In vitro self assembly of supermolecular structures after spontaneous disassembly of carotovoricins

The self-assembly of supramolecular structures (empty sheaths and polysheaths of the macromolecular Erwinia carotovora bacteriocins) was studied by electron microscopy in the course of 1- to 2-year incubation of phage particles at 4 degrees C. This study showed that the empty sheaths and polysheaths of the bacteriocins of eight E. carotovora strains spontaneously assemble at the self-assembly centers (or crystallization centers), which have a diameter of 26-65 nm and contain a dense proteinaceous material. The self-assembly center consists of two components, a primer and the structural protein of contracted sheaths. Empty sheaths assembled in the crystallization centers are polar structures synthesized through the stepwise head-to-tail polymerization of monomeric units. The supramolecular structures of two E. carotovora 62A bacteriocins are assembled in a different way. At the early stages of their self-assembly, a reticular structure is formed, which then transforms into very long polysheaths composed of monomers. Along with polysheaths, rounded or lamplike structures 33-117 nm in size composed of the subunits of contracted sheath are produced. Carotovoricins may serve as suitable objects for the study of the self-assembly of elementary biological structures.     

Defective Lysogeny in Erwinia carotovora

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128–192 nm in length and 13–21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55–59, 66–75, and 92–98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid–induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.     

A Study of Erwinia carotovora Phage Resistance with the Use of Temperate Bacteriophage ZF40

The causes of the unique phage resistance of the pectinolytic phytopathogenic strains of Erwinia carotovora were studied with the use of temperate bacteriophage ZF40. It was shown that, in these bacteria, the bacteriophage–cell interaction can be substantially blocked at the adsorption level. An adequate indicator for studying the temperate bacteriophages of erwinias was developed on the basis of mutants resistant to macromolecular bacteriocins. Various restriction–modification systems, which influence cell resistance to bacteriophages, were revealed for the first time in E. carotovora. The phage resistance was shown to be determined by the wide occurrence of homoimmune temperate viruses in pectinolytic erwinias.     

Kinetics of the cooperative association of T4 tail sheath protein P18 to polysheaths

The polymerization of the monomeric sheath protein P18 to polysheath was followed by light scattering in 1 mM sodium phosphate buffer, pH 7 at a MgCl2 concentration of 5 mM. Sigmoidal kinetics were observed in the case of spontaneous nucleation. These were well fitted by a mechanism involving a slow nucleation step (rate constant kN = 10(-2) M-1 S-1) followed by propagation steps (k = 10(5) M-1 S-1) in which P18 protomers are added to the ends of the polysheath particles. When sonicated polysheaths or contracted sheaths were added as seeds exponential time courses were observed. From the pseudo first order rate constant and the concentration of seeds the above value for the rate constant of propagation was confirmed. The ability of contracted sheaths to nucleate polysheath formation lends support to the conclusion that polysheaths and contracted sheaths have identical structures and differ in their length distributions only. These were measured from electromicrographs and from the distribution of sedimentation coefficients. Poisson type, kinetically controlled size distributions were found after polymerization of polysheath. An extremely slow redistribution towards an exponential distribution was detected. The spontaneous slow formation of polysheaths is much slower than the formation of extended sheath are core baseplates. Extended sheath is a metastable assembly produce of P18 which either dissociates of contracts to form contracted sheath. Polysheaths and contracted sheaths are extremely stable products but their immediate formation is hindered by high nucleation difficulties.     

Changes in the secondary structure bacteriophage T4 envelope protein after its polymerization

Circular dichroism (CD) spectra of the structural protein of the bacteriophage T4 sheath (gp 18) in a monomeric native state, helices, polysheaths and contracted sheaths were measured in the range 184-310 nm. The secondary structure of the protein studied was calculated from the spectra in the range 190-240 nm according to Provencher and Gl?ckner. It has been shown that the polymerization is proceeded without change of the alpha-helical content in the secondary structure of gp 18: estimated alpha-helix in monomeric gp 18, helices and polysheaths was 39%. The beta-form content in monomeric gp 18, helices and polysheaths was 33, 32 and 37%, respectively. Tail sheath contraction is attended by a 14% decrease in gp 18 alpha-helicity and a 5% increase in its beta-form content.     

Sensitivity variations of Listeria strains to the bacteriocins, lactocin 705, enterocin CRL35 and nisin

Five strains of Listeria monocytogenes, four strains of Listeria innocua and a strain of Listeria seeligeri showed different sensitivities to lactocin 705 (17 000 AU ml^–1), enterocin CRL35 (8500 AU ml^–1) and nisin (2500 IU ml^–1) at different pHs (5, 6 and 7). The susceptibility of Listeria strains to bacteriocins at each pH was strain dependent, and it was enhanced at the low pH. L. monocytogenes had enhanced nisin tolerance while the non-nisin bacteriocins were more inhibitory with viability losses of 3–3.4 in contrast with 1.5–1.8 log cycles, respectively. Lower viability loss values were obtained with L. innocua strains with all three bacteriocins while L. seeligeri was more sensitive to nisin than to lactocin 705 or enterocin CRL35.     

The Study of Erwinia carotovora Bacteriocins with the Aid of Nalidixic Acid–Resistant Bacterial Indicator Cells

A novel approach is proposed for the study of the macromolecular bacteriocins of Erwinia carotovora (MCTVs). The approach lies in the bacteriocinogeny of pectolytic erwinia being studied using a lawn of a bacterial mutant resistant to nalidixic acid, an inducer of MCTVs. The high efficiency of this approach was demonstrated by studying carotovoricins in 104 different E. carotovora strains, 88% of which bear MCTVs, distinguished by the morphology of zones of induced lysis on a lawn of susceptible cells, the lysis pattern, and some other characteristics. Preliminary studies using this approach showed that there is no correlation between the occurrence of MCTVs in particular E. carotovora strains and the habitat of the host plants from which these strains were isolated. There are grounds to believe that the approach proposed can also be used for investigating bacterial lysogeny.     

Nucleotide Sequences and Organization of the Genes for Carotovoricin (Ctv) from Erwinia carotovora Indicate That Ctv Evolved from the Same Ancestor as Salmonella typhi Prophage

Carotovoricin Er (CtvEr), which is produced by a plant soft rot disease causative agent, Erwinia carotovora subsp. carotovora Er, is a high-molecular-weight bacteriocin showing Myoviridae phage-tail-like morphology with contractile sheath and plural tail fibers. We determined the complete nucleotide sequences of CtvEr genes on the E. carotovora Er chromosome and report that CtvEr genes consist of lysis cassette, major and minor structural protein gene clusters. Four promoters were identified. The lysis gene cassette, which is composed of the genes for lysis enzyme and holin, was also identified and characterized. The nucleotide sequences and organization of the genes for CtvCGE, which is produced by E. carotovora strain CGE234-M403 with the morphology similar to CtvEr, were also determined and compared to that of CtvEr, and it was found that CtvCGE is almost identical to CtvEr except for tail fibers which are involved in the killing spectra of both bacteriocins. We also explain that the gene organization and the deduced amino acid sequences of both carotovoricins are very close to those of prophage, which is lysogenized in the chromosome on Salmonella enterica serovar Typhi CT18. These findings strongly suggest that Ctv evolved as a phage tail-like bacteriocin from a common ancestor with Salmonella typhi prophage.     

Enhanced nisin and pediocin production on whey supplemented with different nitrogen sources

Production of nisin and pediocin were followed, respectively, in Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 grown with lactose and four different nitrogen sources. Neither NH₄Cl nor glycine improved production of the bacteriocins. Both yeast extract and Casitone increased pediocin production from 55 BU ml^–1 to 195 BU ml^–1 and 185 BU ml^–1, respectively. Nisin increased from 21 BU ml^–1 to 74 BU ml^–1 and 59 BU ml^–1 with these nitrogen sources.     

Ultrastructure of freeze-substituted hyphae of the basidiomyceteLaetisaria arvalis

Summary The ultrastructure of freeze-substituted (FS) hyphae ofLaetisaria arvalis is described and compared to that of similar hyphae preserved by conventional chemical fixation (CF). The outline of membrane-bound organelles as well as the plasma membrane was smooth in FS cells. In contrast, hyphae preserved by CF exhibited membrane profiles that were extremely irregular. Centers of presumed Golgi activity were best preserved by FS. Microvesicles, 27–45 nm diameter and hexagonal in transverse section, were observed most readily in FS cells. Filasomes (= microvesicles within a filamentous matrix) were only observed in FS cells. Apical vesicles, 70–120 nm diameter, associated with the centers of Golgi activity and within the Spitzenkörper region exhibited finely granular matrices in FS hyphae, whereas in CF hyphae the contents were coarsely fibrous and less electron-dense. Microvesicles were present at hyphal apices and regions of septa formation. Filasomes were also found at regions of septa formation as well as along lateral hyphal tip cell walls. Microvesicles, but not filasomes, were observed in membrane-bound vesicles (= multivesicular bodies) and in larger vacuoles. Filaments, 5.2–5.4 nm wide, were juxtaposed with centripetally developing septa. Cytoplasmic inclusions, 20–40 m in length, composed of bundles of 6.7–8.0 nm wide filaments were observed in both FS and CF hyphae.     

The structure of Gloeobacter violaceus and its phycobilisomes

The fine structure of the atypical cyanobacterium Gloeobacter violaceus has been studied on frozen-etched replicas and compared to that of a typical unicellular strain: Synechocystis 6701. The complementary fracture faces of G. violaceus cytoplasmic membrane contain particles less numerous and more heterogenous in size than either the cytoplasmic membrane or the thylakoid membranes of Synechocystis. The most frequently observed particles of the exoplasmic fracture (EF) face of the G. violaceus cytoplasmic membrane are 11 nm in diameter and occasionally form short alignments. This particle class is similar in appearance to the numerous, aligned EF particles of Synechocystis thylakoid membranes. In replicas of cross-fractured G. violaceus, a layer 50–70 nm thick, composed of rod-like elements, underlies the inner surface of the cytoplasmic membrane. The rods, 12–14 nm in diameter, are oriented perpendicularly to the cytoplasmic membrane and show a 6 nm repeat along their length.Isolated phycobilisomes of G. violaceus appear, after fixation and negative staining, as bundles of 6 parallel rodshaped elements connected to an ill-defined basal structure. The bundles are 40–45 nm wide and 75–90 nm long. The rods are 10–12 nm in width; their length varies between 50 and 70 nm. These rods are morphologically similar to those observed at the periphery of hemidiscoidal phycobilisomes of other cyanobacteria, with a strong repeat at 6 nm intervals and a weaker one at 3 nm intervals along their length.The calculated molar ratio of phycobiliproteins in isolated G. violaceus phycobilisomes corresponds to 1:3.9:2.9 for allophycocyanin, phycocyanin and phycoerythrin respectively. When excited at 500 nm, isolated phycobilisomes exhibit a major fluorescence emission band centered at 663 nm.Abbreviations PBS phycobilisome(s) - PBP phycobiliprotein(s) - AP allophycocyanin - PC phycocyanin - PE phycoerythrin - K–PO₄ buffer KH₂PO₄ titrated with KOH to a given pH     

The integumental ultrastructure of Lamippe rubra Bruzelius and Enalcyonium rubicundum Olsson (Copepoda,Poecilostomatoida)

The integument of Lamippe rubra Bruzelius and of Enalcyonium rubicundum Olsson has been studied with the electron microscope.Most of the cuticle covering the body of Lamippe is represented by the epicuticle, which shows an average thickness of about 2.0 µm, but in sclerified zones it consists of a thin epicuticle (0.2 µm) and a stratified laminated procuticle (0.5–1.5 µm) without bow-shaped structure. A complex system of epithelial microvilli or a well-developed system of membranes running parallel to the cuticle is also present.The cuticle of Enalcyonium consists of a thin procuticle (0.4–0.5 µm) covered with a uniform fibrillar coat (0.5 µm), whereas in sclerotized areas it is composed of a stratified procuticle (0.7–3.5 µm) with bow-shaped structures.In both species, cuticular hairs and gland vents occur at the dorsal and ventral surfaces. Some of the hairs are considered to be sensory in nature.The cuticular ultrastructure of L. rubra and of E. rubicundum is compared with that of some other copepods.     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Isolation and Preliminary Characterization of Cryptic Plasmids from Erwinia carotovora

Of the fifty-two Erwinia carotovorastrains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size. Some E. carotovorastrains bore two to five different plasmids. Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins. At the same time, one of the E. carotovorastrains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, the 47.7-kbp pCA 6-2. Three E. carotovorasubsp.carotovorastrains and one E. carotovorasubsp.atrosepticastrain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia.     

Decolorization kinetics of a recombinant Escherichia coli strain harboring azo-dye-decolorizing determinants from Rhodococcus sp.

A 6.3 kb DNA fragment containing genes responsible for azo-dye decolorization was cloned and expressed in Escherichia coli. The resulting recombinant strain E. coli CY1 decolorized 200 mg azo dye (C.I. Reactive Red 22) l^–1 at 28 °C at 8.2 mg g cell^–1 h^–1, while the host (E. coli DH5) had no color-removal activity. Addition of 0.5 mM isopropyl--d-thiogalacto-pyranoside (IPTG) increased the decolorization rate 3.4-fold. The dependence of the decolorization rate on initial dye concentration essentially followed Monod-type kinetics and the maximal rate occurred with the dye at 600 mg l^–1. The decolorization rate of E. coli CY1 was optimal at 40 °C and pH 11. Aeration (increased dissolved O₂ level) strongly inhibited the decolorization, but decolorization occurred effectively under static incubation conditions (no agitation was employed). The CY1 strain also exhibited excellent stability during repeated-batch operations.     

Statistics of canonical RNA pseudoknot structures

In this paper we study canonical RNA pseudoknot structures. We prove central limit theorems for the distributions of the arc-numbers of k-noncrossing RNA structures with given minimum stack-size τ over n nucleotides. Furthermore we compare the space of all canonical structures with canonical minimum free energy pseudoknot structures. Our results generalize the analysis of Schuster et al. obtained for RNA secondary structures [Hofacker, I.L., Schuster, P., Stadler, P.F., 1998. Combinatorics of RNA secondary structures. Discrete Appl. Math. 88, 207–237; Jin, E.Y., Reidys, C.M., 2007b. Central and local limit theorems for RNA structures. J. Theor. Biol. 250 (2008), 547–559; 2007a. Asymptotic enumeration of RNA structures with pseudoknots. Bull. Math. Biol., 70 (4), 951–970] to k-noncrossing RNA structures. Here k2 and τ are arbitrary natural numbers. We compare canonical pseudoknot structures to arbitrary structures and show that canonical pseudoknot structures exhibit significantly smaller exponential growth rates. We then compute the asymptotic distribution of their arc-numbers. Finally, we analyze how the minimum stack-size and crossing number factor into the distributions.     

Architecture of the cell wall of a green alga,Oocystis apiculata

Summary The cell wall of a green alga,Oocystis apiculata, was visualized by electron microscopy after preparation of samples by rapid-freezing and deep-etching techniques. The extracellular spaces clearly showed a random network of dense fibrils of approximately 6.4 nm in diameter. The cell wall was composed of three distinct layers: an outer layer with a smooth appearance and many protuberances on its outermost surface; a middle layer with criss-crossed cellulose microfibrils of approximately 15–17 nm in diameter; and an inner layer with many pores between anastomosing fibers of 8–10 nm in diameter. Both the outer and the inner layer seemed to be composed of amorphous material. Cross-bridges of approximately 4.2 nm in diameter were visualized between adjacent microfibrils by the same techniques. The cross-bridges were easily distinguished from cellulose microfibrils by differences in their dimensions.     

Development and function of Pisolithus and Scleroderma ectomycorrhizas formed in vivo with Allocasuarina,Casuarina and Eucalyptus

The effect of inoculating seedlings of Eucalyptus grandis, Allocasuarina littoralis and Casuarina equisetifolia with two isolates of Pisolithus and two isolates of Scleroderma from under eucalypts was examined in a glasshouse trial. Ectomycorrhizas formed extensively on Eucalyptus (23–46% fine roots ectomycorrhizal) and Allocasuarina (18–51% fine roots ectomycorrhizal). On Casuarina, the fungi were either unable to colonize the rhizosphere (one isolate of Pisolithus), or sheathed roots, resembling ectomycorrhizas, formed on 1–2% of the fine roots. Colonization of roots by one isolate of Scleroderma resulted in the death of Casuarina seedlings. Inoculation with fungi increased shoot dry weight by up to a factor of 32 (Eucalyptus), 4 (Allocasuarina) and 3 (Casuarina). Ectomycorrhizas formed in associations with Eucalyptus and Allocasuarina had fully differentiated mantles and Hartig nets in which the host and fungal cells were linked by an extensive fibrillar matrix. Sheathed roots in Casuarina lacked a Hartig net, and the epidermis showed a hypersensitive reaction resulting in wall thickening and cell death. The sheaths are described as mantles since the density and arrangement of the hyphae in the sheaths was similar to that in mantles of the eucalypt ectomycorrhizas. The intercellular carbohydrate matrix was not produced in the Casuarina mantle in association with Pisolithus, hence the mantle was not cemented to the root. These structures differ from poorly compatible associations described previously for Pisolithus and Eucalyptus. The anatomical data indicate that ectomycorrhizal assessment based on surface morphological features may be misleading in ecological studies because compatible and incompatible associations may not be distinguishable.     

Gene stacking in <Emphasis Type="Italic">Phalaenopsis</Emphasis> orchid enhances dual tolerance to pathogen attack

Cymbidium Mosaic Virus (CymMV) and Erwinia carotovora have been reported to cause severe damage to orchid plants. To enhance the resistance of orchids to both viral and bacterial phytopathogens, gene stacking was applied on Phalaenopsis orchid by double transformation. PLBs originally transformed with CymMV coat protein cDNA (CP) were then re-transformed with sweet pepper ferredoxin-like protein cDNA (Pflp) by Agrobacterium tumefaciens, to enable expression of dual (viral and bacterial) disease resistant traits. A non-antibiotic selection procedure in the second transformation minimized the potential rate of ‘stacking’ antibiotic genes in the orchid gene pool. Transgene integration in transgenic Phalaenopsis lines was confirmed by Southern blot analysis for both CP and pflp genes. Expression of transgenes was detected by northern blot analysis, and disease resistant assays revealed that transgenic lines exhibited enhanced resistance to CymMV and E. carotovora. This is the first report describing a transgenic Phalaenopsis orchid with dual resistance to phytopathogens.     

Electron redistribution in mixed valence cytochrome oxidase following photolysis of carboxy-oxidase

Absorbance changes at 446 nm in purified cytochrome oxidase following flash photolysis of carboxy-oxidase poised in the mixed valence state at +220 mV show biphasic kinetics. One phase corresponds to CO recombination to ferrous cytochromea ₃ with an energy of activation of 9 kcal/mol; the second phase is 3–5 times faster with an energy of activation of 9.15 kcal/mol. Following flash photolysis at approximately –60°C, cytochromesa andc and the 840-nm CuA species are observed to undergo reduction as electrons from ferrous unliganded cytochromea ₃ equilibrate with the equipotential redox centers of the oxidase; as CO recombines with ferrous cyochromea ₃, these centers are oxidized and the mixed valence carboxy-oxidase is regenerated. Electron redistribution between centers of the oxidase in the forward and reverse directions occurs faster than does the binding of CO.