Microbiology of Saturated Salt Solutions and Other Harsh Environments. II. Ribonucleotide Dependency in the Growth of a Salt-Habituated Penicillium notatum in Salt-Free Nutrient Media

A strain of Penicillium notatum has been found growing upon media containing saturated calcium acetate which upon passage through media containing saturated KC1 loses its ability to grow at normal rates in salt-free glucose-peptone medium. This salt-habituated form can be restored to a near-normal growth rate by adding yeast extract, RNA or RNA-hydrolysates, but not by DNA or its hydrolysate. The 4 RNA nucleotides in combination replace yeast extract whereas riboside and base combinations do not. Adenylic, guanylic or cytidilic acids, singly or in various combinations, are even more effective, but uridylic acid alone is not active at all. Ribonucleotide pairs including uridylic acid are less active, but 3 ribonucleotide combinations are highly effective, even in the presence of uridylic acid. Differential effects were evident in mycelial form and sporulation, as well as growth. In addition to a direct role in Penicillium RNA metabolism, the significance of ribonucleotides in energy metabolism or cell wall synthesis is mentioned as alternatives to explain their restorative effects.     

Microbiology of Saturated Salt Solutions and Other Harsh Environments. IV. New Observations of Ribonucleotide-Induced Recovery of KCl-Habituated Penicillium notatum

Salt-tolerant mutant Penicillium notatum sub-cultured in a glucose-peptone broth saturated with KCl shows continued attenuated growth when transferred to salt-free broth. Additional tests have shown E. coli S-RNA to be inferior to yeast RNA preparations, that base-free phosphate sources are inactive, but that nicotinamide adenine dinucleotide and flavine adenine dinucleotide are moderately active. All phosphate derivatives of adenine, cytosine and guanosine and inosine were active including 5'-polyphosphates, 3'(2')-monophosphates 5'-monophosphates, and adenine 3', 5'-cyclic monophosphate. Uracil derivatives were of low activity at best.Among base precursors, orotic acid was moderately active whereas imidazoles were not. The high activity of inosine 5'-phosphate a precursor of other purine nucleotides suggested that one mode of KCl action might involve a block in conversion of 4-amino-5-imidazole carboxamide ribonucleoside to the hypoxanthine nucleotide.     

Salt Tolerance of In Vitro Established Salt-Tolerant Rice Plants during Further Growth in Soil

In vitro salt tolerant rice plants established by step up treatment with 0.5, 1.0, 1.5 and 2.0 % NaCl at 3-week intervals were examined to determine whether they could grow in potted paddy soil containing 0, 0.55 or 0.75 % NaCl till harvesting. All the control plants were necrotic by the 4^th week in the culture. At the 10^th week of culture, 100 % of the salt-tolerant plants subjected to 0 or 0.55 % NaCl survived, and 78 % of the plants at 0.75 % NaCl. The Na⁺ and Cl^– contents in the leaves of salt-tolerant plants grown at 0.55 and 0.75 % NaCl were about 4 times of those without NaCl. The ion contents in non-tolerant plants and seedling plants were 10 to 12 times of those in 0 % NaCl treatment. One of the hypotheses to explain the present data is that the in vitro step up salt selection induces the capability to maintain no lethal concentration of NaCl in the leaves.     

Evidence for two distinct intracellular pools of inorganic sulfate in Penicillium notatum.

A strain of Penicillium notatum unable to metabolize inorganic sulfate can accumulate sulfate internally to an apparent equilibrium concentration 10(5) greater than that remaining in the medium. The apparent Keq is near constant at all initial external sulfate concentrations below that which would eventually exceed the internal capacity of the cells. Under equilibrium conditions of zero net flux, external 35SO42- exchanges with internal, unlabeled SO42- at a rate consistent with the kinetic constants with the sulfate transport system. Efflux experiments demonstrated that sulfate occupies two distinct intracellular pools. Pool 1 is characterized by the rapid release of 35SO42- when the suspension of preloaded cells is adjusted to 10 mM azide at pH 8.4 (t 1/2, 0.38 min). 35SO42- in pool 1 also rapidly exchanges with unlabeled medium sulfate. Pool 2 is characterized by the slow release of 35SO42- induced by azide at pH 8.4 or unlabeled sulfate (t 1/2, 32 to 49 min). Early in the 35SO42- accumulation process, up to 78% of the total transported substrate is found in pool 1. At equilibrium, pool 1 accounts for only about 2% of the total accumulated 35SO42-. The kinetics of 35SO42- accumulation is consistent with the following sequential process: medium----pool 1----pool 2. Monensin (33 microns) accelerates the transfer of 35SO42- from pool 1 to pool 2. Valinomycin (0.2 microM) and tetraphenylboron- (1 mM) retard the transfer of 35SO42- from pool 1 to pool 2. At the concentrations used, neither of the ionophores nor tetraphenylboron- affect total 35SO42- uptake. Pool 2 may reside in a vacuole or other intracellular organelle. A model for the transfer of sulfate from pool 1 to pool 2 is presented.     

Growth Potential of Halophilic Bacteria Isolated from Solar Salt Environments: Carbon Sources and Salt Requirements

Eighteen strains of extremely halophilic bacteria and three strains of moderately halophilic bacteria were isolated from four different solar salt environments. Growth tests on carbohydrates, low-molecular-weight carboxylic acids, and complex medium demonstrated that the moderate halophiles and strains of the extreme halophiles Haloarcula and Halococcus grew on most of the substrates tested. Among the Halobacterium isolates were several metabolic groups: strains that grew on a broad range of substrates and strains that were essentially confined to either amino acid (peptone) or carbohydrate oxidation. One strain (WS-4) only grew well on pyruvate and acetate. Most strains of extreme halophiles grew by anaerobic fermentation and possibly by nitrate reduction. Tests of growth potential in natural saltern brines demonstrated that none of the halobacteria grew well in brines which harbor the densest populations of these bacteria in solar salterns. All grew best in brines which were unsaturated with NaCl. The high concentrations of Na⁺ and Mg²⁺ found in saltern crystallizer brines limited bacterial growth, but the concentrations of K⁺ found in these brines had little effect. MgSO₄ was relatively more inhibitory to the extreme halophiles than was MgCl₂, but the reverse was true for the moderate halophiles.     

Kinetics of sulfate transport by Penicillium notatum. Interactions of sulfate, protons, and calcium.

The active transport of inorganic sulfate by an ATP sulfurylase-negative strain of Penicillium notatum is promoted by H+ ions and metal ions (divalent metal ions being more effective than monovalent metal ions). Initial velocity studies suggest that H+ and SO4(2-) add to the carrier in an ordered sequence (H+ before SO4(2-)), with H+ at equilibrium with free carrier and carrier-H+ complex. The linear reciprocal plots and replots suggest a 1:1 stoichiometry between H+ and SO4(2-). Ca2+ and other divalent metal ions stimulate sulfate transport markedly in buffered suspensions of low ionic strength. The kinetics of the Ca2+/SO4(2-) interaction suggest that Ca2+ (like H+) adds to the carrier before SO4(2-) and is at equilibrium with free carrier and carrier-Ca2+ complex. The linear reciprocal plots and replots indicate a 1:1 stoichiometry between Ca2+ and SO4(2-). Thus the fully loaded carrier-SO4(2-) -Ca2+ -H+ complex has a net positive charge relative to that of the free carrier, a fact consistent with the chemiosmotic hypothesis of membrane transport. The kinetics of the H+/Ca2+ interaction point to a random A-B (rapid equilibrium), ordered C sequence with A = H+, B = Ca2+, and C = SO4(2-). Selenate (an alternate substrate competitive with sulfate) is an uncompetitive inhibitor with respect to Ca2+, in agreement with the suggested mechanism. Internal charge balance is not accomplished by a stoichiometric coaccumulation of Ca2+ and SO4(2-). Sulfate transport does, however, promote 45Ca2+ uptake. A significant fraction of the added Ca2+ is bound by the mycelial surface. Binding is extremely rapid, but reversible.     

Brassinosteroids Regulate Growth in Plants Under Stressful Environments and Crosstalk with Other Potential Phytohormones

Brassinosteroids (BRs) are an important group of plant steroidal hormones that are actively involved in a myriad of key growth and developmental processes from germination to senescence. Moreover, BRs are known for their effective role in alleviation of stress-induced changes in normal metabolism via the activation of different tolerance mechanisms. Efforts to improve plant growth through exogenous application of BRs (through different modes such as foliar spray, presowing seed treatment, or through root growing medium) have gained considerable ground world over. It has been widely demonstrated that the exogenous application of BRs to stressed plants imparts the stress tolerance mechanisms. In BR-induced regulation of physio-biochemical processes in plants, interaction (crosstalk) of BRs with other phytohormones has been reported. This crosstalk may fine-tune the effective roles of other hormones in regulating stress tolerance. The multifaceted role of BRs consolidated so far has reflected their immense potential to help plants in counteracting the stress-induced changes. The effects of introgression and up- and down-regulation of BR-related genes reported so far to improve crop productivity have been presented here. Strong evidence exists that BRs are involved in signal transduction particularly in the regulation of the mitogen-activated protein kinase (MAPK) cascade, which in turn is involved in controlled development, cell death, and the perception of pathogen-associated molecular pattern (PAMP) signaling. How far BRs are involved in signal transduction pathways operative under stressful environments has also been comprehensively discussed in this review.     

Studies on a phospholipase B from Penicillium notatum. Purification, properties, and mode of action.

1. Phospholipase B which hydrolyzes both the acyl ester bonds of diacylphospholipids (diacyl-hydrolase) and the acyl ester bond of monoacylphospholipids or lysophospholipids, [monoacyl-hydrolase or lysophospholipase, EC 3.1.1.5] was purified from Penicillium notatum about 2000-fold over the crude extract. The final preparation was homogeneous on disc electrophoresis. The apparent molecular weight, determined by gel filtration on Sephadex G-200, was about 116,000. The isoelectric point was pH 4.0. 2. The purified enzyme was a glycoprotein. The carbohydrate content was approximately 30%, consisting of mannose, glucose, and glucosamine. The amino acid composition was also determined. 3. The ratio of monoacyl-hydrolase to diacyl-hydrolase activities was influenced by the physical state of the substrate in the assay system. It was about 1 : 1 or 100 : 1 in the presence of absence of Triton X-100, respectively, and the latter value remained constant throughout the purification procedures. 4. Both enzyme activities had the same pH optimum, 4.0, and were heat-labile. None of the metals tested had any effect on either activity except for Fe2+ and Fe3+. Diisopropyl fluorophosphate at relatively high concentrations completely inhibited both enzyme activities. 5. The Michaelis-Menten constants (Km) of the enzyme for egg lecithin were about 1.5 and 25 mM in the absence and presence of Triton X-100, respectively. The Km value for dicaproyllecithin was 9.8 mM in the absence of Triton X-100. 6. Using a mixture of 1-[14C]stearoyl-lecithin and 2-[14C]oleoyl-lecithin in the presence of Triton X-100 as a substrate, it was found that the P. notatum phospholipase B attacked the acyl ester bonds sequentially, first the 2-acyl and then 1-acyl groups.     

Effects of Harsh and Unpredictable Environments in Adolescence on Development of Life History Strategies

The National Longitudinal Study of Adolescent Health data were used to test predictions from life history theory. We hypothesized that (1) in young adulthood an emerging life history strategy would exist as a common factor underlying many life history traits (e.g., health, relationship stability, economic success), (2) both environmental harshness and unpredictability would account for unique variance in expression of adolescent and young adult life history strategies, and (3) adolescent life history traits would predict young adult life history strategy. These predictions were supported. The current findings suggest that the environmental parameters of harshness and unpredictability have concurrent effects on life history development in adolescence, as well as longitudinal effects into young adulthood. In addition, life history traits appear to be stable across developmental time from adolescence into young adulthood.

The Sporulation of Penicillium notatum Westling in Submerged Liquid Culture: II. THE INITIAL SPORULATION PHASE

The initial sporulation time of Penicillium notatum Westlinggrown in shaken submerged culture inoculated with spores orvegetative mycelia is inversely proportional to the logarithmof the inoculum load, with a minimum time of 17.5 hours in theformer and 8 hours in the latter cultures. The development ofcultures is divisible into two stages. Firstly, there is a periodof vegetative growth, the duration of which depends on the inoculumload, after which the culture can be described as mature. Calciumis not required for the development of this maturity. Secondly,the cultures when mature develop phialides and spores if calciumis added to the medium. The development of phialides and thefirst spores takes 6 hours from the time of adding calcium andthis period is not influenced by the inoculum load of the culture.The medium from a mature culture promoted more rapid sporulationof vegetative mycelia placed in it than did a fresh medium,indicating the presence of a sporulation factor(s) in maturemedium. Similar activity was also demonstrated in media whichhad supported growth of any one of five other Penicillium speciesor Aspergillus niger. The nature of the sporulation factor isso far unknown.     

Staphylococci in Competition: III. Influence of pH and Salt on Staphylococcal Growth in Mixed Populations

Previous results showed definite repressive effects on the growth of staphylococci in mixed cultures due to the competitive growth of psychrophilic saprophytes. This study was continued, and the influence of other environmental factors, pH and salt, on the competition between staphylococci and saprophytes was investigated. Initial pH values varied from 5 to 9. At the extremes of the pH range, staphylococci failed to grow, while the saprophytes grew under all of the conditions tested. At pH 5, the growth curves for the saprophytes were markedly altered from those obtained at neutral pH. The lag phases were greatly lengthened at and below 20 C, but normal numbers of saprophytes were reached in the stationary phase. At pH 6 and 8, staphylococcal growth showed the same inhibition observed at pH 7, at and below 20 C; normal multiplication was observed above this temperature, but with accelerated death phases. Thus, pH did not primarily effect staphylococcal growth through its influence on saprophyte growth and competition, but rather directly affected the growth of Staphylococcus cultures. Salt concentrations from 3.5 to 9.5% were investigated for influence on staphylococcal growth in mixed populations. Above 3.5% salt, staphylococcal inhibition at and above 20 C was not as marked as in the controls, although normal numbers were never reached. The saprophytes were increasingly inhibited, and their lag phases materially lengthened as salt concentration was increased. Salt acted directly on the Staphylococcus population and also, by repressing saprophyte growth, decreased competition, which allowed the staphylococci to grow.