Extraction and preliminary characterizaion of a mycobacteriolytic preparation (stazyme) from aStaphylococcus strain: mycobactericidal activity and its use in rapid extraction of mycobacterial DNA

The clear culture filtrate from 1-L culture of a laboratory contaminant ofStaphylococcus (coagulase^– strain, designated Clavelis) was filtered, concentrated, dialyzed, and the proteins were precipitated. The precipitate was washed, concentrated, and aliquoted (about 4 mg of total proteins/ml, designated as Stazyme). The crude preparation was subjected to gel filtration on Sephadex G-75, and the fractions were screened for their lytic ability againstMycobacterium smegmatis. Native proteins in the stazyme were electrophoretically separated, electroeluted, and their lytic activity againstM. smegmatis was compared with parallel controls (partially purified proteins extracted from the same quantity of the uninoculated bacterial growth medium). Only stazyme preparations caused significant growth inhibition ofM. smegmatis, M. chelonae, M. xenopi, M. tuberculosis, andM. kansasii. Stazyme essentially possessed a lytic activity measured with purifiedM. smegmatis andMicrococcus lysodeikticus cell walls and showed high bactericidal activity againstM. smegmatis, M. chelonae, andM. tuberculosis. It was also able to rapidly lyse intactM. smegmatis organisms, permitting significant yield of mycobacterial DNA.     

Activity of dihydrofolate reductase in the mollicutesAcholeplasma laidlawii andMycoplasma gallisepticum and their susceptibility to antifolates

Mycoplasma gallisepticum andAcholeplasma laidlawii were found to possess dihydrofolate reductases exhibiting similar specific activities and kinetics, with values in the range of those reported for other microorganisms. The apparent Km values for dihydrofolate in enzymes ofM. gallisepticum andA. laidlawii are 7.95±0.13 and 7.50±0.11 M, and for NADPNH 8.46±0.25 and 9.32±0.18 M, respectively.M. gallisepticum is 3300-fold more resistant to methotrexate than isA. laidlawii; concentrations causing 50% inhibition were 200.00 and 0.06 M, respectively. This is in contrast to almost the same sensitivity to that drug exhibited by the dihydrofolate reductases of both microorganisms.M. gallisepticum is also 3600-fold more resistant to trimethoprim than isA. laidlawii, and the concentrations for 50% inhibition of growth were 1800.0 and 0.5 M, respectively. The high resistance was found to be due partially to a 130-fold lower affinity of the target enzyme for this antifolate, but another mechanism, presumably impaired transport, must also be involved. This is the first report of dihydrofolate reductase activity in Mollicutes.     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Microtubules and actin filaments co-localize in pollen tubes ofNicotiana tabacum L. andLilium longiflorum Thunb.

Summary Double labeling with fluorescent probes showed that in the cortical cytoplasm of pollen tubes ofNicotiana tabacum andLilium longiflorum, the microtubules and actin filaments co-localize for the most part. They displayed complex net-axial or helical distributions. They structural association of microtubules and actin filaments implies a functional relationship with respect to organelle movement and/or the organization of the cortical cytoplasm and cell surface.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetra-acetic acid - FITC fluoresceine iso-thiocyanate     

Fine structure ofMicrococcus denitrificans andM. halodenitrificans in relation to their taxonomy

A study of ultrathin sections ofMicrococcus denitrificans andM.halodenitrificans has shown similar cell structures. The cell wall consists of several layers corresponding to those of the cell wall of gram-negative bacteria. The thickness of the cell wall is 250 – 350 Å; that of the cytoplasmic membrane 70 Å. The cytoplasm in both species contains ribosomes and inclusions of polymetaphosphate. Comparison with ultrathin sections ofThiobacillus novellus shows too much difference to consider the former two species to be identical with the latter one. The taxonomic position ofM.denitrificans andM.halodenitrificans is discussed.Deceased 3 July 1967.     

Life-history consequences of resource allocation of two bdelloid rotifer species

Two bdelloid species,Macrotrachela quadricornifera (aquatic species) andPhilodina vorax (terrestrial moss species), with similar survival but different age-specific fecundity schedules, were analyzed daily to determine growth rates and the volume invested in reproduction. The two species had similar growth patterns and started reproduction while still growing. In both, the size at maturity was independent of age.M. quadricornifera resumed growth after reaching a size plateau when reproduction was over, whileP. vorax continued to reproduce until death. Although the net reproductive rate ofP. vorax was consistently lower than that ofM. quadricornifera, the same percent of adult volume was invested in reproduction over its life time because its eggs were relatively bigger. The difference in reproductive rates is probably related to different partitioning of equal amounts of relative biomass: more small eggs for the aquatic rotifer vs. fewer big eggs for the terrestrial rotifer. Egg size might be related to the selective pressures of the environments.     

A submicroscopical study of leaves of alfalfa,Basil, and tobacco experimentally infected with lucerne mosaic virus

Summary The leaves of three species of plants (Medicago sativa L.,Ocymum basilicum L., andNicotiana tabacum L., cv.Samsun), previously infected with lucerne mosaic virus (LMV), were studied in the electron microscope. In the leaves ofM. sativa andO. basilicum the infection was systemic, while in the leaves ofN. tabacum the infection was local. In all the three species LMV particles were always detected only in the cytoplasm. Apart from the presence of virus particles in the cytoplasm, no ultrastructural alterations were found inM. sativa andN. tabacum. InO. basilicum, the green leaf areas did not show any prominent alterations, while the yellow leaf areas showed marked alterations of the chloroplasts. In these, the lamellar system was scarcely developed, and the few thylakoids tended to fragment, curl and disappear. In the chloroplast stroma, there was an abnormal development of filamentous structures, resembling the stromacentre.Publication n. 53 of Centro Nazionale Virus Vegetali, Consiglio Nazionale delle Ricerche, sections I and II.     

Reduction of chromate by bacteria isolated from the cooling water of an electricity generating station

Summary Chromate-reducing bacteria were isolated from the cooling water of an electricity generating station where reduction of chromate had caused blockage of pipes by precipitation of chromium(III) oxide. Isolates identified included the generaAlcaligenes, Vibrio, Bacillus, Micrococcus, Staphylococcus andCorynebacterium. Isolate VMC-2 with the highest chromate-reducing activity was tentatively identified asComanonas testosteroni. The concentration of added chromate (K₂CrO₄, 20 M)_decreased by 95% during 45 min incubation with whole cells of VMC-2. In comparison, two Fe(III)-reducing isolates,Vibrio metschnikovii andAeromonas hydrophila, from lake sediments, showed similarly high chromate-reducing activities, and were able to reduce 99% of added chromate (20 M) in 45 min. Moderate Cr(VI)-reducers included strains ofBacillus, Vibrio andCorynebacterium. Micrococcus andStaphylococcus did not reduce Cr(VI). Sulfate (0.5 and 1.0 mM) inhibited the reduction of chromate by VMC-2 suggesting competition between the two oxyanions. Chromate-reducing activity was located in the soluble fraction of this isolate. The intermediacy of Cr(V)_in the reduction of chromate was confirmed by EPR spectroscopy. The bactericidal activity of hypochlorite towards isolate VMC-2 was determined.     

Comparative study of the hemolysins ofTreponema hyodysenteriae andTreponema innocens

The nucleic acid-bound hemolysins from two strains ofTreponema hyodysenteriae (-hemolytic) and two strains ofT. innocens (weakly -hemolytic) were purified and characterized. All four hemolysins shared similar molecular weights, stability to oxygen, and pH and heat lability. The hemolysins were inactivated by pronase and lipase and inhibited by trypan blue. Although lacking detectable proteolytic or phospholipase activity,T. hyodysenteriae andT. innocens hemolysins differ in their sensitivity to phospholipids; the former was insensitive to cardiolipin, which completely inhibited the latter. Furthermore, both groups of hemolysins differed in their hemolytic spectra and in their specific activities on rabbit erythrocytes. The results showed that the observed hemolytic patterns ofT. hyodysenteriae andT. innocens on blood agar were due to different hemolysins.     

Fluorescence microscopy of plastid nucleoids and a survey of nuclease C in higher plants with respect to mode of plastid inheritance

Summary Plastid nucleoids (pt nucleoids) were observed during pollen formation, or in generative cells of mature pollen grains using fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI). Nuclease C activity was surveyed using SDS-PAGE and agarose gel nuclease assay methods. InMirabilis jalapa, pt nucleoids were observed both in pollen mother cells and the monocellular pollen grains after meiosis, followed by the complete disappearance both in the generative and vegetative cells at the bicellular pollen grain stage. This observation is a direct evidence of maternal plastid inheritance. By contrast, in the generative cells of mature pollen grains fromRhododendron kaempferi, Zygocactus truncatus, Oenothera laciniata, andO. speciosa, pt nucleoids were clearly observed. Thus cytological evidence convinces the mode of biparental plastid inheritance. Nuclease C activity was clearly detected both in the stamen and pistil ofM. jalapa. InR. kaempferi low nuclease C activity was detected in both organs, but the activity in the stamen was much less than in the pistil. InZ. truncatus, O. laciniata, andO. speciosa, the activities were difficult to detect in both organs. These results suggest a significant role of nuclease C for the digestion of pt nucleoids in the generative cells.Abbreviations EGTA ethylene-glycol-bis-(2-aminoethyl ether)-N, N, N, N-tetraacetic acid - DAPI 4,6-diamidino-2-phenylindole - Nuclease C Ca²⁺ dependent nuclease - SDS-PAGE SDS-polyacrylamide gel electrophoresis - pt nucleoids plastid nucleoids     

Transmission of Lyme disease spirochetes (Borrelia burgdorferi)

The field and laboratory evidence incriminating nymphalIxodes dammini as the main vectors ofBorrelia burgdorferi is substantial. Furthermore, other members of theIxodes (Ixodes) ricinus complex, includingI. ricinus, I. persulcatus, I. pacificus, andI. scapularis, are competent vectors of the Lyme disease spirochete. Although ticks in other genera are also naturally infected withB. burgdorferi, experimental evidence suggests thatAmblyomma andDermacentor ticks are inefficient vectors of these spirochetes. Current research on the kinetics ofB. burgdorferi growth within ticks demonstrates that Lyme disease spirochetes are dramatically influenced by physiological events during the tick's life-cycle.     

Interactions between freshwater bacteria andAnkistrodesmus braunii in batch and continuous culture

Batch and continuous cultures ofAnkistrodesmus braunii were established in an inorganic medium with growth rate limited by P. In batch culture, inoculation of lake water bacterial isolates ofPseudomonas sp. andFlavobacterium sp. showed that thePseudomonas isolate was capable of more rapid growth on algal exudates of lytic products than was theFlavobacterium isolate. When inoculated singly into a continuous culture (D=0.267 day^–1; P level, 2M), theFlavobacterium isolate initially caused a decrease in the population density of the alga, but then steady states for both organisms were obtained. ThePseudomonas isolate under the same conditions caused a rapid washout of the algal culture, and steady-state conditions were never obtained. When thePseudomonas isolate was added to the two-member, steady-state system ofA. braunii andFlavobacterium, the algal population again washed out of the vessel, followed by theFlavobacterium and then thePseudomonas isolate. A transient increase in the P concentration to 200M in the culture vessel caused the low algal population level to increase, followed by increases in the bacterial isolates when the algal population was high enough to supply the required organic carbon source. The system demonstrated that competition for P between the alga and the bacteria can occur, and the results were dependent on the algal and bacterial relative growth rates. The bacterial growth rates were limited initially by organic substrates produced by the alga, and the different bacterial isolates competed for these substrates.     

Developmental times and oviposition rates of predatory mitesTyphlodromus pyri andAmblyseius andersoni (Acari: Phytoseiidae) reared on different foods

The developmental times and the reproduction of two resistant Italian strains ofTyphlodromus pyri Scheuten andAmblyseius andersoni (Chant) were studied in the laboratory by rearing them on the spider mitesPanonychus ulmi (Koch) andEotetranychus carpini (Oud.), on the eriophyidColomerus vitis (Pgst.) and on pollen ofMesembryanthemum criniflorum. The response ofT. pyri andA. andersoni females to a spider mite supply (P. ulmi orE. carpini) of 4, 8 and 16 adult female prey per female predator per day was also studied.Development ofT. pyri onE. carpini andC. vitis required a shorter period than onM. criniflorum pollen, while intermediate values were recorded forP. ulmi. When the highest number of prey was offered, the influence of different foods on oviposition rates ofT. pyri was not significant. An increase in spider mite supply favoured a shorter pre-oviposition period and higher oviposition rates.Development ofA. andersoni was faster on pollen than on spider mites, while intermediate values were found concerningC. vitis. Differences statistically significant were recorded for development onP. ulmi andC. vitis. Colomerus vitis proved to be the more suitable food in terms of oviposition. The oviposition rate decreased when feeding uponP. ulmi, but reached intermediate values onE. carpini andM. criniflorum. Increasing spider mite densities caused shorter pre-oviposition times and higher oviposition rates. Using a given number ofE. carpini females, rather than those ofP. ulmi, resulted in higher oviposition rates and shorter pre-oviposition times.For both predators, the results suggest a higher intrinsic rate of population increase onE. carpini orC. vitis than onP. ulmi.The research was supported by a grant from Regione Veneto (Lotta biologica ed integrata nel controllo di insetti ed acari dannosi).The general lines of the research have been planned by C. Duso. The authors contributed equally to the experimental work.     

Lamellate bodies in the nuclei of dragonfly (odonata) oocytes

Summary Concentrically lamellate spherical bodies have been observed in the oocyte nuclei of the anisopterous dragonflyCordulia aenea. In their ultrastructure, these bodies resemble the cortical granules reported from the cytoplasm of the sea urchin oocytes. The lamellate bodies ofCordulia are compared with other lamellate systems known to exist in nuclei. It is suggested that the nuclear lamellate spheres ofCordulia may be homologous with the somewhat less regular lamellate bodies described from maize microspores.The competent help of MissRiitta Lallukka, M. A., is acknowledged with many thanks. We are grateful to ProfessorsESko Suomalainen andAntti Telkkä for helpful suggestions. Grants for the study have been received from the National Research Council for Sciences, from the University of Helsinki and from the Emil Aaltonen Foundation.     

Rhizobium inoculation trials designed to support a tropical forage legume selection programme

Summary Three phases of Rhizobium inoculation trials were carried out as part of a programme to select forage legume germplasm adapted to acid, infertile Oxisols of tropical America. Firstly, a range of tropical forage legumes were evaluated for their response to N fertilization or inoculation with strains previously shown to be effective in Leonard jars, using cores of undisturbed soil or in the field at Carimagua, Meta, Colombia. In pure legume stands onlyCentrosema macrocarpum andC. pubescens showed increases in N yield due to both inoculation and N fertilization;C. brasilianum responded only to N fertilization;Zornia latifolia, Z. brasiliensis andStylosanthes capitata responded to neither treatment. Trials in cores and in grass-legume mixtures showed responses ofDesmodium ovalifolium, Pueraria phaseoloides andS. capitata to N fertilization but not to inoculation. In the second phase of experiments strains were screened in soil cores with 16 ecotypes ofDesmodium, Centrosema, Stylosanthes andPueraria spp. Significant increases in N yield due to inoculation occurred with at least one strain in all the legumes exceptS. guianensis tardio, and in some trials withS. capitata. In the third phase of trials the most effective strains were tested in the field. Significant response ofP. phaseoloides andC. macrocarpum to inoculation at two sites and in the second year after establishment were shown. Further screening trials and field trials at different sites are needed in order to provide better recommendations for inoculation of grazing trials being set up in the region under study.     

Ultrastruktur und Entwicklungsgeschichte des Pollenkitts vonEuphorbia cyparissias,E. palustris undMercurialis perennis (Euphorbiaceae)

Development, fine structure and distribution of pollenkitt is investigated inEuphorbia cyparissias, E. palustris, andMercurialis perennis. The predominantly anemophilousM. perennis produces a great amount of strictly homogeneous pollenkitt, which is deposited in the exine caves. In contrast to this and to all other angiosperms so far investigated, bothEuphorbia species produce large quantities of an extremely inhomogeneous and particular pollenkitt. Its ultrastructure is quite different, both during its development and after its deposition on the exine surface: Lipid particles with different electron density and size are wrapped in a strictly homogeneous electron transparent matrix. This can be considered as new and additional proof for the secondary entomophily ofEuphorbia.

     

Genetic interactions indicate a role for Mdg1p and the SH3 domain protein Bem1p in linking the G-protein mediated yeast pheromone signalling pathway to regulators of cell polarity

The pheromone signal in the yeastSaccharomyces cerevisiae is transmitted by the and subunits of the mating response G-protein. TheSTE20 gene, encoding a protein kinase required for pheromone signal transduction, has recently been identified in a genetic screen for high-gene-dosage suppressors of a partly defective G mutation. The same genetic screen identifiedBEM1, which encodes an SH3 domain protein required for polarized morphogenesis in response to pheromone, and a novel gene, designatedMDG1 (multicopy suppressor ofdefectiveG-protein). TheMDG1 gene was independently isolated in a search for multicopy suppressors of abem1 mutation. TheMDG1 gene encodes a predicted hydrophilic protein of 364 amino acids with a molecular weight of 41 kDa that has no homology with known proteins. A fusion of Mdg1p with the green fluorescent protein fromAequorea victoria localizes to the plasma membrane, suggesting that Mdg1p is an extrinsically bound membrane protein. Deletion ofMDG1 causes sterility in cells in which the wild-type G has been replaced by partly defective G derivatives but does not cause any other obvious phenotypes. The mating defect of cells deleted forSTE20 is partially suppressed by multiple copies ofBEM1 andCDC42, which encodes a small GTP-binding protein that binds to Ste20p and is necessary for the development of cell polarity. Elevated levels ofSTE20 andBEM1 are capable of suppressing a temperature-sensitive mutation inCDC42. This complex network of genetic interactions points to a role for Bem1p and Mdg1p in G-protein mediated signal transduction and indicates a functional linkage between components of the pheromone signalling pathway and regulators of cell polarity during yeast mating.     

Plant growth regulatory metabolites from novel actinomycetes

Metabolites from 796 isolates of aerobic actinomycetes were screened for plant growth regulatory properties using an algal bioassay. These included 266 isolates ofStreptomyces, 28 unidentified actinomycetes, and 502 isolates of novel actinomycetes represented by 18 genera. Algal growth inhibition of 30% was observed with 60 isolates, 37 of which belonged to the genusStreptomyces. Among other inhibitors were 8 isolates ofActinomadura, 6 ofActinoplanes, 2 each of the generaThermomonospora, Streptoverticillium, andPromicromonospora, and 3 unidentified. Metabolites from 70 isolates promoted algal growth by 20%. These included 13 isolates ofMicromonospora, 11 ofStreptomyces, 6 ofNocardia, 5 ofActinomadura, and 4 each ofRhodococcus andThermomonospora. Sixteen unidentified isolates; 3 isolates ofPromicromonospora; 2 isolates each ofActinoplanes, Streptosporangium, andOerskovia; and 1 of Thermoactinomyces peptonophilus-like organism andSaccharomonospora viridis also promoted the algal growth by 20%. The plant growth inhibitory properties of 9 actinomycetes and the growth promoting properties of 6 were demonstrable during the secondary screening on higher plants using chemicals extracted from the culture broth. The metabolites fromMicromonospora, Nocardia, Rhodococcus, Streptosporangium, andOerskovia isolates were associated with plant growth promotion only; those fromStreptomyces were most frequently involved with the growth inhibition.This is Michigan Agriculture Experiment Station Journal Article No. 12191.     

Comparison of the effect of antibiotic substances on sclerotia of Mycosphaerella ligulicola and Colletotrichum coccodes

Summary Sclerotia ofColletotrichum coccodes tolerated much higher concentrations of actidione in agar than did sclerotia ofMycosphaerella ligulicola. With increase in concentration of the antibiotic sclerotia of both species took longer to germinate. Increased resistance of both species to actidione developed after growth of a single generation on media containing the antibiotic. Sclerotia ofC. coccodes survived 5 days immersion in a bacterial culture filtrate whereas scleroia ofM. ligulicola ceased to be viable after a similar period.Sclerotia ofC. coccodes andM. ligulicola exhibited strand and loose types of formation respectively. The degree of resistance of these sclerotia to antibiotic substances was correlated with both longevity in soil and type of formation, but, in general, there is unlikely to be a relationship between structure of the sclerotium and longevity.