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Mild acid hydrolysis of the glycolipid derived @om a Salmonella Rd2 mutant led to the liberation of three major split products which, after separation by electrophoresis, were identified as free 2-keto-3-deoxyoctonate (KDO), KDO-7-phosphorylethanolamine, and L-glycero-α-D-mannoheptosyl-l,5-KDO. [ABSTRACT FROM AUTHOR]

The mureins of several micrococi and coryneform organisms contain more than one mole of glutamic acid per mole of meso-diaminopimelic acid (Dpm). The amino acid sequence of these mureins was studied. The peptide subunit consists of Mur-L-Ala-γ-D-Glu-meso-Dpm-D-Ala, where Mur = muramic acid. The γ-carboxyl group of glutamic acid is substituted in most cases by a glycinc amide. The cross-linkage of the peptide subunits is performed by the dipeptide γ-D-glutamyl-D-glutamic acid. This peptide is bound by the γ-carboxyl group of the C-terminal glutamic acid to the amino group of diaminopimelic acid and to the carboxyl group of D-alanine of an adjacent peptide subunit by the amino group of the N-terminal glutamic acid. The mureins of the micrococci studied contain only one mole of amide ammonia per mole of diaminopimelic acid and this is bound to glycine. In the mureins of the coryneform organisms a second mole of amide ammonia was found which is probably bound to diaminopimelic acid. The murein of Staphylococcus lactis CCM 2432 lacks amide ammonia as well as glycine. [ABSTRACT FROM AUTHOR]

This paper presents several lines of evidence indicating that the synthesis of vitamin-B12-dependent 5-methyltetrahydrofolate-homocysteine transmethylase ("methionine synthetase") is closely correlated to the intracellular level of vitamin B12 in manners different between bacteria which do and do not produce vitamin B12. 1. The intracellular level of vitamin B12 in Rhodospirillum rubrum was reduced to one fifth that of the normal cells when the organism was cultivated in a medium lacking cobalt. In accord with the decrease in vitamin B12 production, the specific activity of methionine synthetase was lowered to about 50%. It was of much interest that the enzyme was always present as the holoenzyme and the apoenzyme could not be detected even in the vitamin-B12-deficient cells containing a markedly decreased level of the holoenzyme. When the vitamin-B12-deficient cells were incubated in a cyano- cobalamin-supplemented medium, the specific activity of methionine synthetase began to increase after 2 h lag time and reached the maximum in about 6 h. This enhancement of the specific activity was suppressed if an appropriate inhibitor of protein synthesis was added to the medium. These facts strongly suggest that synthesis of protein de novo is essential for the enhancement of the specific activity by incubation with cyanocobalamin. Similar results were obtained in other vitamin B12 producers such as Streptomyces olivaceus and Corynebacterium simplex. 2. Chromatium D which is known to form cobalt-free corrinoids but not produce complete vitamin B12, was found to possess methionine synthetase as apoenzyme. When the vitamin-B12-free cells were incubated in the presence of cyanocobalamin, all the apoenzyme was readily converted to the holoenzyme within 2 h. As the formation of the holoenzyme proceeded, the level of the total enzyme (holo + apo) increased to a certain degree. The enhancement, however, was not affected by the inhibitors of protein synthesis, indicating that the increment could not have resulted from de novo synthesis of protein. A similar phenomenon was observed in Escherichia coli which does not produce vitamin B12 but possesses apoprotein of methionine synthetase. It is suggested that vitamin B12 synthesis is closely correlated to that of methionine synthetase in bacteria producing the vitamin, such as Rhodospirllum rubrum, Streptomyces olivaceus and Corynebacterium simplex. On the contrary, the apoprotein of the enzyme is synthesized independently of vitamin B12 in bacteria not producing the vitamin, such as Chromatium D and Escherichia coli. [ABSTRACT FROM AUTHOR]

Properties of murein hydrolases, assumed to play an essential role in bacterial morphogenesis, are described. Evidence is presented that in the intact cell these enzymes are prevented from uncontrolled reaction. It is shown that the ‘barrier’ between the enzymes and their substrate sacculus, a shape-maintaining macromolecule within the cell envelope, can be artificially broken. Assay systems to measure murein hydrolase activity in the intact cell, in isolated cell envelopes and after solubilization are described. A combination of Triton X.100 and sodium chloride is shown to liberate membrane-bound hydrolases effectively. [ABSTRACT FROM AUTHOR]

A rapid de novo synthesis of mitochondrial phospholipids can be shown following the incubation of different radioactively labeled precursors ([14C]choline, L-α-[14C]glycerophosphate, [14C]serine and [14C]ethanolamine) with isolated rate liver mitochondria. The data provide strong evidence that mitochondria are able to synthesize their own phospholipids in situ with little or no contribution from contaminating microsomes or bacteria. Some conditions for this incorporation are described which are relevant to the known pathways for phospholipid biosynthesis. [ABSTRACT FROM AUTHOR]

The synthesis of L-pyrrolidonyl-α- and -naphthylamides has been described. The -napththylamide was used for the colorimetric determination of pyrrolidonyl peptidase activity. This enzyme was purified from Bacillus subtilis by precipitation of pyrrolidonyl peptidase activity. This enzyme was purified from bacillus subtilis by precipitation with ammonium sulphate, using a DEAE-cellulose column and finally on a Sephadex G-200 column. The activity of the purified enzyme preparation was 530-fold that of the crude homogenate. No other peptidase activity in this preparation was found. It was demonstrated that the enzyme liberates pyrrolidonecarboxylic acid from human seromucoid. The results obtained suggest that pyrrolidonyl peptidase is an SH-enzyme. The occurrence of the enzyme in some groups of gram-positive and gram-negative bacteria was noted. [ABSTRACT FROM AUTHOR]

We are searching for biochemical events related to the process of bacterial division. Data obtained with intact bacteria, using inhibitors primarily, august that protein synthesis must occur through most of the division cycle in order that a bacterium can divide at the end of the cycle. Hence, one approach is to look for a protein that Is made throughout the division cycle and whose production is related to ability of the bacteria to divide. Dr Masayorl Inouye modified the ability of the bacterium Escherichia coli to divide, using mutants, inhibitors or nutritional deficiencies, and then looked for alterations in the pattern formed by electropharesis of membrane proteins on polyacrylamide gels. One protein peak on the gels was strongly increased whenever the cells Were prevented from dividing. We named this protein "X"; it has a molecular weight of about 50000. A role was sought for the protein made throughout the cycle but showing its effect only at the end, Earlier observations by Dr Helene Smith showed that brief heating of the bacteria, (16 min at 45 °C) produced synchronous cell division; heating delayed division in a way suggesting that a protein needed for division had been damaged and must be resynthesized before division occurs. Damage to this protein causes the celis to swell; this swelling appears to be the prime basis far the delay of cell division, as shown recently by Dr Po Chi Wu. The swelling caused by an osmotic shift similarly delayed cell division; a result that is support for this hypothesis. A model is proposed for the role of the X protein as a sort af "girdle" that must be built up during the cycle, and that constricts the cells at the time of division. [ABSTRACT FROM AUTHOR]

Nitrate reductase of Escherichia coli has been solubilized from particle fractions by a double treatment: first an alkali-acetone precipitation and then a solubilization in a buffered sodium deoxycholate. The enzyme has been purified 50-fold with a yield of I to 2%. Polyacrylamide-gel electrophoresis and ultracentrifugation show the preparation to be nearly homogeneous. The protein has a molecular weight of 320000 and an iso-electric point at pH 4.2. The absorbance which increases continuously from 600 to 280 nm does not reveal the presence of a heine or a flavin group but the spectrum resembles that of some bacterial ferroproteins. The estimation of metals indicates 1.5 atoms Mo and 20 atoms Fe per mole. Approximately one labile sulfide is found per iron atom. It is likely that nitrate reductase A is an iron.sulfur protein containing molybdenum. The purified protein uses as substrates NO3- and ClO3- and as electron donors reduced benzyl- and methyl-viologens, FMNH2 and FADH2 but not NADH or NADPH. It should be pointed out that the solubilization does not modify the enzymatic properties of nitrate reductase. CN- and N3- are strong inhibitors. Azide is a competitive inhibitor and the nitrate reductase affinity for this inhibitor is 1000 times greater than for nitrate. The type of inhibition observed and the metal chelating nature of the inhibitor suggest that a metal, Fe or Mo, or both, play a role in the formation of enzyme-substrate complex. [ABSTRACT FROM AUTHOR]

No pullulanase activity could be detected in maltose-grown cultures of 11 different strains of Escherichia coli. Neither these strains, nor 40 other strains of the same species would grow on pullulan, while all of them grew on maltose and some were shown to grow on maltotriose the exclusive product of pullulanase action on pullulan. These results are in contradiction with those reported last year in this journal by Palmer, Wöber and Whelan. On the other hand pullulanase activity could be detected, as reported by Bender and Wallenfels in 1966, in two strains of Aerobacter aerogenes. [ABSTRACT FROM AUTHOR]

The DNA synthesis in Bacillus amyloliquefaciens infected with wild-type phage φ29 and with temperature-sensitive mutants has been studied in the presence of the drug 6-(p-hydroxy- phenylazo)-uracil. The drug inhibits DNA synthesis in uninfected bacteria while it does not affect DNA synthesis and phage development in φ29-infected bacteria. DNA synthesis was blocked by infection at the restrictive temperature with the mutants F99 and K132, representatives of the two early functions identified so far, as well as with mutant C17, which affects a late function. The remaining mutants, belonging to eight complementation groups, synthesized DNA at the restrictive temperature. The proportion of the DNA resistant to DNAase was different, depending on the mutant. [ABSTRACT FROM AUTHOR]

Two strains of hydrogen-oxidizing bacteria were used: Hydrogenomonas eutropha strain H16 and strain 11/x. The former is a gram-negative flagellated rod and has been relatively well known with respect to its morphological, physiological, and biochemical properties. The latter is a gram positive, non spore-forming, motile small rod the definite classification of which is unknown as yet. Both strains were grown chemolithotrophically in submerged culture with a limited nitrogen source and supplied with a gas mixture of H2 02, and CO2. The lipid composition of the bacteria was determined at the end of the exponential phase (I), at the storage phase (II), and after reutilization of storage material (III). Along with the accumulation of poly(3-hydroxybutyric acid) as the major storage material in strain H16 the amount of other lipids was shown to increase 1.8-fold at phase II and to decrease at phase III. There is little or no change in the lipid pattern in various growth phases. It is suggested that the 1.8.fold increase of the latter lipids during phase II is due to the synthesis of membrane lipids that are required for the formation of membranes surrounding the intra cellular granules of accumulating poly(3-hydroxybutyric acid). In strain II/x the amount of lipids increases 7 times along with an equal increase of carbohydrates at phase II. The majority of accumulated lipids consists of triglycerides free of significant amounts of unusual acyl group. It is suggested that there is a true storage of triglycerides that are reutilized during phase III. The lipid pattern of strain H16 is characterized by a high proportion of "strongly bound lipids" that consist predominantly of phospholipids. Characteristic lipid components are phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol, while coenzyme Q8 (0.41 μmol/g bacterial dry weight) has been found among the neutral lipids. A C17 cyclopropane acid (probably cis-9,10-methylene hexadecanoic acid) is present in all saponifiable lipids. The lipid pattern of strain 11/x is characterized by a high proportion of "loosely bound lipids" that consist predominantly of neutral lipids, particularly of triglycerides. Among the polar lipids, diphosphatidylglycerol and phosphoinositides are most abundant, and several unknown phospholipids are also present. No cyclopropane fatty acid has been found. The possible use of strain 11/x as a source of nutritional fat is discussed. [ABSTRACT FROM AUTHOR]

A bacteriophage-induced DNA polymerase was purified from T7-infected bacteria. This enzyme is composed of one single polypeptide chain with a molecular weight of about 100000. The enzyme requires single-stranded DNA with a 3'-OH end as a primer. In addition to its polymerising activity the enzyme has a 3-ex nucleolytic activity. [ABSTRACT FROM AUTHOR]

1. Electron-exchange rates from the reduced redox proteins, cytochrome c551 and azurin to horse-heart ferricytochrome c have been measured. Values of 8 × 104 M-1 sec-1 and 1.1 × 10³ M-1 sec-1 for the cytochrome - cytochrome exchange and azurin - cytochrome exchange have been found with activation energies of 51 and 58.5 kJ (12.3 and 14 kcal), respectively. 2. A method is described whereby electron-exchange rates can be measured between specifically different cytochromes which are nevertheless almost identical in their spectral characteristic and where, consequently, the direct determination would be difficult. 3. Some observations on the oxidation of horse-heart ferrocytochrome c by mammalian cytochrome oxidase are presented which suggest this to be a complicated mechanism markedly influenced by ionic strength and cytochrome c concentration. [ABSTRACT FROM AUTHOR]

Caecal contents from conventional rats has been shown to have steroid sulphatase and glucuronidase activities. Whereas the sulphatase activity was of microbial origin the glucuronidase activity was also present m caecal contents from germfree rats. In vitro incubations showed that sulphuric acid esters of the 3α-, 3β-, 17β- and 21-hydroxyl groups were all hydrolyzed by the caecal bacteria. The ratio between free and conjugated metabolites of [4-14C]corticosterone administered to germfree and conventional rats was much higher in the conventional than in the germfree rats. This shows that microbial hydrolysis of steroid sulphates takes place also in vivo. [ABSTRACT FROM AUTHOR]

When Leptospira canicola was grown in a synthetic medium in the presence of 14C-labeled acetate, the acetate was readily oxidized and incorporated into non-lipid components of the cells. Only very small amounts of 14C-acetate were incorporated into long chain fatty acids. The effect of the addition, to the growth medium, of different long chain fatty acids, on the fatty acid composition of phosphatidyiethanolamine and triglycerides of L. canicola was studied. When grown on palmitate, palrnitic acid, cis-Delta;9 - and cis-Delta;11-hexadecenoic acid were isolated. %'hen grown on stearate, stearic, palmitic, oleic and cis-Delta;11-hexadecenoic acid were found. When grown on oleate the later was the major component of the cell's lipids. Phosphatidylethanolamine isolated from L. canicola which was grown on Tween, contained oleic acid at the alpha; position and palmitic, stearic and 11-hexadecenoic acid at the β position. When L. canicola was grown in the presence of [1-14C]palmitate or [1-14C]stearate the corresponding monounsaturated acids were also labeled. Oxidative decarboxylation of 14C. labeled 11-hexadecenoic acid isolated from L. canicola which was grown on Tween plus [1-14C]-palmitate indicated that the monoenoic acid was labeled exclusively at the carboxy-carbon. [ABSTRACT FROM AUTHOR]

The binding of Zn2+ to Escherichia coli alkaline phosphatase has been studied by pH titrations and equilibrium dialysis using 1,10-phenanthroline as competing chelating agent. Measurements of the strength of binding under different experimental conditions have been performed: 1. At constant pH with, different concentrations of chelating agent. 2. At different pH with constant concentration of chelating agent. 3. In the presence of denaturing agent at pH 8.0. It was found on the basis of equilibrium dialysis experiments that the binding of the two zinc atoms to alkaline phosphatase may be described as coordination to the two equivalent and independent sites. The high values of the binding constants, and the fact that denaturation destroys the specific binding of zinc to alkaline phosphatase indicates chelate formation with the protein, unless some unknown prosthetic groups are involved. [ABSTRACT FROM AUTHOR]