STIMULATORY PROPERTIES OF FILTRATE FROM THE GREEN ALGA HORMOTILA BLENNISTA. I. DESCRIPTION1,2

A laboratory phenomenon involving autostimulation of growth by filtrates of the green alga Hormotila blennista is described, and stimulation attributed primarily to the secretion of organic metabolites. Filtrates obtained from actively growing cultures 1 through 4 weeks old showed maximum growth rate stimulation values in excess of 100%. Stimulatory properties of filtrate were heat labile, were not closely controlled by the starting pH within the limits normally encountered in culture, and did not result from depletion of essential nutrients. Concomitant with growth rate stimulation, filtrates characteristically extended the lag phase of culture growth. H. blennista filtrate can support bacterial growth and selectively stimulate or inhibit 2 planktonic green algae. It was suggested that extracellular organic products secreted by H. blennista during active growth could be of survival value to the organism, and could also play regulatory roles among other microorganisms in nature.     

Selected muscle and nerve extracts contain an activity which stimulates myoblast proliferation and which is distinct from transferrin

Extracts from normal chicken anterior latissimus dorsi and dystrophic pectoralis major muscles and from normal chicken sciatic nerves induce a growth stimulation in chicken and rat myogenic cell cultures. Transferrin is only partially responsible for the observed stimulation since the addition of the extracts to transferrin-saturated cultures induces a further growth response and extracts from which transferrin has been removed by immunoabsorption still retain a substantial portion of their stimulation activity. The active fractions of muscle and nerve extracts display heat, acid, and organic solvent inactivation. Gel filtration of ammonium sulfate fractionated activity from the anterior latissimus dorsi muscle suggests the presence of a growth factor in the molecular weight range of 10,000 to 30,000.     

Fatty Acid Induced Leaking of Organic Compounds from Boletus variegatus

Addition of octanoic acid (2· 10^-3M) to the suspending medium (final pH 4.85) of Boletus variegatus mycelium induced a marked leaking of UV absorbing substances from the cells. The material had an absorption maximum at 260 nm, a minimum at 240 nm, and the absorption ratios 250: 260 and 280:260 were 0.81 and 0.49. The material released immediately after addition of the acid consisted mainly of low molecular weight substances. These substances, listed according to decreasing rates of leaking, were identified as pentoses, pentosephosphates, nucleosides, and mono- and di-nucleotides. Also, purine and pyrimidine bases were released at this early stage of treatment. After 90 minutes' treatment, an outflux of oligoribonucleotides was observed. The oligoribonucleotides did not occur as single substances, but were forming complexes with peptides. Minor amounts of ribonucleic acid were also leaking out from the cells. Deoxyribose containing substances were never observed in the filtrates. The compounds were subjected to enzymatic degradation after they had left the cells. This was shown by a marked increase with time of inorganic phosphorus, pentose/pentosephosphates, and nucleosides in the filtrate. The leaking of low molecular weight substances immediately after acid addition is correlated to seriously reduced growth. However, the growth was wholly restored after a three days' lag period. On the other hand, when considerable amounts of oligoribonucleotide peptides had been released from the cells, growth could not be re-established.     

The stimulation of extracellular carbohydrases of edible mushrooms by the hot water-soluble fraction from corn fiber

Corn fiber (CNF) is an abundant by-product of the wet corn milling process in the production of cornstarch. We have shown that the hot water-soluble fraction (HWSF) from CNF has a promoting effect on the mycelial growth of various edible mushrooms, including mycorrhizal fungi. To reveal the promoting mechanisms, the effect of CNF-HWSF on the stimulation of extracellular enzymes was examined. The production of extracellular carbohydrases such as amylase, CMCase, and xylanase was markedly enhanced by the addition of low molecular weight fractions (less than MW 500) prepared from CNF-HWSF. The enzymatic stimulation and enhancement of mycelial growth appeared during 3–15 days after inoculation. Furthermore, a fraction of less than MW 500 was separated by gel filtrate chromatography into five fractions (A–E), and the effect of each fraction was investigated. Promoting effects were shown from C and D fractions; mycelial growth and enzyme production of Lentinula edodes were indicated although fraction D has no sugars and amino acids in CNF-HWSF. From these results, the promoting effect of CNF-HWSF seems to be a two-step reaction. The first step could be achieved by rich nutrients such as free amino acids and monosaccharides from CNF-HWSF. The second step (during 3–15 days) is considered to be that the marked promoting effect was caused by the stimulation of extracellular enzymes.     

GROWTH AND EXCRETION IN PLANKTONIC ALGAE AND BACTERIA1

In short-term experiments using cultures of Chlorella pyrenoidosa, Anabaena flos-aquae, Asterionella formosa, and Navicula pelliculosa, both the proportion of photosynthetic products released from cells and the composition of these products altered, with age. In the first 3 species, percentage extracellular release values increased with increasing growth rates but the reverse trend was shown by Navicula. Fractionation of filtrates using Sephadex indicated that, in general, larger molecular weight compounds became predominant as cultures aged. Also a time-dependent shift in a similar direction occurred in cultures of all ages. In several lakes a predominance of large molecular weight compounds was apparent in filtrates even from short-term experiments. Filtrates of mixed cultures of planktonic bacteria growing on ¹⁴C glycolate were found to contain, large molecular weight organic compounds. It was demonstrated that in nonaxenic cultures of algae and in lake water, bacteria utilize low molecular weight extracellular metabolites of algal origin and larger molecular weight compounds are formed.     

Germination-Arrest Factor (GAF): 3. Determination that the herbicidal activity of GAF is associated with a ninhydrin-reactive compound and counteracted by selected amino acids

A novel, naturally-occurring herbicide (Germination-Arrest Factor, GAF), produced by Pseudomonas fluorescens WH6 and several related isolates of rhizosphere bacteria, irreversibly arrests germination of the seeds of a wide range of graminaceous species, including a number of important grassy weed species. GAF activity has been shown previously to be associated with a hydrophilic, low molecular weight compound that contains an acid group. In the present study, thin-layer chromatography (TLC) of extracts of WH6 culture filtrate demonstrated that GAF activity migrates on TLC plates with a particular ninhydrin-reactive compound. This compound was found to be present in GAF-producing P. fluorescens isolates and absent in P. fluorescens strains that lack the ability to produce GAF. Treatments, including mutagenesis, which resulted in the loss of GAF activity in culture filtrates from P. fluorescens WH6 were shown to result in the disappearance of this ninhydrin-reactive compound from extracts of WH6 culture filtrates or in alteration of its appearance on TLC chromatograms. The ninhydrin-reactivity of GAF indicates that it probably contains an amino group, as well as the acid group previously demonstrated, and suggests that GAF may be a small peptide or amino acid analog. Biological investigations motivated by this conclusion demonstrated that the effects of GAF in inhibiting the germination of seeds of annual bluegrass (Poa annua L.) could be counteracted by treatment with alanine or glutamine and, to lesser extent, by several other amino acids, suggesting that this compound may act by interfering with some aspect of amino acid metabolism or function.     

棕鞭藻及其培养滤液对铜绿微囊藻生长及生理特性的影响

为探究藻类之间的可能存在的信息传递, 研究了棕鞭藻(Ochromonas sp.)及其培养滤液对铜绿微囊藻的生长及生理特性的影响。结果发现, 3种不同接种比例(1﹕4、1﹕1和4﹕1)的棕鞭藻与微囊藻共培养下, 微囊藻细胞密度到第4天均下降到最低值, 而棕囊藻细胞密度则显著增加。同时, 棕鞭藻培养滤液能够抑制微囊藻的生长、导致丙二醛(MDA)含量和过氧化氢酶(CAT)活性。此外, 棕鞭藻培养滤液也能促进微囊藻胞外多糖(EPS)含量显著增加。这表明棕鞭藻不仅能吞噬微囊藻, 而且可能释放某些化感物质抑制微囊藻生长及生理参数。这暗示了棕鞭藻可作为潜在的藻类水华控制生物, 抑制早期藻类大量增殖。     

p-Cresol formation by cell-free extracts of Clostridium difficile

Cell-free extracts of Clostridium difficile were shown to form p-cresol by decarboxylation of p-hydroxyphenylacetic acid. This activity required both high and low molecular weight fractions. The active component of the low molecular weight fraction had properties of an amino acid and could be replaced by serine, threonine or the corresponding alpha keto acids. Pyruvate was shown to function catalytically. Since the high molecular weight fraction was O₂-sensitive and since dithionite was as effective as pyruvate with some high molecular weight fractions, the alpha keto acids probably serve as low potential reducing agents in this system. Because of instability, the p-cresolforming enzyme could not be purified.     

Isolation of a factor stimulatory to Bradyrhizobium japonicum in broth culture

Previous experiments indicated that water extracts of lambsquarters (Chenopodium album) and green foxtail (Setaria viridis), among others, stimulated growth of Bradyrhizobium japonicum in broth culture. Water extracts of lambsquarters shoots collected before or after anthesis were equally stimulatory. The stimulatory effect of extracts of lambsquarters when heated to 100°C for 30 min or autoclaved for 15 min was reduced by about 20% compared to untreated extracts. Extracts of green foxtail were less affected by higher temperature under similar conditions. Extracts of both green foxtail and lambsquarters completely lost their stimulatory effect following exposure to aerial microflora for 120 h. Water extract of lambsquarters shoots was more stimulatory than methanol extract, and neither ether nor butanol extracts resulted in stimulation. Both shoots and roots of lambsquarters and green foxtail were sequentially extracted first by water followed by methanol and vice-versa. The bioassay of these extracts indicated that there could be two components of the growth factor-one, larger component is soluble in water, the other, smaller component is soluble in methanol. After fractionation of the crude aqueous extract of lambsquarters shoots by four organic solvents, the residual aqueous extract retained the growth factor. Dialysis of the residual aqueous extract of lambsquarters shoots through a membrane (MWCO 1000) indicated that the molecular weight of the growth factor was less than 1000. The fraction having molecular weight <1000 was separated by paper chromatography using 6% acetic acid as developer. The fraction with Rf 0.91 showed the highest stimulation of the bacterium.     

Rapid induction of alpha-amylase by nongrowing mycelia of Aspergillus oryzae.

A rapid induction system for synthesis of alpha-amylase by the funga Aspergillus oryzae M-13 was established. The mycelia were prepared from 20-h cultures grown on a peptone-glycerol medium and starved for 5 h; maltose was the optimum inducer tested. During h 1 of induction, formation of both intra- and extracellular alpha-amylases occurred at an almost identical rate (70 to 80 microgram/g of cells-h) without a detectable lag period. After a 1-h induction period, a remarkable increase in the extracellular concentration of the enzyme occurred, and a maximum rate (330 microgram/g of cells-h) was reached after 1.5 h of induction. During h 2 of induction, no significant change in mycelial weight was observed. Purified samples of intra- and extracellular enzymes formed in the induction system showed identical properties as examined by behavior in diethylaminoethyl-cellulose column chromatography, gel filtration, discontinuous gel electrophoresis, electrofocusing, optimal conditions for the reaction, heat stability, and molecular weight.     

Rapid induction of alpha-amylase by nongrowing mycelia of Aspergillus oryzae.

A rapid induction system for synthesis of alpha-amylase by the funga Aspergillus oryzae M-13 was established. The mycelia were prepared from 20-h cultures grown on a peptone-glycerol medium and starved for 5 h; maltose was the optimum inducer tested. During h 1 of induction, formation of both intra- and extracellular alpha-amylases occurred at an almost identical rate (70 to 80 microgram/g of cells-h) without a detectable lag period. After a 1-h induction period, a remarkable increase in the extracellular concentration of the enzyme occurred, and a maximum rate (330 microgram/g of cells-h) was reached after 1.5 h of induction. During h 2 of induction, no significant change in mycelial weight was observed. Purified samples of intra- and extracellular enzymes formed in the induction system showed identical properties as examined by behavior in diethylaminoethyl-cellulose column chromatography, gel filtration, discontinuous gel electrophoresis, electrofocusing, optimal conditions for the reaction, heat stability, and molecular weight.     

Human fibrosarcoma cells produce fibronectin-releasing peptides

A sensitive radioimmunoassay, specific for human fibronectin, was used to measure the ability of certain biologically active polypeptides to release fibronectin from cultured human lung fibroblasts into their culture media. Concentrated, serum-free culture supernatant from a human fibrosarcoma cell line was fractionated by gel filtration chromatography in the presence of acetic acid. Various polypeptides with molecular weights between 46,000 and 6,000 were tested for their ability to release fibronectin from cells. The column fraction, containing polypeptides with an apparent molecular weight of 10,000, exhibited the ability to rapidly release fibronectin from target cells. The activity could be inhibited by phenylmethyl sulphonylfluoride. Several other hormonal factors, tested in parallel with the column fractions, failed to show this effect. The 10,000 dalton molecular weight polypeptides may represent a family of cellular gene products responsible for maintenance of low levels of surface associated fibronectin in fibrosarcoma cells and thus be related to their infiltrating properties by preventing the formation of the extracellular matrix.     

The influence of different nitrogen sources on the production of volatile compounds by Dipodascus aggregatus

Summary The yeast fungus Dipodascus aggregatus was grown aerobically on 9 different nitrogen sources and the production of volatile compounds determined by a gas chromatographic head-space technique. Excellent growth was supported by glutamine, aspartic acid, asparagine, (NH₄)₂-tartrate and NH₄H₂PO₄. Valine, leucine, and particularly isoleucine were utilized with a somewhat lower growth rate. Lysine was rapidly utilized after a prolonged lag phase.The highest production of volatile compounds was obtained from leucine and isoleucine. At least 20 volatile compounds were formed from each of them and many products were detected in high concentrations. Intermediate amounts of volatile compounds were produced from asparagine, the ammonium salts and valine, and low amounts from lysine, glutamine and aspartic acid.Ethyl acetate was a major product irrespective of the nitrogen source used. Regarding the pattern of volatile compounds produced, leucine, isoleucine and valine had much in common. Most of the volatile products formed from these amino acids contained a branched carbon chain and at least three high-boiling components eluted later than n-amyl acetate from the gas chromatographic column. The other six nitrogen sources could be grouped together. In general the same volatile compounds were formed from these sources, but the quantities of the individual compounds differed. Only one component eluted later than n-amyl acetate. No basic difference in production of volatile compounds was observed between the ammonium salts and -amino compounds like lysine and asparagine.     

Identification of intracellular amylase activity in Streptococcus bovis and Streptococcus salivarius

The ruminal bacterium Streptococcus bovis has been demonstrated to produce an extracellular amylase activity. We previously reported on the cloning of a gene from S. bovis encoding for what was initially believed to be the extracellular amylase. DNA sequence analyses indicated that the amylase produced by the cloned gene did not match the N-terminus amino acid sequence of the purified extracellular amylase and contained no apparent leader sequence for secretion. Analyses of crude extracts demonstrated the presence of an intracellular amylase in S. bovis JB1 that differed in molecular weight (56,000) from that of the extracellular amylase (70,000). The 56,000 molecular weight amylase was identical to the amylase produced by Escherichia coli containing the cloned amylase gene. Low levels of intracellular amylase activity were also detected in other strains of S. bovis and also Streptococcus salivarius. Introduction of the plasmid pVA838 containing the cloned amylase gene into S. bovis and S. sanguis resulted in enhanced intracellular amylase production by both organisms. The amylase gene has been sequenced, and analysis of the deduced amino acid sequence for the amylase indicates a high degree of similarity with secreted amylases from Bacillus species.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Molybdenum cofactor from the cytoplasmic membrane of Proteus mirabilis

Molybdenum cofactor was extracted from membranes of Proteus mirabilis by three methods: acidification, heat treatment and heat treatment in the presence of sodium-dodecylsulphate (SDS). Extracts prepared by the latter method contained the highest concentration of molybdenum cofactor. In these extracts molybdenum cofactor was present in a low molecular weight form. It could not penetrate an YM-2 membrane during ultrafiltration suggesting a molecular weight above 1000. During aerobic incubation of cofactor extracts from membranes at least four fluorescent species were formed as observed in a reversed-phase high performance liquid chromatography (HPLC) system. The species in the first peak was inhomogeneous while the species in the others seem to be homogenous. In water, all fluorescent products had an excitation maximum at 380 nm and an emission maximum at 455 nm. Their absorption spectra showed maxima at around 270 nm and 400 nm. Fluorescent compounds present in the first peak could penetrate an YM-2 membrane during ultrafiltration, whereas the compounds in the other peaks hardly did. Using xanthine oxidase from milk as source of molybdenum cofactor apparently identical cofactor species were found. Cytoplasmic nor membrane extracts of the chlorate resistant mutant chl S 556 of P. mirabilis could complement nitrate reductase of Neurospora crassa nit-1 in the presence of 20 mM molybdate. However, fluorescent species with identical properties as found for the wild-type were formed during aerobic incubation of extracts from membranes of this mutant.Non-common Abbreviations HPLC high performance liquid chromatography - I.D. internal diameter - SDS sodium dodecyl sulphate     

Effect of extracellular products ofAulosira fertilissima on the growth of rice seedlings

Summary The effect of extracellular products fromAulosira fertilissima on the growth of rice seedlings (IR-8) has been studied. There is indication of liberation of a root-promoting hormone from the alga. The growth pattern of rice seedlings, treated with algal filtrate, parallels that of seedlings treated with gibberellic acid. An increased growth of rice seedlings results when they are treated with algal filtrate obtained from the exponential phase of the alga. The amino acid-treated seedlings do not show any marked increase in growth. Filtrate fromAnacystis nidulans, on the other hand, do not show any growth promoting response. The possibility of liberation of kinins byA. fertilissima does not appear to be involved.     

INFLUENCE OF GROWTH STATUS AND NUTRIENTS ON EXTRACELLULAR POLYSACCHARIDE SYNTHESIS BY THE SOIL ALGA CHLAMYDOMONAS MEXICANA (CHLOROPHYCEAE)1

Increased levels of nitrogen in liquid growth medium bring about increased growth and a delay in extracellular polysaccharide production by Chlamydomonas mexicana Lewin on a per-cell basis. Addition of nitrogen to stationary phase cultures causes renewed growth and a temporary lag in polysaccharide synthesis until growth again ceases. Removal of nitrogen terminates growth, causing an immediate increase in polysaccharide synthesis. Phosphate-starved cells show a response similar to nitrogen-starved cells, indicating that the beginning of stationary phase and not nitrogen depletion causes the stimulation in extracellular polysaccharide synthesis. As similar results are assumed to occur on soil, the significance of this response is discussed.     

The Maceration of Vegetable Tissue by a Strain of Bacillus subtilis

Pectate lyase (PAL EC 4.2.2.2), pectinesterase (PE EC 3.1.1.11), L-arabinanase, D-xylanase, D-galactanase and neutral protease activities were identified in culture filtrates prepared from a strain B3 of Bacillus subtilis isolated from carrot. The PAL was purified by ion-exchange chromatography and iso-electric focusing and its properties examined. PAL had a pI of 9·85 and a molecular weight of 33000. Optimum activity occurred at pH 8–9 and 60–65°C. Calcium and to a lesser extent strontium were stimulatory while ethylenediamine tetraacetic acid led to inactivation. Thin layer chromatography separations of the end products of reactions and viscosity measurements suggested that the enzyme acted in a random manner. When examined over a range of pH values both culture filtrate and the purified PAL produced two distinct peaks of maceration (pH 6–6·5 and 8–9) against carrot or potato tissues. Evidence was obtained that although the presence of lyase was the sole external factor responsible for the maceration of carrot at pH 6·0, it acted in conjunction with a heat-labile, high molecular weight factor extractable from carrot tissue. Carrot extracts were unable to macerate carrot but liberated reducing groups from polygalacturonic acid and it is suggested that the factor may be, in part at least, carrot polygalacturonase. Maceration at pH 8·5 was largely accounted for by PAL and PE activities.     

Fraktionierung von Inhaltsstoffen der Zuckerrübenmelasse,die toxisch für die Citronensäureproduktion mit Aspergillus niger sind

The toxic fractions were isolated from unsuitable molasses,not accepted for citrie acid fermentation. When the strains of A. niger: Z-208, R-8, P-16, R-65/4, all good producers of citric acid, were cultivated on the medium containing such molasses, the growth patterns were altered and the strains lost their acid-forming properties. After dialysis of the molasses samples fractions of low and high molecular weight were obtained but the latter had no effect neither on growth nor on fermentation patterns. The low molecular part, responsible for the toxicity of molasses against A. niger, was fractionated with Sephadex G-25. In such a way a low coloured fraction which inhibited the growth of A. niger strains could be separated. However there were no toxic compounds in the fractions with the highest values of absorbancy. The susceptibility of the strains,to some extent, depended on their ability for adaptation to the toxic fractions of molasses.     

ANTIBIOTIC PROPERTIES OF ACRYLIC ACID, A FACTOR IN THE GASTROINTESTINAL ANTIBIOSIS OF POLAR MARINE ANIMALS.

Sieburth, John McNeill (Virginia Polytechnic Institute, Blacksburg, Va.). Antibiotic properties of acrylic acid, a factor in the gastro-intestinal antibiosis of polar marine animals. J. Bacteriol. 82:72-79. 1961.-Observations were made on acrylic acid to study some of the antibiotic, chemical, and physical properties of this volatile acid which occurs at a concentration of 8% (dry weight) in the marine alga Phaeocystis pouchetii. The sodium salt inhibited both gram-positive and gram-negative bacteria by filter paper disc assays at concentrations of 0.012-12.0 mg/ml. Activity was enhanced by acid reactions approximating those of the avian gut. In an attempt to explain the absence of typical strains of Escherichia coli and the suppression of the atypical coliform microflora in the anterior gastrointestinal segments of pygoscelid penguins in areas where the phytoplankton was dominated by P. pouchetii, chick trials were conducted with sodium acrylate. Acrylate supplementation of chicken feed at levels as low as one-fifth (0.01%) of those estimated to be ingested by penguins under natural conditions, suppressed the E. coli population and permitted its partial replacement by Aerobacter aerogenes. This latter phenomenon was explained by an increase in acrylate resistance by the A. aerogenes population. Acrylate feed levels between 0.01 and 1.0% caused an apparent increase in the growth rate of chicks, whereas 10% levels caused anorexia and death.