Endophyte differentiation inCasuarina actinorhizae

Summary This is an ultrastructural study of development of infected cells in nitrogen fixing root nodules ofCasuarina spp. While several aspects of development are similar to those found in many other actinorhizae, unusual aspects of development of the host cell and differentiation of the endophyte inCasuarina are correlated with unusual changes in the wall of the infected cell. Instead of vesicles the endophyte forms atypical hyphae in mature infected cells. These unusual hyphal forms are termed intracellular hyphae. Intracellular hyphae are nonseptate hyphae which originate and terminate within the same host cell, and have a varying diameter and a multidirectional growth and branching pattern. A laminate surface layer previously undescribed on hyphae ofFrankia is a feature common to mostCasuarina endophytic hyphae and is probably similar chemically to the laminae comprising the multilamellate envelope of endophytic vesicles in other actinorhizae.This paper is Florida Agricultural Experiment Station Journal Series No. 7350.     

MPphiWR-2, a cryptic phage associated withMicromonospora purpurea ATCC 15835

Summary A previously undescribed cryptic phage was found associated withMicromonospora purpurea ATCC 15835.     

Effects of the detergent Tween 80 on Thermomonospora curvata

Tween 80 (0.1%, v/v) added to Thermomonospora curvata growing in minimal medium caused a transient lowering of the dry cell mass, decreased the optimal growth temperature of the thermophile from 62 to 54°C, and increased extracellular esterase activity. Cells grown in the presence of Tween 80 had decreased concentrations of branched chain fatty acids and increased concentrations of oleic acid. The detergent removed surface protuberances from mycelia and increased the liberation of enzymes active against crystalline cellulose, but did not stimulate liberation of enzymes active against carboxymethylcellulose, starch or pectin.     

Carbon metabolism of Frankia spp. in root nodules of Alnus glutinosa and Hippophaë rhamnoides

The occurrence and localization of enzymes involved in glycolysis, tricarboxylic acid cycle and glyoxylate cycle in root nodules of Alnus glutinosa (L.) Vill. and Hippophaë rhamnoides L. ssp. rhamnoides were studied. The following enzymes, catalyzing reversible steps in the glycolysis, were found in both the endophyte Frankia spp. and the plant cytosol of Alnus nodules: fructose-1,6-diphosphate aldolase, glyceralde-hyde-3-phosphate dehydrogenase, phosphoglycerate kinase and enolase. The enzymes catalyzing irreversible steps in glycolysis, viz. hexokinase and pyruvate kinase, were detectable only in the plant cytosol. Similar results were obtained with nodule homogenates of Hippophaë. This indicates the absence of a complete glycolysis in the endophyte. Vesicle clusters of the nodule endophyte of Alnus contained various dehydrogenases of the tricarboxylic acid cycle and showed activity of glutamate oxaloacetate transaminase. Respiration studies showed that vesicle clusters take up oxygen when supplied with NAD, glutamate and malate together. No oxygen uptake was found when any of these compounds was omitted. Vesicle clusters from both Alnus and Hippophaë nodules showed no detectable activity of the glyoxylate cycle enzymes isocitrate lyase and malate synthase. Since these enzymes are known to be present in Frankia Avcll, when grown in a medium with Tween 80 as carbon source, it is suggested that the glyoxylate cycle enzymes are repressed in the root-nodule symbioses.     

Polygalacturonate lyase production byThermomonospora curvata on protein-extracted lucerne fiber

Summary Protein-extracted lucerne fiber was used as carbon and energy source for production of extracellular polygalacturonate lyase byThermomonospora curvata. The optimal fiber concentration was 1.5% (w/v); peal lyase activity in culture fluid occurred after 3 days growth at 53°C. During that time, lyase biosynthesis was controlled through induction; production was accelerated by adding small amounts of pectin or by grinding the fiber to 40-mesh particle size to release more inducer. After 3 days growth, lyase activity decreased; inactivation of the enzyme was delayed by the presence of 1 mM Ca or by inhibition of serine proteases with 0.05 mM phenylmethylsulfonyl fluoride. The molecular weight of the lyase produced during growth on the fiber was 35 kDa compared to 56 kDa for the enzyme produced on pure pectin. TheK _m of the 35-kDa form was 0.54% pectin compared to 0.06% for the 56-kDa form. The smaller form was rapidly inactivated at 60°C, the optimal temperature for activity of the larger form.     

Modulation of glycogen and trehalose levels in Micromonospora echinospora (ATCC 15837)

The growth of Micromonospora echinospora was studied in high and low C/N ratio medium using both batch and continuous culture. Asparagine was consumed rapidly in batch cultures where it served as both a nitrogen and carbon source. Glucose consumption was low suggesting that asparagine functions as the major carbon source under these conditions. The effect of nutrient limitation on the accumulation of storage carbohydrate in batch culture revealed an intimate association between nitrogen limitation and the accumulation of carbonaceous reserves. This study revealed that glycogen constituted the major carbohydrate reserve associated with the onset of sporulation. Intracellular trehalose levels were found to be relatively low and may have been affected by the availability of carbon. Continuous culture studies revealed a correlation between glycogen accumulation and increasing growth rate. It was also found that elevated cellular ATP levels correlated with the increase in glycogen, and reduced glycolytic activity. At the higher growth rates cellular ATP levels were elevated and coincided with reduced activity of the key glycolytic enzyme, phosphofructokinase, suggesting that glycogen can act as a convenient energy reservoir when excess carbon flux dictates.     

一株南海红树林底泥抗真菌放线菌(No.H74-18)的化学成分研究

对我国南海红树林底泥中分离的一株放线菌(No.H74-18)的发酵菌丝体采用95%乙醇提取,并对具有抗真菌活性的乙酸乙酯部位进行研究,通过硅胶开放柱色谱分离得到了一个结晶样品。通过制备型反相高效液相色谱分离,从此结晶样品中分离纯化出3个化合物,经NMR、MS等光谱学方法分别鉴定为抗霉素A1(1)、抗霉素A2a(2)、抗霉素A3(3),这些化合物都是抗霉素类化合物。采用LC-MS联用技术分析了此结晶样品中存在的抗霉素类的可能组份。     

The structural analysis and the role of calcium binding site for thermal stability in mannanase

Mannanase is an important enzyme involved in the degradation of mannan, production of bioactive oligosaccharides, and biobleaching of kraft pulp. Mannanase must be thermostable for use in industrial applications. In a previous study, we found that the thermal stability of mannanase from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) is enhanced by calcium. Here, we investigated the relationship between the three-dimensional structure and primary sequence to identify the putative calcium-binding site. The results of site-directed mutagenesis experiments indicated that Asp-285, Glu-286, and Asp-287 of StMan (StDEDAAAdC) and Asp-264, Glu-265, and Asp-266 of TfMan (TfDEDAAAdC) were the key residues for calcium binding affinity. Isothermal titration calorimetry revealed that the catalytic domain of StMan and TfMan (StMandC and TfMandC, respectively) bound calcium with a K_a of 3.02 × 10⁴ M⁻¹ and 1.52 × 10⁴ M⁻¹, respectively, both with stoichiometry consistent with one calcium-binding site per molecule of enzyme. Non-calcium-binding mutants (StDEDAAAdC and TfDEDAAAdC) did not show any calorimetric change. From the primary structure alignment of several mannanases, the calcium-binding site was found to be highly conserved in GH5 bacterial mannanases. This is the first study indicating enhanced thermal stability of GH5 bacterial mannanases by calcium binding.     

A new actinomycete from a Guadalupian vertebrate coprolite from Brazil

Coprolites (fossil feces) are important sources of evidence of ancient food webs and ecosystems. Actinomycetes are a fundamental component in the decay of organic matter, and serve as catalysts for nutrient cycles. Recently, gas vesicles filled with numerous verrucose colonies of substrate mycelium of an actinomycete were discovered inside a fossilized spiral amphipolar fish coprolite recovered from mid–Permian deposits of Brazil. These colonies are composed of masses of substrate hyphae, some of which are undergoing segmentation. Arising from the colonies are chains of spores separated by narrow, elongate connectives. The fossil actinomycete is described below as Palaeostromatus diairetus gen. et sp. nov. and represents the oldest known actinomycete associated with vertebrate deposits. Since the colonies occur only inside the coprolite, either Palaeostromatus diairetus gen. et sp. nov. was part of the gut flora or it was acquired from a food source. The only other remains in the coprolite are eighteen paleoniscoid fish scales, which suggests that the producer was a carnivorous/omnivorous fish. This is the oldest record of a direct interaction between vertebrates and actinomycetes.