Novel natural parabens produced by a Microbulbifer bacterium in its calcareous sponge host Leuconia...

Novel natural parabens produced by a Microbulbiferbacterium in its calcareous sponge host Leuconianiveaemi_1880 1527..1539

Elodie Quévrain,1 Isabelle Domart-Coulon,2

Mathieu Pernice2 andMarie-Lise Bourguet-Kondracki1*1Laboratoire de Chimie et Biochimie des SubstancesNaturelles UMR 5154 CNRS, Muséum Nationald’Histoire Naturelle, 57 rue cuvier (C. P 54), Paris,France.2Laboratoire de Biologie des Organismes Marins et desEcosystèmes UMR 5178 MNHN-CNRS-UPMC, MuséumNational d’Histoire Naturelle, 57 rue cuvier (C. P 51),Paris, France.

Summary

A broad variety of natural parabens, including fournovel structures and known ethyl and butyl parabens,were obtained from culture of a Microbulbifer sp. bac-terial strain isolated from the temperate calcareousmarine sponge Leuconia nivea (Grant 1826). Theirstructures were elucidated from spectral analysis,including mass spectrometry and 1D and 2D nuclearmagnetic resonance. Their antimicrobial activityevaluated against Staphylococcus aureus was char-acterized by much higher in vitro activity of thesenatural paraben compounds 3–9 than commercial syn-thetic methyl and propyl parabens, usually used asantimicrobial preservatives. Compounds 4 and 9revealed a bacteriostatic effect and compounds 6 and7 appeared as bactericidal compounds. Major parabencompound 6 was also active against Gram positiveBacillus sp. and Planococcus sp. sponge isolates andwas detected in whole sponge extracts during allseasons, showing its persistent in situ productionwithin the sponge. Moreover, Microbulbifer sp. bacte-ria were visualized in the sponge body wall usingfluorescence in situ hybridization with a probe specificto L4-n2 phylotypes. Co-detection in the sponge hostof both paraben metabolites and Microbulbifer sp.L4-n2 indicates, for the first time, production of natural

parabens in a sponge host, which may have an eco-logical role as chemical mediators.

Introduction

Although the Porifera phylum continues to be an excellentsource of bioactive and original natural products (Bluntet al., 2008), sponge-associated microorganisms haveemerged as promising candidates for the production ofnatural compounds (Moore, 1999; Piel, 2006; Blunt et al.,2007). Recently, many studies revealed that metabolitespreviously ascribed to sponges were in fact synthesizedby microorganisms (Proksch et al., 2002; Hildebrandet al., 2004; Piel, 2004; Salomon et al., 2004), whileothers demonstrated structural similarities betweennatural products isolated from sponges and bacterialmetabolites (Crews and Bescansa, 1986; Zabriskie et al.,1986; Jansen et al., 1996; Erickson et al., 1997; Kunzeet al., 1998). These data explain the considerable atten-tion paid by scientists over the last decade (Bewley andFaulkner, 1998; Hentschel et al., 2006; Taylor et al., 2007)to the diverse microorganisms associated with marinesponges. Electron microscopy has shown that in somedemosponges these microorganisms constitute up to60% of the biomass (Vacelet and Donadey, 1977; Wilkin-son, 1978). In contrast to siliceous sponges, very fewchemical and microbiological studies have so far beencarried out on calcareous sponges. Those which do existare restricted to the Calcinea subclass, with to our know-ledge no studies of the Calcaronea subclass (Schreiberet al., 2006; Muscholl-Silberhorn et al., 2008).

In a programme devoted to studying the role of sponge-associated bacteria and especially the chemical media-tors of the interactions between microbial associates intheir sponge host, the cultivable heterotrophic bacterialbiota was isolated from the temperate calcareous spongeLeuconia nivea collected off Concarneau (NortheastAtlantic, France). The sponge L. nivea (Grant 1826),which belongs to the Baeriidae family in the Calcaroneasubclass, was chosen because its chloromethylenic(CH2Cl2) crude extract revealed a significant and annuallypersistent antimicrobial activity against Staphylococcusaureus. This activity was localized in the bacterial fractionof the sponge obtained by differential sedimentation

Received 29 August, 2008; accepted 16 December, 2008. *For cor-respondence. E-mail [email protected]; Tel. (+33) 1 40 79 31 35;Fax (+33) 1 40 79 56 06.

Environmental Microbiology (2009) 11(6), 1527–1539 doi:10.1111/j.1462-2920.2009.01880.x

© 2009 The AuthorsJournal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd

mailto:[email protected]

following the method of Richelle-Maurer and colleagues(2001). The cultivable heterotrophic bacterial biota asso-ciated with L. nivea was screened to isolate bioactivestrains, with antimicrobial activity spectra that matchedwhole sponge extracts. This provided a novel model toinvestigate the contribution of bacterial metabolites to thechemical profile and antimicrobial activity of their spongehost.

The antimicrobial activity of heterotrophic bacteria iso-lated from L. nivea was examined. CH2Cl2 crude extractinvestigations of the most active strains, Microbulbifersp., led to the isolation of a broad variety of natural para-bens. In addition to the known ethyl 1 and butyl 2 para-bens, seven natural parabens including four novelstructures 5, 6, 7 and 9 were identified. We also con-firmed the in situ production and annual persistence ofthe major bacterial compound 6 in the L. nivea spongethrough liquid chromatography mass spectrometry (LC/MS) analysis.

This study provides the first chemical report of a familyof bioactive compounds from a bacterium associated to acalcareous sponge belonging to the Baeriidae family, andproves its in situ production within the sponge host.

Results

Antimicrobial activity of the sponge L. nivea

We established that CH2Cl2 extracts of the sublittoralsponge L. nivea collected off Concarneau inhibited thegrowth of the clinical strain S. aureus (ATCC 6538) in theagar diffusion assay. Collection at three month intervalsfrom 2005 to 2007 showed that this activity was retainedthroughout the year (Table 1). Furthermore, the anti-S. aureus activity was reproducibly localized in the bacte-rial fraction of the sponge, obtained by differentialsedimentation of mechanically dissociated sponge sus-pension in three independent experiments corresponding

to three different seasons (data not shown). These resultsprompted us to investigate the microbial community asso-ciated with this marine calcareous sponge.

Description of the strain L4-n2

Among the cultivable heterotrophic bacterial biota asso-ciated with L. nivea, one of the most active strains, namedL4-n2, was isolated in January 2006 on Marine Agarsupplemented with nalidixic acid and grown in pure culturein marine agar or marine broth. Bacteria of the strain L4-n2were Gram negative, rod-shaped, and approximately0.3–0.5 mm wide by 2–4 mm long. They were aerobic andformed smooth, convex and mucoid colonies on marineagar, producing a non-diffusible brown pigment. They wereresistant to ampicillin, penicillin and oxacillin and sensitiveto kanamycin (30 UI) and ceftazidime (30 mg).

Phylogenetic affiliation of L4-n2

Strain L4-n2 was identified by sequencing the partial 16SrRNA gene. Sequences were compared with those in thedatabase by using FASTA (Pearson and Lipman, 1988).The FASTA search affiliated L4-n2 isolated from L. niveawith the RSBr-1 type strain of marine bacterium Micro-bulbifer arenaceous previously isolated from red sand-stone off the coast of Scotland (Tanaka et al., 2003) with99.8% 16S rDNA sequence identity. A 16S rRNA-basedphylogenetic tree constructed using the maximum likeli-hood algorithm (Felsenstein, 1981) affiliated L4-n2 withthe genus Microbulbifer in the Alteromonadales order ofthe Gammaproteobacteria subdivision. Within the Microb-ulbifer genus, the L4-n2 isolate formed a highly supportedclade with the marine sandstone derived speciesM. arenaceous and with the halophile environmentalMicrobulbifer sp. strain YIM C306, with a bootstrap confi-dence level of 99.8% (Fig. 1).

Table 1. Antimicrobial activity of the sponge Leuconia nivea throughout 2005–2007, extraction yield and presence of compound 6 in the CH2Cl2crude extracts.

Season Collection dateWet weight(g)

Dry weight(g)

CH2Cl2 massextract (mg)

CH2Cl2 extract’s yield(% of dry weight)

Activity againstS. aureus ATCC 6538a

Compound 6detection (LC/MS)

Spring 30 March 2006 21.9 5.2 13.5 0.26 + (7.5 mm) ND27 April 2006 517.0 13.7 26.9 0.20 + (7.5 mm) +

Summer 24 June 2005 4.5 1.1 18.0 1.67 + (11 mm) ND24 July 2005 27.0 6.5 55.9 0.86 + (7 mm) +

Fall 09 September 2006 ND ND 12.3 ND + (8 mm) +27 September 2007 5.3 1.3 11.0 0.87 + (10 mm) +17 October 2005 24.8 5.9 22.7 0.38 + (9 mm) +

Winter 31 January 2006 45.6 10.9 13.3 0.12 + (8 mm) +19 February 2007 12.1 2.9 3.3 0.11 + (8 mm) ND

a. Inhibition’s diameter of the CH2Cl2 extract measured by the disc diffusion assay method for 1 mg per disc.ND, not determined.

1528 E. Quévrain, I. Domart-Coulon, M. Pernice and M.-L. Bourguet-Kondracki

© 2009 The AuthorsJournal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539

https://www.researchgate.net/publication/19993060_Pearson_WR_Lipman_DJ_Improved_tools_for_biological_sequence_comparison_Proc_Natl_Acad_Sci_USA_85_2444-2448?el=1_x_8&enrichId=rgreq-d004c224-49cf-4e8b-bb4a-d53212948464&enrichSource=Y292ZXJQYWdlOzI0MDI0MjM0O0FTOjk3MTEwMTgzNjQ1MTk5QDE0MDAxNjQyODA0OTA=

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https://www.researchgate.net/publication/8964266_Microbulbifer_arenaceous_sp_nov_a_New_Endolithic_Bacterium_Isolated_from_the_Inside_of_Red_Sandstone?el=1_x_8&enrichId=rgreq-d004c224-49cf-4e8b-bb4a-d53212948464&enrichSource=Y292ZXJQYWdlOzI0MDI0MjM0O0FTOjk3MTEwMTgzNjQ1MTk5QDE0MDAxNjQyODA0OTA=

Bacterial paraben compounds

Cultures of the L4-n2 strain were extracted with CH2Cl2.As extracts of both culture supernatant and pellet dis-played identical chemical profile and activity, they werecombined to constitute the CH2Cl2 crude extract of theculture, which was further fractionated using a bio-guidedassay for its antimicrobial activity. Successive chromato-graphies of the CH2Cl2 crude extract on silica gel andreverse-phase columns, guided by antimicrobial activity

against S. aureus, yielded nine compounds belonging tothe paraben series: the known ethyl- and butyl-parabens1–2 and the seven natural analogues 3–9, including thefour new natural parabens 5, 6, 7 and 9 (Fig. 2). Com-pounds 1, 3, 4, 8 appeared for the first time as naturalproducts. The structures of these isolated natural com-pounds were determined by mass and nuclear magneticresonance (NMR) (1D and 2D) spectral analysis.

Compound 1 was obtained as a white powder. Electro-spray ionization–mass spectrometry (ESI-MS) esta-

0.1

Bacillus algicola SC3 (DQ001308)

Moritella marina NCIMB 1144T (X82142)

Alteromonas macleodii DSM 6062 (Y18228)

Pseudoalteromonas flavipulchra NCIMB 2033 (AF297958)

Microbulbifer maritimus TF-17 (AY377986)

L4-n2 (FM200853)

Microbulbifer sp. YIM C306 (EU135714)

Microbulbifer arenaceous RSBr-1T (AJ510266)

Microbulbifer sp. KBB-1 (DQ412068)

Microbulbifer agarilyticus JAMB A3 (AB158515)

Bacterium QM46 (DQ822531)

Microbulbifer elongatus JAMB A7 (AB107975)

Microbulbifer elongatus ATCC 10144T (AB021368)

Microbulbifer elongatus DSM 6810 (AF500006)

Microbulbifer salipaludis SM-1 (AF479688)

Microbulbifer sp. Th/B/38 (AY224196)

Microbulbifer hydrolyticus DSM 11525T (AJ608704)

Microbulbifer sp. A4B-17 (AB243106)

Microbulbifer sp. 17x/A02/240 (AY576773)

Microbulbifer mediterraneus UST 040317-067 (DQ096579)

Microbulbifer cystodytense C1 (AJ620879)

Microbulbifer sp. CJ11049 (AF500206)

Microbulbifer thermotolerans JAMB A94 (AB124836)

99

77

98

50

100

100

93

70

100

98

58

100

51

88

73

(deep sea)

(deep sea)

(deep sea)

(intertidal)

(intertidal)

(intertidal)

(intertidal)

(intertidal)

0.1






L4-n2 (FM200853)

















Microbulbifer thermotolerans JAMB A94 (AB124836)0.1






L4-n2 (FM200853)

















Microbulbifer thermotolerans JAMB A94 (AB124836)

99

77

98

50

100

100

93

70

100

98

58

100

51

88

73

(deep sea)

(deep sea)

(deep sea)

(intertidal)

(intertidal)

(intertidal)

(intertidal)

(intertidal)

Fig. 1. Phylogenetic affiliation of the strain L4-n2 within the genus Microbulbifer in the Alteromonadales order of the Gammaproteobacteria,based on maximum likelihood algorithm using the 16S rRNA gene sequence. The tree was constructed by analysis of 1304 bp of the 1500 bpgene sequence, using the maximum likelihood method from the PHYLIP package (fastdnalml software at http://bioweb.pasteur.fr). Bootstrapvalues (calculated as percentage from 1000 replications) higher than 50% are reported at branch nodes and show the robustness of observedbranching patterns. Scale bar represents 0.1 substitutions per nucleotide position and indicates estimated sequence divergence. Bacillusalgicola (DQ001308) was used as outgroup. Information on the natural environment of the strains is indicated in brackets.

Parabens from sponge associated bacteria 1529


http://bioweb.pasteur.fr

blished the protonated molecular ion [M+H]+ at m/z167.0708 (D -0.019 mmu), indicating the molecularformula C9H11O3. Detailed examination of spectroscopicdata (UV, 1H and 13C NMR spectra) readily establishedthe identity of compound 1 with the ethyl ester of4-hydroxybenzoic acid named ethylparaben, a petro-chemical antimicrobial compound used as preservativefor pharmaceuticals.

1H NMR data of the following compounds 2–9 revealedthat they share two identical sets of coupled protons at d7.94 (d, 8.8) and d 6.83 (d, 8.8) and one deshieldedmethylene proton signal at d 4.25 (t, 6.7). These dataidentified them as analogues of the paraben 1.

Electrospray ionization–mass spectrometry of com-pound 2, a white powder, showed the [M+H]+ ion at m/z195.1028 (D -0.6 mmu), leading to the molecular formulaC11H15O3. Examination of spectroscopic data (UV, 1H and13C NMR spectra) identified compound 2 with the butylester of the 4-hydroxybenzoic acid named butylparaben,another petrochemical antimicrobial compound also usedas preservative in foods and cosmetics.

Compound 3 was obtained as colourless oil. Electro-spray ionization–mass spectrometry analysis suggestedthe molecular formula C15H23O3, for the pseudomolecular[M+H]+ ion peak at m/z 251.1652. In comparison with 1HNMR data of the compound 2, we observed four addi-tional methylene proton signals at d 1.34 (m), and 1.26 (m)suggesting the presence of an octyl alkyl chain in themolecule, which was confirmed with HMBC and COSYspectra. Hence, compound 3 was easily identified asbeing the 4-hydroxybenzoic acid octyl ester or octylpara-ben, already known as a synthetic compound but isolatedfor the first time as natural product.

Compound 4 was obtained as a pale yellow oil.Electrospray ionization–mass spectrometry analysissuggested the molecular formula C17H27O3 for thepseudomolecular [M+H]+ ion peak at m/z 279.1962. Its 1HNMR spectrum displayed analogies with compound 3.The most significant differences observed in the 1H NMRspectrum concerned the alkyl chain. Additional methyleneproton signals at d 1.24 (m) and 1.26 (m) suggesting thepresence of a decyl chain in compound 4, which waseasily identified as being the decyl hydroxybenzoate, anew natural paraben.

Compound 5 was obtained as a pale yellow oil. TheESI-MS showed the [M+H]+ ion at m/z 295.1913 (calc.295.1923 for C17H26O4) suggesting the molecular formulaC17H26O4 for compound 5, which differed from that of 4only by an oxygen atom more. The presence of a parahy-droxybenzoate was easily recognized by characteristicsignals from its 1H NMR spectrum. In the 1H NMR spec-trum, we also observed additional signals indicating thepresence of one methine proton at d 3.71 (m) and fourmethylene signals at d (4.58–4.33, 1.92–1.76, 1.45–1.30,1.40–1.28). The COSY spectrum allowed to delineate thepartial structure C1′–C4′. Finally, HMBC correlationsbetween the deshielded methylene protons at d(4.58–4.33) to carbons at d 36.5 (C2′) and 68.6 (C3′) andbetween the methylene protons at d (1.93–1.76) tocarbons at d 62.0 (C-1′) and 68.6 (C-3′) located thehydroxyl function in position 3′ of the alkyl chain. The finalstructure identified the new natural paraben 5 as the4-hydroxybenzoic acid 3-hydroxy-decyl ester alsonamed hydroxydecylparaben. The very low yield of thiscompound precluded determination of its absoluteconfiguration.

Compound 6 was isolated as a colourless oil. Itsmolecular formula C18H28O3 was deduced from ESI-MS ofthe protonated molecular ion [M+H]+ at m/z 293.2115,indicating the presence of five unsaturations in the mo-lecule. Inspection of the 1H NMR spectrum indicated thatcompound 6 was another 4-hydroxybenzoate (4HBA)alkyl ester. Additional signals for one methine proton at d1.53 (m) and two methyl doublets at d 0.88 (d, 6.6) wereobserved in the 1H NMR spectrum. 1H COSY correlationsbetween this methine proton at d 1.53 and both methylgroups at d 0.88 established the presence of an isopropylgroup. HMBC correlations, as illustrated in Fig. 3,supported the identification of compound 6 as the newmethyldecylparaben.

Compound 7 was obtained as white needles. The[M+H]+ ion at m/z 309.2055 suggesting for compound 7the molecular formula C18H28O4, which differed from thatof 6 only by an oxygen atom more. However, detailedexamination of spectral data indicated similarities withcompound 5. The 1H NMR spectrum also showed thepresence one methine proton at d 3.72 (m) and fourmethylene signals at d (4.57–4.33, 1.93–1.76, 1.47–1.47,

Fig. 2. Structures of the paraben compounds1–9.

1 R = -CH2-CH3

2 R = -(CH2)3- CH3

3 R = -(CH2)7-CH3

4 R = -(CH2)9- CH3

5 R = -(CH2)2-CH(OH)-(CH2)6-CH3

6 R = -(CH2)8-CH(CH3)2

7 R = -(CH2)2-CH(OH)-(CH2)5-CH(CH3)2

8 R = -(CH2)11-CH3

9 R = -(CH2)4-CH=CH-(CH2)5-CH3

HO

O

O

R

12

3

45

6

7

1 R = -CH2-CH3

2 R = -(CH2)3- CH3

3 R = -(CH2)7-CH3

4 R = -(CH2)9- CH3

5 R = -(CH2)2-CH(OH)-(CH2)6-CH3

6 R = -(CH2)8-CH(CH3)2

7 R = -(CH2)2-CH(OH)-(CH2)5-CH(CH3)2

8 R = -(CH2)11-CH3

9 R = -(CH2)4-CH=CH-(CH2)5-CH3

HO

O

O

R

12

3

45

6

7



1.46–1.31), indicating that 7 possessed the same C-1′–C-4′ as compound 5. 1H COSY correlations between themethine proton at d 1.26 and both methyl groups at d 0.83established the presence of an isopropyl group, identify-ing the new paraben 7 as the 4-hydroxybenzoic acid3-hydroxymethyldecyl ester, also named hydroxymethyl-decylparaben. Its absolute configuration could notbe examined due to limited amount of the isolatedcompound.

Compound 8 was isolated as a white amorphous solidof the molecular formula C19H30O3 deduced from ESI-MSof the protonated molecular [M+H]+ ion at m/z 307.2278(C19H31O3). Detailed interpretation of NMR data easilyrevealed the structure of the dodecylparaben.

Compound 9 was isolated as a white amorphous solid.Electrospray ionization–mass spectrometry analysis sug-gested the molecular formula C19H29O3 for the pseudo-molecular [M+H]+ ion peak at m/z 305.2102, differingfrom that of 8 by two hydrogens and indicating the pre-sence of six unsaturations in the molecule. The maindifferences were observed in their NMR data. In 1H and13C NMR spectra of compound 9, we observed the pres-ence of two proton signals at d 5.35 (2H, m) and two

carbon resonances at d 130.6 and 129.2 respectively,suggesting the presence of one additional unsaturationin the alkyl chain. Its localization was determined byHMBC correlations from the both methylene protons at d2.10 (H-4′, m) and d 2.00 (H-7′, m) to the carbons reso-nating at d 129.2 (C-5′) and 130.6 (C-6′). Key COSYcorrelations from the both methylene protons at d 2.10(H-4′, m) and d 2.00 (H-7′, m) to the methine protons atd 5.35 (2H, H-5′, H-6′) delineated the partial structureC4′–C7′ and confirmed the localization of the unsatura-tion. Compound 9 was identified as a new naturalparaben named dodec-5-enylparaben. The geometry ofthe double bond could be determined neither by 13CNMR nor by NOESY spectrum.

Activity of bacterial parabens against S. aureus

Antimicrobial activity of the paraben family isolated fromMicrobulbifer L4-n2 strain was confirmed towardsS. aureus (Gram+) bacterial strain and compared with theantimicrobial activity of the commercial methylparabenand propylparaben (Interchimie). Results of this evalua-tion are summarized in Table 2.

Fig. 3. Selected COSY (–) and HMBC (→)correlations of compound 6.

HO

H

H

H

H

O

O

Table 2 MICs and MBCs of the compounds 1–9 against Staphylococcus aureus.

Compound MIC (mM)a,b MIC (mg ml-1)a,b MBC (mg ml-1)a Ratio MBC/MIC

Methylparaben 328.9–657.8 50–100 > 100 C. Dc

Propylparaben 277.7–555.5 50–100 > 100 C. Dc

1 > 602.0 > 100 > 100 C. Dc

2 128.8–257.7 25–50 > 100 C. Dc

3 100–200 25–50 50–100 2 = bactericidal4 2.8–5.6 0.78–1.56 12.5–25 16 = bacteriostatic5 85–170 25–50 > 100 C. Dc

6 42.8–85.6 12.5–25 25–50 2 = bactericidal7 40.5–81.1 12.5–25 25–50 2 = bactericidal8 81.6–163.3 25–50 > 100 C. Dc

9 20.5–41.1 6.25–12.5 50–100 16 = bacteriostatic

a. Compounds were tested in triplicate.b. By turbidity method.c. C. D: Cannot be determined because MBC is above detection range.Minimal inhibitory concentration (MIC) was expressed as the interval a-b, where a is the highest concentration tested at which microorganisms aregrowing and b the lowest concentration that causes 100% growth inhibition.Minimal bactericidal concentration (MBC) was defined as the lowest concentration of compound yielding colony counts < 0.1% of the initialinoculum.MBC/MIC revealed the nature of compound. If MBC/MIC is less than 4, the compound is bactericidal, If MBC/MIC is between 8 and 16, thecompound is bacteriostatic.



All of the parabens isolated from the culture of Micro-bulbifer sp. strain L4-n2 exhibited significant antimicrobialactivity. Compound 4 exhibited the greatest efficiencyagainst S. aureus with minimal inhibitory concentration(MIC) values of 2.8–5.6 mM.

In order to determine whether the growth inhibitionobserved was reflected a bactericidal or bacteriostaticeffect, minimal bactericidal concentrations (MBCs) weredetermined and the ratio MBC/MIC was calculated. Usingthis method, compounds 4 and 9 appeared as bacterio-static and compounds 3, 6 and 7 appeared as bacteri-cidal. In order to search for a possible synergistic mode ofaction, antimicrobial assays were simultaneously per-formed with mixtures of compounds at equimolar ratio butneither synergistic effects, nor additional effects wereobserved against S. aureus (data not shown).

Antimicrobial activities of the L4-n2 CH2Cl2 crude extractand purified compound 6

The CH2Cl2 crude extract of the strain L4-n2 proved to beactive against marine Gram positive strain isolated fromthe sponge L. nivea and identified as Bacillus sp. L6-3eand also against Gram negative Escherichia coli (ATCC8739) and the environmental bacterium Vibrio splendidus(LGP32) as well as sponge-derived AlphaproteobacteriaPseudovibrio sp. L4-8 (Table 3). These results revealedthe existence of various cross-interactions between strainL4-n2 and other sponge-derived isolates. Compound 6,which was the major bacterial paraben produced byL4-n2, inhibited the growth of the marine L. nivea-derivedGram positive strains Bacillus sp. L6-3e and Planococcussp. L5-4b, but it did not inhibit Gram negative bacteriasuch as E. coli (ATCC 8739), nor marine environmentalV. splendidus (LGP32) and sponge-derived Pseudovibriosp. L4-8 (Table 3). This indicated the presence of antimi-crobial compounds other than parabens in the CH2Cl2extract of L4-n2 Microbulbifer strain, and that these yetundetermined metabolites were being responsible for theactivity against Gram negative bacteria.

In situ chemical detection of compound 6 in the spongeCH2Cl2 extract

Liquid chromatography-mass spectrometry (LC-MS) wascarried out to look for compound 6, the major bacterialparaben produced by the Microbulbifer sp. L4-n2 straincultures. Compound 6 was detected in CH2Cl2 spongeextracts at every season (Table 1 and Fig. 4), indicatingpersistent annual production of this bacterial metabolitewithin the sponge and suggesting a contribution of thismetabolite to the permanent antimicrobial activity ofL. nivea.

In situ localization of the Microbulbifer strain L4-n2 bycatalysed reporter deposition fluorescence in situhybridization in the sponge body wall

A probe was designed against a variable region of the 16SrRNA gene specific to the clade of our Microbulbifer sp.strain L4-n2, M. arenaceous and Microbulbifer sp. strainYIM C306 (Fig. 5). The catalysed reporter deposition fluo-rescence in situ hybridization (CARD-FISH) signal waspositive only for the L4-n2 bacteria (Fig. 5B) and negativefor control Alteromonadales Moritella sp. L7-2nh (Fig. 5C)and Pseudoalteromonas sp. L7-3a (data not shown) aswell as Alphaproteobacteria Pseudovibrio sp. L4-8 (datanot shown) strains also isolated from L. nivea. Tissuesections obtained from L. nivea specimens collected infour seasons were used: July 2005 (two sections),October 2005 (two sections), January 2006 at the time theL4-n2 strain was isolated (two sections) and September2006 when compound 6 detection by LC/MS in wholesponge extracts was highest in two consecutive years(three sections). Examination of sponge tissue sectionsafter hybridization with the specific probe confirmed thepersistent presence of strain L4-n2 with a positive signalobserved in these four seasons, marking a rod-shapedbacteria, in at least five different optical field zones persection. Bacterial distribution was restricted to the extra-cellular mesohyl and preferentially to the ectosome (outerpart of the body wall), although some positive bacteria

Table 3. Antimicrobial activity of the L4-n2 bacterial strain.

Reference strains

Gram positive strains Gram negative strains

S. aureusATCC 6538

Bacillus sp.L6-3ea

Planococcus sp.L5-4ba

E. coliATCC 8739

V. splendidusLGP32

Pseudovibrio sp.L4-8a

CH2CL2 crude extractb +17 mm +11 mm ND +11 mm +7 mm +9 mmCompound 6c +13 mm +9 mm +7.5 mm 0 0 0

a. Leuconia nivea isolates.b. CH2Cl2 crude extract was tested by disc diffusion assay (1 mg per disc).c. Pure compound 6 was tested by disc diffusion assay at 100 mg per disc.ND, not determined.



were also observed in the mesohyl surrounding the cho-anocyte chambers. Signal abundance was less than5% relative to the total number of bacteria stained withthe non-specific DAPI DNA fluorochrome or with thenon-specific eubacterial probe EUB338, indicating alow number of L4-n2 affiliated representatives in the totalbacterial community of L. nivea. Although at low relativeabundance, Microbulbifer sp. L4-n2 was persistentlypresent in situ within its sponge host, throughout the year.

Discussion

The CH2Cl2 crude extracts of the marine sponge L. nivea,selected after antimicrobial screening, revealed perma-nent activity against Gram positive (S. aureus) bacteria inall seasons from June 2005 through February 2007. Thisactivity was localized in the bacterial fraction of thesponge separated by differential sedimentation of the dis-sociated sponge cell suspension. Among the bacterialisolates comprising the cultivable heterotrophic flora, oneof the most active strains against S. aureus was a Gramnegative strain which was phylogenetically affiliated withthe genus Microbulbifer.

The chemical nature of the compounds responsible forthe antimicrobial activity against S. aureus has been

determined as alkyl esters of the 4HBA, also named para-bens. Their antimicrobial activity was evaluated, revealingthat natural parabens 3–9 have better in vitro activity thansynthetic methylparaben and propylparaben and thannatural ethylparaben and butylparaben 1–2, which are thefour parabens usually used as antimicrobial preserva-tives. We found that compounds 3, 4, 6, 8 revealed ahigher potency and appeared about 100-fold more activethan methyl and propyl synthetic parabens as well asethyl and butyl natural parabens (1–2).

Bacteria of the L4-n2 16S rDNA phylotype were loca-lized by fluorescence in situ hybridization with a specificprobe in the sponge body wall, in summer, early fall, andwinter, indicating the persistent presence of these bacte-ria in the L. nivea sponge. Furthermore, the in situ detec-tion of compound 6 by LC/MS in the host sponge extractat every season indicates production of bacterial parabencompounds within its calcareous sponge host.

Recently, Xue Peng and colleagues (2006) reportedthat a strain of Microbulbifer sp. (strain A4B-17), isolatedfrom a tropical ascidian, was able to produce 4HBA andits butyl-, heptyl- and non-yl esters. With the exceptionof butylparaben 2, these compounds are different fromthose produced by our strain L4-n2 derived from a cal-careous temperate sponge. These authors also reported

Fig. 4. Detection of compound 6 by LC/MS in the CH2Cl2 crude extract of L. nivea (January 2006).A. HPLC profile total ion current chromatogram selecting for retention time: 18 min.B. Positive ESI mass spectrum revealing the presence of compound 6.



that out of a total of 23 strains tested, belonging to thegenus Microbulbifer and derived from various marinesources and geographic areas, mostly tropical, none ofthem except the A4B-17 strain was able to produceparabens. Strain A4B-17 shared 95% 16S rRNA genesequence identity with Microbulbifer hydrolyticus IRE-31T and Microbulbifer salipaludis SM-1 T and 94% 16SrRNA gene sequence identity with Microbulbifer elonga-tus ATCC10144 T. Our strain L4-n2 forms a highly sup-ported clade with M. arenaceous, in a subgroup ofMicrobulbifer sp. strains distant from the above men-tioned strains, which reinforces its specific interest. Toour knowledge the antimicrobial activities of M. arena-ceous previously isolated from red sandstone offScotland (Tanaka et al., 2003) and of halophile environ-mental Microbulbifer sp. strain YIM C306 (EMBLEU135714; X.L. Cui, Y.G. Chen, W.J. Li, L.H. Xu andC.L. Jiang, unpublished) have not been evaluated.

All the Microbulbifer sp. strains tested by Peng andcolleagues were able to produce 4HBA, although in loweramounts (less than 20%) than the A4B-17 strain. Com-pound 4HBA which is only soluble in MeOH or H2O wasnot detected in the CH2Cl2 crude extract of our L4-n2Microbulbifer sp. strain but might be present in extractsbased on more polar solvents. Our study confirms that

marine bacteria belonging to the genus Microbulbifer areable to produce natural parabens. Furthermore, we haveshown in preliminary studies that these natural parabenscan inhibit other bacterial strains from the sponge. Theseresults suggest that they might play a role in the interac-tions between bacterial associates within their spongehost and/or might contribute to the chemical defencestrategy of the littoral sponges against opportunistic Grampositive environmental bacteria.

We have shown that strains affiliated to Microbulbifersp. L4-n2 strains were present but not abundant in thesponge L. nivea and mostly restricted to the ectosome ofthe sponge body wall. Our study confirms previousreports of Microbulbifer sp. bacteria in the culturedfraction and among the phylotypes of sponge bacterialcommunities: it adds to the recent discovery in theMediterranean Clathrina clathrus sponge of the Calcineasubclass (Muscholl-Silberhorn et al., 2008) of a Microbul-bifer sp. isolate, based on partial (400 nt) 16S rRNAsequence. We report for the first time the visualization ofthese bacteria within a sponge body wall with a specificoligonucleotidic probe. Future studies targeting other bac-terial groups will be carried out to determine the relativeabundance in the L. nivea sponge host of the differentcultivable bacteria in order to determine which microor-

Fig. 5. CARD-FISH hybridization of bacteria in culture (A, B, C) and in the sponge host Leuconia nivea (D, E, F). Culture of Microbulbifer sp.L4-n2 bacterial cells were hybridized with the universal bacterial probe EUB338 (A) and probe Ma445 specific to the Microbulbifer sp. L4-n2phylotype (B) which does not hybridize with bacterial cells of another sponge-associated Alteromonadales bacteria Moritella sp. L7-2nh (C,and insert C visualized with EUB338 eubacterial probe). Probe NW442 specific to sponge Pseudovibrio sp. Alphaproteobacteria was used asinternal negative control and showed no positive signal with L4-n2 bacterial cells (insert B). Another internal negative control, probe nonEUB,was also used (data not shown). Leuconia nivea tissue sections were hybridized with the universal probe EUB388 (D) and specific probeMa445 with positive signal confirming the presence of Microbulbifer sp. L4-n2 phylotype (arrows) in both samples collected in January 2006(E) and September 2006 (F). In blue, nuclei of L. nivea sponge cells are stained with DAPI. (A, B, C) Scale bar, 10 mm; (D, E, F) Scale bar,20 mm.



ganisms represent stable members of the community. Itopens the way to the isolation and elucidation of otherchemical mediators involved in the interactions betweensponge bacterial associates.

Although parabens are well-known compounds (syn-thetic molecules) widely used as preservatives in cosmet-ics, food, pharmaceutical forms, their natural role in themarine environment has not yet been explored and theirsupply from natural bacterial sources could open newperspectives in the field of marine natural productschemistry.

Conclusion

From the sponge L. nivea, a Microbulbifer sp. strain L4-n2was isolated and shown to produce novel parabens withantibacterial activity against Gram positive referencebacteria S. aureus as well as against marine Bacillus sp.and Planococcus sp. isolates derived from the sponge. Itsmajor paraben metabolite was also detected in the cal-careous sponge at all seasons, contributing to its year-round antimicrobial activity. The Microbulbifer strain L4-n2was localized by fluorescence in situ hybridization in thesponge body wall. Our study is the first to show that aparaben metabolite is persistently produced by a Micro-bulbifer bacterial strain within its sponge host.

These results suggest that the L4-n2 Microbulbiferstrain contributes to the chemical profile of the Calci-sponge L. nivea, and that natural parabens might have anecological role in the balance between bacterial popula-tions within the sponge.

Currently, studies are being carried out in order to cha-racterize the interactions between the L4-n2 Microbulbiferstrain and the other strains in the sponge-associated cul-tivable heterotrophic microbiota, especially to elucidatethe structure of active compounds involved in the mainte-nance of the diversity of the microbial community charac-teristic of marine sponges.

Experimental procedures

General experimental procedures

Optical rotations were obtained on a Perkin Elmer 341 pola-rimeter. UV spectra were obtained in EtOH, using a Kontron-type Uvikon 930 spectrophotometer. Silica gel columnchromatographies were carried out using Kieselgel 60 (230–400 mesh, E. Merck). Fractions were monitored by thin-layerchromatography (TLC) using aluminium-backed sheets (Sigel 60 F254, 0.25 mm thick) with visualization under UV (254and 365 nm) and Liebermann spray reagent. Analyticalreversed-phase high-performance liquid chromatography(HPLC) (Kromasil RP18 column K2185, 4.6 ¥ 250 mm) wereperformed with a L-6200 A pump (Merck-Hitachi) equippedwith a UV-vis detector (l = 254 nm) L-4250C (Merck-Hitachi)and a chromato-integrator D-2500 (Merck-Hitachi). Mass

spectra were recorded on an API Q-STAR PULSAR I ofApplied Biosystem.

13C NMR spectra were obtained on a Bruker AC300 at75.47 MHz, 1H NMR spectra 1D and 2D (COSY, HSQC,HMBC, NOESY) were obtained on a Bruker AVANCE 400.HMQC and HMBC experiments were acquired at 400.13 MHzusing a 1H-13C Dual probehead. The delay preceding the 13Cpulse for the creation of multiple quanta coherences throughseveral bounds in the HMBC was set to 70 ms.

Sponge material and preparation of crude extract

Live specimens of the calcareous sponge L. nivea (Porifera,Calcarea, Calcaronea, Baeriidae) were collected off Concar-neau (France) from 2005 to 2007. Samples of fresh sponge(4–40 g wet weight corresponding to 1–10 g dry weight) wereimmersed after collection in a CH2Cl2/MeOH 1/1 mixture atroom temperature for 2 days, concentrated under reducedpressure and extracted with CH2Cl2.

Antimicrobial assays

They were performed by the disk diffusion assay method:1 mg of each extract (from bacteria or sponge) was solubi-lized in CH2Cl2 and applied directly onto a 6 mm paper diskor deposited in 6 mm wells cut out from agar plates seededwith reference S. aureus (ATCC 6538), E. coli (ATCC 8739),marine V. splendidus LGP32 (provided by Dr F. Leroux 2005)strains. Native Gram positive marine strains, Bacillus sp. andPlanococcus sp. isolated from the cultured fraction of L. niveaheterotrophic bacterial flora were also tested. Antimicrobialactivity was determined by measuring the diameter of theinhibition zones after 24 h incubation at 37°C for S. aureusand E. coli or 23°C for the marine strains. Activity testing wasrepeated in at least two to three independent experiments toensure reproducibility of results.

Isolation of the L4-n2 marine bacterial strain

Sponge-associated bacteria were isolated following themethod outlined by Hentschel and colleagues (2001). Briefly,a 1 ¥ 1 cm section of sponge was rinsed three times in fil-tered (0.2 m) sea water and pressed through a 40 mm pore-size plankton net in calcium and magnesium-free artificial seawater. Serial dilutions at 10-2 and 10-4 of the maceration werespread onto Marine Agar Difco 2216 supplemented with nali-dixic acid (10 mg ml-1) in order to inhibit fast growing Vibriosand increase the bacterial diversity recovered. All the plateswere incubated between 12°C and 20°C. After 14 days ofincubation, a brown pigmented spherical colony (L4-n2) wasisolated from the 10-2 dilution and propagated in pure cultureon marine agar.

Phylogenetic affiliation of strain L4-n2

PCR-based analyses of bacterial 16S ribosomal RNA gene.Bacterial DNA was obtained from the strain isolated in pureculture with reproducible antibacterial activity in the soft agardiffusion assay. Following the method modified from Sritha-ran and Barker (1991), colonies grown on Marine Agar were



suspended in 60 ml sterile water, boiled for 5 min, centrifuged(15 000 g, 5 min), and stored in aliquots at 4°C or -20°C priorto 16S rRNA gene PCR amplification, purification and directsequencing.

Bacterial 16S rRNA gene amplification was performedusing PCR primers: 27F-1385R pair (respectively E. coliposition 9:5′-GAGTTTGATCCTGGCTCA-3′ and position1385:5′-CGGTGTGTRCAAGGCCC-3′). The reaction mixturecontained 50 pmol of each primer, 2.5 mmol of each of deoxy-nucleoside triphosphate, 10¥ ATGC SuperTaq buffer, and0.4 U of ATGC SuperTaq and the volume was adjusted withsterile water to 50 ml. PCR reactions were conducted in aBiotherma PCR system with an initial denaturing step (94°Cfor 5 min) followed by 32 cycles of 94°C for 1 min, 52°C for30 s and 72°C for 30 s and a final elongation step at 72°C for7 min. Amplified DNA was checked by 1.5% agarose gelelectrophoresis and purified with a QIAquick PCR purificationkit (Qiagen).

Gene sequencing and phylogenetic affiliation. Purified 16SrRNA gene amplicons were directly sequenced in both direc-tions by Genome Express (Meylan, France). After sequenceassembly, almost full-length sequences (1304 nt) were com-pared with those in the 16S DNA database (EMBL Prokary-ote) by using FASTA (Pearson and Lipman, 1988) and highlysimilar sequences of marine and sponge origin were includedin the analysis. The 16S rRNA gene sequence data wereedited using the BioEdit software and aligned with theDialign2 software (http://mobyle.pasteur.fr). A phylogenetictree was then constructed by Maximum Likelihood (dnaml)methods in the Phylip software package (Felsenstein, 2002)with bootstrap analyses (1000 replicates) to test the robust-ness of each topology.

Media and growth conditions of the strainMicrobulbifer sp.

The bacterial strain was grown in Marine Broth Difco 2216(37.4 g l-1) in the dark and shaken at 350 r.p.m. at tempera-ture ranging from 8°C to 30°C for 1–3 days. The production ofbioactive compounds was maximized between 12°C and20°C in pigmented culture arrested at the beginning of thestationary phase.

Preparation of bacterial crude extract

A 25 ml inoculum, prepared from five strains in marine brothand incubated during 2 days between 12°C and 20°C withshaking, was transferred in three 2 l Erlenmeyer flasks. Eachbacterial culture was incubated for 2 days between 12°C and20°C with shaking. Cells were separated by centrifugation at4°C during 15 min at 10 000 r.p.m. The supernatant wasimmediately poured in CH2Cl2 overnight. The pellet was sub-mitted to six runs of freezing and sonication before to bepoured in CH2Cl2 for extraction after 48 h. The organic layerswere concentrated in vacuum. Examination by TLC revealedidentical chemical profiles for both supernatant and pelletCH2Cl2 extracts of the culture. As they also showed identicalactivity, they were combined to constitute the CH2Cl2 crudeextract of the culture, which was further extracted.

Extraction and isolation of active compounds

The CH2Cl2 extract (100 mg) was chromatographed on asilicagel (Merck silica gel 70–230 mesh) column using cyclo-hexane with increasing amounts of AcOEt as eluent.Bioassay-guided fractionation retained a maximum of activityin fractions eluted with 20% and 30% AcOEt.

Fraction eluted with 20% AcOEt (17 mg) was subjectedto reversed-phase HPLC column (C18 Uptisphere250 ¥ 7.8 mm) with increasing amount of CH3CN/H2O aseluent (flow rate: 1 ml min-1, wavelength: of 254 nm) to yieldcompound 1 (retention time: 9 min 79, 1.2 mg), compound 2(retention time: 12 min 38, 1.0 mg), compound 3 (retentiontime: 20 min 98, 0.4 mg), compound 4 (retention time: 30 min78, 1.2 mg), compound 6 (retention time: 33 min 68, 2.0 mg),compound 8 (retention time: 39 min 74, 0.6 mg), compound 9(retention time: 31 min 94, 0.8 mg).

In the same manner, fraction eluted with 30% AcOEt(4.6 mg) was subjected to reversed-phase HPLC (C18 Upti-sphere 250 ¥ 7.8 mm) using increasing amount of CH3CN/H2O as eluent (flow rate: 1 ml min-1, wavelength of 254 nm) toyield compound 5 (retention time: 6 min 54, 0.6 mg), com-pound 7 (retention time: 7 min 54, 0.6 mg).

Four cultures of 2.5 l were required to obtain enough quan-tities of pure compounds for structural elucidation and evalu-ation of antimicrobial activities.

The gradient of elution used to purify parabens was appliedfor LC/MS studies to detect compound 6 (retention time:18 min) using a C18 Kromasil (250 ¥ 4.6 mm) column. Massspectra were obtained in positive ESI mode.

Compound 1. Ethylparaben, 4-hydroxybenzoic acid ethylester (0.64 mg per lire of culture, 0.80% of dry weight CH2Cl2crude extract of the bacteria culture), white powder; mp 114–115°C; UV (EtOH) lmax nm (e): 286 (364); ESI-MS [M+H]+

found at m/z 167.0708 (D -0.019 mmu), calc. 167.0708 forC9H11O3.Compound 2. Butylparaben, 4-hydroxybenzoic acid butylester (0.436 mg per litre of culture, 1.78% of dry weightCH2Cl2 crude extract of the bacteria culture), white powder;mp 68–69°C; UV (EtOH) lmax nm (e): 285 (575); ESI-MS[M+H]+ found at m/z 195.1028 (D -0.6 mmu), calc. 195.1093for C11H15O3.Compound 3. Octylparaben, 4-hydroxybenzoic acid octylester (0.109 mg per litre of culture, 0.43% of dry weightCH2Cl2 crude extract of the bacteria culture), white amor-phous solid; UV (EtOH) lmax nm (e): 283 (175); ESI-MS[M+H]+ found at m/z 251.1652 (D -0.48 mmu), calc. 251.1647for C15H23O3.Compound 4. Decylparaben, 4-hydroxybenzoic acid decylester (0.254 mg per litre of culture, 1.04% of dry weightCH2Cl2 crude extract of the bacteria culture), pale yellow oil;UV (EtOH) lmax nm (e): 285 (966); ESI-MS [M+H]+ found atm/z 279.1962 (D -0.18 mmu), calc. 279.1960 for C17H27O3.Compound 5. Hydroxydecylparaben, 4-hydroxybenzoic acid3-hydroxy-decyl ester (0.145 mg per litre of culture, 0.59% ofdry weight CH2Cl2 crude extract of the bacteria culture), paleyellow oil, [a]D

20 = -18° (c 0.14, MeOH), UV (EtOH) lmax nm(e): 286 (1378); ESI-MS [M+H]+ found at m/z 295.1913 (D-0.4 mmu), calc. 295.1923 for C17H27O4.Compound 6. Methyldecylparaben, 4-hydroxybenzoic acidmethyl-decyl ester (0.509 mg per litre of culture, 2.08% of dry



http://mobyle.pasteur.fr

weight CH2Cl2 crude extract of the bacteria culture), paleyellow oil; UV (EtOH) lmax nm (e): 286 (687); ESI-MS [M+H]+

found at m/z 293.2115 (D -0.07 mmu), calc. 293.2116 forC18H29O3.Compound 7. Hydroxymethyldecylparaben, 4-hydroxyben-zoic acid 3-hydroxy-methyl-decyl ester (0.218 mg per litre ofculture, 0.89% of dry weight CH2Cl2 crude extract of thebacteria culture), white amorphous solid; [a]D

20 = -7° (c 0.10,MeOH); UV (EtOH) lmax nm (e): 287 (2000); ESI-MS [M+H]+

found at m/z 309.2055 (D -0.5 mmu), calc. 309.2000 forC18H29O4.Compound 8. Dodecylparaben, 4-hydroxybenzoic aciddodecyl ester (0.109 mg per litre of culture, 0.43% of dryweight CH2Cl2 crude extract of the bacteria culture),whiteamorphous solid; UV (EtOH) lmax nm (e): 285 (966); ESI-MS[M+H]+ found at m/z 307.2278 (D -0.4 mmu), calc. 307.2273for C19H31O3.Compound 9. Dodec-5-enylparaben, 4-hydroxybenzoic aciddodec-5-enyl ester (0.145 mg per litre of culture, 0.59% of dryweight CH2Cl2 crude extract of the bacteria culture), whiteamorphous solid, UV (EtOH) lmax nm (e): 285 (334); ESI-MS[M+H]+ found at m/z 305.2102 (D -1.4 mmu), calc. 305.2116for C19H29O3

Determination of MIC and MBC

Minimal inhibitory concentration determination was carriedout by using a minor modification to the method described byCasteels and colleagues (1993). Minimal inhibitory concen-trations were determined by a twofold dilution methodfrom the started concentration of 100 mg ml-1 of compound.Hence, each compound was tested at 100 mg ml-1 through0.048 mg ml-1. Plates were incubated during 24 h at 30°C withshaking (500 r.p.m.).

Minimal bactericidal concentrations were determined bysubculturing onto agar plates each well with no visible growthafter 24 h incubation. The MBC was defined as the lowestconcentration of compound yielding colony counts < 0.1% ofthe initial inoculums, determined by colony counts from thegrowth control well immediately after inoculation (Fuchset al., 2002). Minimal bactericidal concentration or minimalinhibitory concentration revealed the nature of compound. IfMBC/MIC is less than 4, the compound is bactericidal, ifMBC/MIC is between 8 and 16, the compound is bacterio-static (Bartlett, 2008, http://www.medscape.com/viewarticle/478151_6).

In situ localization of a Microbulbifer strain withantimicrobial activity by CARD-FISH

Fragments 1 cm3 from L. nivea specimens were fixed forCARD-FISH in Bouin fixative overnight, then washed exten-sively and stored at 4°C in 70% ethanol. Fixed tissues weredehydrated in an increasing series of ethanol and xylene andembedded in paraffin. Histological sections (4 mm) were col-lected on gelatine coated slides and deparaffinized withxylene and rehydrated in a decreasing ethanol series. Sec-tions were pretreated with HCl, lyzozyme and proteinase K,to provide accessibility to the complementary 16S rRNA oli-gonucleotide probes. Hybridization was performed for 3 h in

preheated chambers wetted with hybridization buffer (0.9 MNaCl, 0.02 M Tris-HCl, 0.01% SDS, 1% blocking reagent,10% dextran sulfate, 35–50% formamide). Temperature andstringency were evaluated at different formamide concentra-tions (35–50%), to optimize the signal (high specific signaland low background fluorescence) for each oligonucleotidicprobe. The eubacterial probe EUB338 (5′-GCT GCC TCCCGT AGG AGT-3′) (Amann et al., 1990) was used as apositive control for a major part of the eubacterial biota(Daims et al., 1999). A 18 nt probe, named Ma445 (5′-AGCTTA TAG CCT TCC TCC-3′, on position 445 of E. coli 16SrRNA gene) was designed for the Microbulbifer phylogeneticcluster (L4-n2 strain and M. arenaceous AJ510266), usingthe PROBE_DESIGN function of ARB and the 16S rDNAaccessibility information provided by Behrens and colleagues(2003). Its specificity was checked with BLAST (Altschul et al.,1994), FASTA and probecheck (http://www.microbial-ecology.net/probecheck) yielding two hits M. arenaceousAJ510266 and the recently published Microbulbifer sp. YIMC306 EU135714. Specificity of Ma445 to Microbulbifer L4-n2was checked with several Gammaproteobacteria strainsmears, as negative controls, including two AlteromonadalesMoritella sp. L7-2nh and Pseudoalteromonas sp. L7-3a andwith one Alphaproteobacteria Pseudovibrio sp. L4-8 strainsisolated from L. nivea. For the internal negative controls weused the 17 nt nonEUB probe (5′-CTC CTA CGG GAG GCAGC-3′) (Amann et al., 1990) and the 24 nt NW442 probe(5′-AGT TAA TGT CAT TAT CTT CAC TGC-3′) raised againstAlphaproteobacteria strain NW001 which cross-reacts withL4-8 and other demosponge-derived Pseudovibrio sp. strains(Enticknap et al., 2006; Taylor et al., 2007). Reactivity ofMa445 to L4-n2 was tested on pure bacterial smears of theL4-n2 strain, with EUB338 as internal positive control andnonEUB probe and NW442 probe as internal negative con-trols. All probes were labelled with Alexa488 fluorochrome(Invitrogen, Carlsbad, CA). Each section was covered with50 ng of horseradish peroxidase-labelled probe in 150 ml ofhybridization buffer and then hybridized as described byPernice and colleagues (2007). Stringency conditions were35% formamide for the EUB338, nonEUB338 and NW442probes and 50% for the Ma445 probe and temperature waslowered from 46°C to 42°C to improve signal with the strain-specific probes.

The slides were washed with 2¥ PBS for 10 min, MilliQwater for 1 min, 96% ethanol for 1 min, air dried, and thencoverslipped in Prolong Antifade mounting medium with DAPI(Promega). Two sections per tissue in two different seasonsof collection were visually examined for bacterial distributionand relative abundance using a DMLB epifluorescencemicroscope (Leica Microsystèmes SAS, France).

Nucleotide sequence accession numbers

The EMBL accession number of the 16S rRNA sequence ofL4-n2 strain with antimicrobial activity described in this paperis: FM200853 (Gammaproteobacteria Microbulbifer L4-n2).

Supplementary data

1H and 13C NMR data for all compounds are available onrequest (Tables S1 and S2).



http://www.medscape.com/viewarticle/478151_6


http://www.microbial-ecology.net/probecheck

http://www.microbial-ecology.net/probecheck

Acknowledgements

This work is part of Elodie Quévrain’s thesis, supported by agrant from the French Ministère de l’Enseignement Supérieuret de la Recherche. Funding was partly provided by a B.Q.R.grant from the MNHN (2005–2006). We thank Alain Blond,Alexandre Deville, Arul Marie and Lionel Dubost (MNHN,Paris) for NMR and MS measurements, Arlette Longeon,Gérard Gastine and Manon Vandervennet (MNHN, Paris) fortheir assistance in the antibacterial tests and MadeleineMartin for preparation of histological sections. The authorsalso thank the director and personnel of the Laboratoire deBiologie Marine du MNHN, Concarneau, for providing facili-ties during their stays and especially Dr S. Auzoux-Bordenave for help in field work.

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Supporting information

Additional Supporting Information may be found in the onlineversion of this article:

Table S1. 1H NMR data of compounds 1–9 recorded inCDCl3 [dH in ppm (multiplicity, J in Hz)].Table S2. 13C NMR data of compounds 1–9 in CDCl3 (dC inp.p.m.).

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Novel natural parabens produced by a Microbulbifer bacterium in its calcareous sponge host Leuconia...

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