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Occurrence of phosphonosphingolipids in Bdellovibrio bacteriovorus strain UKi2.

The major phospholipids of two strains of Bdellovibrio bacteriovorus were characterized. Both strain UKi1, which is obligately saprophytic, and strain UKi2, which is facultatively parasitic, contained phosphatidylethanolamine and phosphatidylglycerol as their major glycerophosphatides. A branched, 15-carbon fatty acid is the major component of these alkali-labile lipids. Absent from UKi1 but present in UKi2 were three alkali-stable lipids (compounds 8, 9, and 11) which appear to be phosphosphingolipids. After acid hydrolysis, both compound 8 and 9 yield the identical phosphorus-containing substance that is water soluble, dipolar ionic, and ninhydrin positive. This substance appears to contain a C-P bond since P(i) could not be released from this substance by treatment with alkaline phosphatase or by very harsh mineral acid treatment. Based on chromatographic comparisons, this phosphonate appears to be a novel lipid constituent. Upon degradation, compound 8 yields 1 mol of dihydroxy long-chain base and compound 9 yields 1 mol of a trihydroxy long-chain base. These bases appear to have a 17-carbon, possibly branched, structure based on gas-liquid chromatography retention times. Degradation of both sphingolipids yields a mixture of hydroxy fatty acids, the major component being a branched, 15-carbon hydroxy acid.

Alkaline Phosphatase↗

Membrane permeability and the loss of germination factor from Neurospora crassa at low water activities.

Neurospora crassa conidia incubating in buffer at low water activities (a(w)) release a germination-essential component as well as 260-nm absorbing and ninhydrin-positive materials, regardless of whether an electrolyte or non-electrolyte is used to reduce a(w). Chloroform and antibiotics known to increase cell-membrane permeability have a similar effect. This suggests that membrane damage occurs in media of low a(w) and that an increase in permeability is responsible for the release of cellular components. The damage caused in media of low a(w) is nonlethal in most cases, and the conidia recover when transferred to nutrient medium.

Cell Membrane Permeability↗

Characterization of the lipids of mesosomal vesicles and plasma membranes from Staphylococcus aureus.

Mesosomal vesicles and plasma membranes were isolated from Staphylococcus aureus ATCC 6538P by protoplasting and differential centrifugation. The lipids of each of the two membrane fractions were extracted with pyridine-acetic acid-N-butanol, and the nonlipid contaminants were removed by Sephadex treatment. The lipids were then separated by passage through diethylaminoethyl-cellulose columns and characterized by thin-layer chromatographic, chemical, and spectral analyses. The lipids were separated into four discrete diethylaminoethyl fractions: (i) vitamin K2, carotenoids, C55 isoprenoid alcohol, and monoglucosyl diglyceride; (ii) cardiolipin, carotenoids, phosphatidyl glycerol, diglucosyl diglyceride, and an unidentified ninhydrin-positive component; (iii) cardiolipid and phosphatidyl glyderol; (iv) cardiolipin, phosphatidyl glycerol, and phosphatidyl glucose. Qualitatively, no difference in lipid composition between mesosomal vesicles and plasma membranes was found. However, based on equal dry weights of membrane materials, a relative quantitative difference in the amount of specific lipids in mesosomal vesicles and plasma membranes was observed. There are 4 times more monoglucosyl diglyceride, 2.6 times more diglucosyl diglyceride, 3.8 times more phosphatidyl glucose, 2 times more carotenoids, and 2 times more vitamin K2 found in mesosomal vesicles than in plasma membranes. The concentration of cardiolipin and phosphatidyl glycerol is 3.6 and 6 times greater, respectively, in mesosomal vesicles.

Cardiolipins↗

Production of molybdenum-coordinating compound by Bacillus thuringiensis.

Bacillus thuringiensis (ATCC 10792) produces a molybdenum reactive compound (given the trivial name chelin) during growth on iron-deficient medium. This compound accumulates in the culture medium in direct relation to the amount of L-arginine added and reaches a maximum concentration 24 to 48 h after the stationary phase of growth. Chelin absorbs light in the ultraviolet region with absorption maxima at 315 and 248 nm and minima at 284 and 240 nm. Chelin reacts with Na2MoO4, but not with Mo2O4(H2O)6-2+, to form a bright yellow molybdo-chelin complex which absorbs light with an absorption maximum at 330 nm, a minimum at 288 nm, and shoulders at 255 and 400 nm. The differential absorption of molybdo-chelin versus chelin at 425 nm can be used to quantify chelin. This differential absorbance is linear with increasing concentrations of Na2MoO4 and was used to calculate the molar extinction coefficient of molybdochelin at 425 nm (epsilon similar to 6,200). Chelin binds MoO4-2 minus to form a complex (molybdochelin) which migrates as a single band and elutes as a single peak, during acrylamide gel electrophoresis and Sephadex G-15 gel filtration. Molecular weight determinations using Sephadex G-15 gel filtration resulted in an estimated molecular weight of 550 for chelin and an estimated molecular weight of 760 for molybdo-chelin. The peptide nature of chelin is indicated by its positive ninhydrin reaction on thin-layer chromatography plates and by the presence of amino acids in acid-hydrolyzed samples. The major amino acid residues detected were threonine, glycine, and alanine.

Amino Acids↗

Neurospora crassa conidial germination: role of endogenous amino acid pools.

The levels of the endogenous amino acid pools in conidia, germinating conidia, and mycelia of wild-type Neurospora crassa were measured. Three different chromatographic procedures employing the amino acid analyzer were used to identify and quantitatively measure 28 different ninhydrin-positive compounds. All of the common amino acids were detected in conidial extracts except proline, methionine, and cystine. The levels of these three amino acid pools were also very low in mycelia. During the first hour of germination in minimal medium, the levels of most of the free amino acid pools decreased. The pool of glutamic acid, the predominant free amino acid in conidia, decreased 70% during the first hour. Very little glutamic acid or any other amino acid was excreted into the medium. During the first 20 min of germination, the decrease in the glutamic acid pool was nearly equivalent to the increase in the aspartic acid pool. The aspartic acid and lambda-aminobutyric acid pools were the only amino acid pools that increased to maximum levels within the first 20 min of germination and then decreased. It is proposed that an important metabolic event that occurs during the early stages of conidial germination is the production of reduced pyridine nucleotides. The degradation of the large glutamic acid pool existing in the conidia (2.5% of the conidial dry weight) could produce these reduced coenzymes.

Amino Acids↗

Characterization of autolysins from Mycobacterium smegmatis.

This study demonstrates, for the first time, the autolytic enzymes associated with mycobacterial cell walls. Based on the release of radioactivity and ninhydrin-reactive material from isolated cell walls, it was shown that maximum activity occurs during the late log phase of growth and at a buffer pH of about 8.0. Chemical analyses of autolytic digests of isolated cell walls indicated that at least three autolysins are active under the conditions used. These are N-glycolylmuramic acid-L-alanine amidase, an aminopeptidase that releases L-alanine, and an endopeptidase that solubilizes and L-alanyl-D-glutamic acid dippetide. No other endopeptidase, carboxypeptidase, or glycosidase activity was detected.

Alanine↗

Lipophilic O-antigens in Rhodospirillum tenue.

Lipopolysaccharides of eight wild-type strains of the phototrophic bacterium Rhodospirillum tenue have been analyzed. All of the lipopolysaccharides are highly lipophilic. The compositions of preparations obtained by the phenol-water or by the phenol-chloroform-petroleum ether procedure are very similar. The polysaccharide moiety, obtained by mild acid hydrolysis of lipopolysaccharide, consists mainly of aldoheptoses: L-glycero-D-mannoheptose is present in all strains, whereas D-glycero-D-mannoheptose is an additional constituent in some strains. Galactosaminuronic acid and two unknown ninhydrin-positive components were detected in the lipopolysaccharides of six strains. Spermidine and putrescine are present in large amounts in a salt-like linkage in the lipopolysaccharides from three strains. 2-Keto-3-deoxyoctonate forms the linkage between the polysaccharide moiety and lipid A. The lipid A fraction contains all the glucosamine and all the D-arabinose present in the lipopolysaccharide. D-Arabinose is an invariable constituent of the lipid A from the Rhodopseudomonas tenue lipopolysaccharides investigated. The principal fatty acids are beta-hydroxycapric, myristic, and palmitic acids. The isolated R. tenue lipopolysaccharides (O-antigens) react with rabbit antisera prepared against homologous cells. The titers in passive hemagglutination are low, similar to those found with enterobacterial R-lipopolysaccharides. R. tenue O-antigens containing only L-glycero-D-mannoheptose and those containing both the L- and D-epimers of glycero-D-mannoheptose could not be differentiated by serological means.

Amines↗

Cadaverine is covalently linked to peptidoglycan in Selenomonas ruminantium.

Cadaverine was found to exist as a component of cell wall peptidoglycan of Selenomonas ruminantium, a strictly anaerobic bacterium. [14C]cadaverine added to the growth medium was incorporated into the cells, and about 70% of the total radioactivity incorporated was found in the peptidoglycan fraction. When the [14C]cadaverine-labeled peptidoglycan preparation was acid hydrolyzed, all of the 14C counts were recovered as cadaverine. The [14C]cadaverine-labeled peptidoglycan preparation was digested with lysozyme into three small fragments which were radioactive and were positive in ninhydrin reaction. One major spot, a compound of the fragments, was composed of alanine, glutamic acid, diaminopimelic acid, cadaverine, muramic acid, and glucosamine. One of the two amino groups of cadaverine was covalently linked to the peptidoglycan, and the other was free. The chemical composition of the peptidoglycan preparation of this strain was determined to be as follows: L-alanine-D-alanine-D-glutamic acid-meso-diaminopimelic acid-cadaverine-muramic acid-glucosamine (1.0:1.0:1.0:1.0:1.1:0.9:1.0).

Alanine↗

Novel peptidoglycans in Caulobacter and Asticcacaulis spp.

Peptidoglycan sacculi free of poly-beta-hydroxybutyric acid were prepared from whole cells of four species of Caulobacter and two species of Asticcacaluis and from morphological mutants of Caulobacter crescentus and Caulobacter leidyi. Acid hydrolysates of the sacculi were analyzed quantitatively, and each of the hydrolysates was found to contain significant amounts of only five ninhydrin-reactive compounds: alanine, glutamic acid, alpha , omega-diaminopimelic acid, muramic acid, and glucosamine. Four types of peptidoglycans were distinguishable on the basis of the molar ratios among these five compounds. The respective ratios were as follows: in C. leidyi, 2:1:1:1:0.8; in Asticcacaulis biprosthecum, 1.7:1.6:1.1:0.7; in the cells of the remaining species, 2:1:1:1.2:0.8; and in stalks shed by the abscission mutant 2NY66, 2:1:1:1:1.67. Thus, in addition to some species differences among these caulobacters, it was found that the peptidoglycan sacculus of the stalked C. crescentus cell is chemically differentiated; the cellular peptidoglycan is richer in muramic acid than is the peptidoglycan of typical gram-negative bacteria, and the peptidoglycan of the stalk is correspondingly rich in glucosamine. Empirical formulas for the repeating units of the peptidoglycans have been inferred on the basis of the molar ratios of their amino components.

Alanine↗

Isolation and characterization of Phycomyces blakesleeanus ferritin.

Ferritin was isolated from the fungus Phycomyces blakesleeanus and compared biochemically and immunologically with horse spleen ferritin. Phycomyces and horse spleen ferritins were shown to exhibit similar electrophoretic patterns on polyacrylamide gels. Both preparations yielded an identical single band on sodium dodecyl sulfate-containing polyacrylamide gels. Tryptic digests of Phycomyces ferritin yielded 17 ninhydrin-positive spots as compared to 26 for horse spleen ferritin tryptic digests. Phycomyces ferritin was immunologically unrelated to horse spleen ferritin.

Animals↗

Biosynthesis and degradation of nodule-specific Rhizobium loti compounds in Lotus nodules.

Two nodule-specific Rhizobium loti compounds were identified in Lotus tenuis and Lotus pedunculatus nodules induced by strain NZP2037. One, a silver nitrate-positive cation called rhizolotine, has been characterized as the riboside of a novel alpha-hydroxyimino acid containing a 1,4,5,6-tetrahydropyrimidine ring (G. J. Shaw, R. D. Wilson, G. A. Lane, L. D. Kennedy, D. B. Scott, and G. J. Gainsford, J. Chem. Soc. Chem. Commun., p. 180-181, 1986), and the other, yellow-1, stains yellow with ninhydrin. Both compounds were degraded by R. loti NZP2037 but not by strains of Rhizobium meliloti, Rhizobium trifolii, or Agrobacterium tumefaciens. Under the conditions tested neither compound was able to serve as a sole source of C or N for growth of R. loti NZP2037. Rhizolotine and yellow-1 were found in nodules from a range of different legumes inoculated with NZP2037, suggesting that the Rhizobium and not the host plant determines their synthesis. Neither compound was found in nodulelike structures of L. pedunculatus induced by transposon Tn5-induced noninfectious (Inf-) mutants of NZP2037 or in similar structures induced by a transconjugant of NZP2037 containing the symbiotic (Sym) cointegrate plasmid pPN1 of R. trifolii. Both compounds were also absent in the ineffective nodules induced by the bacterial-release-negative (Bar-) mutant, strain PN239. However, both compounds were present in nodules induced by the fixation-negative (Fix-) mutant PN235 and in Fix+ nodules formed by a plasmid-cured derivative of NZP2037. These results would suggest that infection and bacterial release from the infection thread are necessary for nodule (symbiotic) synthesis of these compounds.

Bacterial Proteins↗

Citrate as a siderophore in Bradyrhizobium japonicum.

Under iron-limiting conditions, many bacteria secrete ferric iron-specific ligands, generically termed siderophores, to aid in the sequestering and transport of iron. One strain of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, 61A152, was shown to produce a siderophore when 20 B. japonicum strains were screened with all six chemical assays commonly used to detect such production. Production by strain 61A152 was detected via the chrome azurol S assay, a general test for siderophores which is independent of siderophore structure. The iron-chelating compound was neither a catechol nor a hydroxamate and was ninhydrin negative. It was determined to be citric acid via a combination of thin-layer chromatography and high-voltage paper electrophoresis; this identification was verified by a specific enzymatic assay for citric acid. The inverse correlation which was observed between citric acid release and the iron content of the medium suggested that ferric citrate could serve as an iron source. This was confirmed via growth and transport assays. Exogenously added ferric citrate could be used to overcome iron starvation, and iron-deficient cells actively transported radiolabeled ferric citrate. These results, taken together, indicate a role for ferric citrate in the iron nutrition of this strain, which has been shown to be an efficient nitrogen-fixing strain on a variety of soybean cultivars.

Citrates↗

Some products of the bacterial dissimilation of streptomycin.

Klein, Dolph (Rutgers, The State University, New Brunswick, N. J.) and David Pramer. Some products of the bacterial dissimilation of streptomycin. J. Bacteriol. 83:309-313. 1962.-Bacterial dissimilation of streptomycin resulted in products detectable with Folin-Ciocalteu's phenol reagent, ninhydrin, and Ehrlich's reagent; these products absorbed ultraviolet light, with a maximum at 265 mmu. Evidence was also obtained for the production of a volatile base, possibly methylamine. Urea and streptamine were formed by growing cultures and washed-cell suspensions supplied with streptomycin. More than 90% of the amidine-nitrogen of the antibiotic was recovered as urea-nitrogen in culture filtrates. It was evident that the bacterial dissimilation of streptomycin involved hydrolysis of the guanido groups of the streptidine moiety of the antibiotic molecule. The site of action was the bond between the amidine group and the secondary amino-nitrogen of each guanido group, and the products of the reaction were urea and streptamine.

Anti-Bacterial Agents↗

IDENTIFICATION OF GROWTH STIMULANTS FOR STREPTOCOCCUS LACTIS.

Koburger, J. A. (North Carolina State College, Raleigh), M. L. Speck, and L. W. Aurand. Identification of growth stimulants for Streptococcus lactis. J. Bacteriol. 85:1051-1055. 1963-The growth of Streptococcus lactis in milk was accelerated by pancreas extract, and three distinct components that were detected by bioautography were isolated and identified. These were purified by precipitation with silver ions, and their silver salts were regenerated in acidic solution. After adsorption and elution from characoal, contaminating alpha-amino compounds were destroyed by treatment with ninhydrin. Final purification was by chromatography. Identification of the purified fractions was by paper chromatography, and by ultraviolet and infrared spectra. The active compounds were found to be inosine, hypoxanthine, and adenine.

Acids↗

PROTEOLYTIC ATTACK OF THE CHROMOPROTEINS OF PORPHYRA BY MARINE BACTERIAL ENZYMES.

Merkel, Joseph R. (College of Charleston, Charleston, S.C.), Gordon D. Braithwaite, and Henry Kritzler. Proteolytic attack of the chromoproteins of Porphyra by marine bacterial enzymes. J. Bacteriol. 88:974-980. 1964.-Three chromoproteins were isolated from the marine red alga, Porphyra leucosticta (Thuret), and were purified by adsorption chromatography on calcium phosphate gel and by ammonium sulfate fractionation. The algal chromoproteins were subjected to the proteolytic action of cultures of actively growing marine bacteria, marine bacterial culture filtrates, trypsin, and various marine bacterial enzyme preparations. Digestion of the chromoproteins was followed through various incubation intervals by measuring the loss of chromophore color and the increase in either ninhydrin-positive material (570 mmu) or 280-mmu light-absorbing material. There was good correlation between the loss of color and enzymatic hydrolysis of the proteins. this was particularly evident when a marine pseudomonad proteinase preparation was used as the proteolytic agent. In every digestion mixture, phycoerythrin was more resistant to degradation than was phycocyanin or allophycocyanin. Phycoerythrin was also more resistant to heat than was phycocyanin or allophycocyanin.

Eukaryota↗

PYRUVATE METABOLISM, CARBON DIOXIDE ASSIMILATION, AND NITROGEN FIXATION BY AN ACHROMOBACTER SPECIES.

Hamilton, I. R. (University of Wisconsin, Madison), R. H. Burris, P. W. Wilson, and C. H. Wang. Pyruvate metabolism and carbon dioxide assimilation by an Achromobacter species. J. Bacteriol. 89:647-653. 1965.-Carbon dioxide fixation by washed whole cells of Achromobacter N4-B has been observed during anaerobic pyruvate metabolism with both nitrogen- and NH(4) (+)-grown cells. Labeled sodium bicarbonate-C(14) was assimilated into cells by a mechanism requiring pyruvate under conditions of nitrogen fixation, nitrogenase induction, and assimilation of NH(4) (+). Of the assimilated radioactivity, 89% appeared in six amino acids and two ninhydrin-positive unknown compounds, with the distribution of the label essentially independent of the nitrogen nutritional state of the organism. Aspartic and glutamic acids were the most highly labeled, with lesser amounts in glycine, alanine, ornithine, arginine, and the unknowns. All of the radioactivity extracted from these cells by ethanol-boiling water appeared in a protein fraction precipitated by 20% trichloroacetic acid. Radiorespirometric experiments with individually labeled pyruvate substrates demonstrated the preferential decarboxylation of the C-1 of pyruvate by this organism in a flowing helium gas phase. This decarboxylation was almost completely inhibited by using flowing nitrogen in place of helium; the addition of 0.5% CO(2) to the flowing nitrogen prevented inhibition and allowed 70% of the expected CO(2) evolution. These results, coupled with those from growth experiments, indicate a carbon dioxide requirement for anaerobic growth and pyruvate metabolism, which appears to be coupled to the formation of protein precursors.

Achromobacter↗

Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae.

Yoch, D. C. (South Dakota State University, Brookings), and R. M. Pengra. Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae. J. Bacteriol. 92:618-622. 1966.-The effect of exogenous amino acids and the free amino acid pool on the synthesis of the nitrogenase system of Klebsiella pneumoniae M5al (formerly Aerobacter aerogenes M5al) was investigated. When an actively N(2)-fixing culture was used to inoculate a medium containing a limiting concentration of NH(4) (+), an induction lag period was observed. When either a single amino acid or a mixture of amino acids was substituted at the same nitrogen concentration, growth was uninterrupted by the induction period. It appears that a step or steps in the formation of the nitrogenase system are repressed by NH(4) (+) and are not affected by amino acid N. The amino acids, far from repressing formation of nitrogenase as does NH(4) (+), actually stimulate its formation. It appears that both free and amino nitrogen are used simultaneously. The amino acids that served concomitantly with N(2) as a source of nitrogen were: aspartic acid, serine, threonine, leucine, and histidine. Of these amino acids, it was shown that aspartic acid is readily taken up by the cells. Of the amino acids not serving as an immediate nitrogen source, isoleucine is not taken up by the cells. The free amino acid pool of the cells was measured at the onset and termination of the induction period. Ninhydrin-positive material in the amino acid pool was depleted by 35% during the induction period.

Amino Acids↗

Proline as an intermediate in the reductive deamination of ornithine to delta-aminovaleric acid.

Fresh extracts of cells of Clostridium botulinum reduced a limited amount of ornithine to delta-aminovaleric acid, but at high substrate concentrations a considerable amount of an amino compound accumulated which was neutral at pH 4.2. Aging of the extracts at -10 C or freezing and thawing resulted in the loss of the ability to produce delta-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 delta-aminovaleric acid by cell extracts. The intermediate role of proline in the reduction of ornithine to delta-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 delta-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 delta-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-gamma-semialdehyde and its equilibrium product, Delta(1)-pyrroline-5-carboxylic acid.

Adenine Nucleotides↗