Broad diversity of conjugative plasmids in integron-carrying bacteria from wastewater environments

In this study we assessed the occurrence, diversity and conjugative potential of plasmids in integron-carrying Aeromonas and Enterobacteriaceae from wastewaters. Sixty-six strains were included as donors in mating assays using rifampicin-resistant Escherichia coli and Pseudomonas putida recipient strains. The diversity of plasmids from donors and transconjugants (resistant to tetracycline or streptomycin) was evaluated by restriction analysis and replicon typing targeting 19 incompatibility groups. Restriction patterns revealed a diverse plasmid pool present in these strains. Plasmids were assigned to FrepB (Aeromonas salmonicida, Aeromonas veronii, Aeromonas sp., E.?coli, Enterobacter sp.), FIC (A.?salmonicida, Aeromonas sp.), FIA (Shigella sp.), I1 (A.?veronii, Aeromonas sp., E.?coli), HI1 (E.?coli) and U (Aeromonas media) replicons. Nevertheless, 50% of the plasmids could not be assigned to any replicon type. Among integron-positive transconjugants, FrepB, I1 and HI1 replicons were detected. Results showed that wastewaters enclose a rich plasmid pool associated with integron-carrying bacteria, capable of conjugating to different bacterial hosts. Moreover, replicons detected in this study in Aeromonas strains expand our current knowledge of plasmid diversity in this genus.     

Evolution of camalexin and structurally related indolic compounds

Structurally related secondary products are rather rarely shared by organisms from different kingdoms. Consequently, the evolution of biosynthetic pathways of defence metabolites between distantly related organisms has not been broadly investigated. Thiazolylindoles are found in Arabidopsis thaliana, as the phytoalexin camalexin, and in a Streptomyces strain, which synthesizes a tumour-inhibitory derivative, designated BE-10988. Camalexin originates from cysteine and tryptophan, which is converted to indole-3-acetaldoxime and subsequently dehydrated to indole-3-acetonitrile. The metabolic origin of BE-10988 was determined by retrobiosynthetic NMR analysis and incorporation studies with direct precursors. Like camalexin, it is derived from tryptophan and cysteine. However, as BE-10988 is synthesized via indole-3-pyruvic acid, not via indole-3-acetaldoxime, independent mechanisms of tryptophan modification have evolved.     

Antifeedant effects of azadirachtin and structurally related compounds on lepidopterous larvae

The antifeedant activity of azadirachtin, azadirachtin-derivatives and related limonoids was assessed in choice and no-choice bioassays against four species of Lepidoptera: Spodoptera littoralis, Spodoptera frugiperda, Heliothis virescens and Heliothis armigera. The choice bioassay showed that the feeding behaviour of S. littoralis was affected by more of the compounds than that of either S. frugiperda or H. virescens. H. armigera was the least affected. Azadirachtin and dihydroazadirachtin were the most potent of the 40 compounds tested. The results showed that hydrogenation of the C-22,23 double bond did not decrease antifeedant activity and the nature of the substitutes at C-1, C-3 and C-11 were important. Molecules with bulky substitutes at either C-22 or C-23 were usually ineffective antifeedants as were compounds lacking an epoxide. Compounds recorded as active antifeedants in the choice bioassay were not always as active in the no-choice test. The value of the bioassays in assessing the mode of action of the compounds is discussed.

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Résumé L'activité phagodissuadante de l'azadirachtine, de ses dérivés et des limonoïdes voisins sur 4 espèces de lépidoptères: Spodoptera littoralis, S. frugiperda, Heliothis virescens et H. armigera a été évaluée par des expériences avec et sans choix. Les expériences de choix ont montré que le comportement alimentaire de S. littoralis était modifié par plus de substances que celui de S. frugiperda ou H. virescens. Celui de H. armigera était le moins modifié. Les 2 substances les plus puissantes parmi les 40 examinées, ont été l'azadirachtine et le dihydroazadirachtine. Ces résultats montrent que l'hydrogénation de la double liaison C-22,23 ne réduit pas l'activité phagodissuadante et que la nature des substitutions en C-1, C-3 et C-11 sont importantes. Les molécules avec des substitutions volumineuses en C-22 ou C-23 sont généralement des phagodissuadants aussi inefficaces que ceux ayant perdu un époxide. Les substances notées comme phagodissuadants actifs dans les expériences de choix ne sont pas toujours aussi actives en absence de choix. La valeur des tests dans l'évaluation du mode d'action des substances est discuté.

     

Teratogenicity and embryolethality of acrolein and structurally related compounds in rats

Acrolein, a metabolite of the anticancer agent cyclophosphamide, is teratogenic to rats after intraamniotic administration. It is not known whether acrolein or a metabolite of acrolein is responsible for the teratogenicity of this compound. We assessed the teratogenicity and embryolethality of acrolein and five structurally related compounds: acrylic acid, allyl alcohol, glycidol, glyceraldehyde, and propionaldehyde by intraamniotic injections in Sprague-Dawley rats on day 13 of gestation. All compounds tested were significantly embryolethal with at least one concentration of the drug. Acrolein was the most embryolethal of the drugs, causing a significant increase in resorptions with as little as 0.1 micrograms/fetus; the other drugs were embryolethal at doses 100-10,000 times that of acrolein. Acrolein was also the most teratogenic of the drugs tested; a dose as low as 5 micrograms/fetus caused a significant increase in the incidence of fetal malformations. Of the other compounds tested, only glycidol at a dose of 1,000 micrograms/fetus induced a significant number of malformed fetuses compared to control. These results suggest that it is acrolein itself that is responsible for its teratogenicity.     

Effects of some tricyclic psychopharmacons and structurally related compounds on motility ofProteus vulgaris

A simple test for the evaluation of drugs interfering with bacterial motility was established withProteus vulgaris. With this model, promethazine, 7-hydroxy-chlorpromazine, imipramine, 7,8-dioxochlorpromazine and acridine orange were shown to exert significant motility and swarming inhibitory action onProteus vulgaris strains at subinhibitory concentrations. Quinidine enhanced the antimotility effect of promethazine. The antimotility effect of promethazine was synergized by proton pump inhibitors omeprazole and abscissic acid, but antagonized by extracellular potassium and sodium ions.     

Effects of cysteine and structurally related compounds on ochratoxin production by Aspergillus ochraceus

Addition of 10(-2) M L-cysteine, L-cystine, or S-ethyl-L-cysteine to a synthetic medium containing xylose as the sole carbon source did not decrease ochratoxin production by Aspergillus ochraceus. At that concentration, DL-homocysteine thiolactone HC1, DL-cysteine HC1, L-ethionine, S-methyl-L-cysteine, and glutathione (reduced) strongly inhibited ochratoxin production. DL-Homocysteine thiolactone HC1, DL-cysteine HC1, and L-ethionine also strongly inhibited fungal growth. At lower concentrations (10(-3) and 10(-4) M) only L-ethionine decreased the toxin production. Ochratoxin inhibition caused by DL-homocysteine thiolactone HC1, DL-cysteine HC1, and glutathione was observed only in cases where the pH of the media was below 5.0. The inhibition caused by 10(-3) M ethionine was partially prevented by the addition of 10(-3) M methionine but this was not the case after the addition of S-methyl-L-cysteine to the medium.     

Inhibition of malate dehydrogenase by thyroxine and structurally related compounds.

The inhibition of pig heart mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) by the thyroxine and structurally related compounds was studied to resolve a longstanding question about the exact nature of the inhibition. Thyroxine, in freshly prepared solution, was found to be a "pure" competitive inhibitor relative to the nucleotide cofactor. Upon standing in diffuse daylight, solutions of thyroxine showed increased ability to inhibit the enzyme, presumably as a result of oxidation of enzyme sulfhydryl groups by free iodine that is released photochemically. This behavior probably accounts for earlier reports of irreversible inactivation by thyroxine. Comment is made on the implications of these findings to the mechanism of thyroid hormmone action.     

Replication and conjugative mobilization of broad host-range IncQ plasmids

The IncQ plasmids have a broader host-range than any other known replicating element in bacteria. Studies on the replication and conjugative mobilization of these plasmids, which have mostly been focused on the nearly identical RSF1010 and R1162, are summarized with a view to understanding how this broad host-range is achieved. Several significant features of IncQ plasmids emerge from these studies: (1) initiation of replication, involving DnaA-independent activation of the origin and a dedicated primase, is strictly host-independent. (2) The plasmids can be conjugatively mobilized by a variety of different type IV transporters, including those engaged in the secretion of proteins involved in pathogenesis. (3) Stability is insured by a combination of high copy-number and modulated gene expression to reduce metabolic load.     

The integration-excision system of the conjugative transposon Tn 1545 is structurally and functionally related to those of lambdoid phages

Excision of Tn1545 and related conjugative transposons of Gram-positive bacteria occurs by reciprocal site-specific recombination between non-homologous regions of the transposon-target junctions. Excisive recombination requires two transposon-encoded proteins designated Xis-Tn and Int-Tn. We have shown that, following excision, Tn1545 is a circular structure with ends separated by either of the two hexanucleotides that were present at the transposon-target junctions. Using a trans-complementation assay, we have demonstrated that Int-Tn is able to catalyse in vivo integration of a circular intermediate of Tn1545 defective for integration and excision. comparison of integration sites suggests that limited sequence homology at the vicinity of the recombining sites is required for integration of the element. These data support the hypothesis that the integration/excision systems of conjugative transposons from Gram-positive cocci and of lambdoid phages from Gram-negative bacilli have evolved from a common ancestor.     

Specification of the conjugative pili and surface mating systems of Pseudomonas plasmids

Conjugative pili were identified for representative Pseudomonas plasmids of incompatibility groups P-2, P-3, P-5, P-7, P-8, P-10, P-11, and P-13, pili for groups P-1 and P-9 having already been described in detail. FP5 pili (unclassified) were also found. In most cases pili could be characterized by electron microscopy as rigid or flexible. The majority of Pseudomonas plasmids transferred significantly better on a surface than in a liquid. Examples of all incompatibility groups were tested.     

Phenothiazines and structurally related compounds as inhibitors of adenosylmethionine decarboxylase: effects on the purified enzyme

The effects of chlorpromazine, imipramine, thioridazine, chlorprothixene, amitriptyline, desipramine and triflupromazine on adenosylmethionine decarboxylase purified from rat liver have been studied. The compounds caused competitive inhibition of the enzyme at 10(-5) - 10(-3) M concentrations. For chlorprothixene and triflupromazine the inhibition was linear, while the other drugs showed increasing, nonlinear inhibition at higher concentrations. Apparent Ki's for the compounds were between 6.8 X 10(-5) M (for chlorprothixene) and 6.4 X 10(-4) M (for desipramine). Inhibition of 50% under optimal assay conditions was achieved between drug concentrations of 1.3 X 10(-4) M (thioridazine) and 1.3 X 10(-3) M (imipramine).     

Enzymology of type IV macromolecule secretion systems: the conjugative transfer regions of plasmids RP4 and R388 and the cag pathogenicity island of Helicobacter pylori encode structurally and functionally related nucleoside triphosphate hydrolases

Type IV secretion systems direct transport of protein or nucleoprotein complexes across the cell envelopes of prokaryotic donor and eukaryotic or prokaryotic recipient cells. The process is mediated by a membrane-spanning multiprotein assembly. Potential NTPases belonging to the VirB11 family are an essential part of the membrane-spanning complex. Three representatives of these NTPases originating from the conjugative transfer regions of plasmids RP4 (TrbB) and R388 (TrwD) and from the cag pathogenicity island of Helicobacter pylori (HP0525) were overproduced and purified in native form. The proteins display NTPase activity with distinct substrate specificities in vitro. TrbB shows its highest specific hydrolase activity with dATP, and the preferred substrate for HP0525 is ATP. Analysis of defined TrbB mutations altered in motifs conserved within the VirB11 protein family shows that there is a correlation between the loss or reduction of NTPase activity and transfer frequency. Tryptophan fluorescence spectroscopy of TrbB and HP0525 suggests that both interact with phospholipid membranes, changing their conformation. NTPase activity of both proteins was stimulated by the addition of certain phospholipids. According to our results, Virb11-like proteins seem to most likely be involved in the assembly of the membrane-spanning multiprotein complex.     

Effects of some tricyclic psychopharmacons and structurally related compounds on motility of Proteus vulgaris.

A simple test for the evaluation of drugs interfering with bacterial motility was established with Proteus vulgaris. With this model, promethazine, 7-hydroxy-chlorpromazine, imipramine, 7,8-dioxochlorpromazine and acridine orange were shown to exert significant motility and swarming inhibitory action on Proteus vulgaris strains at subinhibitory concentrations. Quinidine enhanced the antimotility effect of promethazine. The antimotility effect of promethazine was synergized by proton pump inhibitors omeprazole and abscissic acid, but antagonized by extracellular potassium and sodium ions.     

Effect of carbohydrates and related compounds on the long-term preservation of freeze-dried bacteria

A selection of bacteria were freeze-dried in horse serum containing various carbohydrates and related compounds. An accelerated storage test at elevated temperatures was used to determine the long-term viability of the dried organisms with the assumption that the results of accelerated storage reflect long-term viability. The results suggest that meso-inositol, non-reducing disaccharides and certain polyalcohols are the most suitable of the compounds tested for incorporation into suspending media for use in freeze-drying.