Organic nutrition of Beggiatoa sp.

Culture OH-75-B of Beggiatoa sp. differed significantly from any described previously in its utilization of organic carbon and reduced sulfur compounds. It deposited internal sulfur granules characteristic of Beggiatoa sp. with either sulfide or thiosulfate in the medium. This strain (OH-75-B, clone 2a) could be grown in agitated liquid cultures on mineral medium with acetate as the only source of organic carbon. The resultant growth yields and rates were comparable to those for typical heterotrophs. Of the other simple organic compounds tested, only pyruvate, lactate, or ethanol could singly support the growth of this strain. Single sugars or amino acids neither supported growth nor enhanced it when added to acetate-containing medium. In contrast, compounds of the tricarboxylic acid cycle enhanced growth yields when tested in concert with acetate. These and fluoroacetate inhibition results indicate that Beggiatoa sp. possesses a functional tricarboxylic acid cycle. Poor yields characterized the growth of this strain on dilute yeast extract medium, and higher concentrations of yeast extract proved inhibitory. The enzyme catalase, contrary to the findings of others, had no synergistic influence on growth yields when added to medium containing yeast extract or acetate or both.     

Amino acid sequence of ferredoxin II from the phototroph Rhodospirillum rubrum: Characteristics of a 7Fe ferredoxin

The complete sequence of amino acids of ferredoxin II (FdII) from Rhodospirillum rubrum was determined by repetitive Edman degradation using pyridylethylated-ferredoxin and oxidized, denatured ferredoxin. Peptides derived from trypsin, pepsin, Glu-C endoproteinase, Arg-C endoproteinase, tryptophan specific cleavage and partial acid hydrolysis and C-terminal sequence from carboxypeptidase digestion were used to construct the total sequence. RrFdII is a polypeptide of 104 amino acids having a calculated molecular weight of 11556 excluding the iron and sulfur atoms. The complete amino acid sequence was: PYVVTENCIKCKYQDCVEVCPVDCFYEGENFLVINPDECIDCGVCNPECPAEAIAGKWLEINRKFADLWPNITRKGPAL ADADDWKDKPDKTGLLSENPGKGTV. Sequence comparisons, EPR characteristics and iron analyses indicate that RrFdII has structural features in common with ferredoxins containing [3Fe-4S], [4Fe-4S] centers. Of 104 amino acids, 60 (58%) including all 9 cysteines, are found in identical locations in the 7Fe ferredoxin prototype, Azotobacter vinelandii FdI.The protein sequence data reported in this paper will appear in the SWISS-PROT database and EMBL Data Library under the accession number P80448.     

Nutritional and Hormonal Requirements for the Growth of Vigna sinensis Callus in vitro

Nutritional and hormonal requirements for in vitro growth of callus tissue of Vigna sinensis Endl. were studied. Callus was formed on hypocotyl and root sections, when they were cultured on Linsmaier and Skoog's basal medium solidified with 10 g/1 agar and supplemented with only 0.5 mg/12,4-D or 2 mg/1 IAA. Further addition of 0.2 mg/1 kinetin and 1 g/l yeast extract resulted in more active callus formation. For unlimited vigorous growth of subcultured callus which was originally isolated from root sections, yeast extract was indispensable besides 2,4-D and kinetin. Such growth-promoting activity was observed also in malt extract and Ebios (dried cell powder of brewery yeast). Of known compounds tested, nicotinic acid, nicotinamide., methyl nicotinate and NAD were promotive to the growth of the callus, although much less effective than yeast extract. Other pyridine derivatives, vitamins and amino acids tested were ineffective or slightly effective. Sucrose was the most suitable carbon source. Fructose, glucose and maltose also supported the growth. Kinetin stimulated cell proliferation of the callus and cell differentiation to tracheary element.     

Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes.

Although wheat mashes contain only growth-limiting amounts of free amino nitrogen, fermentations by active dry yeast (Saccharomyces cerevisiae) were completed (all fermentable sugars consumed) in 8 days at 20 degrees C even when the mash contained 35 g of dissolved solids per 100 ml. Supplementing wheat mashes with yeast extract, Casamino Acids, or a single amino acid such as glutamic acid stimulated growth of the yeast and reduced the fermentation time. With 0.9% yeast extract as the supplement, the fermentation time was reduced from 8 to 3 days, and a final ethanol yield of 17.1% (vol/vol) was achieved. Free amino nitrogen derived in situ through the hydrolysis of wheat proteins by a protease could substitute for the exogenous nitrogen source. Studies indicated, however, that exogenously added glycine (although readily taken up by the yeast) reduced the cell yield and prolonged the fermentation time. The results suggested that there are qualitative differences among amino acids with regard to their suitability to serve as nitrogen sources for the growth of yeast. The complete utilization of carbohydrates in wheat mashes containing very little free amino nitrogen presumably resulted because they had the "right" kind of amino acids.     

Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes

Although wheat mashes contain only growth-limiting amounts of free amino nitrogen, fermentations by active dry yeast (Saccharomyces cerevisiae) were completed (all fermentable sugars consumed) in 8 days at 20 degrees C even when the mash contained 35 g of dissolved solids per 100 ml. Supplementing wheat mashes with yeast extract, Casamino Acids, or a single amino acid such as glutamic acid stimulated growth of the yeast and reduced the fermentation time. With 0.9% yeast extract as the supplement, the fermentation time was reduced from 8 to 3 days, and a final ethanol yield of 17.1% (vol/vol) was achieved. Free amino nitrogen derived in situ through the hydrolysis of wheat proteins by a protease could substitute for the exogenous nitrogen source. Studies indicated, however, that exogenously added glycine (although readily taken up by the yeast) reduced the cell yield and prolonged the fermentation time. The results suggested that there are qualitative differences among amino acids with regard to their suitability to serve as nitrogen sources for the growth of yeast. The complete utilization of carbohydrates in wheat mashes containing very little free amino nitrogen presumably resulted because they had the "right" kind of amino acids.     

Molecular structure of a yeast gene, PDI1, encoding protein disulfide isomerase that is essential for cell growth.

A genomic DNA clone for protein disulfide isomerase (PDI) of Saccharomyces cerevisiae was isolated by hybridization with synthesized oligonucleotide probes based on a partial amino acid sequence of yeast PDI. The introduction of a multiple copy plasmid carrying this fragment into yeast caused a tenfold increase in PDI specific activity and in the amount of PDI antigen in the extract. The gene on this fragment was named PDI1. The nucleotide sequence of the gene predicts a polypeptide of 522 amino acids with about 30% identity to mammalian PDIs. The predicted amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum retention signal of yeast (HDEL), and two putative active site sequences of PDI (WCGHCK). The predicted polypeptide is acidic and contains five putative glycosylation sites, consistent with the molecular properties of the purified yeast PDI [T. Mizunaga et al. (1990) J. Biochem. 108, 846-851]. The PDI1 gene was mapped on chromosome III. A gene disruption experiment revealed that the PDI1 gene is essential for cell growth.     

Amino acid sequence of a four-iron-four-sulphur ferredoxin isolated from Bacillus stearothermophilus.

1. The primary structure of a 4Fe-4S ferredoxin from Bacillus stearothermophilus was determined and shown to consist of a single polypeptide chain of 81 amino acid residues. The molecular weight of the holoprotein is about 9120. 2. There are only four cysteine residues in the molecule; three of these are located near the N-terminus as a Cys-X-X-Cys-X-X-Cys segment, and the fourth cysteine residue is followed by a proline and located in the C-terminal half. 3. The Fe-S chromophore in B. stearothermophilus ferredoxin was previously well characterized and was shown to consist of a single 4Fe-4S cluster. This ferredoxin sequence establishes for the first time the relative location of the four cysteine residues necessary to bind the 4Fe-4S cluster of a 4Fe ferredoxin, and is in agreement with the criteria for the relative positions of the cysteines proposed from X-ray-crystallographic studies on an 8Fe (two 4Fe-4S clusters) ferredoxin. 4. The sequence of B. stearothermophilus ferredoxin is homologous in many segments to that of other bacterial ferredoxins, the degree of homology being greater towards ferredoxins from Desulfovibrio gigas and photosynthetic bacteria than to Clostridial ferredoxins. 5. The presence of a relatively higher number of glutamic acid and lower number of cysteine residues in the molecule may explain the greater thermal stability and oxygen-insenstivity of this ferredoxin.     

Isolation and some properties of a mesophilic and mixotrophic iron-oxidizing bacterium, OKM-9

An iron-oxidizing bacterium strain, OKM-9, isolated from mud obtained from the bottom of a pond, Minamikata Ohike, in Okayama prefecture, Japan, grew well in an FeSO4 x 7H2O (3%)-medium (pH 2.5) with 0.03% yeast extract. However, the strain could not grow either in an FeSO4 x 7H2O (3%)-medium without yeast extract or in a yeast extract (0.03%)-medium (pH 2.5) without Fe2+. The strain did not use elemental sulfur as an energy source and did not have the activity to fix carbon dioxide. Strain OKM-9 could grow in an FeSO4 x 7H2O (3%)-medium with twenty different L-amino acids instead of yeast extract. Incorporation of [U-14C] glutamic acid into the cells was dependent on the energy produced by the oxidation of Fe2+. Strain OKM-9 did not grow heterotrophically using amino acids and hexoses as a sole energy and carbon source. The results that strain OKM-9 absolutely required ferrous iron (Fe2+) as a sole energy source and yeast extract or L-amino acids as a carbon source for growth strongly suggest that the strain is a mixotrophic iron-oxidizing bacterium. Strain OKM-9 was a gram-negative and rod-shaped bacterium (0.4-0.6 x 1.6-2.2 microm) and the mean G + C content of the DNA of the bacterium was 59.6 mol%. The optimum temperature and pH for growth were 30 degrees C and 2.1, respectively. However, the strain could not grow at temperatures above 45 degrees C. Iron-oxidizing activities of strain OKM-9 measured with intact cells and the plasma membrane were 14.3 and 5.7 microl O2 uptake/mg protein/min, respectively. The pyridine ferrohemochromes prepared from the plasma membrane of this strain showed absorption peaks characteristic of alpha-bands of heme a and b, but not heme c, at 587 and 557 nm, respectively. The results suggest that the cytochromes composing an iron-oxidation system of strain OKM-9 are different from those of the well-known mesophilic iron-oxidizing bacteria Thiobacillus ferrooxidans and Leptospirillum ferrooxidans.     

Ability of casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461

The ability of casamino acids and vitamin-assay casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461 was examined in a medium containing glucose or corn syrup as the carbon source relative to yeast extract supplementation. When glucose or corn syrup served as the carbon source, the presence of yeast extract in the growth medium stimulated gellan production by strain ATCC 31461 on casamino acids. Using vitamin-assay casamino acids as the nitrogen source, the addition of vitamins lowered gellan synthesis by glucose-grown cells regardless of yeast extract supplementation while gellan elaboration by corn syrup-grown strain ATCC 31461 cells could only be increased by supplementing vitamins into medium lacking yeast extract. Independent of carbon source, the absence of yeast extract in the medium reduced biomass production. Biomass production by the strain grown on either carbon source was increased by supplementing vitamins in the medium containing yeast extract.     

Physiological and nutritional features of Corynebacterium pyogenes

Growth and acid metabolic products were similar when Corynebacterium pyogenes was grown aerobically or anaerobically in a serum-free medium (SFM). This indicated that C. pyogenes obtains energy for growth primarily by fermentative metabolism even under aerobic growth conditions. Growth yield was reduced by 90% in SFM minus glucose, 50% in SFM minus NaHCO3, 90% in SFM minus yeast extract, 100% in SFM minus Trypticase and yeast extract, and 30% in SFM minus haemin or Trypticase. Growth was not detectable when a known mixture of amino acids, vitamins, and nucleic acid bases were substituted for Trypticase and yeast extract in SFM; addition to the latter medium of a peptide source such as Trypticase or casitone supported good growth of the organism. When NaHCO3 was omitted from SFM and dissolved CO2 in the medium was rigorously excluded, growth was undetectable indicating that C. pyogenes has an obligate requirement for CO2 for growth. Succinate, formate and acetate were the major fermentation products in SFM, whereas in SFM minus HCO-3 or haemin, lactate was the major product and only small quantities of other acids accumulated.     

Characterization and cloning of an extremely thermostable, Pyrococcus furiosus-type 4Fe ferredoxin from Thermococcus profundus.

An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid sequence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100 degrees C, while the half-life of the aerobically-purified ferredoxin is 10 h at 100 degrees C. Both the anaerobically- and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.     

海带水提液和酵母提取物促大肠杆菌生长研究*

通过细菌培养液DD值测量和平板计数技术观察不同物质对大肠杆菌生长的影响;对微量元素、稀土元素、动、植物激素、细菌提取物、真菌提取物、藻类提取物、植物提取物和动物提取物等13大类120余种物质在一定浓度范围的促大肠杆菌生长作用进行了观察。海带水提液和酵母提取物对大肠杆菌生长具有明显的促进作用;两的最佳作用浓度分别为20g/L和2％,DD值最大可分别达到对照组的2．93倍和5．06倍;平板计数可达3．94倍和5．39倍。     

Development of meridic and oligidic diets for rearing the aphid Myzus persicae

Meridic and oligidic diets suitable for the continuous culture of the aphid Myzus persicae were developed through modifications of a holidic diet. These included the addition of various amounts of crude nutrients and the replacement of defined nutrients by crude nutrients. The highest level of aphid growth (mean weights of 600 to 800 μg) was maintained (for 45 successive generations) on a meridic diet made by supplementing a holidic diet with 2.0% yeast extract (NBC).Continuous culture, at a level of growth (400 to 600 μg) comparable to that on the unsupplemented holidic diet (formulated with 34 defined nutrients), was supported by an oligidic diet containing only 10 weighed ingredients: 15 g sucrose, 2.5 g enzymatic casein hydrolysate (NBC), 2 g yeast extract, 123 mg MgSO₄·7H₂O, 100 mg ascorbic acid, 10 mg niacin, 5 mg Ca pantothenate, 2.5 mg pyridoxine, 2.5 mg thiamine, and appx. 1.5 gm K₂HPO₄·3H₂O (pH 6.8) per 100 ml of diet.Yeast extract at 2.0% provided adequate amounts of choline chloride, biotin, folic acid and inositol, but not of niacin, pantothenate, pyridoxine, and thiamine. Enzymatic casein hydrolyzate at 2.5% could replace the 20 defined amino acids of the holidic diet. Diets with both yeast extract and casein hydrolysate did not have to be supplemented with trace minerals (Cu, Fe, Mn and Zn). Yeast extract at 2.5% provided sufficient amounts of trace minerals, amino acids, and B-vitamins to sustain numerous successive generations, albeit at a low level of growth (200 to 300 μg). The simplicity and low cost of oligidic diets makes them attractive for aphid studies in which nutrition is not the primary consideration.     

Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA which was sequenced. Two non-overlapping open reading frames (ORFs) were identified. The largest, encoding a polypeptide of 382 amino acids (relative molecular mass (Mr) 43,483), was shown to be the viral nucleoprotein gene. The second ORF, found 3' to the larger ORF, encodes a polypeptide of 78 amino acids (Mr 9068) which has yet to be assigned to a viral product. The nucleoprotein gene was expressed in yeast cells under the control of two types of yeast promoters: the constitutive PGK promoter, and the inducible GAL1 promoter. Yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of Mr 47,000, identical to the viral product, that reacted with a specific monoclonal antibody.     

The protein responsible for center A/B in spinach photosystem I: isolation with iron-sulfur cluster(s) and complete sequence analysis

The 9 kDa polypeptide from spinach photosystem I (PS I) complex was isolated with iron-sulfur cluster(s) by an n-butanol extraction procedure under anaerobic conditions. The polypeptide was soluble in a saline solution and contained non-heme irons and inorganic sulfides. The absorption spectrum of this iron-sulfur protein was very similar to those of bacterial-type ferredoxins. The amino acid sequence of the polypeptide was determined by using a combination of gas-phase sequencer and conventional procedures. It was composed of 80 amino acid residues giving a molecular weight of 8,894, excluding iron and sulfur atoms. The sequence showed the typical distribution of cysteine residues found in bacterial-type ferredoxins and was highly homologous (91% homology) to that deduced from the chloroplast gene, frxA, of liverwort, Marchantia polymorpha. The 9 kDa polypeptide is considered to be the iron-sulfur protein responsible for the electron transfer reaction in PS I from center X to [2Fe-2S] ferredoxin, namely a polypeptide with center(s) A and/or B in PS I complex. It is noteworthy that the 9 kDa polypeptide was rather hydrophilic and a little basic in terms of the primary structure. A three-dimensional structure was simulated on the basis of the tertiary structure of Peptococcus aerogenes [8Fe-8S] ferredoxin, and the portions in the molecule probably involved in contacting membranes or other polypeptides were indicated. The phylogenetic implications of the structure of the present polypeptide as compared with those of several bacterial-type ferredoxins are discussed.     

Isolation and partial characterization of aClostridium species transforming para-hydroxybenzoate and 3,4-dihydroxybenzoate and producing phenols as the final transformation products

Organisms present in methanogenic freshwater lake sediments from the vicinity of Athens, Georgia, were adapted to mineralize 2,4-dichlorophenol. Repeated addition of 0.5 to 2.7 mmol/liter of phenol, and later of 0.5–6.2 mmol/liter p-hydroxybenzoate (p-OHB), to such enrichments led to the conversion of p-OHB to phenol at a rate of up to 100 mmol p-OHB per liter per day. Subsequently, a spore-forming, obligately anaerobic bacterium, strain JW/Z-1, was isolated which transformed p-OHB to phenol and 3,4-dihydroxybenzoate (3,4-OHB) to catechol (1,2-dihydroxybenzene) stoichiometrically without further metabolism of the phenols. The strain did not transform benzoate, 4-chlorophenol, 2,4-dichlorophenol, 4-chlorobenzoate, o- and m-hydroxybenzoate, 2,4- and 3,5-dihydroxybenzoate, 2,3,4- and 3,4,5-trihydroxybenzoate, or 4-aminobenzoate. Yeast extract was required for growth of strain JW/Z-1 and only high concentrations of casein hydrolysate or tryptone could substitute it, to some extent. Except for sodium acetate, and some amino acids together with a 20-fold increased concentration of vitamins, no single carbohydrate or defined organic compound has been found to support growth of this strain in the presence (or in the absence) of 0.2 to 0.5% (w/v) yeast extract. The fermentation products during growth on yeast extract indicated that the metabolism of amino acid degradation was the major source for growth. The decarboxylating activity was inducible by p-OHB for the decarboxylation of p-OHB, and at a lower rate for 3,4-OHB, and by 3,4-OHB only for 3,4-OHB, suggesting that two different enzyme systems exist. The addition of the aromatic amino acids phenol or benzoate did not induce the decarboxylation activity in cultures growing with yeast extract. Growth was observed at temperatures ranging from 12–41°C (T_opt, 33–34°C) and at pH-values ranging from 6.0–10.0 (pH_opt, 7.2–8.2). The shortest doubling time observed for strain JW/Z-1 was 3.2 hours.     

Effect of some amino acids and complex organic nitrogenous substances on vegetative growth and gametangial formation in the female clone of Riccia frostii aust.

Investigations in vitro have been carried out with the female clone of Riccia frostii to study the effect of some amino acids and complex organic nitrogenous substances on vegetative growth and gametangial formation. Of the three amino acids tried, aspartic acid and threonine stimulated vegetative growth as well as archegonial production at all levels, whereas tryptophan did so only at lower levels. Aspartic acid was most effective in promoting vegetative growth and threonine the least. At optimum levels addition of threonine initiated maximum archegonia and was followed, in order of effectiveness, by addition of aspartic acid and tryptophan. Complex organic nitrogenous substances (casein hydrolysate, peptone and yeast extract), in general, did not favour normal growth and development, especially at higher levels. Peptone proved inhibitory for archegonial formation, but casein hydrolysate and yeast extract stimulated production of archegonia at 50 ppm and 0.5% (w/v), respectively.     

Primary structure of a 7Fe ferredoxin from Streptomyces griseus

The complete primary structure of a Streptomyces griseus (ATCC 13273) 7Fe ferredoxin, which can couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450_soy for NADPH-dependent substrate oxidation, has been determined by Edman degradation of the whole protein and peptides derived by Staphylococcus aureus V8 proteinase and trypsin digestion. The protein consists of 105 amino acids and has a calculated molecular weight, including seven irons and eight sulfurs, of 12291. The ferredoxin sequence is highly homologous (73%) to that of the 7Fe ferredoxin from Mycobacterium smegmatis. The N-terminal half of the sequence, which is the FeS clusters binding domain, has more than 50% homology with other 7Fe ferredoxins. In particular, the seven cysteines known from the crystal structure of Azotobacter vinelandii ferredoxin I to be involved in binding the two FeS clusters are conserved.     

Simultaneous Growth on Citrate Reduces the Effects of Iron Limitation during Toluene Degradation in Pseudomonas

Rhizoremediation has been suggested as an attractive bioremediation strategy for the effective breakdown of pollutants in soil. The presence of plant root exudates such as organic acids, sugars, and amino acids that may serve as carbon sources or biosynthetic building blocks and the limited bioavailability of iron may influence the degradation of pollutants in the rhizosphere. To test the effect of such compounds on hydrocarbon degradation, trace concentrations of yeast extract or mixtures of organic acids and amino acids were added to continuous cultures of Pseudomonas putida mt2 and P. putida WCS358 (TOL) growing on toluene. By addition of these compounds increased growth yields and higher specific growth rates on toluene were obtained. The effects of iron limitation on the substrate utilization pattern of both strains were tested by growing the strains on a mixture of toluene and the readily degradable carbon source citrate while the iron concentration was varied. Simultaneous use of both substrates under carbon-limited as well as iron-limited conditions was observed. Growth yields were less reduced and iron requirement was lower during iron-limited growth in the toluene + citrate grown cultures compared to cultures in which toluene was used as the sole carbon source. The kinetic properties of the cells for toluene degradation were less hampered by the lack of iron when citrate was used as an additional carbon source. The results indicate that the availability of low concentrations of natural organic compounds, such as produced in the rhizosphere, may positively influence the degradative performance of hydrocarbon-degrading bacteria.