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Ikawa, Miyoshi (University of New Hampshire, Durham), James W. Morrow, and Sheila J. Harney . Paper chromatographic system for the identification of glycerol in bacterial cell walls. J. Bacteriol. 92: 812–814. 1966.—The solvent system consisting of isopropanol-5% boric acid (7:1, v/v) separates glycerol from the other carbohydrate constituents which are found in hydrolysates of bacterial cell walls. This system is useful for the identification of glycerol even when anhydroribitol and rhamnose are both present, and has been found to be applicable on cell wall hydrolysates as well as on synthetic mixtures.

Bartholomew , J. W. (University of Southern California, Los Angeles), Thomas Cromwell, and Richard Gan . Analysis of the mechanism of Gram differentiation by use of a filter-paper chromatographic technique. J. Bacteriol. 90: 766–777. 1965.—Data are presented which demonstrate that the mechanism of gram-positivity could not be due solely to factors such as a single, specific gram-positive substrate, specific affinities of crystal violet for certain cellular components, a specific crystal violet-iodine-substrate complex, or to any specific characteristic of the dye, iodine, or solvent molecules. Ruptured cells of gram-positive organisms stain gram-negatively when subjected to a standard Gram-stain procedure. However, when stained fragments of broken cells were deposited in thick layers on the surface of filter-paper strips and exposed to decolorizers, the rate of dye release correlated with the Gram characteristic of the intact cell. Therefore, the intact cell in itself is not an absolute requirement for Gram differentiation. The data are interpreted as indicating that the mechanism of Gram differentiation primarily involves the rate of permeation of molecules (dye, iodine, solvent) through the interstitial spaces of cell-wall material.

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.

Kobayashi, Yasuo (University of Tokyo, Tokyo, Japan) and Kei Arima . Bacterial oxidation of dipicolinic acid. II. Identification of α-ketoglutaric acid and 3-hydroxydipicolinic acid and some properties of cell-free extracts. J. Bacteriol. 84: 765–771. 1962—When a dipicolinic acid (DPA)-decomposing bacterium, Achromobacter strain 1–2, was incubated at 30 C with shaking in a DPA solution containing 10 −3 m arsenite, a keto acid was accumulated. The 2,4-dinitrophenylhydrazone of this acid was synthesized and identified as α-ketoglutaric acid by paper chromatography, visible absorption spectrum, infrared analysis, elemental analysis, and mixed melting point. During this incubation, oxalic acid equivalent to the consumed dipicolinic acid was produced. A fluorescent material was also isolated from culture fluid and identified as 3-hydroxydipicolinic acid by paper chromatography and the ultraviolet absorption spectrum. Further, cell-free extracts were prepared by sonic oscillation. Ferrous ion and a reduced di- or triphosphopyridine nucleotide-generating system were proven to be required for enzymic oxidation of DPA. And 3-hydroxydipicolinic acid was also oxidized by this preparation. From the results obtained, a possible metabolic pathway of dipicolinic acid was proposed.

Wax D prepared from the P-6 strain of the scotochromogenic species of Mycobacterium scrofulaceum constituted 0.3% of the dry bacilli. In the acid hydrolysates, alanine, glutamic acid, glycine, and mesodiaminopimelic acid were found as amino acid constituents. Mannose, galactose, and glucosamine were detected by paper chromatography. However, arabinose could not be detected. The quantity of hexosamine was 0.2 to 0.3%. Wax D of P-6 was found to be adjuvant-active, as revealed by a positive corneal reaction. The antigenicity of wax D of P-6 was shown by the agglutination reaction between the suspension of wax D and the anti-wax D antiserum. Periodate oxidation of the hydrosoluble portion reduced the inhibitory effect of the hydrosoluble portion on agglutination of the suspension of wax D. The light fraction of wax D had a peptide portion consisting of alanine, glutamic acid, glycine, and diaminopimelic acid and was found to be adjuvant-active in an amount of 1,000 μg.

Resting cells of Corynebacterium sp. E17 formed O -ethylhomoserine from ethyl alcohol for a few hours. Addition of l -homoserine greatly enhanced its formation. Thus, the formation of O -ethylhomoserine from ethyl alcohol by 27 bacteria, 6 yeasts, and 4 fungi was investigated by using growing cultures and resting cells in the presence of l -homoserine. The O -ethylhomoserine formed in the culture supernatant fluids or supernatant fluids of the reaction mixtures was identified by paper chromatography. Many organisms which were incapable of forming O -ethylhomoserine with growing cultures formed it with resting cells. The formation of O -ethylhomoserine appears to be restricted to strains of Brevibacterium, Corynebacterium, Bacillus, Mycobacterium, Nocardia , and Streptomyces .

High activity of taurine:α-ketoglutarate aminotransferase was found exclusively in cell-free extracts of Achromobacter superficialis and A. polymorph . The former was chosen for characterization of the enzymatic reaction. The enzyme activity was enhanced by addition of β-alanine to the growth medium. The product from α-ketoglutarate was identified as l -glutamate. Another product has been isolated, purified, and identified as sulfoacetaldehyde (2-oxoethanesulfonate), a deamination product from taurine, by comparison between the 2,4-dinitrophenylhydrazones of the synthetic and enzymatic products on the basis of studies by paper chromatography, by visible, infrared, and nuclear magnetic resonance spectrophotometries, and by elemental analysis. This enzymatic transamination was found to proceed stoichiometrically and reversibly as follows: NH 2 ·CH 2 ·CH 2 ·SO 3 H + HOOC·CH 2 ·CH 2 ·CO·COOH ⇌ OHC·CH 2 ·SO 3 H + HOOC·CH 2 ·CH 2 ·CH(NH 2 )·COOH.

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.

Cell-free extracts of Acetobacter suboxydans were prepared which were capable of condensing α-ketoisovalerate with 14 C-labeled acetyl-coenzyme A to yield 14 C-labeled α-isopropylmalate. The product of the reaction was isolated by paper and column chromatography and was characterized by recrystallization with synthetic α-isopropylmalic acid to constant specific radioactivity. The formation of α-isopropylmalate by extracts of A. suboxydans plus the ability of the organism to grow in a simple glucose-glycerol medium containing glutamic acid as the only amino acid indicate that the pathway for leucine biosynthesis shown to exist in yeast and Salmonella typhimurium also occurs in A. suboxydans . As a comparison, the condensation of oxalacetate and ( 14 C) acetyl-coenzyme A to yield ( 14 C) citric acid was shown, by similar means, to occur in A. suboxydans . This is of interest since the existence of this classical condensing enzyme has hitherto not been demonstrated in this organism. This reaction was further demonstrated in cell-free extracts of A. suboxydans by means of a spectrophotometric assay at 232 mμ which measured the cleavage of the carbon-sulfur bond of acetyl-coenzyme A in the presence of oxalacetate. Comparison of the specific activities of crude cell-free extracts indicated a much more extensive occurrence of this reaction in yeast than in A. suboxydans .

Uptake of valine by Arthrobotrys conoides was an active process and was independent of its incorporation into cellular protein. Chemical fractionation of cells supplied with 14 C- l -valine for different time intervals revealed that the amino acid initially entered a pool of metabolic intermediates and was extractable with cold trichloroacetic acid. After a 4-min interval, some intracellular valine was incorporated into cell proteins, but most underwent metabolic transformation to a variety of products that included carboxylic acids and other amino acids. Carbon derived from valine was not localized in the lipid or nucleic acid fraction of cells, but some was completely oxidized and recovered as metabolic 14 CO 2 . Autoradiograms of paper and thin-layer chromatograms of acid hydrolysates of cellular protein identified the following amino acids as having originated from valine: glutamate, aspartate, alanine, and leucine. Similar analysis of cold trichloroacetic acid extracts established that 14 C supplied as l -valine had been transformed also to α-ketoisovalerate, isobutyrate, propionate, succinate, malate, oxalacetate, pyruvate, and α-ketoglutarate. Pathways for transformation of the carbon skeleton of valine to various metabolic products are proposed.

Dahiya , R. S. (North Carolina State College, Raleigh) and M. L. Speck . Identification of stimulatory factor involved in symbiotic growth of Streptococcus lactis and Streptococcus cremoris . J. Bacteriol. 85: 585–589. 1963.—Single-strain cultures of Streptococcus lactis and S. cremoris isolated from a commercial starter culture showed symbiotic growth in milk. This study dealt with identification of the main component responsible for high acid production resulting from the combined growth of these cultures. Paper chromatography and bioautography were adapted to isolate the main stimulatory factor excreted by the slower-growing culture ( S. lactis ) in the culture media. It was identified as adenine on the basis of ultraviolet absorption spectra and paper chromatographic R F value. Pure adenine possessed the same properties and was also stimulatory to the faster-growing culture ( S. cremoris ) when added to milk.

The polar lipids of the autotrophically grown, obligately anaerobic, photosynthetic bacterium Chromatium strain D were separated by paper chromatography. Four major phospholipids were identified: lysophosphatidylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In addition, three glycolipids were observed and characterized, namely, monoglucosyldiglyceride, which is found in other biological systems, and (mannosyl, glucosyl)-diglyceride and (dimannosyl, glucosyl)-diglyceride, which heretofore have not been observed in nature.

DeWitt, Charles W. (The Upjohn Co., Kalamazoo, Mich.) and Janet A. Rowe . Sialic acids ( N ,7-O-diacetylneuraminic acid and N -acetylneuraminic acid) in Escherichia coli . I. Isolation and identification. J. Bacteriol. 82: 838–848. 1961.—Two sialic acids, N -acetylneuraminic acid and N ,7-O-diacetylneuraminic acid, were obtained in crude mixtures from whole cells of Escherichia coli and from its endotoxin by weak acid hydrolysis followed by anion exchange resin chromatography. Yields from whole cells were 0.1 to 0.2% (dry weight) with 50 to 60% purity. Identification of the sialic acids was by comparative paper chromatography and colorimetric assays using the acidic p -dimethylaminobenzaldehyde (direct Ehrlich), resorcinol and thiobarbituric acid reactions. The N -acetyl derivative was also shown to be susceptible to hydrolysis by clostridial N -acetylneuraminic aldolase and the end products identified, N -acetylamannosamine by paper chromatography and pyruvic acid by oxidation of DPNH with lactic acid dehydrogenase. The two sialic acids were separated on paper chromatograms, eluted, and assays for total and ester acyl groups showed the suspected N -acetyl derivative to contain 0.11 O-acyl and 1.16 N -acetyl groups per mole sialic acid and the diacetyl derivative to have 1.10 O-acyl and 0.93 N -acetyl groups per mole. The O-acyl group was identified as acetyl by preparation of the hydroxamate.

A heat-stable endogenous CO 2 acceptor has been found in extracts of Rhodospirillum rubrum grown photoheterotrophically on acetate. Evidence is presented which suggests that this factor is propionic acid. Thus, paper and gas chromatographic analyses have indicated that propionic acid is present in boiled extracts prepared from R. rubrum cells. The products of 14 CO 2 fixation obtained with either the boiled extract or propionic acid as the CO 2 acceptor were identical and were identified as methylmalonic acid and succinic acid by paper chromatography. The enzyme which catalyzes the carboxylation of propionyl-coenzyme A (propionyl-CoA carboxylase) was purified from R. rubrum cells grown on acetate and its properties were studied. The enzyme is similar to propionyl-CoA carboxylases isolated from mammalian sources.

Fresh extracts of cells of Clostridium botulinum reduced a limited amount of ornithine to δ-aminovaleric acid, but at high substrate concentrations a considerable amount of an amino compound accumulated which was neutral at p H 4.2. Aging of the extracts at −10 C or freezing and thawing resulted in the loss of the ability to produce δ-aminovaleric acid, but the ability to produce the neutral compound was retained. This compound was separated by column chromatography, and was found to be identical to dl -proline with respect to (i) R F upon paper chromatography, (ii) migration rates upon paper ionophoresis, (iii) spectrum of the product of the ninhydrin reaction, (iv) oxidation with d -amino acid oxidase, and (v) rate of reduction to δ-aminovaleric acid by cell extracts. The intermediate role of proline in the reduction of ornithine to δ-aminovaleric acid was indicated by (i) rate studies with and without an added electron donor and with and without inhibitors of proline reductase, (ii) the initial accumulation of radioactive proline to the exclusion of radioactive δ-aminovaleric acid from 14 C- l -ornithine in the presence of low levels of carrier proline, and (iii) the initial accumulation of proline at low levels prior to a significant accumulation of δ-aminovaleric acid in reaction mixtures in which the latter compound was the primary product after a longer incubation time. The conversion of ornithine to proline was the rate-limiting step in the presence of a good electron donor (alanine). The mechanism of the conversion of ornithine to proline has not been established. Preliminary data indicated that it may involve an oxidation to glutamic-γ-semialdehyde and its equilibrium product, Δ 1 -pyrroline-5-carboxylic acid.

Crude preparations of Desulfotomaculum nigrificans were found to reduce bisulfite to trithionate, thiosulfate, and sulfide. The bisulfite reductase of this organism was partially purified and observed to reduce bisulfite to trithionate as the major product and with thiosulfate and sulfide as minor products. The enzyme exhibited spectral properties identical to the carbon monoxide-reacting pigment (P582) isolated from this organism. It is concluded that the bisulfite reductase of D. nigrificans is P582 and that this organism utilizes a pathway which involves trithionate during the reduction of bisulfite to sulfide.

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.

The slime polysaccharides produced by Pseudomonas aeruginosa isolated from a variety of human infections were investigated. Slime production in culture seemed optimal when adequate amounts of carbohydrate were present and under conditions of either high osmotic pressure or inadequate protein supply. The polysaccharides produced by the organisms were similar to each other, to the slime of Azotobacter vinelandii , and to seaweed alginic acids. They were composed of β-1,4-linked d -mannuronic acid residues and variable amounts of its 5-epimer l -guluronic acid. All bacterial polymers contained o -acetyl groups which are absent in the alginates. The polysaccharides differed considerably in the ratio of mannuronic to guluronic acid content and in the number of o -acetyl groups. The particular composition of the slime was not found to be characteristic for the disease process from which the mucoid variants of P. aeruginosa were obtained.

For the first time, an O-antigenic lipopolysaccharide (LPS) has been isolated from a filamentous blue-green alga ( Anabaena variabilis ). It was extractable with phenol-water, resulting in extraction of the bulk of the LPS into the phenol phase. The polysaccharide moiety of this LPS consists of l -rhamnose, its 3-O-methyl ether l -acofriose, d -mannose, d -glucose, and d -galactose. l -Glycero- d -mannoheptose and 2-keto-3-deoxyoctonate, the two characteristic sugar components of enteric LPS, and phosphate groups are absent from the A. variabilis O antigen. The only amino sugar present is d -glucosamine. Three hydroxy fatty acids were identified, namely, β-hydroxymyristic, β-hydroxypalmitic and β-hydroxystearic acids, in addition to palmitic and unidentified fatty acid. The LPS of A. variabilis is localized in the outermost cell wall layer and behaves like a bacterial O antigen in serological tests. The passive hemagglutination yielded high titers with isolated LPS (pretreated by heat or by alkali) and rabbit antisera prepared against living or heat-killed cells. The position of the precipitation arcs after immunoelectrophoresis of the O antigen indicates the lack of charged groups. The water phase of the phenol-water extract contains, in high yield, a glucose polymer. It is serologically inactive as shown by the passive hemagglutination test and by agar-gel precipitation.

Lipopolysaccharide prepared from cells of Yersinia ( Pasteurella ) pseudotuberculosis of serogroups I, II, III, IV, and V is known to contain the 3,6-dideoxyhexose (DDH) paratose, abequose, paratose, tyvelose, and ascarylose in its respective O-specific side chains. Lipopolysaccharides or lipid-free polysaccharides of all of the 10 known serogroups and subgroups were subjected to methylation analysis and determined as alditol acetates by gas-liquid chromatography and mass spectrometry. The results indicated that the O-specific side chains of nine serotypes are composed of oligosaccharide repeating units in the form of four alternative general structures in which a terminal DDH may vary. These structures are DDH [Formula: see text] 6-deoxy- d - manno -heptose [Formula: see text] d -galactose (serogroups IA, IIA, and IVB), DDH [Formula: see text] d -mannose [Formula: see text] l -fucose (serogroups IB and IIB), and two configurations similar to the latter except that the 4-position of l -fucose was either linked to the d -mannose residue (serogroups VA and VB) or to the DDH residue (serogroups III and IVA). In contrast, O-groups in lipopolysaccharide of the newly discovered serogroup VI contained the DDH colitose and 2-acetamido-2-deoxy- d -galactose. Accordingly, all five known types of DDH have now been detected in lipopolysaccharides of Y. pseudotuberculosis . The sugar 6-deoxy- d - manno -heptose, present in O-specific side chains of serogroups IA, IIA, and IVB, has not yet been reported to occur elsewhere in nature.

A polysaccharide-rich particulate fraction was isolated from cytoplasmic extracts of Candida albicans by a procedure using differential centrifugation. The polysaccharide particles obtained after purification with deoxycholate treatment were essentially free of nitrogen and were identified chemically as polyglucosan, in which the glucosidic links were of alpha type. Both the response to amylolytic enzymes and the spectral characteristics of the iodine complexes of the polysaccharide particles were similar to those of rabbit liver glycogen. They also precipitated with concanavalin A, the glycogen value being assessed at 1.04. These data strongly indicated that the polysaccharide particles have the macromolecular structure characteristic of glycogen. The sedimentation analysis revealed that they were polydisperse, with a weight average sedimentation coefficient of 340 S . In negatively stained specimens, the glycogen particles were seen to form rosette-like structures consisting of a complex unit 40 to 150 nm in diameter. Such complex particles were composed of smaller globules that were fairly uniform in size with an average diameter of 32 nm.

A stabilized L-form ofStreptococcus pyogenescontinues to synthesize glycerol teichoic acid. This polymer was obtained fromS. pyogenesand its L-form, treated in identical fashion, and compared. Highly purified glycerol teichoic acid from only the L-form was found to be devoid ofd-alanine and to have a shorter chain length. Otherwise, the glycerol teichoic acid from these two organisms was found to be a 1,3-phosphodiester-linked glycerophosphate polymer substituted withd-glucose. Evidence is presented that most, if not all, of the glycerol teichoic acid in this streptococcus lies between the wall and membrane. A possible need for the continued synthesis of a minute amount of glycerol teichoic acid by this L-form for survival is discussed in terms of the known function of teichoic acids in bacteria.

Aquaspirillum ( Spirillum ) gracile is one of the few spirilla that cause acidification of the medium when cultured with sugars. Acidic reactions have been reported only for d -glucose, d -galactose, and l -arabinose, and the mode of attack of these sugars has not been previously investigated. The soluble portion of extracts of glucose-cultured cells of A. gracile ATCC 19624 was found by spectrophotometric methods to contain enzyme activities characteristic of the Entner-Doudoroff and Embden-Meyerhof-Parnas pathways. No activity for 6-phosphogluconate dehydrogenase (EC 1.1.1.44) was detected. Pyridine nucleotide-linked dehydrogenase activities for l -arabinose and d -galactose (EC 1.1.1.46 and EC 1.1.1.48) occurred in the soluble fraction of cells cultured with either sugar. Glucose-cultured cells contained not only glucokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activities but also glucose dehydrogenase (EC 1.1.1.47) activity. Enzymes capable of oxidizing gluconate were not detectable, but gluconokinase (EC 2.7.1.12) activity was present. Paper chromatographic analysis of the spent culture supernatant media from glucose-cultured cells indicated an accumulation of gluconic acid, and this was confirmed by enzymatic methods. Evidence is presented for the production of d -galactonic and l -arabonic acids in cultures containing d -galactose or l -arabinose, respectively.

Six glycerolphosphate-containing tetraheteroglycans, a, b-1, b-2, b-3, b-4, and b-5, have been purified from the formamide extracts ofStreptococcus sanguisby alcohol and acetone precipitations, Sephadex G-75, and diethylaminoethyl-cellulose column chromatography. The polysaccharides were judged as at least 95% pure by analytical disc gel electrophoresis and immune double diffusion against rabbit antiserum. They were shown to be cell wall polysaccharides, since they formed a single band of identity in immune double diffusion with partially purified polysaccharide extracted from a purified cell wall preparation ofS. sanguis. The polysaccharides were composed ofl-rhamnose,d-glucose, andN-acetyld-glucosamine in a similar molar ratio, but varied in their glycerol and phosphate contents. They exhibited four different mobilities in polyacrylamide disc gel electrophoresis at pH 8.9. When they were treated with formamide at 170 C for 20 min, the faster moving polysaccharide(s) yielded polysaccharides with mobilities corresponding to the other slower moving polysaccharides. These results indicate that the polysaccharides originated from the same cell wall polysaccharide and were produced as a result of breakage in the phosphodiester bonds during the formamide extraction procedure. A preliminary structural study shows that the terminal reducing sugar isl-rhamnose and that the glycerol moiety is probably linked to the polysaccharide through a phosphodiester bond.

Yukioka , M. (University of Hawaii, Honolulu), and T. Winnick . Synthesis of malformin by an enzyme preparation from Aspergillus niger . J. Bacteriol. 91: 2237–2244. 1966.—An enzyme fraction derived from disrupted Aspergillus cells was able to utilize each of the component labeled amino acids of malformin for the synthesis of this cyclic pentapeptide. The process was stimulated by adenosine triphosphate, K + , and Mg ++ , and was optimal at approximately p H 8.5. It was not affected by inhibitors of protein synthesis (ribonuclease, chloramphenicol, puromycin). There is evidence that cysteine, rather than cystine, was incorporated into peptide linkage, so that the disulfide bridge of malformin was formed subsequently. Although only the d isomers of cysteine and leucine occur in the malformin molecule, the l , as well as the d form of these amino acids, was readily utilized by the enzyme preparation. As in the case of several other microbial peptide systems, it appears that the d enantiomorph can arise from the l isomer at an intermediate stage of polypeptide synthesis.

An asporogenous mutant of Bacillus subtilis Sp − H12-3, which is considered to have a block at stage 0, showed general growth characteristics similar to those of sporulating cultures. However, a sudden increase in the total amount of acid-soluble nucleotides observed at t 2 in sporulating bacteria was completely absent in this mutant. In sporulating cells, a marked increase in two nucleotides which were identified to be uridine diphosphate (UDP)-galactose and UDP- N -acetylglucosamine was noted, whereas UDP-glucose appeared to be accumulated in the mutant cells at t 2 . No unusual nucleotides were found in the strains of B. subtilis examined. The possible role of these UDP derivatives in early stages of sporulation in B. subtilis is discussed.

Observation of the growth of some adenineless mutants of Schizosaccharomyces pombe on six substituted purine analogs leads to the hypothesis that an enzyme is present which catalyzes the conversion of these analogs into hypoxanthine. The enzyme adenase (adenine aminohydrolase, EC 3.5.4.2) has been found to be active in cell-free extracts of S. pombe . Results are reported which are in agreement with the hypothesis that this enzyme is responsible for the in vivo utilization of 6-chloropurine. This evidence comes mainly from a study of adenine aminohydrolase in two mutants selected for partial inability to grow on 6-chloropurine.

The amino acid composition of the murein (peptidoglycan) of 75 strains of enterococci was investigated. All strains designated Streptococcus faecalis according to their physiological properties contained the lysine-alanine type of murein; all strains classified as S. faecium contained the lysine-aspartic acid type of murein.

Incubation of resting cells of Agrobacterium tumefaciens with glucose-1-phosphate resulted in the accumulation of a new sugar phosphate in the suspending medium. Approximately 80% of the glucose-1-phosphate consumed was converted to the new compound, which was identified as α- d - ribo -hexopyranosyl-3-ulose-1-phosphate (3-ketoglucose-1-phosphate). Both utilization of glucose-1-phosphate and accumulation of 3-ketoglucose-1-phosphate were inhibited by 2,4-dinitrophenol, polymyxin, and d -glucose, which are inhibitors of the glucoside transport system of this bacterium but are not inhibitors of d -glucoside-3-dehydrogenase, which is the 3-ketoglucose-1-phosphate-forming enzyme. Consequently, it was concluded that glucose-1-phosphate penetrates into intracellular space by means of an active transport system. The glucose-1-phosphate is converted to 3-ketoglucose-1-phosphate by d -glucoside-3-dehydrogenase, and the 3-ketoglucose-1-phosphate formed reaches the extracellular space by passing through the surface layer of the bacterium.

Cell-free extracts of Neurospora crassa mycelium catalyze the methylation of l -histidine to form hercynine (histidine betaine). The partially methylated compounds, α- N -methyl- l -histidine and α- N,N -dimethyl- l -histidine, are also methylated by the same enzyme preparation to form hercynine. All three methylation reactions are stimulated by the addition of S -adenosylmethionine.

Bolaños, Roger (Tulane University, New Orleans, La.), and Charles W. DeWitt . Isolation and characterization of the K1 (L) antigen of Escherichia coli . J. Bacteriol. 91: 987–996. 1966.—An acidic polysaccharide with the serological properties of the K1 (L) antigen has been isolated from Escherichia coli O2:K1:H4 by means of phenol-water extraction, fractionation with hexadecyltrimethylammonium bromide (Cetavlon), filtration through Sephadex G-200, and chromatography on anion-exchange cellulose columns. Nucleic acid and protein content were reduced to a nondetectable level. There is no contamination with O antigen. The active material appears in two different positions in the Cetavlon fractionation, each with a slightly different serological specificity, as followed by the inhibition of passive hemagglutination. By paper chromatography, the polysaccharide moiety of the O antigenic fraction is composed of glucose, galactose, hexosamine and rhamnose. The absence of colanic acid in either K1 fraction was not proven, although its participation in our assay system, as well as the participation of the common or cross-reacting antigen, was ruled out by serological means.

The oxidation of decane by a cell-free particulate preparation from Candida intermedia was studied. Decane is oxidized to decanoate via decanol and decanaldehyde. Oxidation of decane to decanol requires molecular oxygen. Decanol is oxidized to decanaldehyde by a nicotinamide adenine dinucleotide-linked dehydrogenase differing greatly in specificity from ordinary yeast alcohol dehydrogenase. Decanaldehyde is oxidized to decanoate by a nicotinamide adenine dinucleotide-linked dehydrogenase that oxidizes long-chain aldehydes but not short-chain aldehydes. The enzymes that oxidize decane, decanol, and decanaldehyde are all induced when decane is present in the medium. These enzymes are apparently located in the cell membrane.

Cohen, Gary H. (University of Vermont, Burlington), and Donald B. Johnstone . Extracellular polysaccharides of Azotobacter vinelandii . J. Bacteriol. 88: 329–338. 1964.—Extracellular polysaccharides synthetized by Azotobacter vinelandii strains 155, 102, and 3A were shown to be carboxylic acid heteropolysaccharides of apparent high molecular weight. Cells were grown in a nitrogen-free, mineral broth medium with 2% sucrose. Extracellular slime was recovered by centrifugation and purified by repeated alcohol precipitation and Sevag deproteinization. Capsular polysaccharide was recovered from washed cells by mild alkaline digestion. Methods of isolation and purification appeared to provide polysaccharide showing no evidence of heterogeneity when examined by chemical and physical methods. Infrared analysis of purified slime from the three strains suggested fundamental structural similarities. Colorimetric, paper chromatographic, and enzymatic analyses on both intact and acid-hydrolyzed slime polysaccharide indicated that the polymers contained in common galacturonic acid, [α] d -glucose, and rhamnose at a ratio of approximately 43:2:1, as well as a hexuronic acid lactone, probably mannurono-lactone. However, as shown by chemical and infrared analysis, minor differences did exist; namely, slime from strain 155 and 102 contained o -acetyl groups, whereas slime from strain 3A contained none. A sialic acid-like component (1.5% of dry weight of the polysaccharide, calculated as N -acetyl neuraminic acid), was found only in the slime of strain 155. Capsular polysaccharide composition closely resembled that for slime. It is of interest that the major slime components were identical whether the energy source provided for the cells was sucrose, glucose, fructose, or ethanol.

The lipid composition of the chemoautotroph Ferrobacillus ferrooxidans has been examined. Fatty acids represent 2% of the dry weight of the cells and 86% of the total are extractable with organic solvents. About 25% of the total fatty acids are associated with diacyl phospholipids. Polar carotenoids, the benzoquinone coenzyme Q-8, and most of the fatty acids are present in the neutral lipids. The phospholipids have been identified as phosphatidyl monomethylethanolamine (42%), phosphatidyl glycerol (23%), phosphatidyl ethanolamine (20%), cardiolipin (13%), phosphatidyl choline (1.5%), and phosphatidyl dimethylethanolamine (1%) by chromatography of the diacyl lipids, by chromatography in four systems of the glycerol phosphate esters derived from the lipids by mild alkaline methanolysis, and by chromatographic identification of the products of acid hydrolysis of the esters. No trace of phosphatidylserine (PS), glycerolphosphorylserine, or serine could be detected in the lipid extract or in derivatives of that extract. This casts some doubt on the postulated involvement of PS in iron metabolism. After growth in the presence of 14 C and 32 P, there was essentially no difference in the turnover of either isotope in the glycerolphosphate ester derived from each lipid in cells grown at p H 1.5 or 3.5.

Wang, Marian (University of Pennsylvania, Philadelphia), Edward Steers, and Robert F. Norris . Extracellular polysaccharide of mucoid Lactobacillus bifidus . J. Bacteriol. 86: 898–903. 1963.—The extracellular polysaccharide produced by the “Jackson-mucoid” strain of Lactobacillus bifidus was isolated and purified by three different procedures. The component sugars of the polysaccharide were identified by paper chromatography and confirmed by infrared analysis. Galacturonic acid, galactose, glucose, and 6-deoxy-talose were identified. Contrary to a previous report from this laboratory, fucose was not found to be a component of this polysaccharide.

Dihydroxyacetone was quantitatively produced from glycerol during the primary exponential growth phase and depleted during the secondary exponential phase. Although no growth was detected on the basal medium, growth occurred upon addition of dihydroxyacetone.