Tommy Harder Nielsen

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.

Production of Cyclic Lipopeptides by Pseudomonas fluorescens Strains in Bulk Soil and in the Sugar Beet Rhizosphere

ABSTRACT The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 μg g−1) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ∼2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.

O2 uptake, C metabolism and denitrification associated with manure hot-spots

O2, C and N metabolism in organic hot-spots (sites where the intensity of microbial respiration creates a high O2 demand) was studied with fresh or anaerobically digested liquid cattle manure as substrates. A gel-stabilized mixture of soil and manure, 16 mm thick, was sandwiched between layers of soil with a water content adjusted to field capacity, and incubated at 15°C for up to 3 wk. When fresh manure was used, O2 microprofiles demonstrated an O2 penetration into the hot-spot of < 1 mm after 1–3 d, increasing to ca. 2 mm after 3 wk. During this time, O2 uptake rates decreased from 100–150 to ca. 50 nmol O2 cm−2 h−1. With digested manure, the lower C availability in this substrate resulted in O2 penetration depths of 3–4 mm and O2 uptake rates of <30 nmol O2 cm−2 h−1 throughout the 3 wk. Maximum denitrification rates were also consistently lower with digested manure (4 nmol N cm−2 h−1) than with fresh manure (18 nmol N cm−2 h−1). A numerical model of NO3− transport indicated that denitrification was limited by the availability of NO3− during the first week in the fresh manure treatment, and that the soil was the only significant source of NO3− during at least 3 d; after this time nitrification at the soil-manure interface became increasingly important. After the first week with fresh manure, and throughout the experiment with digested manure, C availability apparently regulated denitrification.

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.

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.

Microbial community‐level toxicity testing of linear alkylbenzene sulfonates in aquatic microcosms

Complex microbial communities may serve as ideal and ecologically relevant toxicity indicators. We here report an assessment of frequently used methods in microbial ecology for their feasibility to detect toxic effects of the environmentally important surfactant linear alkylbenzene sulfonate (LAS) on microbial communities in lake water and treated waste water. The two microbial communities were evaluated for changes in community structure and function over a period of 7 weeks in replicated aquatic microcosms amended with various levels of LAS (0, 0.1, 1, 10 or 100 mg l(-1)) and inorganic nutrients. In general, the two communities behaved similarly when challenged with LAS. Following lag periods of 1-3 weeks, LAS was degraded to non-toxic substances. Denaturing gradient gel electrophoresis of 16S rRNA gene fragments and [3H]leucine incorporation were the most sensitive assays with effect levels of 0-1 and 1-10 mg LAS l(-1), respectively. Community-level physiological profiles and pollution-induced community tolerance determinations using Biolog microplates demonstrated less sensitivity with effect levels of 10-100 mg LAS l(-1). Total cell counts and net uptake of inorganic N and P were unaffected even at 100 mg LAS l(-1). Interestingly, different microbial communities developed in some replicate microcosms, indicating the importance of stochastic events for community succession. We conclude that microbial community-level toxicity testing holds great promise and suggest a polyphasic approach involving a range of independent methods targeting both the structure and function of the tested microbial communities.

Genes Involved in Cyclic Lipopeptide Production Are Important for Seed and Straw Colonization by Pseudomonas sp. Strain DSS73

ABSTRACT Survival in natural bulk soil and colonization of sugar beet seeds and barley straw residues were determined for Pseudomonas sp. strain DSS73 and Tn5 mutants in amsY (encoding a peptide synthetase involved in production of the cyclic lipopeptide amphisin) and gacS (encoding the sensory kinase of the two-component GacA/GacS regulatory system). No differences in survival or growth in response to carbon amendment (citrate) were observed in bulk soil. However, both mutants were impaired in their colonization of sugar beet seeds and barley straw residues by an inoculum established in the bulk soil. The two mutants had comparable colonization phenotypes, suggesting that amphisin production is more important for colonization than other gacS-controlled traits.

Cyclic lipoundecapeptide tensin from Pseudomonas fluorescens strain 96.578

The crystal structure of the non-ribosomal lipoundecapeptide tensin from Pseudomonas fluorescens has been solved as an ethyl acetate/bis-water solvate (tensin ethyl acetate dihydrate, C67H115N12O20·C4H8O2·2H2O) to a resolution of 0.8 A. The primary structure of tensin is β-hydroxydecanoyl-d-Leu-d-Asp-d-allo-Thr-d-Leu-d-Leu-d-Ser-l-Leu-d-Gln-l-Leu-l-Ile-l-Glu. The peptide is a lactone linking the Thr3 Oγ atom to the C-terminal C atom. The stereochemistry of the β-hydroxy acid has been shown to be S. The peptide shows structural resemblance to the non-ribosomal cyclic lipopeptide fengycin from Bacillus subtilis. The structure of tensin is essentially helical (310-helix), with the cyclic peptide wrapping around a hydrogen-bonded water molecule. The lipopeptide is amphipathic in good agreement with its function as a biosurfactant.

Cyclic lipoundecapeptide lokisin from Pseudomonas sp. strain DSS41

Lokisin was isolated from Pseudomonas sp. strain DSS41 as part of a study of prospective anti-fungal bio-control agents. Based on NMR and MS studies, lokisin was tentatively identified as pholipeptin. However, detailed analysis of the amino acid constituents by chiral gas chromatography revealed a different d-/l-leucine ratio of 3:2 and the allo-isomer of threonine. Lokisin represents a new structural variation in the cyclic lipoundecapeptide class.

Direct measurements of oxygen microprofiles and distribution of phospholipid-P in a two-phase soil—manure system

Abstract Concentrations of phospholipid-P (Plip-P) were used as an estimate of biomass in soil. Plip-P was correlated with biomass-C, as determined by chloroform fumigation-direct extraction, in soil from a fertilizer experiment, although a background of non-biomass Plip-P was suggested. The distribution of Plip-P was followed during three weeks in a two-phase model system designed to study microbial processes in and around a manure-saturated zone. Concentrations of Plip-P in the soil were unaffected by the presence of manure at ⩾ 4 mm distance from the soil-manure interface. A sharp gradient between manure and soil phases was maintained throughout the three weeks. Oxygen microelectrodes (i.d. ca. 5 μm, o.d. 150–200 μm) were used for direct measurements of oxygen penetration into the manure-saturated zone. No decrease in oxygen concentration was recorded in the soil phase. By Day 1 oxygen penetrated only 0.15 mm into the manure, increasing to 2-2.5 mm after 21 days. During this period the diffusive flux of oxygen decreased five-fold. Aerobic decomposition of dissolved C in the manure was apparently restricted to a thin layer of 0–2 mm thickness below the soil-manure interface.