Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling^-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.     

The cyclohexane moiety of rapamycin is derived from shikimic acid inStreptomyces hygroscopicus

Summary Although the addition of shikimic acid to the medium had no effect on the level of production of rapamycin byStreptomyces hygroscopicus,¹⁴C-shikimic acid was incorporated into rapamycin to a very high degree.¹³C-Shikimic acid was successfully prepared from 1-[¹³C]-glucose using a mutant ofKlebsiella pneumoniae, and used to label rapamycin. It was found that¹³C-shikimic acid was incorporated into the cyclohexane moiety of rapamycin, thereby establishing the shikimic acid pathway origin of the seven-carbon starter unit.     

Carbon source nutrition of rapamycin biosynthesis inStreptomyces hygroscopicus

Summary Chemically-defined media were developed for rapamycin production byStreptomyces hygroscopicus. Thirty-five carbon sources were tested for their effect on production. Eight failed to support growth and seven appeared to repress or inhibit rapamycin formation. The best combination of two carbon sources were 2% fructose and 0.5% mannose. Acetate and propionate, which are known to contribute most of the carbon atoms of the lactone ring, were unsatisfactory for growth and/or rapamycin production.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Production and nutritional quality of the rotifer Brachionus plicatilis fed marine Chlorella sp. at different cell densities

The effect of different cell densities of marine Chlorella sp. on the growth rate, doubling time and production of the rotifer Brachionus plicatilis was investigated. A significant increase in rotifer production was achieved at a density of 50 × 10⁶ Chlorella cells ml^–1. The nutritional quality of rotifers grown at different concentrations of Chlorella is discussed.     

Rhodospirillum salinarum sp. nov., a halophilic photosynthetic bacterium isolated from a Portuguese saltern

A new species of halophilic photosynthetic bacteria, Rhodospirillum salinarum, has been isolated and described. Its natural habitat are the terminal crystallization ponds of solar salt production plants. R. salinarum grows optimally at 42°C in the presence of 6–18% NaCl (w/v). Growth requirements are complex, yeast extract and peptone being required both for aerobic heterotrophic and for anaerobic phototrophic growth. Increasing concentrations of NaCl in the growth media did not give rise to any corresponding increase in intracellular concentrations of K⁺, Na⁺, polyalcohols or amino acids. Malate dehydrogenase from R. salinarum is not halophilic, being inhibited even at low concentrations of Na⁺ or K⁺. The GC mol % of DNA from R. salinarum is markedly higher than that for DNA from R. salexigens, the only previously described halophilic species of the genus Rhodospirillum.     

Liquid-culture production of blastospores of the bioinsecticidal fungus<Emphasis Type="Italic"> Paecilomyces fumosoroseus</Emphasis> using portable fermentation equipment

The production of fungal spores using on-site, non-sterile, portable fermentation equipment is technically constrained. Very little information is available on the production requirements, such as medium concentration, inoculum stabilization, required fermentation times, and maintenance of axenic growth. In this study, we developed a two-part, liquid concentrate of the production medium that remains stable and soluble at room temperature. We also examined inoculum stability and showed that freeze- or air-dried blastospore preparations were stable for 7 days after rehydration when stored at 4 °C. The use of a low-pH (pH 4), relatively rich complex medium provided a growth environment deleterious to bacterial growth yet conducive to rapid sporulation by Paecilomyces fumosoroseus. High concentrations of blastospores (7.9×10⁸/ml) of P. fumosoroseus were produced in a 40-h fermentation with very low levels of bacterial contamination when the fermentor was charged with a blastospore production medium with a starting pH of 4 and inoculated with blastospore concentrations greater than 1×10⁶ spores/ml. These studies demonstrate that the use of disinfected, portable fermentation equipment has potential for on-site production of high concentrations of blastospores of the bioinsecticidal fungus P. fumosoroseus.     

Cationic responses of organs and haemolymph of Biomphalaria pfeifferi (Krauss), Biomphalaria glabrata (Say) and Helisoma trivolvis (Say) (Gastropoda: Planorbirdae) to cationic alterations of the medium

The cationic responses of haemolymph, mantle collar, headfoot, gut + digestive diverticulum and ovotestis + albumin gland of three planorbids to cationic changes in the media are explored. Body organs exhibited cationic homeostasis, although attained with difficulty under very low Ca : Mg and Ca : Na ratios. Haemolymph imbalances were obtained at very low calcium concentrations and at very low Ca : Mg and Ca : Na ratios in the medium; this may be linked to competition for Ca²⁺ uptake sites in the epithelium by Mg²⁺ and possibly Na⁺ ions.Fecundity and shell growth in relation to calcium concentrations and to Ca : Mg and Ca : Na ratios are examined.Normal internal cationic levels, under optimal conditions, were obtained for each species. Significant interspecific differences for haemolymph sodium were found; magnesium levels were slightly higher in Biomphalaria spp. than in Helisoma trivolvis; potassium levels were all similar. Amounts of tissue calcium decreased as follows: Mantle collar head-foot ovotestis + albumin gland-gut + diverticulum. Tissue magnesium levels in the gut were low. H. trivolvis had highest tissue calcium and sodium; B. glabrata had highest tissue potassium.     

Improvement in cell yield of Methylobacterium sp. by reducing the inhibition of medium components for poly-β-hydroxybutyrate production

The inhibitory effect of the concentrations of medium components on the growth of Methylobacterium sp. for poly--hydroxybutyrate production was investigated by measuring the specific growth rates for various concentrations of each medium component. When the methanol concentration was increased, the cell growth decreased and was strongly inhibited above 6% (v/v) methanol. Ammonia, calcium and iron ion did not significantly inhibit the cell growth while there were some inhibitory effects at high concentrations of sodium, potassium, and magnesium. In particular, phosphate gave most significant inhibition at concentrations higher than 75 mM. By using an automatic feeding control system of methanol, ammonia, phosphate, and minerals, their concentrations were maintained within the level necessary to reduce the inhibition of medium components. The finial dry cell weight of Methylobacterium sp. in such a system was 172 g/l at 84 h.     

Enhancement of sporulation in species of Bipolaris, Curvularia, Drechslera, and Exserohilum by growth on cellulose-containing substrates

Nine species of Bipolaris, Curvularia, Drechslera, and Exserohilum were compared for sporulation on agar media and for enhancement of sporulation by growth on four cellulose-containing substrates (index card, filter paper, cheesecloth, cotton fabric). On two natural and one synthetic agar media, sporulation varied from profuse to nonexistent among three isolates of each species. Growth of all species on cellulose substrates resulted in large and significant increases in sporulation. Growth on index card pieces often provided the greatest increases, but no single substrate was superior for all species, and significant substrate × isolate interactions were observed within species. Overlay of filter paper onto whole colonies in agar plates resulted in 2 to 18-fold increases in sporulation for eight of nine species and production of spores in sufficient quantity for most experimental purposes. Overlay of soil dilution plates with filter paper to promote sporulation of colonies enabled detection of B. spicifera, B. hawaiiensis, C. lunata, and E. rostratum at relatively low population levels (≤1.3 × 10³ colony-forming units per gram of soil) in samples of a naturally infested soil. Results indicate that enhancement of sporulation by growth of species of Bipolaris, Curvularia, Drechslera, and Exserohilum on cellulose substrates may facilitate (i) their identification in culture, (ii) production of spores at relatively high concentrations, and (iii) detection and enumeration of these fungi in soil.     

Squamocin mode of action to stimulate biofilm formation of Pseudomonas plecoglossicida J26, a PAHs degrading bacterium

Squamocin, an annonaceous acetogenin (ACG) extracted from Annona cherimolia (Annonaceae), has been shown to increase biofilm production of Pseudomonas plecoglossicida J26 (closely related to P. plecoglossicida), a polycyclic aromatic hydrocarbon degrading bacterium. PAHs have become priority pollutants for bioremediation due to their carcinogenicity and toxicity. The effect of various stressful stimuli (naphthalene, octanol, HCl, and NaCl) on cell growth, biofilm formation and autoinducer production of P. plecoglossicida were evaluated and compared with the effect of squamocin to establish its mode of action on biofilm formation. All stressors that inhibited growth stimulated autoinducer production while squamocin was growth stimulant at concentrations above 2.5 μg ml⁻¹. Despite structural similarities, squamocin is not an autoinducer agonist. It indirectly stimulates autoinducer production and increases P. plecoglossicida J26 cell growth. This is the first report on an ACG mode of action in the formation of biofilm in a naphthalene-degrading strain.     

Relationship Between Effect of Ethanol on Proton Flux Across Plasma Membrane and Ethanol Tolerance, inPichia stipitis

Pichia stipitisefficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. InSaccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. InP. stipitisthe passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H⁺-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H⁺-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H⁺-extrusion. Comparison of the rates of H⁺-flux with the relatedin vitroH⁺-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) ofP. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H⁺-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grownP. stipitiscells.     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

In Vitro CuIture of the Tropical Sponge <Emphasis Type="Italic">Axinella corrugata</Emphasis> (Demospongiae): Effect of Food Cell Concentration on Growth,Clearance Rate,and Biosynthesis of Stevensine

In vitro culture is one possible method for supplying sponge metabolites for pharmaceutical applications, but appropriate feeding regimens that maximize both growth and metabolite biosynthesis are largely unknown. According to the natural concentration (NC) of cells 1 to 50 µm in size that are available to wild Axinella corrugata, we fed explants a multispecific diet of bacteria, microalgae, and yeast at 4 different concentrations: 1NC, 3NC, 5NC, and 5+1NC (the last consisted of 5 NC of bacteria and 1 NC of microalgae and yeast). Explants fed a 3NC diet had the best culture response, growing on average from 8.5 g to 10.3 g in 8 weeks, and showing a 110% increase in concentration (milligrams per gram of dry weight) of the antitumor compound stevensine. Stevensine production in 3NC explants, representing the total milligrams of metabolite per explant, increased by 157% over the study. Explants fed at 1NC had relatively stable weights, indicating that the diet met metabolic costs only. Explants fed at the two highest concentrations lost weight after 4 weeks, possibly because long-term high cell concentration blocked their aquiferous system, reducing their ability to feed efficiently. Stevensine production in explants fed the 1NC, 5NC, or 5+1NC diets were similar, and varied little from the initial amount. A separate experiment showed that the clearance rate for A. corrugata is similar between the examined food types and cell concentrations over 5 hours, averaging 766 ml h^–1 g DW^–1.Overall, this study demonstrates that relatively small changes in food abundance can greatly affect both sponge growth and metabolite biosynthesis. The good growth and increased production of the target metabolite stevensine for A. corrugata explants fed a 3NC diet suggests that in vitro culture is a viable method of supplying some sponge metabolites.     

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l^–1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 10⁸ spores ml^–1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 10⁸ spores ml^–1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 10⁸ spores ml^–1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.     

In vitro production of microtubers for conservation of potato germplasm: Effect of genotype,abscisic acid,and sucrose

Summary With the objective of using microtubers for conservation of potato germplasm, the main effects of genotype, abscisic acid (ABA), and sucrose level, and of their interactions on biomass production, microtuberization, microtuber dormancy, and dry matter content, were studied. ABA decreased both microtuber production and microtuber dormancy, whereas higher concentrations (60–80 gl⁻¹) of sucrose promoted biomass production, microtuber production as well as microtuber dry matter content. Microtubers stored under diffused light had longer dormancy than those kept continuously in the dark. Interactions among various factors conditioned the main effects for some characters. In vitro performance of the genotypes studied was related to their known performance under in vivo conditions for most of the characters. Microtubers produced on media devoid of ABA and containing high sucrose concentrations and N⁶-benzyladenine (44.38 μM) could be stored for 12 mo. under diffused light at 6±1°C.     

The DNA gyrase inhibitors,nalidixic acid and oxolinic acid,prevent iron-mediated repression of catechol siderophore synthesis inAzotobacter vinelandii

Summary Low concentrations of nalidixic acid and oxolinic acid that were just inhibitory toAzotobacter vinelandii growth promoted the production of the catechol siderophores azotochelin and aminochelin, in the presence of normally repressive concentrations of Fe³⁺. There was a limited effect on the pyoverdin siderophore, azotobactin, where low concentrations of Fe³⁺ were rendered less repressive, but the repression by higher concentrations of Fe³⁺ was normal. These drugs did not induce high-molecular-mass iron-repressible outer-membrane proteins and similar effects on the regulation of catechol siderophore synthesis were not produced by novobiocin, coumermycin, or ethidium bromide. The timing of nalidixic acid and Fe³⁺ addition to iron-limited cells was critical. Nalidixic acid had to be added before iron-repression of catechol siderophore synthesis and before the onset of iron-sufficient growth. Continued production of the catechol siderophores, however, was not due to interference with normal iron uptake. These data indicated that nalidixic acid prevented normal iron-repression of catechol siderophore synthesis but could not reverse iron repression once it had ocurred. The possible roles of DNA gyrase activity in the regulation of catechol siderophore synthesis is discussed.     

Magnesium deficiency of kiwifruit (Actinidia deliciosa)

Magnesium deficiency was associated with large yield reductions in a five-year-old commercial kiwifruit (Actinidia deliciosa) orchard. The effect on yield resulted primarily from a reduction in fruit numbers, there being no difference in mean fruit weight between fruit harvested from affected and unaffected vines. Magnesium deficiency had no deleterious effect on postharvest storage characteristics of fruit stored at 0.5–1°C for 18 weeks; fruit from deficient vines were firmer but had slightly lower soluble solids than fruit from control vines. Although deficiency symptoms were first observed on the basal leaves of the non-fruiting shoots mid season, indications of the impending deficiency could be established very early in the season using foliar analysis. Magnesium concentrations in youngest fully expanded leaves (YFEL) on the affected vines were less than 2.0 g kg⁻¹ DM four weeks after budbreak and remained below this value for the rest of the season; concentrations in YFEL on unaffected vines did not decrease below this value and gradually increased after fruitset to 4.5 g kg⁻¹ DM at harvest. To avert potential production losses, it is suggested that soluble magnesium fertilizers (containing at least 200 kg ha⁻¹ Mg) should be broadcast early in the season if foliar magnesium concentrations less than 2.0 gkg⁻¹ DM are measured four–six weeks after budbreak.     

Production of a hypoglycemic, extracellular polysaccharide from the submerged culture of the mushroom, Phellinus linteus

Mycelial growth and extracellular polysaccharide production of Phellinus linteus were optimal at pH 5 and 25 °C. Maximum biomass production (14.2 g l^–1) was after 15 d of cultivation, whereas, extracellular polysaccharide was maximal (3.5 g l^–1) after 21 d. The hypoglycemic effect of the polysaccharide, investigated in streptozotocin-induced diabetic rats, decreased plasma glucose, total cholesterol and triacylglycerol concentrations by 49%, 32%, and 28%, respectively, and aspartate aminotransferase activity by 20%. The results indicate the potential of this polysaccharide to prevent hyperglycemia in diabetic patients.     

Influence of silicon on cobalt, zinc, and magnesium in baker's yeast, Saccharomyces cerevisiae

Silicon (Si, as silicate) is involved in numerous important structure and function roles in a wide range of organisms, including man. Silicate availability influences metal concentrations within various cell and tissue types, but, as yet, clear mechanisms for such an influence have been discovered only within the diatoms and sponges. In this study, the influence of silicate on the intracellular accumulation of metals was investigated in baker's yeast (Saccharomyces cerevisiae). It was found that at concentrations up to 10 mM, silicate did not influence the growth rate of S. cerevisiae within a standard complete medium. However, an 11% growth inhibition was observed when silicate was present at 100 mM. Intracellular metal concentrations were investigated in yeast cultures grown without added silicate (−Si) or with the addition of 10 mM silicate (+Si). Decreased amounts of Co (52%), Mn (35%), and Fe (20%) were found within +Si-grown yeast cultures as compared to −Si-grown ones, whereas increased amounts of Mo (56%) and Mg (38%) were found. The amounts of Zn and K were apparently unaffected by the presence of silicon. +Si enhanced the yeast growth rate for low-Zn²⁺ medium, but it decreased the growth rate under conditions of a low Mg²⁺ medium and did not alter the growth rates in high Zn²⁺ and Co²⁺ media. +Si doubled the uptake rate of Co²⁺ but did not influence that of Zn²⁺. We propose that a possible explanation for these results is that polysilicate formation at the cell wall changes the cell wall binding capacity for metal ions. The toxicity of silicate was compared to germanium (Ge, as GeO₂), a member of the same group of elements as Si (group 14). Hence, Si and Ge are chemically similar, but silicate starts to polymerize to oligomers above 5 mM, whereas Ge salts remain as monomers at such concentrations. Ge proved to be far more toxic to yeast than Si and no influence of Si on Ge toxicity was found. We propose that these results relate to differences in cellular uptake.     

Influence of ultraviolet radiation, copper, and zinc on microcystin content in Microcystis aeruginosa (Cyanobacteria)

Cyanobacterial toxin production is allied to some unknown trigger resulting in the production of toxins such as microcystin. We hypothesize that microcystins serve as metal ligands to control bioavailability and toxicity of ambient metals. Since ultraviolet radiation (UVR) promotes photo-oxidation of organic metal ligands and influences trace metal bioavailability, the present study aimed to investigate the influence of UVR, Cu, and Zn on specific growth rates, biomass, photosynthetic capacity, and microcystin content in Microcystis aeruginosa. Two toxigenic strains of Microcystis were cultivated using either Lake Erie filtered water or a chemically defined medium, with realistic concentrations of Cu and Zn combined with natural or artificial UVR exposure. Cu was more toxic than Zn on the basis of free ion concentration of trace metals in synthetic medium, although in Lake Erie water total added Zn (10 nM) or Zn plus Cu (10 nM) had a more detrimental effect on biomass and specific growth rate. Natural UVR delivered at 25% ambient levels caused no decrease on the parameters measured (chlorophyll-a, photosynthetic rate), yet artificial levels of UVR (up to 5.9 μmol UVB photons m⁻² s⁻¹) negatively affected biomass and specific growth rate. Cellular levels of microcystin (per unit chlorophyll-a) were concomitant with specific growth rather than being triggered in response either of these stressors (UVR, Zn, and Cu) alone or in combination, in agreement with a purported constitutive production of microcystins.