19/09/2012

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Pseudomonas fluorescens αC119 strain, originally isolated from alfalfa roots, has ex cellent plant growth promoting traits and root-colonizingabilities. It has shown to promote alfalfa growth and protec t against seedling diseases caused by oomycetes such as Pythium spp.. The mechanisms for alfalfagrowth promotion have not been characterized yet. On the othe r hand, the mechanisms for pathogen growth inhibition inclu des production of proteases, volatilesuch as cyanhidric acid, siderophores (iron competition) a nd a biosurfactant. The surfactant compound is a novel cycli c lipopeptide (CLP) whit strong antifungalactivity. CLP are composed by a lipid moiety linked to a pepti de chain cyclized into a lactone ring. A considerable structu ral diversity is observed within CLP ofdifferent bacterial species due to the differences in the le ngth and composition of the lipid moiety as well as in the type , number and configuration of the amino acidsin the peptide chain. Because of this these metabolites have different natural roles such as antagonism towards other mi croorganisms, motility and attachment tosurfaces. The proposed primary mode of action of LPC in antag onisms is pore formation in membranes, leading to an imbalan ce in transmembrane ion fluxes andcell death. Most LPs are synthesized by large nonribosomal pe ptide synthetases (NRPSs) via a thiotemplate process.

� The cyclic lipo-octapeptide synthesized by P. fluorescens strain αC119 might play a role in antimicrobial activity of the strai n and in motility which would be of bigimpact in their root colonization ability.

The main objective of this work was to determine the structur e of the cyclic lipopeptide produced by P. fluorescens αC119 and to characterize the genesinvolved in the biosurfactant synthesis.

PURIFICATION: The surfactant compound was extracted form culturesupernatant with organic solvent. Thin layer chromatograp hy demonstratedthat antifungal activity was linked to surfactant activity . CLP analysis andpurified by high pressure liquid chromatography. Surfacta nt activity wastraced by drop collapse test.

ANALYSIS: MALDI ToF mass spectrometry was applied to elucidate CLPcomposition. The compound had a m/z of 1079,7 and a fractioni ng spectracorresponding to 8 amino acids.

Fig. 3- Ion masses obtained by MALDI-ToF MS/MS espectrometry of the methylated cyclic lipopeptide.Proposed amino acid sequence according to fragmentation products observed is indicated (top). Leu/Ilelinked to the lipidic tail needs to be confirmed.

Diverse chemical reactions were applied to characterize the compound byMALDI Tof :

Fig. 2- A) Reverse phase gradientHPLC profile of an analyticalseparation of the crude surface-activeextract obtained from cell culturesupernatant of P. fluorescens αC119.Mobile phase consisted MeOH PO4buffer (pH6) at 1 ml/min. Detectionwas performed with UV at 210 nm.Arrow indicate surface active fraction.B) Drop collapse test: (i) waterdroplet, (ii) front run and (iii) activefraction.

Chemical reaction Reagents Mass shift Result Interpret ation of result

Ettan CAF (N-terminal sulfonation)

CAF reagent , CAF buffer + 136 (-) N-t not free, Absence of Lys

Methylation Methyl alcohol and AcylChloride

+14 (+) One methylation, not at C-terminal (not free). Presence of Asp.

Hydrolysis of lactone ring Ammonia and Methyl alcohol +17 (+) Linear peptide was obtained

9.0 238.6 468.2 697.8 927.4 1157.0Mass (m/z)

6779.8

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

811.5388

86.1248

981.6580609.4334740.5336

682.5002

599.4621284.2945 722.5202496.349623.0013 964.6304256.2953 853.626738.9771 486.3740 794.5250355.2776242.2005 868.6104 1094.3937985.5778514.3901102.0889 909.0676852.5258611.2832326.1964 979.6597238.2624 698.4856413.3427468.3650 844.6638567.3939344.2337 676.5042185.150474.0846 245.2122 310.2672131.1609 189.1504

Leu/IleLeu/IleGlnLeu/IleLeu/IleThrAsp-met(Leu/Ile + C10HO Acid) “b series”

(Leu/Ile + C10HO Acid)Asp-metThrLeu/IleLeu/IleGlnLeu/Ile Leu/Ile “y series”

Methylated CLP

RANDOM MUTAGENESIS: Mutants unable to produce the CLP were obtained by random mut agenesis using Tn5 transposon. Transposon insertion wasmapped in an amino acid adenylation domain of a non ribosomal peptide synthetase for three mutants and a hypothetical pro tein which seems to beinvolved in flagellum assembly in one mutant. . These mutant s failed to inhibit Pythium debaryanum growth on dual cultures assays and lost theirswimming and swarming ability.

A B

(-) (WT)

(Adn -) (Adn -)

(Adn -)

(Flg -)

Fig. 4- A) Swarming assay on King`s Bmedium (0,3% agar) of two mutants: And-

is a amino acid adenylation mutant; Flg- isa mutant in a flagellum related protein.Plates were inoculated with 10µl of an ONculture of mutants and wild type strain.Plates were incubated during 8h. B)Antifungal activity of culture supernatant offour mutants unable to produce CLP.Supernatant were mixed with potatodextrose agar medium. Wells wereinoculated with Pythium debaryanum.Negative control was not supplemented.

(Adn -)

(Flg -)

(WT)

Mutants will be complemented by introduction ofthe intact gene obtained from a clone of agenomic library of P. fluorescens αC119.Genomic library was assessed usingCopyControl™ Fosmid Library production kit.Fosmid clones failed to express surfactantactivity when screened by dropcollapse test. A PCR approach willbe optimized for NRPS screening.

A B

(i) (ii) (iii)

•P. fluorescens αC119 produces a novel Leucine rich cyclic lipopeptid e. •Purification methodology was optimized for this com pound.•According to mass spectrometry analysis the compositi on of the CLP is comprised for a peptide chain of 8 amino acids and a lipidic tail presumably 3-hidroxydecanoic acid. •This cyclic lipo-octapeptide might play a role in an timicrobial activity of the strain and in motility as mutants unable to produce the sufactant compound cannot inhibit P. debaryanum growth and are impaired in swarming and swimming mo tility.

Financial support: ANII and FCE grant, PEDECIBA

5

4

3

2

1

control

TLC

1

4 5 MeOH

32

Fig. 1- A) Thin layerchromatography of crudeextract. Fractions were elutedand concentrated in Methylalcohol. Then they weresubject to drop collapse testand oomycete growthinhibition assay. Wells wereinoculated with P. debaryanumagar plug.