Engineering of complex polyketide biosynthesis — insights from sequencing of the monensin biosynthetic gene cluster

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 360–367. Received 18 March 2001/ Accepted in revised form 09 July 2001     

Enhanced deoxyribozyme‐catalyzed RNA ligation in the presence of organic cosolvents

Deoxyribozyme and aptamer selections are typically conducted in aqueous buffer solutions. Using nonaqueous cosolvents in selection experiments will help expand the activity of deoxyribozymes with non‐oligonucleotide substrates and will allow identification of new aptamers for nonprotein targets. We undertook in vitro selections utilizing a small amount of methanol in the reaction to keep the herbicides alachlor and atrazine in solution with the goal of identifying deoxyribozymes that require these herbicides for activity. The resulting deoxyribozymes successfully catalyze RNA ligation, but do not require alachlor or atrazine. Surprisingly, some of these deoxyribozymes displayed better catalytic activity in the presence of methanol over just aqueous buffer. We investigated several organic cosolvents to see if this enhancement was limited to methanol and found that other cosolvents, including ethanol, DMSO, and DMF, supported activity; in some cases, greater enhancement was observed. On the basis of these results, we tested two other previously identified RNA‐ligating deoxyribozymes to assess their tolerance of cosolvents and determined that different deoxyribozymes showed different responses to the cosolvents. Our results demonstrate that deoxyribozymes can tolerate and, in some cases, display enhanced activity in alternative solvent conditions. These findings will facilitate the development of responsive deoxyribozyme systems utilizing components with limited water solubility. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 382–391, 2013.     

Cyto-nuclear discordance in the phylogeny of Ficus section Galoglychia and host shifts in plant-pollinator associations

Background  

Hybridization events are relatively common in vascular plants. However, the frequency of these events is unevenly distributed across the plant phylogeny. Plant families in which individual species are pollinated by specific pollinator species are predicted to be less prone to hybridization than other families. However, exceptions may occur within these families, when pollinators shift host-plant species. Indeed, host shifts are expected to increase the rate of hybridization events. Pollinators of Ficus section Galoglychia are suspected to have changed host repeatedly, based on several cases of incongruence between plant phylogeny and taxonomy, and insect phylogeny and taxonomy. We tracked cyto-nuclear discordance across section Galoglychia as evidence for hybridization. To achieve a proper global view, we first clarified the monophyly of section Galoglychia as it had been questioned by recent phylogenetic studies. Moreover, we investigated if fig size could be a factor facilitating host shifts.     

Reconsideration of the term ‘vitrification’ as used in micropropagation

The term vitrification is currently used to describe two types of processes related to tissue-cultured plant material. The first is used to describe organs and tissues having an abnormal morphological appearance and physiological function. The second is used to describe the transition from liquid to solid state, i.e. the formation of ice during low temperature storage of in vitro cultured cells, tissues and organs. Use of the same term to define two greatly different processes in the same research area can only lead to confusion, especially for key words. Thus it is appropriate to reconsider the usage of vitrification in the first sense mentioned above. It is recommended that the term vitrification should no longer be used to indicate plant material with an abnormal morphological appearance and physiological function, and should be substituted by the term hyperhydricity.This paper was written as a follow up of the discussions held during the workshop on Vitrification at the IAPTC-Congress in Amsterdam (1990). Different eminent research workers in the field of vitrification were invited to formulate their preference for a new terminology, and they came to a consensus.     

Euseius addoensis addoensis,an effective predator of citrus thrips,Scirtothrips aurantii,in the eastern Cape Province of South Africa

The predacious mite,Euseius addoensis addoensis (McMurtry) (Acari: Phytoseiidae), significantly (P<0.05) reduced scarring on Palmer Navel oranges,Citrus sinensis (L.) Osbeck, caused by the citrus thrips,Scirtothrips aurantii Faure (Thysanoptera: Thripidae). A chemical exclusion method employing dicofol was used to determine the effect of the predator and numbers ofS. aurantii were monitored on sticky yellow traps. At two sites where numbers of citrus thrips and predacious mites were high, the percentage fruit culled from export quality due to damage byS. aurantii was reduced from 25.5 to 15.5% and from 32.7 to 18.7% by predacious mite activity.Euseius a. addoensis sometimes prevented damage from exceeding 1% cull and was most effective when mean numbers exceeded one mite per inside leaf at petal fall. The best correlation between numbers of mites and the percentage fruit culled at harvest was –0.96 at the location with the highest number of citrus thrips. Tartar emetic (0.398% a.i.) plus sugar (0.4% a.i.) treatments for citrus thrips were slightly detrimental toE. a. addoensis populations but their numbers soon recovered. An alternative treatment, methiocarb 0.016% a.i. plus sugar (0.4% a.i.), almost eliminated the predacious mites and resulted in more citrus thrips damage (10.7% cull) than in the adjacent untreated control (0.2% cull) which contained predacious mites.     

A mechanism for the pinocytosis of latex spheres by tomato fruit protoplasts

Plant protoplasts isolated from tomato fruit locule tissue take up 0·12 polystyrene latex spheres by an endocytotic process. Freeze-etching enables the process to be clearly visualized in the electron microscope and provides important data from face view membrane fractures. When protoplasts are glutaraldehyde fixed prior to mixing with latex, the spheres adhere to the plasmalemma and cause some localized membrane distortion. This shows that adhesive forces are sufficient to initiate the invagination process. Freeze-etch membrane fracture faces carry numerous granules, the density of which is lower in the invaginating region. This reduction is in accordance with the expected value if only that part of the membrane which comes in intimate contact with the sphere expands to surround it. There is no change in the granule densities in the surrounding membrane. In fractured membrane surface views distinct phases can be delimited, and their relative occurrence indicates that the second half of the invagination process (i.e. after the diameter of the sphere has reached the membrane plane) is some thirty times quicker than the first half. It is proposed this indicates operation of surface tension forces during the fast phase, and it may be that cellular energy is required only for the initial slow phase of membrane expansion.     

Molecular epidemiological tools for Salmonella Dublin typing

Abstract A total of 32 strains of Salmonella Dublin recovered from cattle were differentiated by electrophoretic typing of their esterases (zymotyping), restriction fragment length polymorphism of ribosomal DNA (ribotyping), arbitrarily primed PCR (AP-PCR) using five primers, PCR based on repetitive extragenic palindromic sequences (REP-PCR) and PCR based on enterobacterial repetitive intergenic consensus sequences (ERIC-PCR). ERIC-PCR and REP-PCR each gave one type, zymotyping gave three, AP-PCR gave five and ribotyping gave seven types. Combination of ribotyping and AP-PCR produced a total of 11 types, whereas 14 different types were obtained by all five methods. Thus a combination of several methods enhanced the discrimination of cattle-adapted strains among the genotypically homogeneous serovar Salmonella Dublin.     

Withanolides of Datura spp. and hybrids

A new steroidal lactone of the withanolide group has been isolated from two Datura species and three hybrids and characterized as (20R,22R-5α,12α-dihydroxy-1-oxo-6α, 7α-epoxywitha-2,24-dienolide. The distribution of six withanolides throughout section Stramonium of the genus has been investigated.