Antibiotic activity and microbial community of the temperate sponge, Haliclona sp.

Accepted Article

Article Type: Original Article

Antibiotic activity and microbial community of the temperate sponge, Haliclona sp.

Amanda Hoppers, Julie Stoudenmire, Shuyan Wu, and Nicole B. Lopanik*

Department of Biology, Georgia State University, Atlanta, GA 30303

Running title: Chemical and microbial ecology of a temperate sponge

*Corresponding author Email: [email protected] Address: PO Box 4010 Atlanta, GA 30302-4010 Phone: 404-413-5430

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12709 This article is protected by copyright. All rights reserved.

Accepted Article

ABSTRACT Aims: Sessile marine invertebrates engage in a diverse array of beneficial interactions with bacterial symbionts. One feature of some of these relationships is the presence of bioactive natural products that can defend the holobiont from predation, competition, or disease. In this study, we investigated the antimicrobial activity and microbial community of a common temperate sponge from coastal North Carolina. Methods and Results: The sponge was identified as a member of the genus Haliclona, a prolific source of bioactive natural products, based on its 18S rRNA gene sequence. The crude chemical extract and methanol partition had broad activity against the assayed Gram-negative and Gram-positive pathogenic bacteria. Further fractionation resulted in two groups of compounds with differing antimicrobial activity, primarily against Gram-positive test organisms. There was, however, notable activity against the Gram-negative marine pathogen, Vibrio parahaemolyticus. Microbial community analysis of the sponge and surrounding seawater via denaturing gradient gel electrophoresis (DGGE)

indicates that it harbors a distinct group of bacterial associates. Conclusions: The common temperate sponge, Haliclona sp., is a source of multiple antimicrobial compounds, and has some consistent microbial community members that may play a role in secondary metabolite production. Significance and Impact: These data suggest that common temperate sponges can be a source of bioactive chemical and microbial diversity. Further studies may reveal the importance of the microbial associates to the sponge and natural product biosynthesis.

Key words: Sponge, natural products, antibiotics, Haliclona sp., microbial symbionts

This article is protected by copyright. All rights reserved.

Accepted Article

RESULTS Sponge identification Universal eukaryotic ribosomal small subunit primers were utilized to amplify the sponge 18S

rRNA gene from metagenomic DNA isolated from freshly collected tissue. The PCR amplicon was cloned, RFLP analysis was used to identify unique clones, and 4 distinct representatives were sequenced. BLAST analysis revealed one of the clones had an insert with high similarity to other sponge 18S rRNA genes (one of the others was a protist, and the other was a fungus). The complete, double-stranded sequence of the clone was obtained (1962 bp), and its closest relative determined to be a Panamanian specimen of Haliclona vansoesti (Accession: KC902323, 99% identity, e-value: 0.0) by BLAST analysis. The sponge 18S rRNA gene sequence obtained in this study is available in GenBank by accession number xxxxxx.

Antibiotic activity of sponge extracts and fractions The crude extract and the subsequent methanol partition of the crude extract inhibited the growth

of all the assay organisms (E. coli, S. aureus, P. aeruginosa, V. parahaemolyticus, vancomycin-resistant E. faecalis, B. subtilis, and M. luteus), whereas the hexane partition of the crude extract was not active in disk diffusion assays (Table 1). Disk diffusion assays of the silica gel column fractioned methanol partition revealed that the 50% hexane/50% EtOAc (fraction 1.2), 100% EtOAc (fraction 1.3), 90% EtOAc/10% MeOH (fraction 1.4), 80% EtOAc/ 20% MeOH (fraction 1.5) fractions were active. This fractionation resulted in loss of activity against the gram-negative strains E. coli and P. aeruginosa, and

gram-positive B. subtilis. The fractions with the greatest activity (fractions 1.2-1.5) were combined, further fractionated, and assayed. Two groups of fractions displayed activity in disk diffusion

bioassays. The group containing fractions 2.2 and 2.3 (75% Hexane/25% EtOAc, and 50% Hexane/50% EtOAc, respectively) were active against S. aureus, V. parahaemolyticus, VRE, and B.


environmental microbes (Wilkinson et al. 1984), it is clear that bacteria may not only use the sponge

Accepted Article

as a habitat, but can also be important partners for the sponge. Culture-independent methods have been used to show that some sponges have stable communities of bacteria associated with them, suggesting that sponges may select for specific types or species of bacteria (Hentschel et al. 2002; Taylor et al. 2004). Bioactive natural products can be produced by the sponge itself (Sipkema et al. 2005), or through

mutualistic and symbiotic relationships with bacteria (Faulkner et al. 1994). The diverse microbial population of sponges often makes identifying the producer of the natural product difficult. Techniques like cell separation followed by chemical analysis (Bewley et al. 1996) and microscopy

with antibodies for the compound (Gillor et al. 2000) can be used to identify likely sources within the

sponge. In some cases, the producing organism can be cultured (Kennedy et al. 2009; Phelan et al.

2013; Sathiyanarayanan et al. 2014), but the majority of sponge-associated bacteria are not yet

cultured, making assessment of their diversity and role in natural product biosynthesis difficult (Hochmuth and Piel 2009). Microbially-produced secondary metabolites can confer benefits to the

sponge such as antimicrobial or antifouling properties, or distasteful chemicals that discourage predation [reviewed in (Paul et al. 2007; Paul et al. 2011; Lopanik 2014)]. In addition, bioactive compounds produced by either the sponge or an associated microbe can affect colonization behaviors such as swarming and attachment of other bacteria (Kelly et al. 2005), which has both biological and medical relevance. In this study, the chemical and microbial ecology of a sponge collected from the temperate waters

of North Carolina (NC) were surveyed. This sponge is frequently seen as a member of fouling communities on floating docks, and is visually distinguishable because of its distinct pink coloration. The sponge was identified using molecular techniques, and the antibiotic activity of the sponge organic extract and fractions was assayed against a variety of Gram-positive and Gram-negative bacterial


pathogens. Finally, the microbial community of the sponge was investigated by denaturing gel

Accepted Article

gradient electrophoresis (DGGE).

MATERIALS AND METHODS Sponge sample collection Sponge tissue was processed separately for DNA analysis and natural product extraction. In

November of 2009 and July 2010, sponges were collected from floating docks in Beaufort, North Carolina. Samples were housed overnight in filtered, flowing seawater facilities at the University of North Carolina’s Institute of Marine Sciences, in Morehead City, NC. A total of 12.15 kg wet weight of sponge tissue was used for natural product extraction. Tissue was stored in 100% methanol and frozen at -20°C. A small portion of several individuals was reserved for sponge identification via 18S rRNA gene sequencing. For microbial community analysis, 14 sponge individuals were collected from different docks in Beaufort and Morehead City, NC, at different times of the year (November 2011, March 2012, and April 2012) and stored in RNALater (Life Technologies, Carlsbad, CA) at -20°C prior

to metagenomic DNA extraction.

Molecular identification of the sponge Freshly collected sponge tissue (November 2009) was homogenized in 0.22 μm filtered seawater

and 10% glycerol. The homogenate was centrifuged at maximum speed for 10 min to pellet cellular material, which included both sponge and associated microorganisms. Total genomic DNA was

extracted from the tissue pellet using the Qiagen (Valencia, CA) genomic-tip 20/G according to the manufacturer’s protocol. The DNA quantity and quality were assessed by spectrophotometry (NanoDrop, ThermoFisher, Waltham, MA). The primers EUKF and EUKR (Medlin et al. 1988) were


used to PCR amplify the sponge 18S rRNA gene sequence. The reaction mixture contained 1X reaction

Accepted Article

buffer (containing 1.5 mmol l-1 MgCl2), dNTPs (200 µmol l-1), 3% DMSO, primers (0.5 µmol l-1 each),

Phire Hot Start DNA polymerase (0.6 U, ThermoScientific, Waltham, MA), and template DNA (25-30 ng). The PCR conditions were as follows: 98°C for 30 s, followed by 36 cycles of 98°C for 10 s, annealing at a gradient of 48°C to 58°C for 10 s, extension at 72°C for 20 s and a final extension of 72°C for 2 min. The PCR amplicons were separated by agarose gel electrophoresis and visualized under UV light after ethidium bromide staining.

Purified PCR products (GeneJET PCR Purification Kit, ThermoScientific) of eukaryotic 18S rRNA

genes (~1,900 bp) were cloned using the pGEM–T Vector cloning system (Promega, Madison, WI), and transformed into E. coli XL-1 Blue (Stratagene, La Jolla, CA) competent cells and plated onto LB plates containing ampicillin (100 mg l-1), X-gal (80 mg l-1), and IPTG (0.5 mmol l-1). Successful transformants

were selected by blue/white screening. Colony PCR with the M13 forward/reverse primers (M13F and R) was used to verify the presence of inserts. PCR amplicons from clones with correctly sized inserts were digested with AluI (New England Biolabs, Iswich, MA) and digestion products were subjected to agarose gel electrophoresis to generate restriction fragment length polymorphism (RFLP) profiles. Plasmids from clones with different RFLP patterns were sequenced (GSU DNA Sequencing Core Facilities) using the M13F and R primers. The resulting sequences were trimmed with SeqMan

(Lasergene 6). All sequences were individually analyzed with the Basic Local Alignment Search Tool (BLAST) algorithm (McGinnis and Madden 2004) and their closest hits were identified. The clone containing an 18S rRNA gene sequence highly similar to that of sponges was selected for complete sequencing. Internal primers were designed to obtain complete double-stranded sequence from the original clone.


Accepted Article

Extraction and fractionation of antibiotic metabolites Sponge tissue preserved at -20°C in 100% methanol (MeOH, Chromasolve, Sigma Aldrich, St. Louis,

MO) was exhaustively extracted with 1x volume (approximately 500 mL) of MeOH three times, 1x volume 50% MeOH/50% dichloromethane (DCM, Fisher Scientific, Fair Lawn, NJ) three times, and 1x volume DCM three times. The extracts were then filtered (Whatman filter paper) to remove debris, pooled, and dried by rotary evaporation resulting in 65.15 g of crude extract. The crude extract was then partitioned between MeOH and hexane (Sigma Aldrich) to separate the metabolites based on polarity. The antimicrobial activity of the crude extract, and polar (MeOH) and non-polar (hexane) extract partitions was assessed using disk-diffusion assays (see below). Sponge metabolites in the active MeOH partition were separated by flash column chromatography.

The MeOH partition was applied to a silica gel column (75 g, Analtech, Newark, DE) and 200 ml of each solvent or solvent combination was used to elute fractions of the crude extract based on a polarity gradient (Table 1). Each eluate was dried using rotary evaporation, and tested for antimicrobial activity by disk diffusion assay (see below). Fractions 1.2 – 2.5 had activity, thus were combined and fractionated a second time by silica gel chromatography using different solvent combinations based on a polarity gradient (Table 1). Each subsequent eluate was tested for antibacterial activity by disk diffusion assay.

Antibacterial assays The antimicrobial activity of the sponge metabolites was determined using disk diffusion and

liquid culture assays with a variety of gram-negative and gram-positive strains, including Bacillus subtilus (ATCC 6633), Escherichia coli (ATCC 10798), Micrococcus luteus (ATCC 4698), Pseudomonas aeruginosa (ATCC10145), Staphlococcus aureus (ATCC 25923), Vibrio parahaemolyticus (ATCC 17802), vancomycin-resistant Enterococcus faecalis (VRE, ATCC 51299). Assay organisms were cultured in


different media: B. subtilis and P. aeruginosa in Difco Nutrient Broth (BD, Sparks, MD), V.

Accepted Article

parahaemolyticus in Difco Nutrient Broth + 3% NaCl, E. coli in Difco Luria Broth (BD), M. luteus and S.

aureus in BBL Trypticase Soy media (BD), and VRE in BBL Brain-Heart Infusion media (BD). For disk diffusion assays, chemical fractions were dissolved in methanol to a concentration of 50 mg ml-1 and applied to 6.5 mm filter paper disks. Positive control disks contained amikacin (10 mg ml-1), except in

the case of VRE where spectinomycin (10 mg ml-1) was used. Methanol was also applied to disks as solvent controls, which never inhibited bacterial growth. After the solvent had evaporated, the dry

disks were placed onto the appropriate agar media plate spread with 100 μl bacterial overnight liquid culture. The plates were incubated for 18 hours at 37°C for all bacteria except B. subtilis and M. luteus, which were incubated at 30°C before measuring zones of inhibition.

Liquid culture assays for all extracts and fractions were tested in triplicate. The test bacteria were

grown in 4 ml media overnight in a 37°C shaking incubator at 250 rpm. The OD600 was measured using a spectrophotometer (BioPhotometer, Eppendorf, Hauppauge, NY) and the bacteria were diluted to OD600 0.100 with the appropriate medium. The bacterial suspension was pipetted into 96-well plates. The extracts and fractions were dried under nitrogen gas and re-dissolved in dimethylsulfoxide (DMSO, ThermoScientific) to a concentration of 50 mg ml-1, 25 mg ml-1, 10 mg ml-1, and 2.5 mg ml-1.

The bacteria were grown overnight in a 37°C shaking incubator at 75 rpm. Blanks were wells with media and extract, and positive controls were bacteria grown in the presence of the antibiotics spectinomycin (5 mg ml-1) and amikacin (0.5 mg ml-1). Solvent control wells contained only DMSO (0 mg ml-1 of extract or fraction). The OD600 for the 96 well plates was measured using the Victor3V 1420

Multilabel counter. SPSS Statistics 21 (IBM) was used to calculate significant differences in inhibitory activity.


Accepted Article

High Performance Liquid Chromatography (HPLC) Reversed-phase HPLC (Shimadzu, Columbia, MD) was used to further separate the combined

active fractions. A gradient of acetonitrile and water was used in combination with a semi-prep C18 column (Gemini 5 μm C18, 250 x 10.00 mm) and a photodiode array detector (200-400 nm). The fractions were collected based on the strength of the peak and time, and then assessed for activity using disk diffusion assays. Peaks were collected while monitoring the chromatogram at spectrum max plot, which shows the most intense peak regardless of wavelength at that specific time point. The collected fractions were dried by rotary evaporation. Because of the low levels of the eluted compounds, the fractions were unable to be tested for antibacterial activity at a concentration of 50 mg ml-1. The dried fractions were instead dissolved in 100 μl of methanol, the same volume of extract

injected, for the subsequent disk diffusion assay.

DGGE The sponge tissue stored in RNALater was thawed on ice before being rinsed with sterile PBS, and

then the tissue was homogenized in separate 1.5 ml microcentrifuge tubes using autoclaved pestles.

Metagenomic DNA was extracted using the Zymo Research Miniprep Fungal and Bacterial DNA Extraction kit (Irvine, CA) according to the manufacturer’s protocol. Quality and quantity of DNA was assessed using a spectrophotometer (NanoDrop ND-1000) and stored at -20°C. The extracted DNA was aliquoted and diluted to 50 ng µl-1 for use in PCR reactions. Seawater (1.7 l) was collected from the area next to the sponges, and filtered through a 0.45 μm polycarbonate filter. The filter was placed into filter-sterilized seawater and shaken vigorously for 5 min to remove the cells from the filter. This process was repeated once more. The cells were pelleted by centrifugation and resuspended in a small amount of PBS buffer. DNA was extracted from the cells, and subjected to the same microbial diversity analysis of the individual sponges.


Bacterial 16S rRNA genes were amplified using PCR from each of the sponge metagenomic DNA

Accepted Article

samples, as well as the seawater environmental DNA sample. Eubacterial primers Eub341f-GC and Eub519r were utilized to amplify the bacterial 16S rRNA genes (Schafer et al. 2001). The reaction

mixture contained 1X reaction buffer (containing 1.5 mmol l-1 MgCl2), dNTPs (200 µmol l-1), 3% DMSO, primers (0.5 µmol l-1 each), Phire HS II DNA polymerase (0.6 U, ThermoScientific), and template DNA (50 ng). Touchdown PCR conditions were 98°C for 30 min, followed by 19 cycles of 98°C for 5 s, 65°C for 15 s decreasing by 0.5°C each cycle, 72°C for 1 min, then 20 cycles of 98°C for 5 s, 58°C for 15 s, 72°C for 20 s with a final extension of 72°C for 5 min. Amplification was assayed by visualization of DGGE PCR amplicons after agarose gel electrophoresis.

Top-loading DGGE gels were produced using the BioRad Model 475 Gradient Delivery System

according to the manufacturer’s specifications (BioRad, Hercules, CA). Gradient gels had a final concentration of 8% bis-acrylamide, contained formamide and urea as denaturants, and had a final concentration of 1% glycerol to make the polymerized gel more flexible and less susceptible to tearing. Gel stocks were degassed for 20 min and then stored shielded from light at 4°C for less than 30 days before use. Gels were polymerized using a final concentration of 0.09% of ammonium persulfate solution (APS) and tetramethylethylenediamine (TEMED). Optimization experiments showed that the best separation of bands was attained with a gradient from 30%-60% denaturant. Gels were loaded with 15 µl of PCR product per lane. DGGE was performed under constant temperature conditions of 60°C for 16 hours at 70V in a 1X TAE running buffer. Pipette tips were used to pick the bands of interest, and then placed in PCR tubes containing 5 µl of water for approximately 5 min. The same PCR conditions described above were used to amplify the DNA from the bands in the DGGE gel. PCR amplicons were purified (Zymo Research PCR Cleanup Kit) according to the manufacturer’s protocol,

and sequenced using one or both PCR primers at the GSU Core Facility (ABI 3100 Genetic Analyzer). Band intensities and DGGE patterns were compared using BioNumerics 7.1.


Accepted Article

RESULTS Sponge identification Universal eukaryotic ribosomal small subunit primers were utilized to amplify the sponge 18S

rRNA gene from metagenomic DNA isolated from freshly collected tissue. The PCR amplicon was cloned, RFLP analysis was used to identify unique clones, and 4 distinct representatives were sequenced. BLAST analysis revealed one of the clones had an insert with high similarity to other sponge 18S rRNA genes (one of the others was a protist, and the other was a fungus). The complete, double-stranded sequence of the clone was obtained (1962 bp), and its closest relative determined to be a Panamanian specimen of Haliclona vansoesti (Accession: KC902323, 99% identity, e-value: 0.0) by BLAST analysis. The sponge 18S rRNA gene sequence obtained in this study is available in GenBank by accession number xxxxxx.

Antibiotic activity of sponge extracts and fractions The crude extract and the subsequent methanol partition of the crude extract inhibited the growth

of all the assay organisms (E. coli, S. aureus, P. aeruginosa, V. parahaemolyticus, vancomycin-resistant E. faecalis, B. subtilis, and M. luteus), whereas the hexane partition of the crude extract was not active in disk diffusion assays (Table 1). Disk diffusion assays of the silica gel column fractioned methanol partition revealed that the 50% hexane/50% EtOAc (fraction 1.2), 100% EtOAc (fraction 1.3), 90% EtOAc/10% MeOH (fraction 1.4), 80% EtOAc/ 20% MeOH (fraction 1.5) fractions were active. This fractionation resulted in loss of activity against the gram-negative strains E. coli and P. aeruginosa, and

gram-positive B. subtilis. The fractions with the greatest activity (fractions 1.2-1.5) were combined, further fractionated, and assayed. Two groups of fractions displayed activity in disk diffusion

bioassays. The group containing fractions 2.2 and 2.3 (75% Hexane/25% EtOAc, and 50% Hexane/50% EtOAc, respectively) were active against S. aureus, V. parahaemolyticus, VRE, and B.


subtilis. The second group of fractions was eluted with higher polarity solvent combinations (fractions

Accepted Article

2.6-2.9), and had activity against S. aureus, P. aeruginosa, V. parahaemolyticus, VRE, B. subtilis, and M.

luteus. Since fractions 2.6-2.9 were active against a broader range of organisms, these were combined and tested at several different concentrations in liquid assays.

Results from the liquid assays were similar to those from the disk diffusion assays. In general, the

gram-positive bacteria were significantly inhibited by the presence of the crude extract, methanol partition, and the combined fractions 2.6-2.9 (see Figure 1A and 1B, and Supporting Information Figure 1), but not by the hexane partition (data not shown). The assay strains were inhibited

differently by the extracts. For instance, S. aureus (Figure 1A) was only inhibited by the highest concentration of the crude extract (minimum inhibitory concentration compared to control of 0 mg ml1,

MIC 50 mg ml-1, P < 0.01, one-way ANOVA, Tukey’s HSD), whereas, for VRE, the MIC of the crude

extract was 10 mg ml-1 (Figure 1B, P < 0.02). For S. aureus, the MICs of the three extracts were

different: 50 mg ml-1 for the crude extract, 25 mg ml-1 for the methanol partition (P < 0.01), and 10 mg

ml-1 for the fractions (P < 0.01). The MICs for the three extracts were the same for VRE (10 mg ml-1;

methanol partition and fractions, P < 0.01). In contrast to the gram-positive bacteria, the extracts did not greatly affect growth of gram-negative bacteria, except for V. parahaemolyticus. The MICs for the crude extract, methanol partition, and fractions are 2.5 mg ml-1 (P < 0.01), 10 mg ml-1 (P = 0.02), and 25 mg ml-1 (P < 0.01), respectively. P. aeruginosa was significantly inhibited by the crude extract, the methanol partition, and the fractions, but at a much lower level (Supporting Information Figure 1A). Interestingly, low concentrations of the crude extract significantly enhanced growth of S. aureus (P < 0.01 for 25, 10, and 2.5 mg ml-1).


Accepted Article

HPLC Analysis HPLC analysis of the combined fractions 2.6-2.9 indicated the presence of multiple compounds

(Supporting Information Figure 2A). The eluent was collected from 0.0-15.5 min, 15.6-23.4 min, 23.543.5 min, 43.6-56.5 min, 56.6-73 min, and 73.1-95.0 min, and then tested in disk diffusion assays (Supporting Information Table 1). The 15.5-23.35 min fraction (3.2) and the 23.25 min to 43.5 min

fractions (3.3) inhibited the growth of S. aureus, V. parahemolyticus, VRE, B. subtilis, and M. luteus (Supporting Information Table 1). As there were a large number of compounds in these two fractions, the HPLC gradient was modified to improve their separation (Supporting Information Figure 2B). The eluent was collected from 0.0-15.0 min, 15.1-48.0 min, 48.1-70.0 min, and 70.1-90.0 min, and then subjected to disk diffusion assays. The compounds collected between 15.1-48.0 min (fraction 4.2) were inhibitory, but sufficient quantities were not available for further isolation and chemical characterization. Reduction in peak size and intensity suggested that compounds in the extract degraded between the first and second HPLC analysis, but the fractions eluted from the second analysis retained antimicrobial activity (Supporting Information Table 1).

Microbial community analysis by DGGE DGGE was performed to identify microorganisms that associate consistently with the sponge

Haliclona sp., and that may be the true source of the bioactive natural products. The 16S rRNA amplicons from metagenomic DNA extracted from fourteen sponge individuals were separated and visualized by DGGE (Figure 2). There are three distinct areas (labeled A, B, and C) in the gel that are similar in most of the sponge samples. A prominent band in area A is present in all sponge individuals except S1, S3, and S6. It is 80% less prominent in sponges S4 and S12, and 33% less prominent in the seawater sample, although its slightly higher position on the gel and sequence (Table 2) suggests that it may be a different bacterial strain. Area B contains a band in the same location for sponges S2, S4,


S6, S7, S11, S12, S13 and perhaps sponge S9, as well as in the seawater sample. In area C, a band is

Accepted Article

abundant in all fourteen sponge individuals, but its intensity is reduced 75% in the seawater sample. Comparison of 16S rRNA gene sequences from the DGGE gel with sequences from the GenBank Bacteria and Archaea 16S rRNA gene database resulted in matches primarily to Proteobacteria (mostly γ, but some α and β, Table 2). There were also some matches for the 16S rRNA gene sequences of

diatom plastids and cyanobacteria. Band similarity matrix comparison of the DGGE fingerprints in sponges collected at different times indicate that there is little apparent seasonality in the microbial community (data not shown). The microbial community in two sponges collected in November 2011 (S3, S4) are divergent from the others, suggesting that the microbial community may be somewhat less

stable in the cooler weather.

DISCUSSION Marine sponges are continual sources of novel natural products, with 283, 296, and 355 new

compounds published in 2010, 2011, and 2012, respectively (Blunt et al. 2012; 2013; 2014). These

compounds can play an important role in the chemical defense of the animal (Paul et al. 2011; Pawlik 2011), as sessile marine invertebrates are unable to flee to escape predators. This selective pressure is thought to drive the evolution of natural products with potent bioactivity that may be exploited for pharmaceutical development (Laport et al. 2009; Sepcic et al. 2010). Sponges in the genus Haliclona are a particularly rich source of natural products, including amino alcohols (Devijver et al. 2000), macrocyclic diamides (Jang et al. 2009), sphingoid bases (Molinski et al. 2013), steroids and

terpenoids (Sakowicz et al. 1998; Blackburn and Faulkner 2000; Volk and Kock 2003; Volk et al. 2004; Yu et al. 2006; Trianto et al. 2011), alkaloids (Charan et al. 1996; Clark et al. 1998; Schmidt et al. 2011;

Schmidt et al. 2012; Kock et al. 2013), hydroquinones (Bokesch et al. 2002), acetylenes (Chill et al.

2000; Nuzzo et al. 2012), cyclic peptides (Randazzo et al. 2001), and nucleosides (Wang et al. 2009). When we began this study, we were interested in natural products from marine invertebrates in


temperate habitats, as they are less studied than their tropical counterparts. After identifying the

Accepted Article

sponge as a Haliclona sp. in the course of this research, it is not surprising that it possesses antimicrobial activity given the large number of compounds isolated sponges in this genus. The crude extract from this Haliclona sp. sponge had activity against all of the gram-negative and

gram-positive bacteria (Table 1) in disk diffusion assays, although the amount of inhibition was low against most of the strains. Similarly, the methanol partition of the crude extract was active against six of the seven bacteria tested, whereas the hexane partition was not inhibitory, suggesting that the compounds are more polar in nature. The fractionated methanol partition was not active against gram-negative E. coli or P. aeruginosa, which suggests that multiple compounds may act synergistically

against these organisms. Interestingly, the gram-negative opportunistic marine pathogen V. parahaemoylticus remained susceptible to these fractions, indicating that these compounds may be important in defense against native pathogens. Many other investigations into the antibacterial activity of extracts from marine sponges demonstrate greater inhibition of the growth of gram-positive bacteria compared to gram-negative bacteria (McCaffrey and Endean 1985; Thompson et al. 1985;

Newbold et al. 1999; Sepcic et al. 2010). We used many clinically relevant pathogens, but many

studies have shown that sponge extracts also have activity against strains isolated from healthy and diseased sponges (Newbold et al. 1999; Kelman et al. 2001; Kelly et al. 2005), demonstrating that these

compounds are likely ecologically important.

The strains responded differently in the liquid assays with the crude extract, methanol partition,

and the active fractions (2.6-2.9) recovered from the silica gel column (Figure 1). For V. parahaemolyticus and S. aureus, the decreased MICs of the three extracts reflect the increasing concentration of inhibitory molecules, as the dilution of the compounds is based on weight. In contrast, the MICs for the three extracts are similar against VRE, suggesting that some compounds that inhibit its growth may be lost during fractionation. HPLC analysis of active fractions indicate the


presence of multiple compounds that could contribute to the antimicrobial activity (Supporting

Accepted Article

Information Table 1, Supporting Information Figure 2). Further, the presence of moderate activity in column fractions 2.2 and 2.3 in addition to fractions 2.6-2.9 indicate that there are less polar compounds that may also be antibacterial. This is not surprising because of the diversity of natural products found in Haliclona spp. Like many natural products, however, low concentrations prevented active compound isolation and structure elucidation. While many bacteria and fungi are known for their ability to produce bioactive compounds, sponge cells have also been implicated in the production of natural products. Several studies examining the true source of specific bioactive natural products have concluded that the sponge is the producer (Garson et al. 1998; Pomponi 1999; Turon et al. 2000;

Richelle-Maurer et al. 2001), and not the microbe. In contrast, some studies have shown that bacteria

and fungi isolated from Haliclona spp. sponges produce extracts with antimicrobial activity (Baker et al. 2009; Kennedy et al. 2009; Zhou et al. 2011). Another approach has identified natural product

biosynthetic genes typically associated with bacteria (primarily polyketide synthases and nonribosomal peptide synthetases) from either sponge metagenomic DNA (Kennedy et al. 2008; Khan et al. 2014) or isolated strains (Jiang et al. 2007; Kennedy et al. 2009; Khan et al. 2011).

Sponges are well known for harboring microbial associates (Taylor et al. 2007; Webster and

Taylor 2012), and can usually be categorized as one of two types: “high-microbial-abundance” and “low-microbial-abundance” (Hentschel et al. 2006). Three species of Haliclona spp. have been described as low-microbial-abundance sponges, including the Caribbean sponge H. vansoesti (RichelleMaurer et al. 2001), H. tubifera from the Gulf of Mexico (Erwin et al. 2011) and Haliclona (?gellius) sp.

from Monterey harbor (CA) (Sipkema et al. 2009). Despite the low abundance of microbes in this sponge, culture independent methods have revealed that those harbored are diverse (Kennedy et al.

2008; Sipkema et al. 2011; Khan et al. 2014). In turn, researchers have also been able to isolate a number of bacteria and fungi from Haliclona spp. For instance, 24 different species of Actinobacteria


were isolated from a Haliclona sp. sponge from the South China Sea, half of which contained PKS or

Accepted Article

NRPS genes, which prescribe the biosynthesis of some classes of natural products (Jiang et al. 2007).

Nineteen different fugal strains were isolated from Haliclona simulans, some of which exhibited antimicrobial activity. Up to 50% of the bacteria from diverse phyla (Bacteriodetes, Actinobacteria, Proteobacteria, and Firmicutes) isolated from Haliclona simulans displayed activity against at least one test organism (Kennedy et al. 2009). In this study, we also examined the microbial community in the sponge to identify stable

associates that could be potential symbionts using DGGE of the 16S rRNA gene amplified from sponge metagenomic DNA (Taylor et al. 2004; Webster et al. 2013). The sponge individuals possess fewer

bands than the seawater sample, indicating that the sponges have an overall lower level of diversity than the surrounding seawater (Figure 2). In general, this could mean that the sponge is preferentially expelling some microorganisms from seawater or consuming them for nourishment (Reiswig 1975), while some microorganisms remain inside its tissue. A lack of clear seasonal patterning in the sponge microbial community from individuals collected in November, March, and April suggests that the microbial associates may be stable. BLAST analysis of DGGE gel band sequences indicate the presence of primarily γ-Proteobacteria, but also members of the α- and β-Proteobacteria and Cyanobacteria (Table 2). There is one band present in all 14 sponge individuals and not in the seawater (Area C in Figure 2) that could be a stable associate. The sequences of Bands 1 and 10 were similar to a strain of Kistimonas scapharcae that was isolated from a marine clam (Lee et al. 2012). This newly-described

organism is a motile, Gram-negative γ-Proteobacterium, facultative anaerobe which is catalase- and oxidase-negative. Another newly-described Kistimonas species was isolated from a starfish (Choi et al.

2010). The sequence of Band 9, most similar (88% identity) to the β-proteobacterium Dechlorosoma suillum (Table 2), was ~76% identical to the sequences of Bands 1 and 10, indicating that more than one organism is represented by the DGGE band. Sequences from bands 5 and 6 were similar to the Gram-negative, facultatively anaerobic member of Enterobacteriaceae, Serratia liquefaciens, which is also a fish pathogen (Austin and Billaud 1990) and forms biofilms (Grimont and Grimont 1978).


Moreover, some Serratia spp. bacteria produce antibacterial and surfactant natural products (Li et al.

Accepted Article

2005; Gerc et al. 2012; Masschelein et al. 2013). The band in area A is more frequent in sponges

collected in the Spring (7/9 in March and April) than in those collected in late Fall (2/5). The sequence from Band 7 is 95% identical to Tranquillimonas alkanivorans, a Gram-negative halophilic bacteria that can degrade short-chain alkanes (Harwati et al. 2008). The increased frequency of this bacterium in sponges in warmer temperatures could be due to increased boat traffic and pollution in the marinas where the sponges were collected, and may represent a seasonal associate.

In summary, we determined that the Haliclona sp. sponge collected from the temperate waters of

North Carolina possesses antimicrobial activity that is due to more than one compound. Moreover, this sponge appears to host some microbes that may be involved in antibiotic production. Further work needs to be done to isolate the antibiotic compounds and elucidate their structure. Next, identifying the antibiotic producing organism (host or microbe) can be a challenging task. While in some instances the producing organism may be cultured, symbiotic microbes are usually difficult to culture because of unknown host factors that promote their growth. The diversity of metabolites and microbes in these abundant, temperate Haliclona spp. sponges make this organism a worthy target for bioprospecting.

ACKNOWLEDGEMENTS We would like to thank Niels Lindquist for generous access to dry and wet laboratory facilities and Jonathan Linneman for assistance in collecting sponge samples. This research was funded by Georgia State University Research Foundation (N.B.L.).


Accepted Article

CONFLICT OF INTEREST No conflict of interest declared.

REFERENCES Austin, B. and Billaud, A.C. (1990) Inhibition of the fish pathogen, Serratia liquefaciens, by an antibioticproducing isolate of Planococcus recovered from sea water. J Fish Dis 13, 553-556.

Baker, P.W., Kennedy, J., Dobson, A.D.W. and Marchesi, J.R. (2009) Phylogenetic Diversity and Antimicrobial Activities of Fungi Associated with Haliclona simulans Isolated from Irish Coastal Waters. Mar Biotechnol (NY) 11, 540-547. Baltz, R.H. (2006) Marcel Faber Roundtable: Is our antibiotic pipeline unproductive because of starvation, constipation or lack of inspiration? J Ind Microbiol Biotechnol 33, 507-513. Bewley, C.A., Holland, N.D. and Faulkner, D.J. (1996) Two classes of metabolites from Theonella swinhoei are localized in distinct populations of bacterial symbionts. Experientia 52, 716-722. Blackburn, C.L. and Faulkner, D.J. (2000) Adociasulfate 10, a new merohexaprenoid sulfate from the sponge Haliclona (aka Adocia) sp. Tetrahedron 56, 8429-8432. Blunt, J.W., Copp, B.R., Keyzers, R.A., Munro, M.H.G. and Prinsep, M.R. (2012) Marine natural products. Nat Prod Rep 29, 144-222. Blunt, J.W., Copp, B.R., Keyzers, R.A., Munro, M.H.G. and Prinsep, M.R. (2013) Marine natural products. Nat Prod Rep 30, 237-323. Blunt, J.W., Copp, B.R., Keyzers, R.A., Munro, M.H.G. and Prinsep, M.R. (2014) Marine natural products. Nat Prod Rep 31, 160-258. Bokesch, H.R., Stull, A.C., Pannell, L.K., McKee, T.C. and Boyd, M.R. (2002) A new pentacyclic sulfated hydroquinone from the marine sponge Haliclona sp. Tetrahedron Lett 43, 3079-3081. Charan, R.D., Garson, M.J., Brereton, I.M., Willis, A.C. and Hooper, J.N.A. (1996) Haliclonacyclamines A and B, cytotoxic alkaloids from the tropical marine sponge Haliclona sp. Tetrahedron 52, 9111-9120.

Chill, L., Miroz, A. and Kashman, Y. (2000) Haliclonyne, a new highly oxygenated polyacetylene from the marine sponge Haliclona species. J Nat Prod 63, 523-526. Choi, E.J., Kwon, H.C., Sohn, Y.C. and Yang, H.O. (2010) Kistimonas asteriae gen. nov., sp nov., a gammaproteobacterium isolated from Asterias amurensis. Int J Syst Evol Microbiol 60, 938-943.


Accepted Article

Clark, R.J., Field, K.L., Charan, R.D., Garson, M.J., Brereton, I.M. and Willis, A.C. (1998) The haliclonacyclamines, cytotoxic tertiary alkaloids from the tropical marine sponge Haliclona sp. Tetrahedron 54, 8811-8826. Devijver, C., Salmoun, N., Daloze, D., Braekman, J.C., De Weerdt, W.H., De Kluijver, M.J. and Gomez, R. (2000) (2R,3R,7Z)-2-Aminotetradec-7-ene-1,3-diol, a new amino alcohol from the Caribbean sponge Haliclona vansoesti. J Nat Prod 63, 978-980. Erwin, P.M., Olson, J.B. and Thacker, R.W. (2011) Phylogenetic Diversity, Host-Specificity and Community Profiling of Sponge-Associated Bacteria in the Northern Gulf of Mexico. PLoS One 6. Faulkner, D.J., Unson, M. and Bewley, C. (1994) The chemistry of some sponges and their symbionts. Pure Appl Chem 66, 1983-1990. Garson, M.J., Flowers, A.E., Webb, R.I., Charan, R.D. and McCaffrey, E.J. (1998) A sponge/dinoflagellate association in the haplosclerid sponge Haliclona sp.: cellular origin of cytotoxic alkaloids by Percoll density gradient fractionation. Cell Tissue Res 293, 365-373.

Gerc, A.J., Song, L.J., Challis, G.L., Stanley-Wall, N.R. and Coulthurst, S.J. (2012) The Insect Pathogen Serratia marcescens Db10 Uses a Hybrid Non-Ribosomal Peptide Synthetase-Polyketide Synthase to Produce the Antibiotic Althiomycin. PLoS One 7. Gillor, O., Carmeli, S., Rahamim, Y., Fishelson, Z. and Ilan, M. (2000) Immunolocalization of the toxin latrunculin B within the Red Sea sponge Negombata magnifica (Demospongiae, Latrunculiidae). Mar Biotechnol (NY) 2, 213-223. Grimont, P.A.D. and Grimont, F. (1978) The Genus Serratia. Annu Rev Microbiol 32, 221-248. Harwati, T.U., Kasai, Y., Kodama, Y., Susilaningsih, D. and Watanabe, K. (2008) Tranquillimonas alkanivorans gen. nov., sp nov., an alkane-degrading bacterium isolated from Semarang Port in Indonesia. Int J Syst Evol Microbiol 58, 2118-2121. Hay, M.E. (2009) Marine Chemical Ecology: Chemical Signals and Cues Structure Marine Populations, Communities, and Ecosystems. Ann Rev Mar Sci 1, 193-212.

Heck, K.L.J. and Wilson, K.A. (1987) Predation rates on decapod crustaceans in latitudinally separated seagrass communities: A study of spatial and temporal variation using tethering techniques. J Exp Mar Bio Ecol 107, 87-100.

Hentschel, U., Hopke, J., Horn, M., Friedrich, A.B., Wagner, M., Hacker, J. and Moore, B.S. (2002) Molecular evidence for a uniform microbial community in sponges from different oceans. Appl Environ Microbiol 68, 4431-4440.

Hentschel, U., Usher, K.M. and Taylor, M.W. (2006) Marine sponges as microbial fermenters. FEMS Microbiol Ecol 55, 167-177.


Accepted Article

Hochmuth, T. and Piel, J. (2009) Polyketide synthases of bacterial symbionts in sponges – Evolutionbased applications in natural products research. Phytochemistry 70, 1841-1849. Hunter, P.A., Darby, G.K. and Russell, N.J. (1995) Fifty years of antimicrobials: past perspectives and future trends: Cambridge University Press. Jang, K.H., Kang, G.W., Jeon, J.E., Lim, C., Lee, H.S., Sim, C.J., Oh, K.B. and Shin, J. (2009) Haliclonin A, a New Macrocyclic Diamide from the Sponge Haliclona sp. Org Lett 11, 1713-1716. Jiang, S.M., Sun, W., Chen, M.J., Dai, S.K., Zhang, L., Liu, Y.H., Lee, K.J. and Li, X. (2007) Diversity of culturable actinobacteria isolated from marine sponge Haliclona sp. Antonie Van Leeuwenhoek 92, 405416. Kelly, S.R., Garo, E., Jensen, P.R., Fenical, W. and Pawlik, J.R. (2005) Effects of Caribbean sponge secondary metabolites on bacterial surface colonization. Aquat Microb Ecol 40, 191-203. Kelman, D., Kashman, Y., Hill, R.T., Rosenberg, E. and Loya, Y. (2009) Chemical warfare in the sea: The search for antibiotics from Red Sea corals and sponges. Pure Appl Chem 81, 1113-1121. Kelman, D., Kashman, Y., Rosenberg, E., Ilan, M., Ifrach, I. and Loya, Y. (2001) Antimicrobial activity of the reef sponge Amphimedon viridis from the Red Sea: evidence for selective toxicity. Aquat Microb Ecol 24, 9-16. Kennedy, J., Baker, P., Piper, C., Cotter, P.D., Walsh, M., Mooij, M.J., Bourke, M.B., Rea, M.C., O'Connor, P.M., Ross, R.P., Hill, C., O'Gara, F., Marchesi, J.R. and Dobson, A.D.W. (2009) Isolation and Analysis of Bacteria with Antimicrobial Activities from the Marine Sponge Haliclona simulans Collected from Irish Waters. Mar Biotechnol (NY) 11, 384-396. Kennedy, J., Codling, C.E., Jones, B.V., Dobson, A.D.W. and Marchesi, J.R. (2008) Diversity of microbes associated with the marine sponge, Haliclona simulans, isolated from Irish waters and identification of polyketide synthase genes from the sponge metagenome. Environ Microbiol 10, 1888-1902.

Khan, S.T., Komaki, H., Motohashi, K., Kozone, I., Mukai, A., Takagi, M. and Shin-ya, K. (2011) Streptomyces associated with a marine sponge Haliclona sp.; biosynthetic genes for secondary metabolites and products. Environ Microbiol 13, 391-403. Khan, S.T., Musarrat, J., Alkhedhairy, A.A. and Kazuo, S. (2014) Diversity of bacteria and polyketide synthase associated with marine sponge Haliclona sp. Ann Microbiol 64, 199-207. Kock, M., Munoz, J., Cychon, C., Timm, C. and Schmidt, G. (2013) The Arctic sponge Haliclona viscosa as a source of a wide array of 3-alkyl pyridine alkaloids. Phytochem Rev 12, 391-406. Laport, M.S., Santos, O.C.S. and Muricy, G. (2009) Marine Sponges: Potential Sources of New Antimicrobial Drugs. Curr Pharm Biotechnol 10, 86-105.


Accepted Article

Lee, J., Shin, N.R., Lee, H.W., Roh, S.W., Kim, M.S., Kim, Y.O. and Bae, J.W. (2012) Kistimonas scapharcae sp nov., isolated from a dead ark clam (Scapharca broughtonii) and emended description of the genus Kistimonas. Int J Syst Evol Microbiol 62, 2865-2869. Li, H., Tanikawa, T., Sato, Y., Nakagawa, Y. and Matsuyama, T. (2005) Serratia marcescens gene required for surfactant serrawettin W1 production encodes putative aminolipid synthetase belonging to nonribosomal peptide synthetase family. Microbiol Immunol 49, 303-310. Lopanik, N.B. (2014) Chemical defensive symbioses in the marine environment. Funct Ecol 28, 328340. Masschelein, J., Mattheus, W., Gao, L.J., Moons, P., Van Houdt, R., Uytterhoeven, B., Lamberigts, C., Lescrinier, E., Rozenski, J., Herdewijn, P., Aertsen, A., Michiels, C. and Lavigne, R. (2013) A PKS/NRPS/FAS Hybrid Gene Cluster from Serratia plymuthica RVH1 Encoding the Biosynthesis of Three Broad Spectrum, Zeamine-Related Antibiotics. PLoS One 8. McCaffrey, E.J. and Endean, R. (1985) Antimicrobial activity of tropical and subtropical sponges. Mar Biol 89, 1-8. McGinnis, S. and Madden, T.L. (2004) BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res 32, W20-W25. Medlin, L., Elwood, H.J., Stickel, S. and Sogin, M.L. (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71, 491-499.

Molinski, T.F., Biegelmeyer, R., Stout, E.P., Wang, X., Frota, M.L.C. and Henriques, A.T. (2013) Halisphingosines A and B, Modified Sphingoid Bases from Haliclona tubifera. Assignment of Configuration by Circular Dichroism and van't Hoff's Principle of Optical Superposition. J Nat Prod 76, 374-381. Newbold, R.W., Jensen, P.R., Fenical, W. and Pawlik, J.R. (1999) Antimicrobial activity of Caribbean sponge extracts. Aquat Microb Ecol 19, 279-284.

Nuzzo, G., Ciavatta, M.L., Villani, G., Manzo, E., Zanfardino, A., Varcamonti, M. and Gavagnin, M. (2012) Fulvynes, antimicrobial polyoxygenated acetylenes from the Mediterranean sponge Haliclona fulva. Tetrahedron 68, 754-760. Paul, V.J., Arthur, K.E., Ritson-Williams, R., Ross, C. and Sharp, K. (2007) Chemical defenses: From compounds to communities. Biol Bull 213, 226-251. Paul, V.J., Ritson-Williams, R. and Sharp, K. (2011) Marine chemical ecology in benthic environments. Nat Prod Rep 28, 345-387. Pawlik, J.R. (2011) The Chemical Ecology of Sponges on Caribbean Reefs: Natural Products Shape Natural Systems. Bioscience 61, 888-898.


Accepted Article

Pennings, S.C. and Silliman, B.R. (2005) Linking biogeography and community ecology: Latitudinal variation in plant-herbivore interaction strength. Ecology 86, 2310-2319. Pettit, G.R., Day, J.F., Hartwell, J.L. and Wood, H.B. (1970) Antineoplastic components of marine animals. Nature 227, 962-963. Pettit, G.R., Herald, C.L., Doubek, D.L., Herald, D.L., Arnold, E. and Clardy, J. (1982) Isolation and structure of bryostatin 1. J Am Chem Soc 104, 6846-6848.

Phelan, R.W., Barret, M., Cotter, P.D., O'Connor, P.M., Chen, R., Morrissey, J.P., Dobson, A.D.W., O'Gara, F. and Barbosa, T.M. (2013) Subtilomycin: A New Lantibiotic from Bacillus subtilis Strain MMA7 Isolated from the Marine Sponge Haliclona simulans. Mar Drugs 11, 1878-1898. Pomponi, S.A. (1999) The bioprocess-technological potential of the sea. J Biotechnol 70, 5-13. Randazzo, A., Bifulco, G., Giannini, C., Bucci, M., Debitus, C., Cirino, G. and Gomez-Paloma, L. (2001) Halipeptins A and B: Two novel potent anti-inflammatory cyclic depsipeptides from the vanuatu marine sponge Haliclona species. J Am Chem Soc 123, 10870-10876.

Reiswig, H.M. (1975) Bacteria as food for temperate-water marine sponges. Can J Zool 53, 582-589. Richelle-Maurer, E., Braekman, J.C., De Kluijver, M.J., Gomez, R., Van de Vyver, G., Van Soest, R.W.M. and Devijver, C. (2001) Cellular location of (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol, a potent antimicrobial metabolite produced by the Caribbean sponge Haliclona vansoesti. Cell Tissue Res 306, 157-165. Sakowicz, R., Berdelis, M.S., Ray, K., Blackburn, C.L., Hopmann, C., Faulkner, D.J. and Goldstein, L.S.B. (1998) A marine natural product inhibitor of kinesin motors. Science 280, 292-295. Sathiyanarayanan, G., Gandhimathi, R., Sabarathnam, B., Kiran, G.S. and Selvin, J. (2014) Optimization and production of pyrrolidone antimicrobial agent from marine sponge-associated Streptomyces sp. MAPS15. Bioprocess Biosyst Eng 37, 561-573. Schafer, H., Bernard, L., Courties, C., Lebaron, P., Servais, P., Pukall, R., Stackebrandt, E., Troussellier, M., Guindulain, T., Vives-Rego, J. and Muyzer, G. (2001) Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in the genetic diversity of bacterial populations. FEMS Microbiol Ecol 34, 243-253. Schemske, D.W., Mittelbach, G.G., Cornell, H.V., Sobel, J.M. and Roy, K. (2009) Is There a Latitudinal Gradient in the Importance of Biotic Interactions? Annu Rev Ecol Evol Syst 40, 245-269. Schmidt, G., Timm, C., Grube, A., Volk, C.A. and Kock, M. (2012) Viscosalines B1,2 and E1,2: Challenging New 3-Alkyl Pyridinium Alkaloids from the Marine Sponge Haliclona viscosa. Chem-Eur J 18, 81808189. Schmidt, G., Timm, C. and Kock, M. (2011) Haliclocyclin C, a New Monomeric 3-Alkyl Pyridinium Alkaloid from the Arctic Marine Sponge Haliclona viscosa. Z Naturforsch (B) 66, 745-748.


Accepted Article

Sepcic, K., Kauferstein, S., Mebs, D. and Turk, T. (2010) Biological Activities of Aqueous and Organic Extracts from Tropical Marine Sponges. Mar Drugs 8, 1550-1566. Sipkema, D., Franssen, M.C.R., Osinga, R., Tramper, J. and Wijffels, R.H. (2005) Marine sponges as pharmacy. Mar Biotechnol (NY) 7, 142-162. Sipkema, D., Holmes, B., Nichols, S.A. and Blanch, H.W. (2009) Biological Characterisation of Haliclona (?gellius) sp.: Sponge and Associated Microorganisms. Microb Ecol 58, 903-920. Sipkema, D., Schippers, K., Maalcke, W.J., Yang, Y., Salim, S. and Blanch, H.W. (2011) Multiple Approaches To Enhance the Cultivability of Bacteria Associated with the Marine Sponge Haliclona (gellius) sp. Appl Environ Microbiol 77, 2130-2140. Siska, E.L., Pennings, S.C., Buck, T.L. and Hanisak, M.D. (2002) Latitudinal variation in palatability of salt-marsh plants: Which traits are responsible? Ecology 83, 3369-3381.

Taylor, M.W., Hill, R.T., Piel, J., Thacker, R.W. and Hentschel, U. (2007) Soaking it up: the complex lives of marine sponges and their microbial associates. ISME J 1, 187-190.

Taylor, M.W., Schupp, P.J., Dahllof, I., Kjelleberg, S. and Steinberg, P.D. (2004) Host specificity in marine sponge-associated bacteria, and potential implications for marine microbial diversity. Environ Microbiol 6, 121-130. Thomas, T.R.A., Kavlekar, D.P. and LokaBharathi, P.A. (2010) Marine Drugs from Sponge-Microbe Association-A Review. Mar Drugs 8, 1417-1468. Thompson, J.E., Walker, R.P. and Faulkner, D.J. (1985) Screening and bioassays for biologically-active substances from forty marine sponge species from San Diego, California, USA. Mar Biol 88, 11-21. Trianto, A., Hermawan, I., de Voogd, N.J. and Tanaka, J. (2011) Halioxepine, a New Meroditerpene from an Indonesian Sponge Haliclona sp. Chem Pharm Bull (Tokyo) 59, 1311-1313.

Trindade-Silva, A.E., Lim-Fong, G.E., Sharp, K.H. and Haygood, M.G. (2010) Bryostatins: biological context and biotechnological prospects. Curr Opin Biotechnol 21, 834-842. Turon, X., Becerro, M.A. and Uriz, M.J. (2000) Distribution of brominated compounds within the sponge Aplysina aerophoba: coupling of X-ray microanalysis with cryofixation techniques. Cell Tissue Res 301, 311-322. Vacelet, J. and Donadey, C. (1977) Electron microscope study of the association between some sponges and bacteria. J Exp Mar Bio Ecol 30, 301-314. Volk, C.A. and Kock, M. (2003) Viscosamine: The first naturally occurring trimeric 3-alkyl pyridiniurn alkaloid. Org Lett 5, 3567-3569. Volk, C.A., Lippert, H., Lichte, E. and Kock, M. (2004) Two new haliclamines from the arctic sponge Haliclona viscosa. European J Org Chem, 3154-3158.


Accepted Article

Wang, B., Dong, J., Zhou, X.F., Lee, K.J., Huang, R.M., Zhang, S. and Liu, Y.H. (2009) Nucleosides from the Marine Sponge Haliclona sp. Z Naturforsch (C) 64, 143-148. Webster, N.S., Luter, H.M., Soo, R.M., Botte, E.S., Simister, R.L., Abdo, D. and Whalan, S. (2013) Same, same but different: symbiotic bacterial associations in GBR sponges. Front Microbiol 3.

Webster, N.S. and Taylor, M.W. (2012) Marine sponges and their microbial symbionts: love and other relationships. Environ Microbiol 14, 335-346. Wilkinson, C.R. (1984) Immunolgical evidence for the Precambrian origin of bacterial symbioses in marine sponges. Proc R Soc Lond B Biol Sci 220, 509-517.

Wilkinson, C.R., Garrone, R. and Vacelet, J. (1984) Marine sponges discriminate between food bacteria and bacterial symbionts: electron microscope radioautography and in situ evidence. Proc R Soc Lond B Biol Sci 220, 519-528.

Yu, S.J., Deng, Z.W., Proksch, P. and Lin, W.H. (2006) Oculatol, oculatolide, and A-nor sterols from the sponge Haliclona oculata. J Nat Prod 69, 1330-1334.

Zhou, K., Zhang, X., Zhang, F.L. and Li, Z.Y. (2011) Phylogenetically Diverse Cultivable Fungal Community and Polyketide Synthase (PKS), Non-ribosomal Peptide Synthase (NRPS) Genes Associated with the South China Sea Sponges. Microb Ecol 62, 644-654.

Table 1 Disk diffusion assay inhibition zones against the extract fractions (mm). Fraction

Solvent

Ec*

Pa

Vp

Sa

VRE

Bs

Ml

0.1

Crude extract

1.5

1.5

2.0

3.0

2.0

8.5

7.0

0.2

Methanol partition

2.0

0.5

2.0

2.0

2.5

4.0

5.0

0.3

Hexane partition

-

-

-

-

-

-

-

1.1

100% Hexane

-

-

-

-

-

-

-

1.2

50% Hexane/50% EtOAc

-

-

3.0

1.0

2.5

-

5.0

1.3

100% EtOAc

-

-

2.5

2.0

2.0

-

5.0

1.4

90% EtOAc/10% MeOH

-

-

2.5

1.0

1.2

-

4.0

1.5

80% EtOAc/20% MeOH

-

-

2.0

0.5

2.0

-

2.0


70% EtOAc/30% MeOH

-

-

-

-

-

-

0.5

1.7

60% EtOAc/40% MeOH

-

-

-

-

-

-

0.5

1.8

50% EtOAc/50% MeOH

-

-

-

-

-

-

-

1.9

25% EtOAc/75% MeOH

-

-

-

-

-

-

-

1.1

100% MeOH

-

-

-

-

-

-

-

2.1

100% Hexane

-

-

-

-

-

-

-

2.2

75% Hexane/25%EtOAc

-

-

3.0

1.0

3.0

3.0

-

2.3


-

-

3.0

2.0

4.0

5.0

-

2.4


-

-

0.5

-

-

-

-

2.5

100% EtOAc

-

-

0.5

-

-

-

-

2.6

95% EtOAc/5% MeOH

-

0.5

3.0

2.0

4.0

4.0

5.0

2.7

90% EtOAc/10% MeOH

-

0.5

2.5

2.0

4.0

5.0

5.0

2.8

85% EtOAc/15% MeOH

-

0.5

4.0

1.0

2.5

3.0

6.0

2.9

80% EtOAc/20% MeOH

-

0.5

3.0

1.0

4.0

6.0

7.0

2.10

75% EtOAc/25% MeOH

-

-

-

-

-

-

-

2.11

50% EtOAc/50% MeOH

-

-

-

-

-

-

-

Accepted Article

1.6

*Ec = E. coli; Pa = P. aeruginosa; Vp = V. parahaemolyticus; Sa = S. aureus; VRE = Vancomycin-resistant Enterococcus; Bs = B. subtilis; Ml = M. luteus. “-“ indicates no inhibition.


Accepted Article

Table 2 Organisms indicated by BLAST analysis of sequences from DGGE gel bands. Analyses performed with the GenBank Bacteria and Archaea 16S rRNA gene database. The band number corresponds to those bands indicated on the DGGE gel (Figure 2).

Band

Top BLAST Hit

Accession number

e-value

% Ident.

Length (bp)

1

Kistimonas scapharcae strain A36

NR_109422

5e-61

94

152

2

Stanieria cyanosphaera strain PCC 7437

NR_102468

1e-55

96

130

3


NR_109422

1e-61

94

154

4

Candidatus Pelagibacter ubique HTCC1062

NR_074224

2e-58

98

126

5

Serratia liquefaciens strain JCM1245

NR_112008

1e-57

96

144

6

Serratia liquefaciens strain JCM1245

NR_112008

1e-57

96

145

7

Tranquillimonas alkanivorans strain A34

NR_041598

3e-47

95

125

8

Endozoicomonas gorgoniicola strain PS125

NR_109685

3e-53

95

145

9

Dechlorosoma suillum PS

NR_074103

2e-44

88

149

10


NR_109422

1e-61

94

156

11

Cyanothece sp. ATCC 51142

NR_074316

1e-41

94

119

12

Candidatus Pelagibacter ubique HTCC1062

NR_074224

3e-57

98

124


FIGURE LEGENDS

Accepted Article

Figure 1 Activity of different concentrations of Haliclona sp. crude and purified extracts against (A) S. aureus, (B) VRE, and (C) V. parahaemolyticus measured via liquid bioassays. Concentrations of the extracts were applied at

0,

2.5,

10,

25, and

50 mg ml-1. Letters indicate significant

differences between cell growth in the presence of differing extract concentrations (one-way ANOVA, Tukey’s HSD, P < 0.05).

Figure 2 DGGE analysis of sponge individuals and surrounding seawater sample. Lanes S1-S14 contain the environmental DNA from sponge individuals 1-14 and lane sw contains the seawater sample. Sequenced bands and areas of interest (A-C) indicated. Black arrows correspond with numbers in Table 2, indicating which bands were chosen for sequencing.


c

1.0

OD600 (+SE)

0.8

a

b

a

a

b

0.6 0.4 0.2

b

c

d 0.0 -0.2

Crude

B

1.2

Methanol

a

a

Fractions

a

OD600 (+SE)

1.0 0.8 0.6

b

b

0.4

c

b

c

0.2 0.0 -0.2

Crude

C

1.0 0.8

OD 600 (+ SE)

Accepted Article

A

a

a

Fractions

a b

b

c

0.6 0.4

Methanol

c

0.2

d

b

0.0 -0.2 -0.4

Crude

Methanol

Figure 1


Fractions

Accepted Article

Nov 2011 S1

Mar 2012

S2 S3 S4 S5

S6

S7

Apr 2012

S8

S9

S10 S11 S12 S13 S14 sw

2

3

7

6

11

A 8

4

12

5

1

10

Figure 2


B

9

C

A new 3-alkylpyridine alkaloid from the marine sponge Haliclona sp. and its cytotoxic activity.

Antibacterial Activity of Metabolites Products of Vibrio Alginolyticus Isolated from Sponge Haliclona sp. Against Staphylococcus Aureus.

Apoptosis-Inducing Activity of Marine Sponge Haliclona sp. Extracts Collected from Kosrae in Nonsmall Cell Lung Cancer A549 Cells.

Effect of copper treatment on the composition and function of the bacterial community in the sponge Haliclona cymaeformis.

Antifungal and antibacterial activity of Haliclona sp. from the Persian Gulf, Iran.

A novel erythromycin resistance plasmid from Bacillus sp. strain HS24, isolated from the marine sponge Haliclona simulans.

Isolation and identification of antitrypanosomal and antimycobacterial active steroids from the sponge Haliclona simulans.

H-3, a new lectin from the marine sponge Haliclona caerulea: purification and mass spectrometric characterization.

Absolute Configuration of the New 3-epi-cladocroic Acid from the Mediterranean Sponge Haliclona fulva.

Variations of Soil Microbial Community Structures Beneath Broadleaved Forest Trees in Temperate and Subtropical Climate Zones.

Antifungal activity of meridine, a natural product from the marine sponge Corticium sp.

Activity and community structures of sulfate-reducing microorganisms in polar, temperate and tropical marine sediments.

Successive nonstatistical and statistical approaches for the improved antibiotic activity of rare actinomycete Nonomuraea sp. JAJ18.

Bacterial community composition and extracellular enzyme activity in temperate streambed sediment during drying and rewetting.

Antibiotic resistance genes and associated microbial community conditions in aging landfill systems.

Effects of manure compost application on soil microbial community diversity and soil microenvironments in a temperate cropland in China.

Microbial community in microbial fuel cell (MFC) medium and effluent enriched with purple photosynthetic bacterium (Rhodopseudomonas sp.).

Cryptic species obscure introduction pathway of the blue Caribbean sponge (Haliclona (Soestella) caerulea), (order: Haplosclerida) to Palmyra Atoll, Central Pacific.

Cytotoxic sesterterpenoids isolated from the marine sponge Scalarispongia sp.

Chemistry and Selective Tumor Cell Growth Inhibitory Activity of Polyketides from the South China Sea Sponge Plakortis sp.

Improved chalcopyrite bioleaching by Acidithiobacillus sp. via direct step-wise regulation of microbial community structure.

Influence of tetracycline on the microbial community composition and activity of nitrifying biofilms.

GP antibiotic overuse and microbial resistance.

Soil microbial community responses to antibiotic-contaminated manure under different soil moisture regimes.