Kenneth J. Curry

The pentameric vertex proteins are necessary for the icosahedral carboxysome shell to function as a CO2 leakage barrier.

Background Carboxysomes are polyhedral protein microcompartments found in many autotrophic bacteria; they encapsulate the CO2 fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) within a thin protein shell and provide an environment that enhances the catalytic capabilities of the enzyme. Two types of shell protein constituents are common to carboxysomes and related microcompartments of heterotrophic bacteria, and the genes for these proteins are found in a large variety of bacteria. Methodology/Principal Findings We have created a Halothiobacillus neapolitanus knockout mutant that does not produce the two paralogous CsoS4 proteins thought to occupy the vertices of the icosahedral carboxysomes and related microcompartments. Biochemical and ultrastructural analyses indicated that the mutant predominantly forms carboxysomes of normal appearance, in addition to some elongated microcompartments. Despite their normal shape, purified mutant carboxysomes are functionally impaired, although the activities of the encapsulated enzymes are not negatively affected. Conclusions/Significance In the absence of the CsoS4 proteins the carboxysome shell loses its limited permeability to CO2 and is no longer able to provide the catalytic advantage RubisCO derives from microcompartmentalization. This study presents direct evidence that the diffusion barrier property of the carboxysome shell contributes significantly to the biological function of the carboxysome.

Strawberry anthracnose: Histopathology of Colletotrichum acutatum and C. fragariae

ABSTRACT Ontogeny of the invasion process by Colletotrichum acutatum and C. fragariae was studied on petioles and stolons of the strawberry cultivar Chandler using light and electron microscopy. The invasion of host tissue by each fungal species was similar; however, each invasion event occurred more rapidly with C. fragariae than with C. acutatum. Following cuticular penetration via an appressorium, subsequent steps of invasion involved hyphal growth within the cuticle and within the cell walls of epidermal, subepidermal, and subtending cells. Both species of fungi began invasion with a brief biotrophic phase before entering an extended necrotrophic phase. Acervuli formed once the cortical tissue had been moderately disrupted and began with the development of a stroma just beneath the outer periclinal epidermal walls. Acervuli erupted through the cuticle and released conidia. Invasion of the vascular tissue typically occurred after acervulus maturation and remained minimal. Chitin distribution in walls of C. fragariae was visualized with gold-labeled wheat germ agglutinin. The outer layer of bilayered walls of conidia, germ tubes, and appressoria contained less chitin than unilayered hyphae in planta.

Aggregation of montmorillonite and organic matter in aqueous media containing artificial seawater

BackgroundThe dispersion-aggregation behaviors of suspended colloids in rivers and estuaries are affected by the compositions of suspended materials (i.e., clay minerals vs. organic macromolecules) and salinity. Laboratory experiments were conducted to investigate the dispersion and aggregation mechanisms of suspended particles under simulated river and estuarine conditions. The average hydrodynamic diameters of suspended particles (representing degree of aggregation) and zeta potential (representing the electrokinetic properties of suspended colloids and aggregates) were determined for systems containing suspended montmorillonite, humic acid, and/or chitin at the circumneutral pH over a range of salinity (0 – 7.2 psu).ResultsThe montmorillonite-only system increased the degree of aggregation with salinity increase, as would be expected for suspended colloids whose dispersion-aggregation behavior is largely controlled by the surface electrostatic properties and van der Waals forces. When montmorillonite is combined with humic acid or chitin, the aggregation of montmorillonite was effectively inhibited. The surface interaction energy model calculations reveal that the steric repulsion, rather than the increase in electronegativity, is the primary cause for the inhibition of aggregation by the addition of humic acid or chitin.ConclusionThese results help explain the range of dispersion-aggregation behaviors observed in natural river and estuarine systems. It is postulated that the composition of suspended particles, specifically the availability of steric polymers such as those contained in humic acid, determine whether the river suspension is rapidly aggregated and settled or remains dispersed in suspension when it encounters increasingly saline environments of estuaries and oceans.

Staining fragrance glands in orchid flowers

STERN , W. L., K. J. CURRY AND W. M. WHITTEN (Department of Botany and Florida State Museum, University of Florida, Gainesville 32611). Staining fragrance glands in orchid flowers. Bull. Torrey Bot. Club 113:288-297. 1986.-Orchid flowers representing a diversity of species within that family were stained in vivo with neutral red for presumptive evidence of the presence of fragrance glands (osmophores). Additional floral material was embedded in epoxy resin, sectioned, and stained with sudan black B or methylene blue-azure II-basic fuchsin for microscopic examination of tissue with presumed involvement in fragrance production. Sudan black B stained the vacuoles in most of the presumptive osmophoric floral tissue. Neutral red was seen as a precipitate in the vacuoles of epoxy-embedded, sectioned tissue. Neutral red and sudan black B both may have an affinity for flavonoids and terpenoids present in the vacuoles of some orchid flowers. Neither staining reaction is considered an absolute indicator of osmophore tissue, but both stains are useful to establish presumptive evidence for the presence of orchid osmophores. Methylene blue-azure II-basic fuchsin is an effective general stain for this epoxy-embedded tissue, but it is not considered an indicator of orchid osmophores.

Comparative study of sand porosity and a technique for determining porosity of undisturbed marine sediment

Porosity is a fundamental property of marine sediment from which wet bulk density can be easily determined and used in a variety of geoacoustic, geotechnical, and sedimentological studies, analyses, and models. However, methods of sampling marine sands suffer from the common problem of core disturbance making the in situ porosity difficult to obtain. Embedding the sediment within an epoxy resin matrix will minimize the disturbance to the microfabric and preserve the in situ sedimentary structure for subsequent study. Image analysis can then be used on thin sections to study the microfabric and porometry. A comprehensive review and analysis of published data on the porosity of predominantly clean sands has been completed and a simple, accurate, and nondestructive technique is described for preparing and measuring the porosity of marine sediment (siliciclastic sand) that has been infiltrated aboard ship immediately upon sample collection and chemically fixed and infiltrated with epoxy shortly thereafter. The average porosity of 36 samples of marine sand collected offshore Fort Walton Beach, Florida, and embedded with resin was determined to be 41.30%. From the review of published data the average porosity of sand was determined to be 37.7%, 42.3%, and 46.3% for packed, natural (in situ), and loose packing conditions, respectively, for a range of sorting coefficients and grain sizes.

Improved Microassays Used to Test Natural Product-Based and Conventional Fungicides on Plant Pathogenic Fungi

Seven important plant pathogenic fungi (Botrytis cinerea, Colletotrichum acutatum, C. fragariae, C. gloeosporioides, Fusarium oxysporum, Phomopsis obscurans, and P. viticola) valuable in screening fungicides were tested. Our procedure included washing conidia to reduce germination times, incorporating Roswell Park Memorial Institute 1640 as a medium of known composition, and using coverslips in the 24-well cell culture clusters to document the effect of fungicides on fungal morphology. The natural product-based fungicide, sampangine, a sampangine analog, 4-bromosampangine, plus seven conventional fungicides (benomyl, captan, cyprodinil, fenbuconazole, fenhexamid, iprodione, and kresoxim-methyl) were tested in vitro for their ability to inhibit germination and growth of the seven fungal species. Sampangine inhibited germination in all fungi except C. acutatum. Comparison of results of germination and morphology microbioassays with results of microtiter assays suggests that some fungicides stop fungal germination, whereas others only slow down fungal growth. We hypothesize that sampangine, except against C. acutatum, has the same physical mode of action, germination inhibition, as the conventional fungicides captan, iprodione, and kresoxim-methyl. 4-Bromosampangine caused morphological anomalies including excessive branching of germ tubes of C. fragariae and splaying and branching of germ tubes of B. cinerea.

Observations of the sediment-water interface: Marine and fresh water environments

The sediment at the interface immediately beneath the water column is distinct from deeper-lying sediments in its properties and, at least quantitatively, in the processes driving diagenesis. Progress in understanding the sediment-water interface can be based on consideration of fundamentals of biogeochemical particle / fluid interactions and on application of certain biological techniques especially suited to this challenging portion of the sediment column. This article reports results achieved by combining theoretical fundamentals and specialized experimental techniques in the study of the interface from selected depositional environments. For fine-grained and sandy deposits from fresh-water to coastal marine environments, the interface is characterized by exaggerated microrelief, great porosity, and significant biological alteration. Additional application of this research approach is poised to further our understanding of engineering, and acoustic and xenochemical responses of sedimentary materials, with special emphasis on the influence of the bio-organic phase of the interface upon its fabric and physical properties.

In situ permeabilities of selected coastal marine sediments

An in situ permeameter probe was deployed off Fort Walton Beach, Florida in shallow-water coastal sandy sediment. Stations were occupied in a 600 m /spl times/ 600 m area. Intrinsic permeability in sand at 17 stations varied from 0.3 to 6.1 /spl times/ 10/sup -11/ m/sup 2/ to subbottom depths of 50 cm. Permeability decreased with increasing subbottom depth and minimal compaction. The sediment is a well-sorted, medium quartz sand with a mean grain size of approximately 0.34-0.52 mm and with 5%-8% carbonate shells and shell fragments. The probe was tested in a slightly finer-grained sediment near the main study area and revealed permeabilities of 0.1-3.2 /spl times/ 10/sup -11/ m/sup 2/. The permeabilities measured are reasonable for the observed sandy and slightly finer-grained sediments.

Role of Chitin in Montmorillonite Fabric: Transmission Electron Microscope Observations

Particle concentration, charge, solution chemistry (i.e. ionic strength), and the nature of organic matter (OM) are the major factors controlling particle flocculation in aqueous environments. In the present study, the nature of clay fabric associated with clay—OM interaction at a range of ionic strengths was the focus. In the flocculation experiments, the aqueous suspension of montmorillonite and chitin was mixed with NaCl/MgSO4 electrolyte solution. Advanced sample-preparation techniques and visualization methods using transmission electron microscopy were used to observe directly the micro- and nano-scale clay—OM fabric of the resulting flocs. Such direct observation elucidated the role of OM in clay flocculation; few attempts have been made in the past due to the technical difficulties in preserving the original structure. A comparison of clay fabric at two different ionic strengths of 0 and 0.14Mrevealed that the individual hexagonal clay particles settled slowly with little intra-aggregate void space (void ratio: 0.07) at 0 M while rapid flocculation and settling of clay particles at 0.14 M, with or without OM, resulted in a more open fabric with greater void space (void ratio: 0.33). The silver-staining technique demonstrated effectively the location of electron-transparent chitin in montmorillonite aggregates. Chitin appeared to link the face-to-face (FF) contacts of clay domains by bridging between negatively charged face surfaces. However, the resultant void ratio and the average hydrodynamic diameter (dH) values were lower than in the OM-free system after flocculation. The results indicated that the interplay between ionic strength and OM content affected the floc architecture and void ratio.

Fungicide Resistance Profiles for 13 Botrytis cinerea Isolates from Strawberry in Southeastern Louisiana

Fungicidal sprays are widely used for control of Botrytis fruit rot; however, the pathogen often develops resistance to frequently used fungicides. A 96-well plate micro-dilution broth bioassay developed for fungicide discovery was used to provide strawberry growers with a rapid assessment of the fungicide sensitivity of Botrytis isolates against 16 fungicides. Three sensitivity phenotypes were identified: benzimidazole and dicarboximide resistant, benzimidazole resistant and dicarboximide sensitive, and an intermediate response to both fungicides. Codon at position 198 in the β-tubulin gene confirmed benomyl resistance. This bioassay rapidly identifies fungicide resistance and allows growers to quickly adjust their disease management strategy.