Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus

A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was originally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide bialaphos as a secondary metabolite. Bialaphos contains phosphinothricin, an analogue of glutamate which is an inhibitor of glutamine synthetase. The bar gene product was purified and shown to be a modifying enzyme which acetylates phosphinothricin or demethylphosphinothricin but not bialaphos or glutamate. The bar gene was subcloned and its nucleotide sequence was determined. Interspecific transfer of this Streptomyces gene into Escherichia coli showed that it could be used as a selectable marker in other bacteria. In the accompanying paper, bar has been used to engineer herbicide-resistant plants.     

Photoheterotrophic growth of Physcomitrella patens

Physcomitrella patens is a model bryophyte representing an early land plant in the green plant lineage. This organism possesses many advantages as a model organism. Its genome has been sequenced, its predominant life cycle stage is the haploid gametophyte, it is readily transformable and it can integrate transformed DNA into its genome by homologous recombination. One limitation for the use of P. patens in photosynthesis research is its reported inability to grow photoheterotrophically, in the presence of sucrose and the Photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which prevents linear photosynthetic electron transport. In this communication we describe the facile isolation of a P. patens strain which can grow photoheterotrophically. Additionally, we have examined a number of photosynthetic parameters for this strain grown under photoautotrophic, mixotrophic (in the presence of sucrose) and photoheterotrophic conditions, as well as the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-inhibited state. The ability to grow P. patens photoheterotrophically should significantly facilitate its use in photosynthetic studies.     

A glutathione-S-transferase (TuGSTd05) associated with acaricide resistance in Tetranychus urticae directly metabolizes the complex II inhibitor cyflumetofen

Cyflumetofen is a recently introduced acaricide with a novel mode of action, acting as an inhibitor of complex II of mitochondrial electron transport chain. It is activated by hydrolysis and the resulting de-esterified metabolite is a much stronger inhibitor. Cyflumetofen represents a great addition for the control of mite species including Tetranychus urticae, a major agricultural pest, which has the ability to develop resistance to most classes of pesticides rapidly. A resistant strain (Tu008R) was recently described and synergism experiments pointed towards the involvement of GSTs. Here, we conducted genome-wide gene expression analysis, comparing Tu008R with its parental susceptible strain, and identified the delta GST TuGSTd05 as the prime resistance-conferring candidate. Docking analysis suggests that both cyflumetofen and its de-esterified metabolite are potential substrates for conjugation by TuGSTd05. Several amino acids were identified that might be involved in the interaction, with Y107 and N103 possibly having an important role. To further investigate interaction as well as the role of Y107 and N103 in vitro, we recombinantly expressed and kinetically characterized the wild type TuGSTd05, TuGSTd05 Y107F and TuGSTd05 N103L mutants. While cyflumetofen was not found to act as a strong inhibitor, the de-esterified metabolite showed strong affinity for TuGSTd05 (IC₅₀ = 4 μM), which could serve as a mechanism of rapid detoxification. Y107 and N103 might contribute to this interaction. HPLC-MS analysis provided solid indications that TuGSTd05 catalyzes the conjugation of ionized glutathione (GS⁻) to cyflumetofen and/or its de-esterified metabolite and the resulting metabolite and possible site of attack were identified.     

Metabolism of Dibenzo-p-Dioxin and Chlorinated Dibenzo-p- Dioxins by a Beijerinckia Species

Whole cells of the parent strain of Beijerinckia, grown with succinate and biphenyl, oxidized dibenzo-p-dioxin and several chlorinated dioxins. The rate of oxidation of the chlorinated dibenzo-p-dioxins decreased with an increasing degree of chlorine substitution. A mutant strain (B8/36) of Beijerinckia oxidized dibenzo-p-dioxin to cis-1,2-dihydroxy-1,2-dihydrodibenzo-p-dioxin. The mutant organism also oxidized two monochlorinated dibenzo-p-dioxins to cis-dihydrodiols. No metabolites were detected from two dichlorinated dibenzo-p-dioxins. Growth of the parent strain of Beijerinckia on succinate was inhibited after 4 h when 0.05% dibenzo-p-dioxin was present in the culture medium. Resting cell suspensions of the parent organism, previously grown with succinate and biphenyl, oxidized dibenzo-p-dioxin to a compound identified as 1,2-dihydroxydibenzo-p-dioxin. Further degradation of this metabolite was not detected, as the compound was found to be a potent mixed-type inhibitor of two ring-fission oxygenases present in this organism.     

Control of protein synthesis in brine shrimp embryos by repression of ribosomal activity

Undeveloped encysted embryos of the brine shrimp, Artemia salina, contain a large quantity of metabolically repressed 80S ribosomes. These ribosomes appear to be inactive or nonfunctional due to the presence of an inhibitor protein on their 60S subunit. During development the inhibitor is released or inactivated and the 80S ribosomes and their constituent subunits become fully functional in a poly(U)-directed protein-synthesizing system. The inefficiency of most 80S ribosomes from undeveloped Artemia embryos appears to be due to their inability to form stable complexes with poly(U) and phe-tRNA in the presence of elongation factor, EF-1. A potent inhibitor of protein synthesis has also been found in the 105,000g supernatant fraction from undeveloped Artemia embryos. The exact nature of this inhibitor has not been ascertained but it appears to be a heat-labile protein devoid of RNase and protease activity. It is not known whether this inhibitor is the same as that associated with 60S ribosomal subunits of undeveloped cyst ribosomes.     

Rhizomastix biflagellata sp. nov., a new amoeboflagellate of uncertain phylogenetic position isolated from frogs

The genus Rhizomastix contains five species of amoeboflagellates with a single anterior flagellum, which live as intestinal symbionts of insects and amphibians. Though established in 1911, Rhizomastix has been neglected for decades and its phylogenetic position is uncertain. This paper describes the morphology of the first cultivated strain of Rhizomastix. The organism was isolated from an argentine horned frog and differs from the known Rhizomastix species by the presence of biflagellate cells. The isolate is described as Rhizomastix biflagellata sp. nov. A possible relationship of Rhizomastix to Archamoebae is discussed.     

Environmental Attributes Influencing the Distribution of Burkholderia pseudomallei in Northern Australia

Factors responsible for the spatial and temporal clustering of Burkholderia pseudomallei in the environment remain to be elucidated. Whilst laboratory based experiments have been performed to analyse survival of the organism in various soil types, such approaches are strongly influenced by alterations to the soil micro ecology during soil sanitisation and translocation. During the monsoonal season in Townsville, Australia, B. pseudomallei is discharged from Castle Hill (an area with a very high soil prevalence of the organism) by groundwater seeps and is washed through a nearby area where intensive sampling in the dry season has been unable to detect the organism. We undertook environmental sampling and soil and plant characterisation in both areas to ascertain physiochemical and macro-floral differences between the two sites that may affect the prevalence of B. pseudomallei. In contrast to previous studies, the presence of B. pseudomallei was correlated with a low gravimetric water content and low nutrient availability (nitrogen and sulphur) and higher exchangeable potassium in soils favouring recovery. Relatively low levels of copper, iron and zinc favoured survival. The prevalence of the organism was found to be highest under the grasses Aristida sp. and Heteropogon contortus and to a lesser extent under Melinis repens. The findings of this study indicate that a greater variety of factors influence the endemicity of melioidosis than has previously been reported, and suggest that biogeographical boundaries to the organisms’ distribution involve complex interactions.     

Elutriation and Coulter Counts of Tetrahymena pyriformis Grown in Peanut and Cottonseed Meal Media

Growth of Tetrahymena pyriformis W has been used to evaluate nutritional quality of peanut and cottonseed meals. An efficient elutriation method is described for separating cells of this organism from particulate matter left in the substrate (enriched with basal medium) after 4 days of incubation. After elutriation the cells can be counted with a Coulter counter by using calibration procedures which are presented. Elutriation and Coulter counting provide a rapid and efficient method of measuring the growth response of T. pyriformis W. Utility of the method is demonstrated by agreement between Coulter counts and visual counts of the cells and by demonstration of a linear response of cell numbers to substrate nitrogen.     

Preservation of algal and higher plant ribosomal RNA integrity during extraction and electrophoretic quantitation

Isolation of high molecular weight ribosomal RNA from the wall-less alga Olisthodiscus luteus and the angiospermous plant Sauromatum guttatum is described. It has been found that a buffer which contains magnesium must be used to successfully isolate Olisthodiscus rRNA whereas the isolation of intact Sauromatum rRNA requires a buffer system containing a high amount of the chelator EDTA, Sauromatum but not Olisthodiseus extracts were contaminated with ribonuclease unless the inhibitor diethylpyrocarbonate was used during the ribonucleic acid extraction procedure. Nuclease levels were monitored by coincubating [³H]-labeled Escherichia coli ribosomal RNA with the experimental RNA samples. The effects of detergents on the isolation and quantitation of RNA are presented, and methods to avoid loss of highly thermolabile plant ribosomal RNA species are discussed.     

Inhibition of inosinate dehydrogenase by metabolites of 2-β-D-ribofuranosylthiazole-4-car☐amide

Addition of 2-β-D-ribofuranosyl-thiazole-4-car?amide (NSC 286193, RTC, T-CAR) to Chinese hamster ovary (CHO) cells in culture drastically reduced the level of IMP dehydrogenase activity in the cell sonicate. Dialysis of the sonicate removed this inhibition. NSC 286193 was phosphorylated to the 5′-monophosphate in vitro in the presence of ATP and Mg⁺⁺. The monophosphate was further converted to another compound in the presence of ATP and Mg⁺⁺ which has been found to be a potent inhibitor of IMP-dehydrogenase. This metabolite is tentatively identified as thiazole-4-car?amide adenine dinucleotide.     

Characterization of genes encoding topoisomerase IV of Mycoplasma genitalium

A type-II topoisomerase (Topo-IV) encoded by the parC and parE genes in Escherichia coli and Salmonella typhimurium is thought to be involvd in cell septation and in the decatenation of newly replicated chromosomes. We have identified parC and parE homologs in the pleomorphic, wall-less organism Mycoplasma genitalium. Since the mechanics of cell septation in conventional eubacterial species is believed to be mediated by cell-wall constituents, there is no clear understanding of what coordinates that process in wall-less species. The presence of par genes in this bacterium, which has the smallest genome of any free-living organism, suggests that Top-IV has been evolutionarily conserved because of an essential role in mediating cell division.     

Isolation and description of a non-motile,fusiform, stalked bacterium,a representatieve of a new genus

A single strain representing the fusiform group of caulobacters first described by Henrici and Johnson has been isolated from a freshwater pond. Like the genusCaulobacter this is a chemo-organotrophic bacterium that has one polar prostheca, a stalk in the sense that its apical holdfast permits the cell to attach to solid substrates. Fine structure studies reveal, however, that the prostheca of this organism contains typical cellular constituents, not the membranous material found in the stalks ofCaulobacter andAsticcacaulis. The organism also differs from the other caulobacters in having no motile stage and no dimorphic life cycle (both daughter cells are stalked at the time of division). Because only one strain has been isolated no nomenclatural proposals are made, but sufficient evidence is presented to indicate that this is a representative of a new genus of the Schizomycetes.     

A natural non-protein low molecular weight cAMP-dependent protein kinase inhibitor from the insect Ceratitis capitata: Isolation and preliminary characterization

Three protein kinase inhibitors have been detected and isolated by gel filtration chromatography from the dipterous Ceratitis capitata. Two of them were proteinaceous; the third one was resistant to proteolytic treatments and its molecular weight ranged from 1000 and 6000. This inhibitor was purified to thin-layer electrophoretic homogeneity and a preliminary analysis carried out to determine its composition showed that it is a complex molecule with a probable peptidyl-purine structure. This new inhibitor acts competitively with ATP on the cAMP-dependent protein kinase activity and has been also found to inhibit the adenylate cyclase system. This metabolite may be important in regulating cAMP mediated responses in the insect.     

Glycogen phosphorylase from Dictyostelium: a kinetic analysis by computer simulation.

A model of carbohydrate metabolism during differentiation in Dictyostelium discoideum has been used to investigate which enzyme kinetic mechanism(s) might be operative for glycogen phosphorylase in vivo. The model, which has been described previously, is capable of simulating experimentally observed changes in metabolite concentrations and fluxes during differentiation under both the standard starvation condition and in the presence of glucose (25 mM). The concentrations of saccharide end products of differentiation under these 2 conditions differ substantially.Glycogen phosphorylase is described in the model by a rapid equilibrium random bi bi mechanism and the effect of substituting 4 other kinetic mechanisms was examined. Each of these mechanisms in the model allows simulations compatible with the saccharide accumulation patterns found during differentiation in the absence of glucose. However, in the presence of glucose, only a reversible mechanism (random or ordered) is compatible with the experimental data. It is concluded that glycogen degradation in vivo is controlled by an enzyme catalyzing a reversible reaction, the rate of which is inversely related to the glucose-1-P concentration.     

Pseudomonas creosotenesis sp. n., a Creosote-tolerant Marine Bacterium

In a study of the marine biological environment in which creosoted pilings are located, a previously unreported species of bacteria was isolated. This species was detected on creosoted piling from 11 widely differing locations and was the predominant species of bacteria found on these piling. The new organism was identified as a gram-negative rod belonging to the genus Pseudomonas and has been named Pseudomonas creosotensis. It has been completely described by the standard morphological and biochemical tests.     

Sensitivity of Mixed Populations of Staphylococcus aureus and Escherichia coli to Mercurials

Staphylococcus aureus was found to have a higher resistance to merbromin and mercuric chloride in the presence of Escherichia coli. The protective effect of the gram-negative organism on S. aureus was due to the production of extracellular glutathione and hydrogen sulfide and to an unequal distribution of the inhibitor between the two species. S. aureus did not significantly influence the resistance of E. coli to mercurials.     

Cloning and characterization of an ftsZ homologue from a bacterial symbiont of Drosophila melanogaster

A 1194 by open reading frame that codes for a 398 amino acid peptide was cloned from a λgt11 library of Drosophila melanogaster genomic DNA. The predicted peptide sequence is very similar to three previously characterized protein sequences that are encoded by the ftsZ genes in Escherichia coli, Bacillus subtilis and Rhizobium meliloti. The FtsZ protein has a major role in the initiation of cell division in prokaryotic cells. Using a tetracycline treatment that eradicates bacterial parasites from insects, the ftsZ homologue has been found to be derived from a bacterium that lives within the strain. However, polymerase chain reaction (PCR) amplification of the gene from treated embryos suggests that it is not derived from a gut bacterium. Nevertheless, by amplifying and characterizing part of the 16S rRNA from this bacterium we have been able to demonstrate that it is a member of the genus Wolbachia, a parasitic organism that infects, and disturbs the sexual cycle of various strains of Drosophila simulans. We suggest that this ftsZ homologue is implicated in the cell division of Wolbachia, an organism that fails to grow outside the host organism. Sequence and alignment analysis of this ftsZ homologue show the presence of a potential GTP-binding motif indicating that it may function as a GTPase. The consequences of this function particularly with respect to its role in cell division are discussed.     

Inhibition of Leuconostoc mesenteroides P-60 by D-tyrosine and its relationship with sodium and potassium content of the medium

Evidence for a growth inhibition of Leuconostoc mesenteroides P-60 by the d-isomer of tyrosine has been obtained by comparison of the relative activities of l- and dl-tyrosine for this organism, and by comparison of tyrosine assay results obtained on acid and alkaline hydrolysates of casein and beef round. It has been further found that this inhibition is significantly greater on a conventional medium buffered with both sodium and potassium salts than on one containing all of the buffer salts in the potassium form. Similar studies with Leuconostoc citrovorum 8081 and Lactobacillus delbrueckii 3 indicate that these organisms are neither inhibited nor stimulated significantly by the presence of d-tyrosine, and are therefore to be recommended as more satisfactory assay organisms for tyrosine whenever the d-isomer is involved in the assays.     

The fine structure of Herpetosiphon,and a note on the taxonomy of the genus

The fine structure of the Gram-negative filamentous gliding bacterium, Herpetosiphon is described. The outer membrane of the cell envelope could not be resolved as a separate structure, probably because it is fused with the underlying dense (peptidoglycan) layer. There was an additional wall layer outside this membrane-peptidoglycan complex, but a sheath in the classical sense, as postulated in the definition of the genus, was lacking. On the cell surface a loose network of fibrils could be seen. Inside the cells 3 types of intracytoplasmic membranes were discernible: a) true mesosomes near cross walls; b) a system of coarser membranes which was not connected with the septa and formed networks or tubular complexes; c) degenerated septa within bulbs. the bulbs are swollen sections of filaments, occurred mainly in ageing cultures, and are probably a degeneration phenomenon. The filaments contained necridia, i.e. dead and empty cells, across which breaks may occur so that empty cell wall cylinders remain attached to the ends of the daughter filaments, falsely suggesting the presence of a sheath. The taxonomy of Herpetosiphon is discussed in detail: The organism has been described before as Flexibacter giganteus. It is proposed to abandon the species H. aurantiacus in favor of H. giganteus, but to retain the genus Herpetosiphon. An improved definition of the genus is given.     

Biosynthesis of the Actinomycin Chromophore: Incorporation of 3-Hydroxy-4-Methylanthranilic Acid into Actinomycins by Streptomyces antibioticus

Actinomycin synthesis by washed mycelia of Streptomyces antibioticus has been conducted in the presence of 3-hydroxy-4-methylanthranilate-(carboxyl-¹⁴C). Incorporation of this compound into actinomycins has been observed, which constitutes further evidence that 3-hydroxy-4-methylanthranilate is an intermediate in actinomycin biosynthesis. The position of the incorporated label has been determined to be within the actinomycin chromophore, and the label appears to be equally distributed between both halves of the chromophore. Incidental to these findings was the observation that the ¹⁴C-labeled actinomycins were subject to rapid reabsorption by the organism with actinomycin V taken up preferentially to actinomycin IV.