UDP-N-acetylglucosamine 1-carboxyvinyl-transferase from Acinetobacter calcoaceticus

Abstract We have analyzed the sequence downstream of rpoN from Zcinetobacter calcoaceticus and identified an open reading frame encoding a protein with high similarity to UDP- N -acetylgucosamine 1-carboxyvinyl-transferase (MurZ). Multicopy plasmids encoding this enzyme conferred phosphomycin resistance to A. calcoaceticus . The polar effect of a rpoN mutation on the phosphomycin resistance level suggests that murZ is, in part, cotranscribed with rpoN . These observations confirm that A. calcoaceticus represents the first exceptin from a conserved genetic context of rpoN observed in several other Gram-negative bacteria.     

Relative growth rate correlates negatively with pathogen resistance in radish: the role of plant chemistry

Plant growth rate has frequently been associated with herbivore defence: a large investment in quantitative defence compounds occurs at the expense of growth. We tested whether such a relationship also holds for growth rate and pathogen resistance. For 15 radish (Raphanus sativus L.) cultivars, we determined the potential growth rate and the resistance to fungal wilt disease caused by Fusarium oxysporum. We subsequently aimed to explain a putative negative relationship between growth rate and resistance based on plant chemical composition. Both growth rate and resistance level varied greatly among cultivars. Moreover, there was a strong negative correlation between growth rate and resistance, i.e. there are costs associated with a high resistance level. Roots of slow-growing, resistant cultivars have a higher biomass density. Using pyrolysis mass spectrometry. we part1y explained variation in both growth rate and resistance in terms of the same change in chemical composition. Leaves of slow-growing, resistant cultivars contained more cell wall material. Surprisingly, roots of slow-growing, highly resistant cultivars contained significantly less cell wall material, and more cytoplasmic elements (proteins). We speculate that this higher protein concentration is related to high construction and turn-over costs and high metabolic activity. The latter in turn is thought to be responsible for a rapid and adequate resistance reaction, in which phenols may be involved.     

Genetics and Mechanism of Resistance to Meloidogyne arenaria in Peanut Germplasm

Segregation of resistance to Meloidogyne arenaria in six BC₅F₂ peanut breeding populations was examined in greenhouse tests. Chi-square analysis indicated that segregation of resistance was consistent with resistance being conditioned by a single gene in three breeding populations (TP259-3, TP262-3, and TP271-2), whereas two resistance genes may be present in the breeding populations TP259-2, TP263-2, and TP268-3. Nematode development in clonally propagated lines of resistant individuals of TP262-3 and TP263-2 was compared to that of the susceptible cultivar Florunner. Juvenile nematodes readily penetrated roots of all peanut genotypes, but rate of development was slower (P = 0.05) in the resistant genotypes than in Florunner. Host cell necrosis indicative of a hypersensitive response was not consistently observed in resistant genotypes of either population. Three RFLP loci linked to resistance at distances of 4.2 to 11.0 centiMorgans were identified. Resistant and susceptible alleles for RFLP loci R2430E and R2545E were quite distinct and are useful for identifying individuals homozygous for resistance in segregating populations.     

Embryo culture of Medicago scutellata and M. sativa

Resistance to the alfalfa weevil (Hypera postica (Gyllenhal)) and the potato leafhopper (Empoasco fabae (Harris)) is lacking in cultivated alfalfa. However, a closely related annual Medicago, Medicago scutellata, possesses dense glandular stem and leaf hairs which provides a mechanism for resistance. Several attempts have been made at transfering the glandular haired traint from M. scutellata to perennial alfalfa with limited success. Earlier studies have shown that one reason for the lack of success is embryo abortion. Therefore, this study was initiated to observe zygotic embryo-genesis and to develop an embryo rescue technique for M. scutellata and M. sativa. Observations of zygotic embryogenesis showed that the two species are similar in morphology and can be described from youngest to oldest as globular, heart, torpedo, and hook shaped embryos. M. sativa embryos are smaller than M. scutellata embryos and develop three to four days later. Self pollinated M. scutellata (PI 307446) and sib mated M. sativa (Saranac AR) embryos were cultivated on Murashige and (2,4-D), indolacetic acid (IAA), 6-benzylaminopurine (BAP), and kinetic (KIN). Embryos from both species were also cultured on Schenk and Hildebrandt's (SH) basal medium with the addition of L-glutamine and L-proline. The experimental design was a completely randomized factorial for each experiment. Heart and torpedo shaped embryos from M. scutellata grew best (27.5% plantlet recovery) when cultured on MS medium with 0.05 mgl^-1 of both IAA and BAP. After 15 to 30 days on this medium, the embryos had only developed shoots. Therefore, it was necessary to transfer the shoots to MS basal medium without phytohormones for rooting. Rooting occurred in 15 to 30 days and the plantlets could be acclimatized to soil within 2 to 4 weeks. M. sativa embryos grew best (31% plantlet recovery) on SH medium with 50 mM L-glutamine. M. sativa embryos developed both shoots and roots on this medium. This information may now be applied to the development of an embryo culture method for recovering insect resistant hybrids between M. scutellata and M. sativa. Disclaimer statement: Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable.     

Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry

 Mammalian metallothioneins (MT) are known to maximally bind 12 copper ions in two six-Cu(I) ion clusters. Using electrospray ionization mass spectrometry of MT at pH 4.5, a four-Cu(I) ion cluster was observed intermediate to a fully formed six Cu(I) in a single domain or a fully formed Cu₁₂MT species. The four-Cu(I) cluster was observed in both MT1 and MT3 isoforms. Addition of increasing amounts of Cu(I) to MT at pH 4.5 resulted in prominent ions whoses masses were consistent with apo-MT, Cu₄MT, Cu₆MT, and Cu₁₂MT. The cooperativity of cluster formation was reduced at pH 2.5. Addition of Cu(I) to apo-MT at a reduced pH resulted in a series of ions consistent with Cu₄ to Cu₁₂MT species. However, formation of the tetracopper MT species remained cooperative at low pH, suggesting that this species is very stable. To determine whether the tetracopper cluster was formed in either the α or β domain, domain peptides of MT3 were used. Addition of Cu(I) to the apo β domain resulted in a peak consistent with the formation of a four-Cu(I) cluster. This is consistent with reports that Cu(I) ions bind preferentially to the β domain of MTs. Received: 2 June 1998 / Accepted: 21 August 1998     

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.     

Trichome-borne and artificially applied acylsugars of wild tomato deter feeding and oviposition of the leafminer Liriomyza trifolii

Oviposition and adult feeding of the leafminer Liriomyza trifollii (Burgess) (Diptera, Agromyzidae) on Lycopersicon pennellii (Corr.) D'Arcy and its F₁ hybrid with Lycopersicon esculentum (Mill.) was significantly less than that on the cultivated tomato, L. esculentum. The resistance of L. pennellii and the F₁ was reduced following rinsing of foliage with ethanol. Resistant attributes of L. pennellii were transferred to L. esculentum through appression of L. pennellii foliage to L. esculentum leaflets. Application of purified 2,3,4-tri-O-acylglucoses (the principal component of type IV glandular trichome exudate of L. pennellii) to L. esculentum significantly decreased feeding and oviposition on L. esculentum leaflets by 61–99%. Therefore the principal mechanism of resistance to this leafminer by L. pennellii is the secretion of these acylglucoses. Dose response analysis of acylglucoses applied to L. esculentum shows that dosages as low as 10% those found on L. pennellii provide large reductions (91%) in leaf punctures and mines.