Construction of pMEKm12, an expression vector for protein production in Pseudomonas syringae

Characterization of the biological roles of proteins is essential for functional genomics of pseudomonads. Heterologous proteins overproduced in Escherichia coli frequently fail to exhibit biological function. To circumvent this problem, vector pMEKm12 was constructed and used to overexpress proteins in Pseudomonas. The vector contains the pRO1600 replication origin, the maltose-binding protein (MBP) fusion system, and an inducible tac promoter. The pMEKm12 was successfully used to overexpress the syringomycin synthetase SyrB1 protein fused to MBP in Pseudomonas syringae pv. syringae. Furthermore, expression of the MBP-SyrB1 protein in the syrB1 mutant BR132A1 resulted in the restoration of syringomycin production. This vector will facilitate confirmation of the biochemical roles of nonribosomal peptide synthetase genes in Pseudomonas syringae, and studies of gene function from a wide spectrum of pseudomonads.     

Initial kinetics of the inactivation of calmodulin by the fungal toxin ophiobolin A

Ophiobolin A, a fungal toxin that affects rice and maize, inhibits calmodulin by reacting with the lysine residues in calmodulin. Previous studies have shown that lysines 75, 77 and 148 in the calmodulin molecule were the binding sites for ophiobolin A, and that lysine 75 was the primary inhibitory site. In this study, we used kinetic analysis and mutated calmodulins to further characterize the inhibition process. The inhibition of bovine-brain calmodulin by ophiobolin A in the presence of excess ophiobolin A occurred rapidly and followed pseudo-first-order kinetics with a second-order rate constant of 3470 M(-1) min(-1). The kinetics data indicated that the binding of a single ophiobolin A molecule was enough to inactivate a calmodulin molecule. Mutant calmodulins in which two of the three aforementioned binding sites for ophiobolin A had been removed by site-directed mutagenesis were examined for the role of each of the three lysines in the inhibition. It was found that when lysine 75 or 77 in the mutant calmodulin was reacted with ophiobolin A, the resulting calmodulin became a poor activator of phosphodiestease. These results provide further evidence that lysine 75 in calmodulin is the primary inhibitory site for ophiobolin A.     

Physiological and ultrastructural changes in “green islands” on Avena sterilis leaves caused by (8R,16R)-(–)-pyrenophorin

The biochemical and ultrastructural changes in "green islands" (GIs) on detached Avena sterilis leaves caused by the macrodiolide (8R,16R)-(-)-pyrenophorin in the dark were examined. In the absence of light, leaf segments retained their photosynthetic pigments for 96 h after treatment with (8R,16R)-(-)-pyrenophorin (70 muM), whereas in the untreated leaves complete senescence, loss of photosynthetic pigments and cell disorganization were observed 72 h after detachment. Proteolytic enzyme activity in treated tissues with pyrenophorin remained at low levels for 96 h after treatment and protein dissipation was lower in the treated than in the untreated. Although tissues in "GIs" seem macroscopically healthy, electron microscopy observations revealed structurally disorganized cells filled with granular, electron-dense material. Chloroplasts were severely damaged and contained a large number of plastoglobuli. Similar ultrastructural changes were also observed in A. sterilis tissues treated with the phytotoxin under illumination, indicating a mechanism operating both under illumination and in the dark.     

Clarifying the Nomenclature in Microbial Weed Control

Correct terminology is essential to promote the concepts of biological control. In the current literature there are many terms used interchangeably and often inappropriately. The purpose of this paper is to examine the overlapping and sometimes contradictory terms used in biological weed control and to suggest a standardized usage to clarify existing terms. This is achieved through a hierarchical classification of existing terms (bioherbicide, mycoherbicide) and the introduction of new terms such as biopestistat (an inundatively applied, living organism which reduces the competitive ability of the target weed to below a desired threshold) and microbially derived phytotoxin (secondary metabolite used in weed management practices as an analogue of a chemical herbicide).     

Relationship of soil hydrogen sulfide level to net carbon assimilation ofPanicum hemitomon andSpartina patens

Panicum hemitomon Schult andSpartina patens (Ait) Muhl. plants from Louisiana Gulf Coast fresh and brackish marshes were subjected to hydrogen sulfide under controlled sediment redox conditions. Net carbon assimilation responses of both species to the combined sediment anaerobiosis and hydrogen sulfide concentrations was measured.Panicum hemitomon was more sensitive to hydrogen sulfide as compared toSpartina patens. Initiation of reduction in net carbon assimilation inP. hemitomon began when H₂S concentrations of soil solution exceeded 0.22 mgl^-1. Reductions in net carbon assimilation inS. patens were also noted at H₂S concentrations exceeding 0.34 mgl^-1. The reduction in net carbon assimilation of both species measured at elevated H₂S concentrations suggests that extreme anaerobiosis and elevated sulfide could contribute to the growth reduction of these species under certain conditions. However based on H₂S concentration in fresh and brackish marsh soil profiles, levels were too low to cause any adverse effects ofPanicum hemitomon. In brackish marsh soils containing hydrogen sulfide of 3.4 mgl^-1 in soil solution, sulfide could be a major factor limiting growth ofS. patens.     

Biosynthesis of coronatine,a thermoregulated phytotoxin produced by the phytopathogen Pseudomonas syringae

Coronatine (COR) is a non-host-specific phytotoxin that is produced by several different pathovars in the species Pseudomonas syringae. COR consists of two distinct components: coronafacic acid (CFA), which is synthesized via the polyketide pathway, and coronamic acid (CMA), a cyclized derivative of isoleucine. Both CFA and CMA function as intermediates in the pathway to COR and must be joined together by an amide bond to form the phytotoxin. Although the mode of action for COR remains obscure, the CFA moiety is a structural and functional analogue of jasmonic acid, a compound that is produced in a variety of plants in response to stress. The COR biosynthetic gene cluster generally occurs on large plasmids in P. syringae, an observation that helps to explain the production of COR by multiple pathovars. Mutagenesis, feeding studies, and complementation analyses have been used to divide the COR biosynthetic gene cluster into functional regions. Nucleotide sequencing of the regions involved in CFA and CMA biosynthesis has revealed relatedness to genes encoding polyketide and peptide synthetases, respectively. The deduced amino acid sequence of the gene responsible for catalyzing amide bond formation between CMA and CFA shows relatedness to enzymes that activate cyclic carboxylic acids by adenylation. Coronatine biosynthesis has been shown to be temperature-sensitive and regulated by a modified two-component regulatory system. Received: 12 February 1996 / Accepted: 8 May 1996