grow on gluconate at 40 C. Similarly, an additional mutation in phosphoglucose isomerase (pgi(-)) restores the ability of these fda(-)gnd(-) strains to grow on glucose at 40 C. Glucose metabolism of these fda(-)gnd(-)pgi(-) strains was investigated. The enzymes of the Entner-Doudoroff pathway are induced to an appreciable extent upon growth of these mutants on glucose medium; further evidence for glucose degradation via this route (which normally is induced only in the presence of gluconate) was provided by following the fate of the C1 label of radioactive glucose in l-alanine. Predominant labeling of the carboxyl-carbon of l-alanine was observed, inciating a major contribution of the Entner-Doudoroff path to pyruvate formation from glucose. Chromatographic analysis of the intermediates of glucose metabolism showed further that glucose apparently is at least partly metabolized via a bypass consisting of the accumulation of extracellular gluconic acid which arises by dephosphorylation of 6-phosphogluconolactone and possibly of 6-phosphogluconate. This extracellular gluconate is then taken up and metabolized in the normal manner via the Entner-Doudoroff enzymes.

Several growth characteristics of Escherichia coli K-12 suggest that growth on l-fucose results in the synthesis of all the enzymes necessary for growth on d-arabinose. Conversely, when a mutant of E. coli is grown on d-arabinose, all of the enzymes necessary for immediate growth on l-fucose are present. Three enzymes of the l-fucose pathway in E. coli, l-fucose isomerase, l-fuculokinase, and l-fuculose-l-phospháte aldolase possess activity on d-arabinose, d-ribulose, and d-ribulose-l-phosphate, respectively. The products of the aldolase, with d-ribulose-l-phosphate as substrate, are dihydroxyacetone phosphate and glycolaldehyde. l-Fucose, but not d-arabinose, is capable of inducing these activities in wild-type E. coli. In mutants capable of utilizing d-arabinose as sole source of carbon and energy, these activities are induced in the presence of d-arabinose and in the presence of l-fucose. Mutants unable to utilize l-fucose, selected from strains capable of growth on d-arabinose, are found to have lost the ability to grow on d-arabinose. Enzymatic analysis of cell-free extracts, prepared from cultures of these mutants, reveals that a deficiency in any of the l-fucose pathway enzymes results in the loss of ability to utilize d-arabinose. Thus, the pathway of d-arabinose catabolism in E. coli K-12 is believed to be: d-arabinose right harpoon over left harpoon d-ribulose --> d-ribulose-l-phosphate right harpoon over left harpoon dihydroxyacetone phosphate plus glycolaldehyde. Evidence is presented which suggests that the glycolaldehyde is further oxidized to glycolate.

The inducer specificity and kinetics of induction of the ara operon were examined in Escherichia coli B/r. A difference in the kinetics of induction was found between our B/r strains and the K-12 strain previously described by Schleif. The roles of active transport and metabolism of inducer, and of cell density, in induction were studied. d-Fucose and beta-methyl-l-arabinoside were competitive inhibitors of induction. No inducer of the wild-type strain other than l-arabinose was found. However, a procedure for selecting mutants with altered inducer affinity or specificity was developed. The properties of one such mutant (inducible by d-fucose) are described.

Temperature-sensitive mutants of Escherichia coli K-12 which cannot form cell wall peptidoglycan at 42 C were isolated. The thermosensitive steps were characterized biochemically, and the genes coding the enzymes of peptidoglycan synthesis were mapped. These genes were in two clusters: one group, located at about 1.5 min between leu and azi, was designated as mra (murein a); the second group, located at about 77.5 min close to argH and metB, was designated as mrb (murein b, with the order metB-argH-mrb). No simple relations were found between the gene location and the order or localization of enzymes involved in the sequence of reactions of cell wall peptidoglycan biosynthesis.

Wild-type Escherichia coli strains (beta-gl(-)) do not split beta-glucosides, but inducible mutants (beta-gl(+)) can be isolated which do so. This inducible system consists of a beta-glucoside permease and an aryl beta-glucoside splitting enzyme. Both can be induced by aryl and alkyl beta-glucosides. In beta-gl(-) and noninduced beta-gl(+) cells, C(14)-labeled thioethyl beta-glucoside (TEG) is taken up by a constitutive permease, apparently identical with a glucose permease (GP). This permease has a high affinity for alpha-methyl glucoside and a low affinity for aryl beta-glucosides. No accumulation of TEG occurs in a beta-gl(-) strain lacking glucose permease (GP(-)). In induced beta-gl(+) strains, there appears a second beta-glucoside permease with low affinity for alpha-methyl glucoside and high affinity for aryl beta-glucosides. Autoradiography shows that TEG is accumulated by the beta-glucoside permease and glucose permease in two different forms (one being identical with TEG, the other probably phosphorylated TEG). In GP(+) beta-gl(+) strains with high GP activity, alkyl beta-glucosides induce the enzyme and the beta-glucoside permease after a prolonged induction lag, and they competitively inhibit the induction by aryl beta-glucosides. The induction lag and competition do not exist in GP(-) beta-gl(+) strains. It is assumed that phosphorylated alkyl and thioalkyl beta-glucosides inhibit the induction, and that this inhibition is responsible for the induction lag.

Four mutants (pop-1, pop-6, pop-10, and pop-14) which accumulate a red water-insoluble pigment were obtained in Escherichia coli K-12 AB1621. For each mutant, the red pigment was shown to be protoporphyrin IX, a late precursor of heme. Mutagenic treatment of mutant pop-1 yielded a secondary mutant, pop-1 sec-20, which accumulated a brown water-soluble pigment. The brown pigment was shown to be coproporphyrin III. Mutant pop-1 resembled the parental strain in its cytochrome absorption spectrum, catalase activity, and ability to grow on nonfermentable carbon and energy sources; therefore, its ability to produce and utilize heme was unimpaired. Judged on the same criteria, the secondary mutant, pop-1 sec-20, was partially heme and respiratory deficient. Growth in anaerobic conditions decreased by 25% the accumulation of protoporphyrin by pop-1; under the same conditions, pop-1 sec-20 did not accumulate coproporphyrin or coproporphyrinogen. The mutations causing protoporphyrin accumulation in all four pop mutants were found to map in the lac to purE (10-13 min) region of the E. coli chromosome. In the case of mutant pop-1, the mutation was shown to be strongly linked to the tsx locus (12 min). In mutant pop-1 sec-20, the second mutation causing coproporphyrin accumulation was co-transducible with the gal locus at a frequency of 88 to 96%. The mechanism of porphyrin accumulation by the mutants is discussed.