Cembella, Allan

Krock, Bernd

Ma, Haiyan

Muck, Alexandr

Svatoš, Aleš

Wielsch, Natalie

English

Electronic Publication Information Center

Bernd Krock1, Haiyan Ma1, Alexandr Muck2,3, Natalie Wielsch2, Aleš Svatoš2, Allan D. Cembella1ExperimentalCell free culture medium of the lytic strain of A. tamarense (Alex2) was used for the purification of lytic compounds by bioassay (against the cryptophyte Rhodomonas salina) driven fractionation and a non-lytic strain (alex5) was used as a negative control. Lytic compounds were purified by reversed phase SPE and hydrophilic interaction liqui d chromatography (HILIC) [3] and subsequently analyzed by several mass spectrometric techniques.  ResultsQqQ and Orbitrap experimentsTwo masses (m/z 1062 and 1292) were detected in the lytic strain Alex2, which were absent in the non-lytic strain Alex5. However, chromatography on different stationary phases showed thatAlfred-Wegener-Institutfür Polar- und Meeresforschungin der Helmholtz-Gemeinschaftwww.awi.de2) Max-Planck-Institut für Chemische ÖkologieHans-Knöll-Str. 807745 Jena, Germany  Mass spectrometric characterization of allelochemicals produced by the marine dinoflagellate Alexandrium tamarense IntroductionAllelopathic effects, including growth inhibition and cell lysis of target protistan species (Fig.1) have been investigated for decades among the toxic and harmful algal bloom (HAB) forming Alexandrium spp. [1,2], and are believed to relate to bloom formation of the producing organisms. However, neither the chemical character nor the mode of action of the compounds excreted into the surrounding medium by the producing organism have been elucidated.References:[1] Hansen, P.J., 1989. MEPS 53:105-116.[2]  Tillmann, U., 2002. MEPS 230, 47-58.[3]  Ma, H., 2009, Mar. Drugs, 7(4), 497-522.these masses only co-eluted with lytic activity on a C18 phase, but neither on C8 nor on HILIC (data not shown). Thus these masses were excluded as lytic compounds.MALDI-TOF experimentsSince no unique masses of Alex2 could be detected in the low molecular range up to 2000 Da, ytic fractions were analyzed by MALDI-TOF in the linear mode. While negative ionization did not show any peaks (Fig. 2C/D), positive ionization revealed four mass clusters in the range between 7 and 15 kDa only in the lytic strain but not in the non-lytic one (Fig. 2A/B).Trypsin digest and SECBiomolecules of large molecular weight usually are proteins. Therefore, the lytic fraction was incubated with trypsin and the digest chromatographed by size exclusion chromatography (SEC). However, lytic activity was unchanged compared to the untreated fraction in terms of lytic intensity and retention time (Fig 3.). Saccharides were also excluded by a photometric sugar assay (data not shown).ConclusionAlexandrium tamarense lytic compounds consist of a suite of 7 to 15 kDa molecular weight compounds, which are neither polysaccharides nor proteins. Further research is necessary to fully characterize their identiy.1) Alfred Wegener InstituteAm Handelshafen 1227570 Bremerhaven, Germany3) current address: Waters Corp.Helfmann-Park 1065760 Eschborn, GermanyFig. 2: MALDI-TOF mass spectra of HILIC fractions. A. Alex5, linear positive mode; B. Alex2, linear positive mode; C. Alex5, linear negative mode; D. Alex2, linear negative mode.1 /1  v /v  w i t h  S in  a c id  m a t r ix ,  s a m p le  in  1 0 0  u l  w a t e r ,  l a s e r  2 3 0 ,  p u ls e  8 0 0 Vm /z2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 1 2 0 0 0 1 4 0 0 0 1 6 0 0 0 1 8 0 0 0 2 0 0 0 0 2 2 0 0 0 2 4 0 0 0 2 6 0 0 0 2 8 0 0 0%01 0 0%01 0 0%01 0 0%01 0 01 0 0 3 2 9 _ A W I_ B K _ A 5 _ 0 2 _ S IN _ p o s l in   2  ( 0 .1 0 0 )  S m  ( M n ,  3 x 1 5 .0 0 ) ;  S b  ( 1 5 ,1 0 .0 0  ) ;  C m  ( 2 :2 5 ) T O F  L D +  1 .0 7 e 3x 43 1 5 76 9 1 03 9 8 2 7 3 4 68 8 4 0 9 8 9 9 1 0 6 8 7 1 2 1 0 0 1 5 8 4 01 3 5 8 9 1 7 3 8 51 0 0 3 2 9 _ A W I_ B K _ A 2 _ 0 2 _ S IN _ p o s l in   9  ( 0 .4 5 0 )  S m  ( M n ,  3 x 1 5 .0 0 ) ;  S b  ( 1 5 ,1 0 .0 0  ) ;  C m  ( 1 :2 6 ) T O F  L D +  1 .0 7 e 3x 43 0 7 99 7 4 63 1 5 47 8 4 24 0 0 2 4 5 5 6 6 8 3 61 2 5 9 91 1 5 5 31 4 8 4 11 0 0 3 2 9 _ B K _ A W I_ A 5 _ 0 2 _ D H B _ n e g r l in   2 0  ( 1 .0 0 1 )  S m  ( M n ,  3 x 1 5 .0 0 ) ;  S b  ( 1 5 ,1 0 .0 0  ) ;  C m  ( 1 :2 0 ) T O F  L D -  1 .0 7 e 3x 48 0 0 47 0 6 0 1 1 6 3 81 0 0 3 2 9 _ B K _ A W I_ A 2 _ 0 2 _ D H B _ n e g r l in   1 1  ( 0 .5 4 8 )  S m  ( M n ,  3 x 1 5 .0 0 ) ;  S b  ( 1 5 ,1 0 .0 0  ) ;  C m  ( 2 :2 4 ) T O F  L D -  1 .0 7 e 3x 41 3 0 8 41 1 1 9 33 2 2 8ABCD%%%%Non-lytic strain Alex5 Pos. Lin.Lytic strain Alex2 Pos. Lin.Non-lytic strain Alex5 Neg. Lin.Lytic strain Alex2 Neg. Lin.Fig. 1: Lytic effect of Alexandrium tamarense on Oxyrrhis marina (small heterotrophic dinoflagellate).Black arrows: AlexandriumRed arrows: Oxyrrhis residuesAlexandrium tamarenseOxyrrhis marinaPhotos: U. TillmannFig. 3: SEC chromatograms of A: lytic SPE 80% methanol fraction; B: Trypsin; C: SPE + Trypsin. Left scale: absorbance at 280 nm, right scale: lytic activity of individual fractionsLytic SPE fractionTrypsinLytic SPE fraction incubated with trypsinR. salina’smortality [%]020406080100020406080100020406080100SPE + TrypsinSPETrypsinR. salina’smortality [%]

Mass spectrometric characterization of allelochemicals produced by the marine dinoflagellate Alexandrium tamarense

Abstract

Similar works

Full text

Available Versions

Electronic Publication Information Center