Environmental Microbiology (2009) 11(6), 1527–1539
doi:10.1111/j.1462-2920.2009.01880.x
Novel natural parabens produced by a Microbulbifer bacterium in its calcareous sponge host Leuconia nivea emi_1880
1527..1539
Elodie Quévrain,1 Isabelle Domart-Coulon,2 Mathieu Pernice2 and Marie-Lise Bourguet-Kondracki1* 1 Laboratoire de Chimie et Biochimie des Substances Naturelles UMR 5154 CNRS, Muséum National d’Histoire Naturelle, 57 rue cuvier (C. P 54), Paris, France. 2 Laboratoire de Biologie des Organismes Marins et des Ecosystèmes UMR 5178 MNHN-CNRS-UPMC, Muséum National d’Histoire Naturelle, 57 rue cuvier (C. P 51), Paris, France. Summary A broad variety of natural parabens, including four novel structures and known ethyl and butyl parabens, were obtained from culture of a Microbulbifer sp. bacterial strain isolated from the temperate calcareous marine sponge Leuconia nivea (Grant 1826). Their structures were elucidated from spectral analysis, including mass spectrometry and 1D and 2D nuclear magnetic resonance. Their antimicrobial activity evaluated against Staphylococcus aureus was characterized by much higher in vitro activity of these natural paraben compounds 3–9 than commercial synthetic methyl and propyl parabens, usually used as antimicrobial preservatives. Compounds 4 and 9 revealed a bacteriostatic effect and compounds 6 and 7 appeared as bactericidal compounds. Major paraben compound 6 was also active against Gram positive Bacillus sp. and Planococcus sp. sponge isolates and was detected in whole sponge extracts during all seasons, showing its persistent in situ production within the sponge. Moreover, Microbulbifer sp. bacteria were visualized in the sponge body wall using fluorescence in situ hybridization with a probe specific to L4-n2 phylotypes. Co-detection in the sponge host of both paraben metabolites and Microbulbifer sp. L4-n2 indicates, for the first time, production of natural
Received 29 August, 2008; accepted 16 December, 2008. *For correspondence. E-mail
[email protected]; Tel. (+33) 1 40 79 31 35; Fax (+33) 1 40 79 56 06.
parabens in a sponge host, which may have an ecological role as chemical mediators. Introduction Although the Porifera phylum continues to be an excellent source of bioactive and original natural products (Blunt et al., 2008), sponge-associated microorganisms have emerged as promising candidates for the production of natural compounds (Moore, 1999; Piel, 2006; Blunt et al., 2007). Recently, many studies revealed that metabolites previously ascribed to sponges were in fact synthesized by microorganisms (Proksch et al., 2002; Hildebrand et al., 2004; Piel, 2004; Salomon et al., 2004), while others demonstrated structural similarities between natural products isolated from sponges and bacterial metabolites (Crews and Bescansa, 1986; Zabriskie et al., 1986; Jansen et al., 1996; Erickson et al., 1997; Kunze et al., 1998). These data explain the considerable attention paid by scientists over the last decade (Bewley and Faulkner, 1998; Hentschel et al., 2006; Taylor et al., 2007) to the diverse microorganisms associated with marine sponges. Electron microscopy has shown that in some demosponges these microorganisms constitute up to 60% of the biomass (Vacelet and Donadey, 1977; Wilkinson, 1978). In contrast to siliceous sponges, very few chemical and microbiological studies have so far been carried out on calcareous sponges. Those which do exist are restricted to the Calcinea subclass, with to our knowledge no studies of the Calcaronea subclass (Schreiber et al., 2006; Muscholl-Silberhorn et al., 2008). In a programme devoted to studying the role of spongeassociated bacteria and especially the chemical mediators of the interactions between microbial associates in their sponge host, the cultivable heterotrophic bacterial biota was isolated from the temperate calcareous sponge Leuconia nivea collected off Concarneau (Northeast Atlantic, France). The sponge L. nivea (Grant 1826), which belongs to the Baeriidae family in the Calcaronea subclass, was chosen because its chloromethylenic (CH2Cl2) crude extract revealed a significant and annually persistent antimicrobial activity against Staphylococcus aureus. This activity was localized in the bacterial fraction of the sponge obtained by differential sedimentation
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd
1528 E. Quévrain, I. Domart-Coulon, M. Pernice and M.-L. Bourguet-Kondracki Table 1. Antimicrobial activity of the sponge Leuconia nivea throughout 2005–2007, extraction yield and presence of compound 6 in the CH2Cl2 crude extracts.
Season
Collection date
Wet weight (g)
Dry weight (g)
CH2Cl2 mass extract (mg)
CH2Cl2 extract’s yield (% of dry weight)
Activity against S. aureus ATCC 6538a
Compound 6 detection (LC/MS)
Spring
30 March 2006 27 April 2006 24 June 2005 24 July 2005 09 September 2006 27 September 2007 17 October 2005 31 January 2006 19 February 2007
21.9 517.0 4.5 27.0 ND 5.3 24.8 45.6 12.1
5.2 13.7 1.1 6.5 ND 1.3 5.9 10.9 2.9
13.5 26.9 18.0 55.9 12.3 11.0 22.7 13.3 3.3
0.26 0.20 1.67 0.86 ND 0.87 0.38 0.12 0.11
+ + + + + + + + +
ND + ND + + + + + ND
Summer Fall
Winter
(7.5 mm) (7.5 mm) (11 mm) (7 mm) (8 mm) (10 mm) (9 mm) (8 mm) (8 mm)
a. Inhibition’s diameter of the CH2Cl2 extract measured by the disc diffusion assay method for 1 mg per disc. ND, not determined.
following the method of Richelle-Maurer and colleagues (2001). The cultivable heterotrophic bacterial biota associated with L. nivea was screened to isolate bioactive strains, with antimicrobial activity spectra that matched whole sponge extracts. This provided a novel model to investigate the contribution of bacterial metabolites to the chemical profile and antimicrobial activity of their sponge host. The antimicrobial activity of heterotrophic bacteria isolated from L. nivea was examined. CH2Cl2 crude extract investigations of the most active strains, Microbulbifer sp., led to the isolation of a broad variety of natural parabens. In addition to the known ethyl 1 and butyl 2 parabens, seven natural parabens including four novel structures 5, 6, 7 and 9 were identified. We also confirmed the in situ production and annual persistence of the major bacterial compound 6 in the L. nivea sponge through liquid chromatography mass spectrometry (LC/ MS) analysis. This study provides the first chemical report of a family of bioactive compounds from a bacterium associated to a calcareous sponge belonging to the Baeriidae family, and proves its in situ production within the sponge host.
Results Antimicrobial activity of the sponge L. nivea We established that CH2Cl2 extracts of the sublittoral sponge L. nivea collected off Concarneau inhibited the growth of the clinical strain S. aureus (ATCC 6538) in the agar diffusion assay. Collection at three month intervals from 2005 to 2007 showed that this activity was retained throughout the year (Table 1). Furthermore, the antiS. aureus activity was reproducibly localized in the bacterial fraction of the sponge, obtained by differential sedimentation of mechanically dissociated sponge suspension in three independent experiments corresponding
to three different seasons (data not shown). These results prompted us to investigate the microbial community associated with this marine calcareous sponge.
Description of the strain L4-n2 Among the cultivable heterotrophic bacterial biota associated with L. nivea, one of the most active strains, named L4-n2, was isolated in January 2006 on Marine Agar supplemented with nalidixic acid and grown in pure culture in marine agar or marine broth. Bacteria of the strain L4-n2 were Gram negative, rod-shaped, and approximately 0.3–0.5 mm wide by 2–4 mm long. They were aerobic and formed smooth, convex and mucoid colonies on marine agar, producing a non-diffusible brown pigment. They were resistant to ampicillin, penicillin and oxacillin and sensitive to kanamycin (30 UI) and ceftazidime (30 mg).
Phylogenetic affiliation of L4-n2 Strain L4-n2 was identified by sequencing the partial 16S rRNA gene. Sequences were compared with those in the database by using FASTA (Pearson and Lipman, 1988). The FASTA search affiliated L4-n2 isolated from L. nivea with the RSBr-1 type strain of marine bacterium Microbulbifer arenaceous previously isolated from red sandstone off the coast of Scotland (Tanaka et al., 2003) with 99.8% 16S rDNA sequence identity. A 16S rRNA-based phylogenetic tree constructed using the maximum likelihood algorithm (Felsenstein, 1981) affiliated L4-n2 with the genus Microbulbifer in the Alteromonadales order of the Gammaproteobacteria subdivision. Within the Microbulbifer genus, the L4-n2 isolate formed a highly supported clade with the marine sandstone derived species M. arenaceous and with the halophile environmental Microbulbifer sp. strain YIM C306, with a bootstrap confidence level of 99.8% (Fig. 1).
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539
Parabens from sponge associated bacteria 1529 Bacillus algicola SC3 (DQ001308) Moritella marina NCIMB 1144T (X82142) Alteromonas macleodii DSM 6062 (Y18228)
99 77
Pseudoalteromonas flavipulchra NCIMB 2033 (AF297958)
Microbulbifer maritimus TF-17 (AY377986) L4-n2 (FM200853)
(intertidal)
(intertidal)
Microbulbifer sp. YIM C306 (EU135714)
100 98
Microbulbifer arenaceous RSBr-1T (AJ510266)
(intertidal)
Microbulbifer sp. KBB-1 (DQ412068) 100 50
(deep sea)
Microbulbifer agarilyticus JAMB A3 (AB158515) Bacterium QM46 (DQ822531)
51 73 58
Microbulbifer elongatus JAMB A7 (AB107975)
(deep sea)
Microbulbifer elongatus ATCC 10144T (AB021368) (intertidal)
88
Microbulbifer elongatus DSM 6810 (AF500006)
Microbulbifer salipaludis SM-1 (AF479688) Microbulbifer sp. Th/B/38 (AY224196)
(intertidal)
98
Microbulbifer hydrolyticus DSM 11525T (AJ608704) Microbulbifer sp. A4B-17 (AB243106) 100 93 70
Microbulbifer sp. 17x/A02/240 (AY576773) Microbulbifer mediterraneus UST 040317-067 (DQ096579) Microbulbifer cystodytense C1 (AJ620879)
Microbulbifer sp. CJ11049 (AF500206) 100
0.1
(deep sea)
Microbulbifer thermotolerans JAMB A94 (AB124836)
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 bp gene sequence, using the maximum likelihood method from the PHYLIP package (fastdnalml software at http://bioweb.pasteur.fr). Bootstrap values (calculated as percentage from 1000 replications) higher than 50% are reported at branch nodes and show the robustness of observed branching patterns. Scale bar represents 0.1 substitutions per nucleotide position and indicates estimated sequence divergence. Bacillus algicola (DQ001308) was used as outgroup. Information on the natural environment of the strains is indicated in brackets.
Bacterial paraben compounds Cultures of the L4-n2 strain were extracted with CH2Cl2. As extracts of both culture supernatant and pellet displayed identical chemical profile and activity, they were combined to constitute the CH2Cl2 crude extract of the culture, which was further fractionated using a bio-guided assay for its antimicrobial activity. Successive chromatographies of the CH2Cl2 crude extract on silica gel and reverse-phase columns, guided by antimicrobial activity
against S. aureus, yielded nine compounds belonging to the paraben series: the known ethyl- and butyl-parabens 1–2 and the seven natural analogues 3–9, including the four new natural parabens 5, 6, 7 and 9 (Fig. 2). Compounds 1, 3, 4, 8 appeared for the first time as natural products. The structures of these isolated natural compounds were determined by mass and nuclear magnetic resonance (NMR) (1D and 2D) spectral analysis. Compound 1 was obtained as a white powder. Electrospray ionization–mass spectrometry (ESI-MS) esta-
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539
1530 E. Quévrain, I. Domart-Coulon, M. Pernice and M.-L. Bourguet-Kondracki O
3 4
HO
2
7 1 6
5
R O
1 2 3 4 5 6 7 8 9
R = -CH2-CH3 R = -(CH2)3- CH3 R = -(CH2)7-CH3 R = -(CH2)9- CH3 R = -(CH2)2-CH(OH)-(CH2)6-CH3 R = -(CH2)8-CH(CH3)2 R = -(CH2)2-CH(OH)-(CH2)5-CH(CH3)2 R = -(CH2)11-CH3 R = -(CH2)4-CH=CH-(CH2)5-CH3
blished the protonated molecular ion [M+H]+ at m/z 167.0708 (D -0.019 mmu), indicating the molecular formula C9H11O3. Detailed examination of spectroscopic data (UV, 1H and 13C NMR spectra) readily established the identity of compound 1 with the ethyl ester of 4-hydroxybenzoic acid named ethylparaben, a petrochemical antimicrobial compound used as preservative for pharmaceuticals. 1 H NMR data of the following compounds 2–9 revealed that they share two identical sets of coupled protons at d 7.94 (d, 8.8) and d 6.83 (d, 8.8) and one deshielded methylene proton signal at d 4.25 (t, 6.7). These data identified them as analogues of the paraben 1. Electrospray ionization–mass spectrometry of compound 2, a white powder, showed the [M+H]+ ion at m/z 195.1028 (D -0.6 mmu), leading to the molecular formula C11H15O3. Examination of spectroscopic data (UV, 1H and 13 C NMR spectra) identified compound 2 with the butyl ester of the 4-hydroxybenzoic acid named butylparaben, another petrochemical antimicrobial compound also used as preservative in foods and cosmetics. Compound 3 was obtained as colourless oil. Electrospray ionization–mass spectrometry analysis suggested the molecular formula C15H23O3, for the pseudomolecular [M+H]+ ion peak at m/z 251.1652. In comparison with 1H NMR data of the compound 2, we observed four additional methylene proton signals at d 1.34 (m), and 1.26 (m) suggesting the presence of an octyl alkyl chain in the molecule, which was confirmed with HMBC and COSY spectra. Hence, compound 3 was easily identified as being the 4-hydroxybenzoic acid octyl ester or octylparaben, already known as a synthetic compound but isolated for the first time as natural product. Compound 4 was obtained as a pale yellow oil. Electrospray ionization–mass spectrometry analysis suggested the molecular formula C17H27O3 for the pseudomolecular [M+H]+ ion peak at m/z 279.1962. Its 1H NMR spectrum displayed analogies with compound 3. The most significant differences observed in the 1H NMR spectrum concerned the alkyl chain. Additional methylene proton signals at d 1.24 (m) and 1.26 (m) suggesting the presence of a decyl chain in compound 4, which was easily identified as being the decyl hydroxybenzoate, a new natural paraben.
Fig. 2. Structures of the paraben compounds 1–9.
Compound 5 was obtained as a pale yellow oil. The ESI-MS showed the [M+H]+ ion at m/z 295.1913 (calc. 295.1923 for C17H26O4) suggesting the molecular formula C17H26O4 for compound 5, which differed from that of 4 only by an oxygen atom more. The presence of a parahydroxybenzoate was easily recognized by characteristic signals from its 1H NMR spectrum. In the 1H NMR spectrum, we also observed additional signals indicating the presence of one methine proton at d 3.71 (m) and four methylene 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 the partial structure C1′–C4′. Finally, HMBC correlations between the deshielded methylene protons at d (4.58–4.33) to carbons at d 36.5 (C2′) and 68.6 (C3′) and between the methylene protons at d (1.93–1.76) to carbons at d 62.0 (C-1′) and 68.6 (C-3′) located the hydroxyl function in position 3′ of the alkyl chain. The final structure identified the new natural paraben 5 as the 4-hydroxybenzoic acid 3-hydroxy-decyl ester also named hydroxydecylparaben. The very low yield of this compound precluded determination of its absolute configuration. Compound 6 was isolated as a colourless oil. Its molecular formula C18H28O3 was deduced from ESI-MS of the protonated molecular ion [M+H]+ at m/z 293.2115, indicating the presence of five unsaturations in the molecule. Inspection of the 1H NMR spectrum indicated that compound 6 was another 4-hydroxybenzoate (4HBA) alkyl ester. Additional signals for one methine proton at d 1.53 (m) and two methyl doublets at d 0.88 (d, 6.6) were observed in the 1H NMR spectrum. 1H COSY correlations between this methine proton at d 1.53 and both methyl groups at d 0.88 established the presence of an isopropyl group. HMBC correlations, as illustrated in Fig. 3, supported the identification of compound 6 as the new methyldecylparaben. Compound 7 was obtained as white needles. The [M+H]+ ion at m/z 309.2055 suggesting for compound 7 the molecular formula C18H28O4, which differed from that of 6 only by an oxygen atom more. However, detailed examination of spectral data indicated similarities with compound 5. The 1H NMR spectrum also showed the presence one methine proton at d 3.72 (m) and four methylene signals at d (4.57–4.33, 1.93–1.76, 1.47–1.47,
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539
Parabens from sponge associated bacteria 1531 H
Fig. 3. Selected COSY (–) and HMBC (→) correlations of compound 6.
O
H O
H
HO H
1.46–1.31), indicating that 7 possessed the same C-1′– C-4′ as compound 5. 1H COSY correlations between the methine proton at d 1.26 and both methyl groups at d 0.83 established the presence of an isopropyl group, identifying the new paraben 7 as the 4-hydroxybenzoic acid 3-hydroxymethyldecyl ester, also named hydroxymethyldecylparaben. Its absolute configuration could not be examined due to limited amount of the isolated compound. Compound 8 was isolated as a white amorphous solid of the molecular formula C19H30O3 deduced from ESI-MS of the protonated molecular [M+H]+ ion at m/z 307.2278 (C19H31O3). Detailed interpretation of NMR data easily revealed the structure of the dodecylparaben. Compound 9 was isolated as a white amorphous solid. Electrospray ionization–mass spectrometry analysis suggested the molecular formula C19H29O3 for the pseudomolecular [M+H]+ ion peak at m/z 305.2102, differing from that of 8 by two hydrogens and indicating the presence of six unsaturations in the molecule. The main differences were observed in their NMR data. In 1H and 13 C NMR spectra of compound 9, we observed the presence 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 unsaturation in the alkyl chain. Its localization was determined by HMBC correlations from the both methylene protons at d 2.10 (H-4′, m) and d 2.00 (H-7′, m) to the carbons resonating at d 129.2 (C-5′) and 130.6 (C-6′). Key COSY correlations from the both methylene protons at d 2.10 (H-4′, m) and d 2.00 (H-7′, m) to the methine protons at d 5.35 (2H, H-5′, H-6′) delineated the partial structure C4′–C7′ and confirmed the localization of the unsaturation. Compound 9 was identified as a new natural paraben named dodec-5-enylparaben. The geometry of the double bond could be determined neither by 13C NMR nor by NOESY spectrum. Activity of bacterial parabens against S. aureus Antimicrobial activity of the paraben family isolated from Microbulbifer L4-n2 strain was confirmed towards S. aureus (Gram+) bacterial strain and compared with the antimicrobial activity of the commercial methylparaben and propylparaben (Interchimie). Results of this evaluation are summarized in Table 2.
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 Propylparaben 1 2 3 4 5 6 7 8 9
328.9–657.8 277.7–555.5 > 602.0 128.8–257.7 100–200 2.8–5.6 85–170 42.8–85.6 40.5–81.1 81.6–163.3 20.5–41.1
50–100 50–100 > 100 25–50 25–50 0.78–1.56 25–50 12.5–25 12.5–25 25–50 6.25–12.5
> 100 > 100 > 100 > 100 50–100 12.5–25 > 100 25–50 25–50 > 100 50–100
C. Dc C. Dc C. Dc C. Dc 2 = bactericidal 16 = bacteriostatic C. Dc 2 = bactericidal 2 = bactericidal C. Dc 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 are growing 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 initial inoculum. 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, the compound is bacteriostatic.
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539
1532 E. Quévrain, I. Domart-Coulon, M. Pernice and M.-L. Bourguet-Kondracki All of the parabens isolated from the culture of Microbulbifer sp. strain L4-n2 exhibited significant antimicrobial activity. Compound 4 exhibited the greatest efficiency against S. aureus with minimal inhibitory concentration (MIC) values of 2.8–5.6 mM. In order to determine whether the growth inhibition observed was reflected a bactericidal or bacteriostatic effect, minimal bactericidal concentrations (MBCs) were determined and the ratio MBC/MIC was calculated. Using this method, compounds 4 and 9 appeared as bacteriostatic and compounds 3, 6 and 7 appeared as bactericidal. In order to search for a possible synergistic mode of action, antimicrobial assays were simultaneously performed with mixtures of compounds at equimolar ratio but neither synergistic effects, nor additional effects were observed against S. aureus (data not shown).
Antimicrobial activities of the L4-n2 CH2Cl2 crude extract and purified compound 6 The CH2Cl2 crude extract of the strain L4-n2 proved to be active against marine Gram positive strain isolated from the sponge L. nivea and identified as Bacillus sp. L6-3e and also against Gram negative Escherichia coli (ATCC 8739) and the environmental bacterium Vibrio splendidus (LGP32) as well as sponge-derived Alphaproteobacteria Pseudovibrio sp. L4-8 (Table 3). These results revealed the existence of various cross-interactions between strain L4-n2 and other sponge-derived isolates. Compound 6, which was the major bacterial paraben produced by L4-n2, inhibited the growth of the marine L. nivea-derived Gram positive strains Bacillus sp. L6-3e and Planococcus sp. L5-4b, but it did not inhibit Gram negative bacteria such as E. coli (ATCC 8739), nor marine environmental V. splendidus (LGP32) and sponge-derived Pseudovibrio sp. L4-8 (Table 3). This indicated the presence of antimicrobial compounds other than parabens in the CH2Cl2 extract of L4-n2 Microbulbifer strain, and that these yet undetermined metabolites were being responsible for the activity against Gram negative bacteria.
In situ chemical detection of compound 6 in the sponge CH2Cl2 extract Liquid chromatography-mass spectrometry (LC-MS) was carried out to look for compound 6, the major bacterial paraben produced by the Microbulbifer sp. L4-n2 strain cultures. Compound 6 was detected in CH2Cl2 sponge extracts at every season (Table 1 and Fig. 4), indicating persistent annual production of this bacterial metabolite within the sponge and suggesting a contribution of this metabolite to the permanent antimicrobial activity of L. nivea.
In situ localization of the Microbulbifer strain L4-n2 by catalysed reporter deposition fluorescence in situ hybridization in the sponge body wall A probe was designed against a variable region of the 16S rRNA gene specific to the clade of our Microbulbifer sp. strain L4-n2, M. arenaceous and Microbulbifer sp. strain YIM C306 (Fig. 5). The catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) signal was positive only for the L4-n2 bacteria (Fig. 5B) and negative for control Alteromonadales Moritella sp. L7-2nh (Fig. 5C) and Pseudoalteromonas sp. L7-3a (data not shown) as well as Alphaproteobacteria Pseudovibrio sp. L4-8 (data not shown) strains also isolated from L. nivea. Tissue sections obtained from L. nivea specimens collected in four seasons were used: July 2005 (two sections), October 2005 (two sections), January 2006 at the time the L4-n2 strain was isolated (two sections) and September 2006 when compound 6 detection by LC/MS in whole sponge extracts was highest in two consecutive years (three sections). Examination of sponge tissue sections after hybridization with the specific probe confirmed the persistent presence of strain L4-n2 with a positive signal observed in these four seasons, marking a rod-shaped bacteria, in at least five different optical field zones per section. Bacterial distribution was restricted to the extracellular mesohyl and preferentially to the ectosome (outer part of the body wall), although some positive bacteria
Table 3. Antimicrobial activity of the L4-n2 bacterial strain. Gram positive strains
Gram negative strains
Reference strains
S. aureus ATCC 6538
Bacillus sp. L6-3ea
Planococcus sp. L5-4ba
E. coli ATCC 8739
V. splendidus LGP32
Pseudovibrio sp. L4-8a
CH2CL2 crude extractb Compound 6c
+17 mm +13 mm
+11 mm +9 mm
ND +7.5 mm
+11 mm 0
+7 mm 0
+9 mm 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.
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539
Parabens from sponge associated bacteria 1533
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.
were also observed in the mesohyl surrounding the choanocyte chambers. Signal abundance was less than 5% relative to the total number of bacteria stained with the non-specific DAPI DNA fluorochrome or with the non-specific eubacterial probe EUB338, indicating a low number of L4-n2 affiliated representatives in the total bacterial community of L. nivea. Although at low relative abundance, Microbulbifer sp. L4-n2 was persistently present 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 permanent activity against Gram positive (S. aureus) bacteria in all seasons from June 2005 through February 2007. This activity was localized in the bacterial fraction of the sponge separated by differential sedimentation of the dissociated sponge cell suspension. Among the bacterial isolates comprising the cultivable heterotrophic flora, one of the most active strains against S. aureus was a Gram negative strain which was phylogenetically affiliated with the genus Microbulbifer. The chemical nature of the compounds responsible for the antimicrobial activity against S. aureus has been
determined as alkyl esters of the 4HBA, also named parabens. Their antimicrobial activity was evaluated, revealing that natural parabens 3–9 have better in vitro activity than synthetic methylparaben and propylparaben and than natural ethylparaben and butylparaben 1–2, which are the four parabens usually used as antimicrobial preservatives. We found that compounds 3, 4, 6, 8 revealed a higher potency and appeared about 100-fold more active than methyl and propyl synthetic parabens as well as ethyl and butyl natural parabens (1–2). Bacteria of the L4-n2 16S rDNA phylotype were localized by fluorescence in situ hybridization with a specific probe in the sponge body wall, in summer, early fall, and winter, indicating the persistent presence of these bacteria in the L. nivea sponge. Furthermore, the in situ detection of compound 6 by LC/MS in the host sponge extract at every season indicates production of bacterial paraben compounds within its calcareous sponge host. Recently, Xue Peng and colleagues (2006) reported that a strain of Microbulbifer sp. (strain A4B-17), isolated from a tropical ascidian, was able to produce 4HBA and its butyl-, heptyl- and non-yl esters. With the exception of butylparaben 2, these compounds are different from those produced by our strain L4-n2 derived from a calcareous temperate sponge. These authors also reported
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539
1534 E. Quévrain, I. Domart-Coulon, M. Pernice and M.-L. Bourguet-Kondracki
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-n2 phylotype (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 as internal 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 probe Ma445 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.
that out of a total of 23 strains tested, belonging to the genus Microbulbifer and derived from various marine sources and geographic areas, mostly tropical, none of them except the A4B-17 strain was able to produce parabens. Strain A4B-17 shared 95% 16S rRNA gene sequence identity with Microbulbifer hydrolyticus IRE31T and Microbulbifer salipaludis SM-1 T and 94% 16S rRNA gene sequence identity with Microbulbifer elongatus ATCC10144 T. Our strain L4-n2 forms a highly supported clade with M. arenaceous, in a subgroup of Microbulbifer sp. strains distant from the above mentioned strains, which reinforces its specific interest. To our knowledge the antimicrobial activities of M. arenaceous previously isolated from red sandstone off Scotland (Tanaka et al., 2003) and of halophile environmental Microbulbifer sp. strain YIM C306 (EMBL EU135714; X.L. Cui, Y.G. Chen, W.J. Li, L.H. Xu and C.L. Jiang, unpublished) have not been evaluated. All the Microbulbifer sp. strains tested by Peng and colleagues were able to produce 4HBA, although in lower amounts (less than 20%) than the A4B-17 strain. Compound 4HBA which is only soluble in MeOH or H2O was not detected in the CH2Cl2 crude extract of our L4-n2 Microbulbifer sp. strain but might be present in extracts based on more polar solvents. Our study confirms that
marine bacteria belonging to the genus Microbulbifer are able to produce natural parabens. Furthermore, we have shown in preliminary studies that these natural parabens can inhibit other bacterial strains from the sponge. These results suggest that they might play a role in the interactions between bacterial associates within their sponge host and/or might contribute to the chemical defence strategy of the littoral sponges against opportunistic Gram positive environmental bacteria. We have shown that strains affiliated to Microbulbifer sp. L4-n2 strains were present but not abundant in the sponge L. nivea and mostly restricted to the ectosome of the sponge body wall. Our study confirms previous reports of Microbulbifer sp. bacteria in the cultured fraction and among the phylotypes of sponge bacterial communities: it adds to the recent discovery in the Mediterranean Clathrina clathrus sponge of the Calcinea subclass (Muscholl-Silberhorn et al., 2008) of a Microbulbifer sp. isolate, based on partial (400 nt) 16S rRNA sequence. We report for the first time the visualization of these bacteria within a sponge body wall with a specific oligonucleotidic probe. Future studies targeting other bacterial groups will be carried out to determine the relative abundance in the L. nivea sponge host of the different cultivable bacteria in order to determine which microor-
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539
Parabens from sponge associated bacteria 1535 ganisms represent stable members of the community. It opens the way to the isolation and elucidation of other chemical mediators involved in the interactions between sponge bacterial associates. Although parabens are well-known compounds (synthetic molecules) widely used as preservatives in cosmetics, food, pharmaceutical forms, their natural role in the marine environment has not yet been explored and their supply from natural bacterial sources could open new perspectives in the field of marine natural products chemistry. Conclusion From the sponge L. nivea, a Microbulbifer sp. strain L4-n2 was isolated and shown to produce novel parabens with antibacterial activity against Gram positive reference bacteria S. aureus as well as against marine Bacillus sp. and Planococcus sp. isolates derived from the sponge. Its major paraben metabolite was also detected in the calcareous sponge at all seasons, contributing to its yearround antimicrobial activity. The Microbulbifer strain L4-n2 was localized by fluorescence in situ hybridization in the sponge body wall. Our study is the first to show that a paraben metabolite is persistently produced by a Microbulbifer bacterial strain within its sponge host. These results suggest that the L4-n2 Microbulbifer strain contributes to the chemical profile of the Calcisponge L. nivea, and that natural parabens might have an ecological role in the balance between bacterial populations within the sponge. Currently, studies are being carried out in order to characterize the interactions between the L4-n2 Microbulbifer strain and the other strains in the sponge-associated cultivable heterotrophic microbiota, especially to elucidate the structure of active compounds involved in the maintenance of the diversity of the microbial community characteristic of marine sponges. Experimental procedures General experimental procedures Optical rotations were obtained on a Perkin Elmer 341 polarimeter. UV spectra were obtained in EtOH, using a Kontrontype Uvikon 930 spectrophotometer. Silica gel column chromatographies were carried out using Kieselgel 60 (230– 400 mesh, E. Merck). Fractions were monitored by thin-layer chromatography (TLC) using aluminium-backed sheets (Si gel 60 F254, 0.25 mm thick) with visualization under UV (254 and 365 nm) and Liebermann spray reagent. Analytical reversed-phase high-performance liquid chromatography (HPLC) (Kromasil RP18 column K2185, 4.6 ¥ 250 mm) were performed with a L-6200 A pump (Merck-Hitachi) equipped with 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 of Applied Biosystem. 13 C NMR spectra were obtained on a Bruker AC300 at 75.47 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 MHz using a 1H-13C Dual probehead. The delay preceding the 13C pulse for the creation of multiple quanta coherences through several 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 Concarneau (France) from 2005 to 2007. Samples of fresh sponge (4–40 g wet weight corresponding to 1–10 g dry weight) were immersed after collection in a CH2Cl2/MeOH 1/1 mixture at room temperature for 2 days, concentrated under reduced pressure 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 solubilized in CH2Cl2 and applied directly onto a 6 mm paper disk or deposited in 6 mm wells cut out from agar plates seeded with 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. and Planococcus sp. isolated from the cultured fraction of L. nivea heterotrophic bacterial flora were also tested. Antimicrobial activity was determined by measuring the diameter of the inhibition zones after 24 h incubation at 37°C for S. aureus and E. coli or 23°C for the marine strains. Activity testing was repeated in at least two to three independent experiments to ensure reproducibility of results.
Isolation of the L4-n2 marine bacterial strain Sponge-associated bacteria were isolated following the method outlined by Hentschel and colleagues (2001). Briefly, a 1 ¥ 1 cm section of sponge was rinsed three times in filtered (0.2 m) sea water and pressed through a 40 mm poresize plankton net in calcium and magnesium-free artificial sea water. Serial dilutions at 10-2 and 10-4 of the maceration were spread onto Marine Agar Difco 2216 supplemented with nalidixic acid (10 mg ml-1) in order to inhibit fast growing Vibrios and increase the bacterial diversity recovered. All the plates were incubated between 12°C and 20°C. After 14 days of incubation, a brown pigmented spherical colony (L4-n2) was isolated from the 10-2 dilution and propagated in pure culture on 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 pure culture with reproducible antibacterial activity in the soft agar diffusion assay. Following the method modified from Sritharan and Barker (1991), colonies grown on Marine Agar were
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539
1536 E. Quévrain, I. Domart-Coulon, M. Pernice and M.-L. Bourguet-Kondracki 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 prior to 16S rRNA gene PCR amplification, purification and direct sequencing. Bacterial 16S rRNA gene amplification was performed using PCR primers: 27F-1385R pair (respectively E. coli position 9:5′-GAGTTTGATCCTGGCTCA-3′ and position 1385:5′-CGGTGTGTRCAAGGCCC-3′). The reaction mixture contained 50 pmol of each primer, 2.5 mmol of each of deoxynucleoside triphosphate, 10¥ ATGC SuperTaq buffer, and 0.4 U of ATGC SuperTaq and the volume was adjusted with sterile water to 50 ml. PCR reactions were conducted in a Biotherma PCR system with an initial denaturing step (94°C for 5 min) followed by 32 cycles of 94°C for 1 min, 52°C for 30 s and 72°C for 30 s and a final elongation step at 72°C for 7 min. Amplified DNA was checked by 1.5% agarose gel electrophoresis and purified with a QIAquick PCR purification kit (Qiagen). Gene sequencing and phylogenetic affiliation. Purified 16S rRNA gene amplicons were directly sequenced in both directions by Genome Express (Meylan, France). After sequence assembly, almost full-length sequences (1304 nt) were compared with those in the 16S DNA database (EMBL Prokaryote) by using FASTA (Pearson and Lipman, 1988) and highly similar sequences of marine and sponge origin were included in the analysis. The 16S rRNA gene sequence data were edited using the BioEdit software and aligned with the Dialign2 software (http://mobyle.pasteur.fr). A phylogenetic tree was then constructed by Maximum Likelihood (dnaml) methods in the Phylip software package (Felsenstein, 2002) with bootstrap analyses (1000 replicates) to test the robustness of each topology.
Media and growth conditions of the strain Microbulbifer 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 temperature ranging from 8°C to 30°C for 1–3 days. The production of bioactive compounds was maximized between 12°C and 20°C in pigmented culture arrested at the beginning of the stationary phase.
Preparation of bacterial crude extract A 25 ml inoculum, prepared from five strains in marine broth and incubated during 2 days between 12°C and 20°C with shaking, was transferred in three 2 l Erlenmeyer flasks. Each bacterial culture was incubated for 2 days between 12°C and 20°C with shaking. Cells were separated by centrifugation at 4°C during 15 min at 10 000 r.p.m. The supernatant was immediately poured in CH2Cl2 overnight. The pellet was submitted to six runs of freezing and sonication before to be poured in CH2Cl2 for extraction after 48 h. The organic layers were concentrated in vacuum. Examination by TLC revealed identical chemical profiles for both supernatant and pellet CH2Cl2 extracts of the culture. As they also showed identical activity, they were combined to constitute the CH2Cl2 crude extract of the culture, which was further extracted.
Extraction and isolation of active compounds The CH2Cl2 extract (100 mg) was chromatographed on a silicagel (Merck silica gel 70–230 mesh) column using cyclohexane with increasing amounts of AcOEt as eluent. Bioassay-guided fractionation retained a maximum of activity in fractions eluted with 20% and 30% AcOEt. Fraction eluted with 20% AcOEt (17 mg) was subjected to reversed-phase HPLC column (C18 Uptisphere 250 ¥ 7.8 mm) with increasing amount of CH3CN/H2O as eluent (flow rate: 1 ml min-1, wavelength: of 254 nm) to yield compound 1 (retention time: 9 min 79, 1.2 mg), compound 2 (retention time: 12 min 38, 1.0 mg), compound 3 (retention time: 20 min 98, 0.4 mg), compound 4 (retention time: 30 min 78, 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 Uptisphere 250 ¥ 7.8 mm) using increasing amount of CH3CN/ H2O as eluent (flow rate: 1 ml min-1, wavelength of 254 nm) to yield compound 5 (retention time: 6 min 54, 0.6 mg), compound 7 (retention time: 7 min 54, 0.6 mg). Four cultures of 2.5 l were required to obtain enough quantities of pure compounds for structural elucidation and evaluation of antimicrobial activities. The gradient of elution used to purify parabens was applied for LC/MS studies to detect compound 6 (retention time: 18 min) using a C18 Kromasil (250 ¥ 4.6 mm) column. Mass spectra were obtained in positive ESI mode. Compound 1. Ethylparaben, 4-hydroxybenzoic acid ethyl ester (0.64 mg per lire of culture, 0.80% of dry weight CH2Cl2 crude 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 for C9H11O3. Compound 2. Butylparaben, 4-hydroxybenzoic acid butyl ester (0.436 mg per litre of culture, 1.78% of dry weight CH2Cl2 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. 195.1093 for C11H15O3. Compound 3. Octylparaben, 4-hydroxybenzoic acid octyl ester (0.109 mg per litre of culture, 0.43% of dry weight CH2Cl2 crude extract of the bacteria culture), white amorphous solid; UV (EtOH) lmax nm (e): 283 (175); ESI-MS [M+H]+ found at m/z 251.1652 (D -0.48 mmu), calc. 251.1647 for C15H23O3. Compound 4. Decylparaben, 4-hydroxybenzoic acid decyl ester (0.254 mg per litre of culture, 1.04% of dry weight CH2Cl2 crude extract of the bacteria culture), pale yellow oil; UV (EtOH) lmax nm (e): 285 (966); ESI-MS [M+H]+ found at m/z 279.1962 (D -0.18 mmu), calc. 279.1960 for C17H27O3. Compound 5. Hydroxydecylparaben, 4-hydroxybenzoic acid 3-hydroxy-decyl ester (0.145 mg per litre of culture, 0.59% of dry weight CH2Cl2 crude extract of the bacteria culture), pale yellow oil, [a]D20 = -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 acid methyl-decyl ester (0.509 mg per litre of culture, 2.08% of dry
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539
Parabens from sponge associated bacteria 1537 weight CH2Cl2 crude extract of the bacteria culture), pale yellow 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 for C18H29O3. Compound 7. Hydroxymethyldecylparaben, 4-hydroxybenzoic acid 3-hydroxy-methyl-decyl ester (0.218 mg per litre of culture, 0.89% of dry weight CH2Cl2 crude extract of the bacteria culture), white amorphous solid; [a]D20 = -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 for C18H29O4. Compound 8. Dodecylparaben, 4-hydroxybenzoic acid dodecyl ester (0.109 mg per litre of culture, 0.43% of dry weight CH2Cl2 crude extract of the bacteria culture),white amorphous solid; UV (EtOH) lmax nm (e): 285 (966); ESI-MS [M+H]+ found at m/z 307.2278 (D -0.4 mmu), calc. 307.2273 for C19H31O3. Compound 9. Dodec-5-enylparaben, 4-hydroxybenzoic acid dodec-5-enyl ester (0.145 mg per litre of culture, 0.59% of dry weight CH2Cl2 crude extract of the bacteria culture), white amorphous solid, UV (EtOH) lmax nm (e): 285 (334); ESI-MS [M+H]+ found at m/z 305.2102 (D -1.4 mmu), calc. 305.2116 for C19H29O3
Determination of MIC and MBC Minimal inhibitory concentration determination was carried out by using a minor modification to the method described by Casteels and colleagues (1993). Minimal inhibitory concentrations were determined by a twofold dilution method from the started concentration of 100 mg ml-1 of compound. Hence, each compound was tested at 100 mg ml-1 through 0.048 mg ml-1. Plates were incubated during 24 h at 30°C with shaking (500 r.p.m.). Minimal bactericidal concentrations were determined by subculturing onto agar plates each well with no visible growth after 24 h incubation. The MBC was defined as the lowest concentration of compound yielding colony counts < 0.1% of the initial inoculums, determined by colony counts from the growth control well immediately after inoculation (Fuchs et al., 2002). Minimal bactericidal concentration or minimal inhibitory concentration revealed the nature of compound. If MBC/MIC is less than 4, the compound is bactericidal, if MBC/MIC is between 8 and 16, the compound is bacteriostatic (Bartlett, 2008, http://www.medscape.com/viewarticle/ 478151_6).
In situ localization of a Microbulbifer strain with antimicrobial activity by CARD-FISH Fragments 1 cm3 from L. nivea specimens were fixed for CARD-FISH in Bouin fixative overnight, then washed extensively and stored at 4°C in 70% ethanol. Fixed tissues were dehydrated in an increasing series of ethanol and xylene and embedded in paraffin. Histological sections (4 mm) were collected on gelatine coated slides and deparaffinized with xylene and rehydrated in a decreasing ethanol series. Sections were pretreated with HCl, lyzozyme and proteinase K, to provide accessibility to the complementary 16S rRNA oligonucleotide probes. Hybridization was performed for 3 h in
preheated chambers wetted with hybridization buffer (0.9 M NaCl, 0.02 M Tris-HCl, 0.01% SDS, 1% blocking reagent, 10% dextran sulfate, 35–50% formamide). Temperature and stringency were evaluated at different formamide concentrations (35–50%), to optimize the signal (high specific signal and low background fluorescence) for each oligonucleotidic probe. The eubacterial probe EUB338 (5′-GCT GCC TCC CGT AGG AGT-3′) (Amann et al., 1990) was used as a positive control for a major part of the eubacterial biota (Daims et al., 1999). A 18 nt probe, named Ma445 (5′-AGC TTA TAG CCT TCC TCC-3′, on position 445 of E. coli 16S rRNA gene) was designed for the Microbulbifer phylogenetic cluster (L4-n2 strain and M. arenaceous AJ510266), using the PROBE_DESIGN function of ARB and the 16S rDNA accessibility information provided by Behrens and colleagues (2003). Its specificity was checked with BLAST (Altschul et al., 1994), FASTA and probecheck (http://www.microbialecology.net/probecheck) yielding two hits M. arenaceous AJ510266 and the recently published Microbulbifer sp. YIM C306 EU135714. Specificity of Ma445 to Microbulbifer L4-n2 was checked with several Gammaproteobacteria strain smears, as negative controls, including two Alteromonadales Moritella sp. L7-2nh and Pseudoalteromonas sp. L7-3a and with one Alphaproteobacteria Pseudovibrio sp. L4-8 strains isolated from L. nivea. For the internal negative controls we used the 17 nt nonEUB probe (5′-CTC CTA CGG GAG GCA GC-3′) (Amann et al., 1990) and the 24 nt NW442 probe (5′-AGT TAA TGT CAT TAT CTT CAC TGC-3′) raised against Alphaproteobacteria strain NW001 which cross-reacts with L4-8 and other demosponge-derived Pseudovibrio sp. strains (Enticknap et al., 2006; Taylor et al., 2007). Reactivity of Ma445 to L4-n2 was tested on pure bacterial smears of the L4-n2 strain, with EUB338 as internal positive control and nonEUB probe and NW442 probe as internal negative controls. All probes were labelled with Alexa488 fluorochrome (Invitrogen, Carlsbad, CA). Each section was covered with 50 ng of horseradish peroxidase-labelled probe in 150 ml of hybridization buffer and then hybridized as described by Pernice and colleagues (2007). Stringency conditions were 35% formamide for the EUB338, nonEUB338 and NW442 probes and 50% for the Ma445 probe and temperature was lowered from 46°C to 42°C to improve signal with the strainspecific probes. The slides were washed with 2¥ PBS for 10 min, MilliQ water for 1 min, 96% ethanol for 1 min, air dried, and then coverslipped in Prolong Antifade mounting medium with DAPI (Promega). Two sections per tissue in two different seasons of collection were visually examined for bacterial distribution and relative abundance using a DMLB epifluorescence microscope (Leica Microsystèmes SAS, France).
Nucleotide sequence accession numbers The EMBL accession number of the 16S rRNA sequence of L4-n2 strain with antimicrobial activity described in this paper is: FM200853 (Gammaproteobacteria Microbulbifer L4-n2).
Supplementary data 1
H and 13C NMR data for all compounds are available on request (Tables S1 and S2).
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539
1538 E. Quévrain, I. Domart-Coulon, M. Pernice and M.-L. Bourguet-Kondracki Acknowledgements This work is part of Elodie Quévrain’s thesis, supported by a grant from the French Ministère de l’Enseignement Supérieur et 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) for their assistance in the antibacterial tests and Madeleine Martin for preparation of histological sections. The authors also thank the director and personnel of the Laboratoire de Biologie Marine du MNHN, Concarneau, for providing facilities during their stays and especially Dr S. AuzouxBordenave for help in field work.
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Supporting information Additional Supporting Information may be found in the online version of this article: Table S1. 1H NMR data of compounds 1–9 recorded in CDCl3 [dH in ppm (multiplicity, J in Hz)]. Table S2. 13C NMR data of compounds 1–9 in CDCl3 (dC in p.p.m.). Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
© 2009 The Authors Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1527–1539
