Sporulation of Streptomyces venezuelae in submerged cultures

Shaken cultures of Streptomyces venezuelae ISP5230 in minimal medium with galactose and ammonium sulphate as carbon and nitrogen sources, respectively, showed extensive sporulation after 72 h incubation at 37 degrees C. The spores formed in these cultures resembled aerial spores in their characteristics. The ability of the spores to withstand lysozyme treatment was used to monitor the progress of sporulation in cultures and to determine the physiological requirements for sporulation. In media containing ammonium sulphate as the nitrogen source, galactose was the best of six carbon sources tested. With galactose S. venezuelae ISP5230 sporulated when supplied with any of several nitrogen sources; however, an excess of nitrogen source was inhibitory. In cultures containing galactose and ammonium sulphate, sporulation was suppressed by a peptone supplement. The onset of sporulation was accompanied by a drop in intracellular GTP content. When decoyinine, an inhibitor of GMP synthase, was added to a medium containing starch and ammonium sulphate, a slight increase in sporulation was seen after 2 d. The suppression of sporulation by peptone in liquid or agar cultures was not reversed by addition of decoyinine. A hypersporulating mutant of S. venezuelae ISP5230 was altered in its ability to assimilate sugars. In cultures containing glucose the mutant sporulated more profusely than did the wild-type and did not acidify the medium to the same extent. However, the suppressive effect of glucose on sporulation was not merely a secondary result of acid accumulation.     

Nitrogen utilization of highly and weakly virulent isolates of Colletotrichum falcatum Went

Nine inorganic nitrogenous compounds and sixteen amino acids were used to study the growth and sporulation of highly and weakly virulent isolates ofColletotrichum falcatum Went. Potassium nitrate, sodium nitrate and calcium nitrate supported good growth, while ammonium phosphate, potassium nitrate and sodium nitrate supported good sporulation of both isolates. Among the amino acids tested, glycine and DL-alanine were best for growth, and DL-threonine, L-leucine, DL-tyrosine, DL-serine, DL-phenylalanine and L-cystine for sporulation. No correlation existed between virulence and growth but the highly virulent isolate sporulated significantly more than the weakly virulent isolate.This work is a part of the author's Ph. D. Dissertation submitted to the Faculty of the Graduate School of the College of the Agriculture, University of the Philippines (1969).     

Sporulation of Streptomyces antibioticus ETHZ 7451 in submerged culture.

Streptomyces antibioticus ETHZ 7451 formed spores in cultures grown in a liquid medium from either a spore or a mycelium inoculum. The spores formed were similar to those formed on surface-grown cultures, except for reduced heat resistance. Both types of spores were sensitive to lysozyme, which is unusual for Streptomyces spores. Glucose and other carbon sources, which promoted different growth rates, did not affect sporulation efficiency. Nitrogen sources, such as casamino acids, that allowed high growth rates suppressed the sporulation. A remarkable repression was also observed in media with some nitrogen sources that promoted noticeably lower growth rates. In permissive media, with nitrogen sources that permitted relatively high growth rates, sporulation was conditioned to the consumption of ammonium in the medium, but not to that of other nitrogen sources, such as asparagine. Phosphate did not show a repressive effect on sporulation in the assayed conditions.     

Biochemical studies on sporulation in blue-green algae. II. Factors affecting glycogen accumulation

Inorganic nitrogens sources like nitrate, nitrite enhanced sporulation and glycogen accumulation in Anabaena sp. but ammonium chloride neither influenced sporulation nor glycogen accumulation. Acetate and citrate also stimulated early sporulation and glycogen level was higher over nitrogen free control. Nitrogen and carbon sources in combination proved to be useful in inducing early sporulation and increased content of glycogen. Phosphate and calcium also affected glycogen accumulation significantly, although, the sporulation was found to be of the same order as in nitrogen free medium. Sulphate initiated early sporulation, the mechanism of which is not known.     

Nitrogen and sulphur requirements of Colletotrichum inamdarii Lal

Summary Nitrogen and sulphur requirements ofColletotrichum inamdarii Lal isolated from the leaves ofCarissa carandas L. have been studied. DL-serine, L-asparagine and L-phenylalanine have been found to be of good nitrogen source followed by potassium nitrate, calcium nitrate, magnesium nitrate, DL-alanine, ammonium nitrate, glutamic acid, ammonium sulphate, DL-valine, aspartic acid, ammonium chloride, ammonium hydrogencarbonate, L-histidine and potassium nitrite. There was no growth in the absence of nitrogen.Sporulation was excellent on calcium nitrate and sodium nitrate, Very good on DL-serine, potassium nitrate, and magnesium nitrate. Good on L-asparagine, L-phenylalanine and ammonium oxalate. Fair on DL-alanine, DL-leucine, ammonium sulphate, DL-valine, ammonium chloride and L-histidine whereas poor on glutamic acid, aspartic acid, ammonium tartarate and ammonium nitrate. Few spores were observed on ammonium hydrogencarbonate but potassium nitrite did not show any sporulation.Amongst the sulphur compounds sodium bisulphate gave the best growth and good sporulation, followed by sodium thiosulphate, magnesium sulphate, ammonium sulphate and potassium sulphate. Thiourea gave negligible growth whereas it failed to grow on zinc sulphate and potassium persulphate.     

The effect of different nitrogen sources on pigment production and sporulation of Monascus species in submerged, shaken culture.

Thirty-nine strains from the genus Monascus were cultivated aerobically to study the relation between nitrogen nutrition and sporulation and pigment production. The effects of yeast extract, nitrate, ammonium, and ammonium nitrate have been compared. During cultivation the pHs of the different media are not the same, resulting in the formation of different coloured pigments. When the source of nitrogen is yeast extract or nitrate the pH is around 6.5 and red pigments are formed, whereas with ammonium or ammonium nitrate the pH is around 2.5 and the pigments are orange. It is proposed that only the orange pigments, monascorubrin and rubropunctatin, are produced biosynthetically and that the other pigments are formed from these by chemical transformations depending on the cultural conditions. The presence of organic nitrogen is optimal for growth and unfavourable for pigment production. Reduced growth and best pigment formation occurs with the three other nitrogen sources. Nitrate stimulates conidiation and sexual reproduction, while ammonium is inhibitory. Pigment production is better when conidiation is reduced. A mechanism is proposed for the control of sporulation and pigment production.     

Nitrogen requirements of certain isolates of alternaria tenuis auct

Three isolates ofA. tenuis isolated from the diseased leaves ofMangifera indica l. Musa paradisiaca l. andPsidium guajava l. were investigated. They were grown on different sources of nitrogen viz., potassium nitrate, sodium nitrate, calcium nitrate, ammonium nitrate, sodium nitrite, ammonium sulphate, ammonium chloride, glycine, DL-valine, L-glutamic acid, urea, thiourea, L-asparagine and peptone. They were also grown on the medium lacking nitrogen. A wide variation was observed in the growth and reproduction of the different isolates. The growth of all of them was good on potassium nitrate, calcium nitrate, glycine, DL-valine, L-glutamic acid, L-asparagine and peptone but the sporulation was satisfactory on calcium nitrate only. Sodium nitrite supported moderate growth of banana leaf isolate whereas there was no growth of the other two isolates. None of the organisms could grow on the medium lacking nitrogen as well as on thiourea. The results obtained with the isolates under study have been compared with those of earlier investigators and it has been clearly established that the different isolates ofA. tenuis could show marked differences in their nitrogen requirements.     

Bacitracin and protease production in relation to sporulation during exponential growth of Bacillus licheniformis on poorly utilized carbon and nitrogen sources.

Growth of Bacillus licheniformis in a chemically defined medium containing glucose and ammonium chloride yielded a doubling time of 1.00 h. Examination of the culture during exponential growth revealed a lack of heat-resistant spores together with a complete absence of detectable concentrations of bacitracin or extracellular serine protease. Replacement of glucose as the sole carbon source by glycerol, pyruvic acid, citric acid, or lactic acid resulted in doubling times of 1.13, 2.00, 3.16, and 3.95 h, respectively. Bacitracin, protease, and heat-resistant spores were produced during exponential growth in amounts related to these doubling times. A qualitatively similar pattern was observed when ammonium chloride was replaced by sodium nitrate, alanine, or glutamic acid which gave doubling times of 1.65, 1.77, and 1.90 h, respectively. Protease, but not bacitracin, concentrations were substantially higher when the growth rate was restricted by use of poor nitrogen rather than poor carbon sources. The relationships between bacitracin production, protease production, and the sporulation process are discussed.     

Utilization of Carbon, Nitrogen and Sulphur Compounds by Kabatiella zeae Narita et Hiratsuka

Abstract Morphogenesis of two K. zeae isolates (V7486 and ZP24986) on carbon, nitrogen and sulphur solid nutritive media after 2, 10, 18 and 28 days of incubation were studied. Highly significant differences were found between the isolates in regard to the use of compounds containing these elements. On carbon sources the fungus generally developed smaller colonies of branchy, loose, creamy-brown appearance, with abundant sporulation and abnormal conidia. Sucrose and D-glucose were the best carbon sources for K. zeae because both isolates had the maximum growth, abundant to medium abundant sporulation and mostly typical conidia. K. zeae colonies on organic and inorganic nitrogen and on sulphur sources too were leathery, creased, predominantly pink. Organic nitrogen sources lead to maximum growth and abundant sporulation, whereas ammonium and nitrate compounds caused intermediary growth and medium to very poor fungus sporulation. Addition of different sulphur sources, except sodium thiosulphate and thiocarbamide, did not affect the growth and sporulation of V7486. The growth of ZP 24986 depended on potassium and ammonium sulphate and sodium bisulphate.     

Nitrate Utilization by Nitrate Reductase-deficient Barley Mutants

Two nitrate reductase-deficient barley mutants were studied for growth on nitrate and ammonium sources of nitrogen and for resistance to chlorate. Although nitrate reductase-deficient mutants in some species are chlorate-resistant (unable to reduce chlorate to chlorite), the barley mutants used in these studies when grown on nitrate and treated with chlorate were only slightly more resistant to chlorate than the control. When grown to maturity on vermiculite supplemented with either nitrate or ammonium nutrient solutions, the mutants produced as much dry weight and reduced nitrogen per plant as the control. The in vivo and in vitro nitrate reductase activities in the roots and shoots of the mutants grown on nitrate were consistently less than 10% of the control. To avoid the possibility that the mutants received reduced nitrogen from microbial sources, excised embryos were cultured under sterile conditions. Again the mutants were capable of growth and reduced nitrogen accumulation with nitrate as the sole source of nitrogen. In spite of the low apparent nitrate reductase activity, the nitrate reductase-deficient mutants are capable of substantial nitrate reduction.     

Studies on the nutrition of Arthrobotrys oligospora Fres. and A. robusta Dudd: The predaceous phase

The effects of substrate on the predaceous activity in sand cultures of two isolates of the genus Arthrobotrys were measured by expressing the number of non-survivors of a free-living nematode population exposed to the fungi for 4 days as a percentage of a comparable population of nematodes not exposed to predation. This percentage is designated the predacity number. By varying the energy source it was shown that nutrient requirements for the extension of mycelium may differ from those for the expression of predacity. There is a greater reduction in the activity of A. oligospora when nitrogen is supplied as ammonium than when it is presented as nitrate or nitrite. The predacity of A. robusta is greatest on media deficient in biotin and with nitrite as nitrogen source. The relationship of results derived from artificial cultures to results from more direct examination of soils is discussed.     

Effect of different carbon and nitrogen sources on the growth and sporulation of Claviceps microcephal (Wallr) TUL.

The effect of different carbon and nitrogen compounds on growth and sporulation ofC. microcephala (Wallr.)Tul. causing ergot disease of Bajra has been studied. Nine different sources of carbon were used but cane sugar was found to be the best source for both, growth and sportulation of the fungus. Glucose, sucrose and maltose gave good growth but fair sporulation. Lactose and sorbitaol proved to be the poor sources. However, fungus failed to utilize starch, dextrin and mannitol.Nineteen nitrogen compounds were tried for the growth and sporulation of the fungus. Best growth and sporulation were supported by peptone and glycine. L-asparagine, DL-valine, Urea, magnesium nitrate and L-proline supported good growth and fair sporulation except DL-valine where it was excellent. Poor growth was obtained on L-isoleucin, ammonium sulphate, potassium nitrate,-alanine, ammonium chloride, DL-aspartic acid and DL-methionine. Fungus failed to utilize thio-urea.     

Influence of glutamate on growth, sporulation, and spore properties of Bacillus cereus ATCC 14579 in defined medium

A chemically defined medium in combination with an airlift fermentor system was used to study the growth and sporulation of Bacillus cereus ATCC 14579. The medium contained six amino acids and lactate as the main carbon sources. The amino acids were depleted during exponential growth, while lactate was metabolized mainly during stationary phase. Two concentrations of glutamate were used: high (20 mM; YLHG) and low (2.5 mM; YLLG). Under both conditions, sporulation was complete and synchronous. Sporulation started and was completed while significant amounts of carbon and nitrogen sources were still present in the medium, indicating that starvation was not the trigger for sporulation. Analysis of amino acids and NH4+ in the culture supernatant showed that most of the nitrogen assimilated by the bacteria was taken up during sporulation. The consumption of glutamate depended on the initial concentration; in YLLG, all of the glutamate was used early during exponential growth, while in YLHG, almost all of the glutamate was used during sporulation. In YLLG, but not in YLHG, NH4+ was taken up by the cells during sporulation. The total amount of nitrogen used by the bacteria in YLLG was less than that used by the bacteria in YLHG, although a significant amount of NH4+ was present in the medium throughout sporulation. Despite these differences, growth and temporal expression of key sigma factors involved in sporulation were parallel, indicating that the genetic time frames of sporulation were similar under both conditions. Nevertheless, in YLHG, dipicolinic acid production started later and the spores were released from the mother cells much later than in YLLG. Notably, spores had a higher heat resistance when obtained after growth in YLHG than when obtained after growth in YLLG, and the spores germinated more rapidly and completely in response to inosine, l-alanine, and a combination of these two germinants.     

DIFFERENCES IN NITROGEN REQUIREMENTS OF TWO CLONES OF CONVOLVULUS ARVENSIS IN VITRO

Various nitrogen sources were shown to alter the growth and modify nitrate reductase activities of stem callus tissue derived from two clones of Convolvulus arvensis L. (field bindweed). Callus from a Washington (S) clone grew better and had a higher level of nitrate reductase activity than callus from a New Mexico (R) clone when nitrate was the only source of nitrogen available in the culture medium. The addition of glycine to the culture medium decreased growth of the R clone and increased growth of the S clone, but glutamic acid repressed growth of both clones. An amide source of nitrogen such as glutamine or asparagine, or ammonium was required to produce maximum growth of both bindweed clones. Glutamine increased nitrate reductase activity in the two clones, and glycine increased nitrate reductase activity in the S clone but decreased it in the R clone. Glutamic acid decreased nitrate reductase activities in both the R and S tissues.     

Waterlogging and fire impacts on nitrogen availability and utilization in a subtropical wet heathland (wallum)

Protein, amino acids and ammonium were the main forms of soluble soil nitrogen in the soil solution of a subtropical heathland (wallum). After fire, soil ammonium and nitrate increased 90- and 60-fold, respectively. Despite this increase in nitrate availability after fire, wallum species exhibited uniformly low nitrate reductase activities and low leaf and xylem nitrate. During waterlogging soil amino acids increased, particularly γ-aminobutyric acid (GABA) which accounted for over 50% of amino nitrogen. Non-mycorrhizal wallum species were significantly (P < 0.05) ¹⁵N-enriched (0.3–4.3‰) compared to species with mycorrhizal associations (ericoid-type, ecto-, va-mycorrhizal) which were strongly depleted in ¹⁵N (-6.3 to -1.8‰). Lignotubers and roots had δ¹⁵N signatures similar to that of the leaves of respective species. The exceptions were fine roots of ecto-, ecto/va-, and ericoid type mycorrhizal species which were enriched in ¹⁵N (0.1–2.4‰). The 5¹⁵N signatures of δ¹⁵N_{total soil N} and δ¹⁵N_{soil NH4+} were in the range 3.7–4.5‰, whereas δ¹⁵N_{soil NO3?} was significantly (P < 0.05) more enriched in ¹⁵N (9.2–9.8‰). It is proposed that there is discrimination against ¹⁵N during transfer of nitrogen from fungal to plant partner. Roots of selected species incorporated nitrogen sources in the order of preference: ammonium > glycine > nitrate. The exception were proteoid roots of Hakea (Proteaceae) which incorporated equal amounts of glycine and ammonium.     

Interactions in the uptake of amino acids, ammonium and nitrate ions in the Arctic salt-marsh grass, Puccinellia phryganodes

The uptake of amino acids and inorganic nitrogen by roots of Puccinellia phryganodes was examined to assess the potential contribution of soluble organic nitrogen to plant nitrogen uptake in Arctic coastal marshes, where free amino acids constitute a substantial fraction of the soil‐soluble N pool. Short‐term excised root uptake experiments were performed using tillers grown hydroponically under controlled conditions in the field. The percentage reductions in ammonium uptake at moderate salinity (150 mm NaCl) compared with uptake at low salinity (50 mm NaCl) were double those of glycine, but glycine uptake was more adversely affected than ammonium uptake by low temperatures. Glycine uptake was higher at pH 5·7 than at pH 7·0 or 8·2. The glycine uptake was up‐regulated in response to glycine, whereas ammonium uptake was up‐regulated in response to ammonium starvation. Nitrate uptake was strongly down‐regulated when tillers were grown on either ammonium or glycine. In contrast to N‐starved roots, which absorbed ammonium ions more rapidly than glycine, the roots grown on glycine, ammonium and nitrate and not N‐starved prior to uptake absorbed glycine as rapidly as ammonium and nitrate ions combined. Overall, the results indicate that amino acids are probably an important source of nitrogen for P. phryganodes in Arctic coastal marshes.     

地衣芽孢杆菌BF-002高产芽孢的氮源流加工艺研究*

目的: 提高地衣芽孢杆菌BF-002的芽孢产量,实现氮源流加过程的自动化控制,降低生产成本,为其他芽孢杆菌提高芽孢产率的研究提供一种思路。方法: 通过摇瓶做单因素实验,筛选最佳温度和碳氮源,在此基础上进行5 L发酵罐实验。初始添加不同浓度的氮源,探索芽孢形成与氮源的关系。提出相对氨基氮的概念,通过恒速补料、间歇补料和基于尾气CO₂浓度反馈流加三个策略控制相对氨基氮浓度水平。采用Python语言编写计算机控制程序,实现基于尾气CO₂浓度反馈流加策略的自动化控制。结果: 摇瓶筛选最佳温度及碳氮源分别为:37℃、葡萄糖、鱼粉蛋白胨、豆粕。上罐结果表明,相对氨基氮浓度越低芽孢率越高,采用基于尾气CO₂浓度反馈流加能将相对氨基氮控制在8.42 mg/OD₆₀₀水平,芽孢量可达4.25×10⁹ cfu/mL。利用计算机程序自动控制低价氮源氯化铵的流加,可以使芽孢量达到1.87×10¹⁰ cfu/mL,是前期最优批次的4.4倍,同时降低原料成本。结论: 将相对氨基氮浓度控制在适宜水平可以得到芽孢量较高的培养液,自动流加氯化铵策略能降低生产成本并实现自动化控制,为研究芽孢杆菌产孢提供一种思路。     

Transformation of Copper Oxychloride Fungicide into Copper Oxalate by Tolerant Fungi and the Effect of Nitrogen Source on Tolerance

Aspergillus niger and Penicillium chrysogenum were able to grow on Czapek Dox medium amended with elevated concentrations [up to 500 ppm active ingredient (ai)] of the fungicide copper oxychloride. Solubilization of the fungicide in solid medium was evident by the appearance of a clear (halo) zone underneath and around the growing colonies. The halo formed with A. niger, grown on fungicide-containing nitrate nitrogen medium, was found subsequently to enclose concentric rings of newly crystalline precipitate. These crystals were extracted, examined by scanning electron microscopy and IR, and identified as copper oxalate. The supplemented nitrogen source to the medium greatly affected both fungicide solubilization and fungal tolerance. Ratios of fungicide solubilization rate (R(S)) in relation to the colony growth rate (R(G)) were significantly higher on ammonium than on nitrate nitrogen medium for both fungal strains. Growth ratios (the colony extension rate in the presence of a given concentration of the fungicide in relation to the control colony growth rate) of A. niger were markedly lower on ammonium than on nitrate nitrogen medium. The cellular copper contents, taken up from the fungicide, and the medium titratable acidity were higher in ammonium than in nitrate medium for both fungi. These results suggested fungal possession of variable tolerance mechanisms to this fungicide by complexation and/or precipitation of copper in the medium. Additionally, this work emphasizes the activity of fungi in transformation of insoluble inorganic metal-containing fungicides into insoluble organic metal compounds, which has a potentiality in metal cycling in biogeochemical and environmental context.     

Homeostasis in Nitrogenous Uptake/Assimilation by the Green Alga Platymonas (Tetraselmis) striata (Prasinophyceae)

When nitrate, ammonium, or urea were the sole nitrogen sourcesfor the growth of Platymonas striata there was a short initiallag period, after which uptake/assimilation occurred at a constantrate. Glycine ‘uptake’ was however non-linear withtime. Addition of ammonium or urea markedly inhibited nitrate‘uptake’ whereas although glycine was initiallytotally inhibitory, nitrate ‘uptake’ recommencedabout 1 h after addition, but at a greatly reduced rate. Ammoniumtotally inhibited urea ‘uptake’, but did not inhibitglycine ‘uptake’. When ammonium, urea, or glycinewere present at the same time as nitrate the total rate of nitrogen'uptake/assimilation was within 40% (or closer) of that of nitratenitrogen ‘uptake’ alone. In the first two casesthis reflected a total switching off of nitrate ‘uptake’whilst the new substrate was assimilated at a rate similar tothe initial nitrate rate, whereas in the case of glycine, bothsubstrates were used simultaneously. Once the concentrationof any of the inhibitory nitrogen sources had been reduced toa low level the ‘uptake’/assimilation of nitraterecommenced. It is suggested that the ability of P. striatato maintain a more or less constant rate total nitrogen ‘uptake’/assimilationirrespective of the mixture of utilizable nitrogen sources presentedto it, is not due to controls at the level of the entry of substrateinto the cell, but to intracellular actions at the enzyme level.The relative constancy of, and high rate of ‘uptake’,irrespective of nitrogen source and over a wide range of extracellularconcentrations, is tentatively considered to be due to saturationof the assimilatory mechanisms under the conditions employed;all the sources lead, either directly or indirectly, to theproduction of amino groupings for entry into nitrogenous metabolism.This homeostasis of nitrogen ‘uptake’/assimilationwould clearly be of considerable benefit to the alga in nature,enabling it to maximize growth in changing environmental conditions. Platymonas striata, nitrogen update, nitrogen assimilation     

Buoyant density heterogeneity in spores of Bacillus subtilis: biochemical and physiological basis

The biochemical and physiological basis of density heterogeneity in Renografin of Bacillus subtilis W23 spores was determined by analysis of metals, macromolecules, and dipicolinic acid in the two density classes of the population. Germination rate and heat resistance were measured in both density classes. Atomic absorption spectrophotometry revealed that heavy spores (density = 1.335 g/ml) have 30% more calcium than light spores (density = 1.290 g/ml). Other metals found in greater amounts in heavy spores were manganese and potassium. However, light spores had more sodium than heavy spores. The amounts of carbohydrates, nucleic acids, and proteins were the same in both types of spores, but light spores contained more lipids, whereas heavy spores had 30% more dipicolinic acid than light spores. Calcium and lipid were excluded as causes of the heterogeneity in density in that alteration of their contents in spores did not detectably affect the density of these spores. Spores of two densities were genetically similar. Furthermore, light density spores arose earlier during sporulation than heavy spores as determined by releasing refractile forespores at various times during sporulation. We concluded that light spores represent an incomplete stage in development because they became heavy when reinoculated into spent sporulation medium. This must involve the additional accretion or synthesis of dipicolinic acid.