Carsten Christophersen

How Delisea pulchra furanones affect quorum sensing and swarming motility in Serratia liquefaciens MG1

Halogenated furanones produced by the benthic marine macroalga Delisea pulchra inhibit swarming motility of Serratia liquefaciens MG1. This study demonstrates that exogenously added furanones control transcription of the quorum sensing regulated gene swrA in competition with the cognate signal molecule N:-butanoyl-L-homoserine lactone. This in turn results in reduced production of the surface-active compound serrawettin W2, which is crucial for surface translocation of the differentiated swarm cells. It is demonstrated that furanones interfere with interspecies communication during swarming of mixed cultures and that the mode of interference in quorum-sensing control and interspecies communication is not through inhibition of autoinducer synthesis.

Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54

Pseudomonas fluorescens DR54 showed antagonistic properties against plant pathogenic Pythium ultimum and Rhizoctonia solani both in vitro and in planta. Antifungal activity was extractable from spent growth media, and fractionation by semi‐preparative HPLC resulted in isolation of an active compound, which was identified as a new bacterial cyclic lipodepsipeptide, viscosinamide, using 1D and 2D 1H‐, 13C‐NMR and mass spectrometry. The new antibiotic has biosurfactant properties but differs from the known biosurfactant, viscosin, by containing glutamine rather than glutamate at the amino acid position 2 (AA2). No viscosin production was observed, however, when Ps. fluorescens DR54 was cultured in media enriched with glutamate. In vitro tests showed that purified viscosinamide also reduced fungal growth and aerial mycelium development of both P. ultimum and R. solani. Viscosinamide production by Ps. fluorescens DR54 was tightly coupled to cell proliferation in the batch cultures, as the viscosinamide produced per cell mass unit approached a constant value. In batch cultures with variable initial C, N or P nutrient levels, there were no indications of elevated viscosinamide production during starvation or maintenance of the cultures in stationary phase. Analysis of cellular fractions and spent growth media showed that a major fraction of the viscosinamide produced remained bound to the cell membrane of Ps. fluorescens DR54. The isolation, determination of structure and production characteristics of the new compound with both biosurfactant and antibiotic properties have promising perspectives for the application of Ps. fluorescens DR54 in biological control.

Antibiotic and Biosurfactant Properties of Cyclic Lipopeptides Produced by Fluorescent Pseudomonas spp. from the Sugar Beet Rhizosphere

ABSTRACT Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent Pseudomonas spp. To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent Pseudomonas spp. from two different agricultural soils by using three different growth media. CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil. Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups. The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety. Production of specific CLPs could be affiliated with Pseudomonas fluorescens strain groups belonging to biotype I, V, or VI. In vitro analysis using both purified CLPs and whole-cell P. fluorescens preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi. The CLP-producing P. fluorescens strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum.

Structure, production characteristics and fungal antagonism of tensin – a new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578

Aim: To study the antagonistic activity by Pseudomonas fluorescens strain 96.578 on the plant pathogenic fungus Rhizoctonia solani.

Secondary metabolites characteristic of Penicillium citrinum, Penicillium steckii and related species

Two new carboxylic acids, tanzawaic acid E (1) and F (2) in addition to the unknown benzopyran 3,7-dimethyl-1,8-dihydroxy-6-methoxy-isochroman (3), and the known mycotoxin 3,7-dimethyl-8-hydroxy-6-methoxyisochroman (4) were produced by a marine-derived strain of Penicillium steckii isolated from an unidentified tunicate. The carboxylic acids and the benzopyran were identified on the basis of mass spectrometry, and one and two dimensional NMR spectroscopic techniques. The structures 1 and 2 resemble tanzawaic acid A-D, previously isolated from Penicillium citrinum. Screening of isolates of species related to P. citrinum and P. steckii showed that P. citrinum (25 isolates) consistently produced citrinin and tanzawaic acid A, P. steckii (18 isolates) produced isochroman toxins (except 2) and tanzawaic acid E, P. sizovae consistently produced tanzawaic acid A, P. corylophilum (10 isolates) produced citreoisocoumarinol and P. sumatrense (15 isolates) always produced curvularin.

Structure of red and orange fluorescein

The structures of colorless, red, and yellow-orange fluorescein have been investigated by a combination of solution and solid state13C NMR. It is demonstrated that the three forms have lactonic, zwitterionic, and quinoid structures, respectively. Conflicting X-ray, NMR, and IR structural evidence is discussed for samples of red fluorescein which cannot readily be obtained in definite crystalline form. It is concluded that solid-state13C NMR spectroscopy is superior by revealing primarily the molecular structure but being fairly insensitive toward lattice variations.

Lipopeptide Production in Pseudomonas sp. Strain DSS73 Is Regulated by Components of Sugar Beet Seed Exudate via the Gac Two-Component Regulatory System

ABSTRACT Pseudomonas sp. strain DSS73 isolated from the sugar beet rhizosphere produces the cyclic lipopeptide amphisin, which inhibits the growth of plant-pathogenic fungi. By Tn5::luxAB mutagenesis, we obtained two nonproducing mutant strains, DSS73-15C2 and DSS73-12H8. The gene interrupted by the transposon in strain DSS73-15C2 (amsY) encoded a protein with homology to peptide synthetases that was designated amphisin synthetase. DSS73-12H8 carried the transposon in a regulatory gene encoding a protein with homology to the sensor kinase GacS. Growth of strain DSS73-15C2 (amsY) was impaired during the transition to stationary phase in a minimal medium amended with an exudate of sugar beet seeds. This growth phenotype could be complemented by purified amphisin. Seed exudate further induced expression of bioluminescence from the amsY::luxAB reporter during the transition to stationary phase. This agreed with an increase in amphisin production by the DSS73 wild-type strain during early stationary phase. Amphisin synthesis in DSS73 was strictly dependent on GacS, and even induction by seed exudate depended on a functional gacS locus. Hence, a signal triggering the GacS/GacA two-component system appeared to be present in the seed exudate.

Glucosylation of human haemoglobin a in red blood cells studied in vitro. Kinetics of the formation and dissociation of haemoglobin A1c.

We examined the stability of the ketoamine adduct, termed HbA1c, formed between the N-terminal valine of the haemoglobin A (HbA) beta chain and D-glucose using an isoelectric focusing method. Prolonged saline incubation of purified HbA1c followed by renewed isoelectric focusing revealed that the HbA1c concentration decreased while a corresponding increase in the HbA concentration occurred. The emergence of haemoglobin A on saline incubation indicates that the non-enzymatic glucosylation of haemoglobin A to HbA1c is a reversible process. This finding was further substantiated by kinetic studies on the formation and breakdown of the ketoamine adduct during incubation of red cells in glucose and saline. It appeared that the rate constant for the formation k2 was 14.2 X 10(-6) X s-1 at 37 degrees C while the rate constant for dissociation k-2 was 1.7 X 10(-6) X s-1. From these data an equilibrium constant K of 8.4 was calculated. This information should be of importance in interpretation of HbA1c levels during abrupt changes in diabetic control.

Cyclic lipoundecapeptide amphisin from Pseudomonas sp. strain DSS73

The crystal structure of the lipoundecapeptide amphisin, presented here as the tetrahydrate, C(66)H(114)N(12)O(20).4H(2)O, originating from non-ribosomal biosynthesis by Pseudomonas sp. strain DSS73, has been solved to a resolution of 0.65 A. The primary structure of amphisin is beta-hydroxydecanoyl-D-Leu-D-Asp-D-allo-Thr-D-Leu-D-Leu-D-Ser-L-Leu-D-Gln-L-Leu-L-Ile-L-Asp (Leu is leucine, Asp is aspartic acid, Thr is threonine, Ser is serine, Gln is glutamine and Ile is isoleucine). The peptide is a lactone, linking Thr4 O(gamma) to the C-terminal. The stereochemistry of the beta-hydroxy acid is R. The peptide is a close analogue of the cyclic lipopeptides tensin and pholipeptin produced by Pseudomonas fluorescens. The structure of amphisin is mainly helical (3(10)-helix), with the cyclic peptide wrapping around a hydrogen-bonded water molecule. This lipopeptide is amphiphilic and has biosurfactant and antifungal properties.

Antibacterial activity of marine-derived fungi

A total of 227 marine isolates of ubiqituous fungi were cultivated on different media and the secondary metabolite content of the extracts (ethyl acetate/chloroform/methanol 3 : 2 : 1) characterized by HPLC. The fungi were secured from animals, plants and sediments of Venezuelan waters (0–10 m) including mangroves and lagoonal areas. The extracts were tested for antibacterial activity. A total of 7 were active towards Vibrio parahaemolyticus and 55 towards Staphylococcus aureus, representing 18 different fungal species from 8 ascomycetous genera. For 61 strains of Penicillium citrinum antibacterial activity correlated well with content of secondary metabolites as measured by HPLC. Thirteen isolates of Penicillium steckii produced very similar profiles of secondary metabolites and 6 of these had activity against either V. parahaemolyticus or S. aureus or both.