NolL of Rhizobium sp. strain NGR234 is required for O-acetyltransferase activity

Following (iso)flavonoid induction, nodulation genes of the symbiotic nitrogen-fixing bacterium Rhizobium sp. strain NGR234 elaborate a large family of lipooligosaccharidic Nod factors (NodNGR factors). When secreted into the rhizosphere of compatible legumes, these signal molecules initiate root hair deformation and nodule development. The nonreducing glucosamine residue of NodNGR factors are N acylated, N methylated, and mono- or biscarbamoylated, while position C-6 of the reducing extremity is fucosylated. This fucose residue is normally 2-O methylated and either sulfated or acetylated. Here we present an analysis of all acetylated NodNGR factors, which clearly shows that the acetate group may occupy position C-3 or C-4 of the fucose moiety. Disruption of the flavonoid-inducible nolL gene, which is preceded by a nod box, results in the synthesis of NodNGR factors that lack the 3-O- or 4-O-acetate groups. Interestingly, the nodulation capacity of the mutant NGROmeganolL is not impaired, whereas introduction of the nod box::nolL construct into the related strain Rhizobium fredii USDA257 extends the host range of this bacterium to Calopogonium caeruleum, Leucaena leucocephala, and Lotus halophilus. Nod factors produced by a USDA257(pnolL) transconjugant were also acetylated. The nod box::nolL construct was also introduced into ANU265 (NGR234 cured of its symbiotic plasmid), along with extra copies of the nodD1 gene. When permeabilized, these cells possessed acetyltransferase activity, although crude extracts did not.     

Structure of the capsular polysaccharide from the Klebsiella K8 reference strain 1015

The structure of the capsular polysaccharide from the Klebsiella K8 reference strain 1015 has been elucidated. The structure was deduced from sugar analysis, different methylation analyses, a uronic acid degradation, and NMR spectroscopy. It is concluded that the polysaccharide is composed of pentasaccharide repeating units with the structure: [formula: see text] The structure differs from that of the previously published structure of the capsular polysaccharide from Klebsiella K8, which originates from another strain and has the following structure: [formula: see text] The serological similarity between the two strains is most likely derived from a common tetrasaccharide which is substituted in different ways in the two strains. Since the strain in the present investigation originates from the Klebsiella K reference strain collection of the International Escherichia and Klebsiella Centre, Copenhagen, Denmark, it is suggested that it should keep the designation K8. The other polysaccharide with Klebsiella K8 specificity should be renamed as K8,52,59 based on the cross-reactivity of the strain (I. Orskov, unpublished).     

The Bradyrhizobium japonicum noeD gene: a negatively acting, genotype-specific nodulation gene for soybean

Bradyrhizobium japonicum strain USDA 110 is restricted for nodulation by soybean genotype PI 417566. We previously reported the identification of a USDA 110 Tn5 mutant, strain D4.2-5, that had the ability to overcome nodulation restriction conditioned by PI 417566 (S. M. Lohrke, J. H. Orf, E. Martínez-Romero, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:2378-2383, 1995). In this study, we report the cloning and characterization of the negatively acting DNA region mutated in strain D4.2-5 that is involved in the genotype-specific nodulation of soybean. The Tn5 integration site was localized to a 5.2-kb EcoRI fragment isolated from wild-type USDA 110 genomic DNA. Saturation Tn5 mutagenesis of this 5.2-kb region and DNA homogenitization studies indicated that a 0.9-kb DNA region was involved in the genotype-specific nodulation of PI 417566. A single open reading frame (ORF) of 474 nucleotides, encoding a predicted protein of 158 amino acids, was identified within this region by DNA sequencing. This ORF was named noeD. Computer comparisons with available data bases revealed no significant similarities between the noeD DNA or predicted amino acid sequence and any known genes or their products. However, comparisons done with the region upstream of noeD revealed a high degree of similarity (about 76% similarity and 62% identity) to the N-terminal regions of the Rhizobium leguminosarum bv. viciae and R. meliloti nodM genes, which have been postulated to encode a glucosamine synthase. Southern hybridization analysis indicated that noeD is not closely linked to the main or auxiliary nodulation gene clusters in B. japonicum and that both nodulation-restricted and -unrestricted B. japonicum serogroup 110 strains contain a noeD homolog. High-performance liquid chromatography and fast atom bombardment-mass spectrometry analyses of the lipo-chitin oligosaccharide (LCO) nodulation signals produced by an noeD mutant showed a higher level of acetylation than that found with wild-type USDA 110. These results suggest that specific LCO signal molecules may be one of the factors influencing nodulation specificity in this symbiotic system.     

Specificity of mutations induced by riboflavin mediated photosensitization in the supF gene of Escherichia coli

Riboflavin-mediated photosensitization has been shown to produce 8-hydroxyguanine (oh8Gua) in DNA. We investigated the specificity of mutation of photosensitized supF gene induced in Escherichia coli. The oh8Gua repair deficient E. coli mutant mutM and mutY were transformed with plasmid pUB3 carrying the supF gene irradiated with white light in the presence of riboflavin. Under these conditions, riboflavin photosensitization increased the amounts of oh8Gua in pUB3 DNA. Three types of a single base substitution occurring at G:C pairs were detected in both wild-type and mutM mutant strains. Almost all base substitutions were transversions to T:A or C:G pairs occurring at a similar extent in both wild-type and mutM strains. Mutations derived from mutY strain transformed with photosensitized DNA were only G:C to T:A transversions. These G:C to T:A transversions observed in the mutY strain were suggested to be the result of mispairing of oh8Gua with adenine. Riboflavin-mediated photosensitization may also produce lesions on DNA causing G:C to C:G changes by unknown mechanisms.     

Fatty acid composition of neutral lipid energy reserves in infective juveniles of entomopathogenic nematodes

The fatty acid composition of neutral lipids from infective juveniles (IJs) of Steinernema carpocapsae strain All, S. riobravis strain Biosys 355, S. feltiae strain UK76, and S. glaseri strain NC stored in distilled water at 25 degrees C was determined. Newly emerged IJs of all four species had similar neutral lipid fatty acid profiles and of the 18 fatty acids identified, C18:1n-9 (43-49 mol %), C16:0 (18-23%), C18:2n-6 (8-14%) and C18:0 (4-8%) were the most abundant. Unsaturated fatty acids predominated, with about 50% being monoene and 14-22% polyene; the unsaturation index ranged from 91.6 in S. glaseri to 111.6 in S. carpocapsae. The fatty acid composition of the total lipid and the free fatty acid fraction mirrored that of the neutral lipids. During storage, the relative levels (%) of C16:0, C18:0, and C18:ln-9 in the neutral lipids declined significantly, suggesting they were preferentially utilised.     

Phospholipid fatty acid profiles in selected members of soil microbial communities

Fatty acids derived from phospholipids and lipopolysaccharides were investigated from 33 taxonomically different organisms (bacteria, fungi and plant cells) known a priori to inhabit soil (except E. coli). The extended extraction procedure used, liberated non-ester-linked fatty acids in addition to ester-linked fatty acids, hydroxy substituted fatty acids in three different fractions. The amount of non-ester-linked fatty acids was as high as 70% of the total phospholipid fatty acids in some fungi and varied considerably in different organisms. The cis vaccenic acid constituted about 50% of phospholipid fatty acids in selected bacteria belonging to the alpha subclass of Proteobacteria. These fatty acids were not found in other selected organisms. A large amounts of branched chain fatty acids were found in various organisms. If the branching are localised on positions other than iso and anteiso they were strong indicators for gram positive bacteria. The cyclopropyl fatty acids are mainly localized in gram negative bacteria. The beta hydroxy fatty acid of the outer membrane are widespread among bacterial taxa and fungi. These fatty acids are not recommended to use as "signature" fatty acids for gram negative bacteria.     

The effect of hard segment size on the hydrolytic stability of polyether-urea-urethanes when exposed to cholesterol esterase

Lipo-oligosaccharide (LOSa) was obtained by phenol-water extraction of bacterial cells of an isolate PG 836, identified as Campylobacter jejuni serotype O:10, from a patient who subsequently developed the Miller-Fisher syndrome (MFS). The product was separated into a water-insoluble gel of low Mr and a water-soluble component of high Mr. The structure of the core oligosaccharide region in LOSa is reported herein for comparison with LOSb from the C. jejuni O:10 reference strain, and is based on investigations carried out on: (1) O-deacylated LOSa; (2) the core oligosaccharide (OS 1a) liberated on acetic acid hydrolysis of the ketosidic linkages to lipid A, with accompanying loss of N-acetylneuraminic acid residues; (3) the product of the removal of phosphate residues from OS 1a to give OS 2a; and (4) the Smith degradation of OS 2a to yield a mixture of Os 3a and OS 4a. The results revealed that the core oligosaccharide region in LOSa from the MFS bacterial isolate had chains (1a), of which some were terminated by an N-acetylneuraminobiose [Neu5Ac(alpha 2-8)Neu5Ac] unit in a GD3 [Neu5Ac-Neu5Ac-Gal] epitope, and the inner regions of which were different from those of other C. jejuni serotypes. Similar experiments on LOSb from bacterial cells of the C. jejuni O:10 reference strain showed that the core oligosaccharide unit [1a, R = P (phosphoric monoester)] of LOSa from the MFS isolate was more uniformly complete than that of the O:10 reference strain [1b, R = AEP (2-aminoethylphosphate)] differing in the nature of the phosphate substituent at the inner heptose residue. The close structural relationship of LOSa from the MFS associated bacterium to LOSb from the O:10 reference strain runs parallel to that of the previously studied Guillain-Barré syndrome (GBS) associated bacterium typed as C. jejuni O:19 in comparison with the lipo-oligosaccharide from the reference strain. Preliminary studies on the high Mr components showed that those from the O:10 strains were indistinguishable from each other, but were structurally unrelated to those from the GBS associated C. jejuni serotype O:19 isolates and the O:19 reference strain [G.O. Aspinall, A.G. McDonald, and H. Pang, Biochemistry, 33 (1994) 250-255].     

Cyclosporin C is the main antifungal compound produced by Acremonium luzulae

A strain of Acremonium luzulae (Fuckel) W. Gams was selected in screening new microorganisms for biological control of fruit postharvest diseases, especially gray and blue mold diseases on apples and strawberries. This strain manifests a very strong activity against a large number of phytopathogenic fungi. In this work, the product responsible for this antifungal activity was isolated from modified Sabouraud dextrose broth cultures of A. luzulae. It was purified to homogeneity by reverse-phase column chromatography. On the basis of UV, infrared, and 1H and 13C nuclear magnetic resonance spectra, mass spectral analysis, and the amino acid composition of the acid hydrolysates, the antibiotic was determined to be cyclosporin C. Cyclosporin C showed a broad-spectrum activity against filamentous phytopathogenic fungi but no activity against bacteria or yeasts. Its antifungal activity is only fungistatic. In contrast to Tolypocladium inflatum, another cyclosporin-producing strain, A. luzulae, did not produce additional cyclosporins. This was confirmed by in vivo-directed biosynthesis.     

Identification of immunoreactive epitopes in proteins coded by gag, env, and pol genes of the type I human T-lymphotropic virus (HTLV-I) using synthetic peptides

Reactivity of 26 synthetic peptides that comprise 12 to 26 amino acid residues corresponding to segments of the gag p19, env gp46, and pol proteins of human T-lymphotropic virus type I toward 31 positive sera was studied using enzyme-linked immunosorbent assay. Specific reactivity with high titers of antibodies (presented in reciprocal dilution values) was detected for the synthetic peptides corresponding to fragments 110-130 and 100-130 (titers up to 4050) of p19, 174-197 (up to 800), 186-201 (up to 4050), 191-215 (up to 1350), 242-257 (up to 800), and 272-292 (up to 450) of gp46. Immunoreactivity of seven peptides, fragments of pol-proteins, was weak. New linear epitopes in the regions 145-158, 272-277, and 292-300 of gp46 were detected. In addition, location of the known linear epitopes in p19 and gp46 was refined on the basis of comparative study of overlapping peptides from these proteins.     

Kinetic constants of alpha-methyl-D-glucoside transport in the chick small intestine during perinatal development

BACKGROUND: Experimental evidence suggests that neutralizing antibodies could constitute an important factor in the control of AIDS progression and that the V3 loop of gp120 constitutes the main target for such purposes. We have previously developed two neutralizing murine monoclonal antibodies (Mabs) against the V3 region of the HIV-1 MN strain. OBJECTIVES: To characterize those Mabs in terms of fine specificity and DNA sequence of their V regions and to study if Fab fragments retain their neutralizing potential in vitro. STUDY DESIGN: A set of 12-mer alanine substituted peptides were employed for epitope mapping using two ELISA procedures: (1) indirect, with each peptide bound to polystyrene plates, and (2) competitive, with co-incubation of peptides and Mabs in solution. The V regions of both Mabs were PCR amplified from cDNA and their nucleotide sequences were determined. Finally, Fab fragments of Mab 10F10 were generated and their neutralizing capacity against the MN isolated was assessed. RESULTS: We first restricted the minimal length of the epitopes recognized by 2C4 and 10F10 to the 12-mer peptide KRIHIGPGRAFY. The core of the epitopes recognized by Mabs 2C4 and 10F10 were IHIGP-R and IHIG-R, respectively. While substitution of proline in position 7 completely abolished the binding of 2C4, it only reduced that of 10F10 by 50%. Finally, Fab fragments of Mab 10F10 were still able to neutralize the HIV-1 MN strain in vitro. CONCLUSION: This subtle distinction in the fine mapping of the epitope recognized by Mabs 2C4 and 10F10 should correspond to three amino acid differences that we found in the heavy chain V-regions. The Fab fragments of Mab 10F10 retained the neutralizing capacity. This indicates that HIV neutralization by anti V3 Mabs is an Fc independent process.     

Electrophoretic behavior and size distribution of the acidic polysaccharides produced by the bacteria Bradyrhizobium (Chamaecytisus) strain BGA-1 and Bradyrhizobium japonicum USDA 110

The electrophoretic behavior in polyacrylamide gels of the acidic polysaccharides produced by the soil bacteria Bradyrhizobium (Chamaecytisus) strain BGA1 and Bradyrhizobiumjaponicum USDA1 10 has been studied. Both polysaccharides were polydisperse, producing a ladder-like pattern after fixation with Alcian Blue and silver staining of the gel. The polysaccharide molecules were separated according to their size, and they behaved as a collection of flexible random coils of different size and similar charge/mass ratio. The electrophoretic behavior was not affected by the presence of acetyl groups in the polysaccharide. The range of molecular weights of the exopolysaccharide produced by B. japonicum USDA110 was wider and with larger molecules than that of the polysaccharide produced by strain BGA1. The resolution was dependent on the electrophoresis buffer; the best results were achieved with Tris-borate; in Tris-glycine buffer, the resolution was worse, and it was not improved by the addition of sodium dodecyl sulfate (SDS).     

Thermal analysis of the compositional shift in a transient liquid phase during sintering of a ternary Cu-Sn-Bi powder mixture

In this work, differential scanning calorimetry (DSC) and microstructural analysis were used to study the transient-liquid-phase sintering (TLPS) of a Cu-Sn-Bi powder mixture. During sintering, the liquid phase shifts from a Sn-rich (i.e., ∼90 wt pct Sn) to a Bi-rich (i.e., >78 wt pct Bi) composition. In addition, the presence of Bi creates two melting events: a Sn:Bi eutectic reaction at 139 °C and a reaction involving the melting of (Bi) at 191 °C. The Sn:Bi eutectic melting event was fully transient. The melting event at 191 °C was consistent with the formation of a terminal Bi-rich liquid phase. The rate of compositional shift toward this terminal liquid phase at 260 °C was dependent on the rate of the reaction of the Sn with the Cu powder to form intermetallic phases. For mixtures made with medium and fine Cu powder, the terminal Bi-rich composition was reached after isothermal hold times of 20 and 15 minutes, respectively. This resulted in a new melting point for the mixture of 191 °C. For coarse Cu powders, the rate of the compositional shift toward a Bi-rich composition was much slower. The liquid phase remained at a hypoeutectic Sn-Bi composition estimated at 80 wt pct Sn, while the mixture maintained a melting point of 139 °C.     

Hereditary isolated renal magnesium loss maps to chromosome 11q23

Dendritic cells (DC) expanded in the presence of GM-CSF from the bone marrow of C57BL/6 mice process Gram-negative bacteria expressing the model antigen Crl-OVA for peptide presentation on MHC class I molecules. Here we show that presentation of OVA(257-264) processed by DC co-incubated with E. coli expressing Crl-OVA, which contains the Kb-binding OVA(257-264) epitope, occurs by a cytosolic MHC-I presentation pathway. First, we demonstrate the requirement for the transporter associated with antigen processing (TAP) by showing that DC from TAP1-/- mice co-incubated with E. coli expressing Crl-OVA did not result in Kb presentation of OVA(257-264). Second, the proteasome inhibitor MG132 abrogated presentation of OVA(257-264) on Kb when C57BL/6 DC phagocytosed and processed E. coli expressing Crl-OVA. Third, inhibiting protein synthesis using cycloheximide or blocking exocytosis of newly synthesized proteins from the endoplasmic reticulum using brefeldin A abrogated presentation of OVA(257-264) processed from bacteria expressing Crl-OVA by C57BL/6 DC. Finally, peptide regurgitation and loading of OVA(257-264) on neighboring bystander Kb-expressing antigen-presenting cells after BALB/c (H-2d) DC phagocytosed E. coli expressing Crl-OVA could not be detected. Together, these data support a cytosolic MHC-I presentation pathway for OVA(257-264) processed from E. coli expressing Crl-OVA by bone marrow-derived DC.     

Serine 257 phosphorylation regulates association of polyomavirus middle T antigen with 14-3-3 proteins

Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.     

Relationship of Neisseria elongata subsp. Glycolytica to other members of the family Neisseriaceae

Neisseria elongata subsp. glycolytica strain 6171/75 is closely similar to the type strain of N. elongata, M2, as regards DNA base composition, fatty acid content and electrophoretic mobility of two glutamate dehydrogenases, one of which showed a reaction of identity with the corresponding enzyme from M2 in double immunodiffusion in agar. The strain showed genetic homologies with strain M2 in genetic transformation at a level suggesting species identity, and with N. meningitidis at a lower level. No affinity to Moraxella species or "false neisseriae" was demonstrated, with the exception of a production of a few transformants in the 6171/75 recipient by DNA from Kingella kingae. The strain showed the same pattern of associated variation of colony type, fimbriation and competence in transformation as that found in other Neisseria and Moraxella species. After continuous subcultivation for some time some clones of the strain appeared to have lost the ability to produce acid from glucose.     

Efficacy of a novel infectious bursal disease virus immune complex vaccine in broiler chickens

Two experiments were conducted to test the efficacy of a novel infectious bursal disease virus (IBDV) vaccine in broiler chickens with maternal IBDV immunity. The IBDV vaccine was formulated by mixing IBDV strain 2512 with bursal disease antibodies (BDA) to produce the IBDV-BDA complex vaccine. In Expt. I, 1-day-old Cobb x Cobb broiler chickens were vaccinated subcutaneously with either IBDV-BDA or commercial live intermediate IBDV vaccine (vaccine A) or were left unvaccinated. In Expt. 2, the vaccine A group was not included; instead, IBDV strain 2512 was included. Chickens were maintained in isolation houses. On day 28 (Expt. 1) and day 32 (Expt. 2) of age, chickens from each group were challenged with a standard USDA IBDV (STC strain) challenge. Challenged and unchallenged chickens were evaluated for their bursa/body weight ratios and antibody titers 3 days post-challenge. Bursae collected from Expt. 2 were examined histologically to evaluate bursal lesions and confirm gross examination. None of the unvaccinated chickens was protected against the challenge virus as evidenced by the presence of acute bursal lesions (edema/hemorrhage). All chickens receiving the IBDV-BDA complex or the IBDV strain 2512 (Expt. 2) were protected from the challenge virus as evidenced by no acute bursal lesions. Additionally, chickens receiving the IBDV-BDA complex vaccine or the IBDV strain 2512 had antibody titers to IBDV, indication the presence of an active immune response. In Expt. 1, chickens vaccinated with vaccine A and challenged had bursal lesions similar to those observed in the unvaccinated, challenged chickens. These chickens also showed no indication of active immunity against the virus. These results suggest that the 1-day-of-age-administered IBDV-BDA complex vaccine can induce active immunity and protection against a standard IBDV challenge in the face of variable levels of maternal IBDV immunity.     

Luteococcus japonicus gen. nov., sp. nov., a new gram-positive coccus with LL-diaminopimelic acid in the cell wall

A new gram-positive, nonmotile coccus is described. Strains IFO 12422T (T = type strain) and IFO 15385 in the Institute for Fermentation, Osaka, culture collection, which were isolated from soil and water, respectively, have the following chemotaxonomic characteristics: menaquinone MK-9(H4); G + C content of DNA of 67 mol%; and LL-diaminopimelic acid, alanine, glycine, and glutamic acid in a molar ratio of ca. 1:2:1:1 (type A3 gamma). Mycolic acids are not present. The taxonomic characteristics of these organisms are different from those of previously described gram-positive, high-G + C-content cocci. The partial 16S rRNA sequence indicated that IFO 12422T represents a distinct line of descent among gram-positive bacteria with a high G + C content. The name Luteococcus japonicus gen. nov., sp. nov. is proposed. The type strain is strain IFO 12422.     

Influence of various heat treatments on the microstructure of polycrystalline IN738LC

IN738LC is a modern, nickel-based superalloy utilized at high temperatures in aggressive environments. Durability of this superalloy is dependent on the strengthening of γ′ precipitates. This study focuses on the microstructural development of IN738LC during various heat treatments. The 1120 °C/2 h/accelerated air-cooled (AAC) solution treatment, given in the literature, already produces a bimodal precipitate microstructure, which is, thus, not an adequate solutionizing procedure to yield a single-phase solid solution in the alloy at the outset. However, the 1235 °C/4 h/water quenched (WQ) solution treatment does produce the single-phase condition. A microstructure with fine precipitates develops if solutionizing is carried out under 1200 °C/4 h/AAC conditions. Agings at lower temperatures after 1200 °C/4 h/AAC or 1250 °C/4 h/AAC or WQ conditions yield analogous microstructures. Agings below ∼950 °C for 24 hours yield nearly spheroidal precipitates, and single aging for 24 hours at 1050 °C or 1120 °C produces cuboidal precipitates. Two different γ′ precipitate growth processes are observed: merging of smaller precipitates to produce larger ones (in duplex precipitate-size microstructures) and growth through solute absorption from the matrix. Average activation energies for the precipitate growth processes are 191 and 350 kJ/mol in the ranges of 850 °C to 1050 °C and 1050 °C to 1120 °C, respectively, calculated using the precipitate sizes from microstructures in the WQ condition, and 150 and 298 kJ/mol in the analogous temperature ranges, calculated from precipitate sizes in the microstructures in the slow furnace-cooled condition.     

Linkage of mutant alleles of CYP2C18 and CYP2C19 in a Japanese population

Prorocentrum lima was found to be distributed on the surface of the algae, Sargassum confusum and Carpopeltis flabellata collected at the Sanriku coast, northern Japan. Chemical analysis of cultured cells revealed that Sanriku strains of P. lima produce okadaic acid, a toxin responsible for diarrhetic shellfish poisoning. The Sanriku strain grew well in T1 medium at 15 degrees C at which tropical strains do not grow, indicating that it is a local strain which adapts to cooler environments.