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Applied and Environmental Microbiology, April 2004, p. 2332-2341, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2332-2341.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Oxygen-Controlled Bacterial Growth in the Sponge Suberites domuncula: toward a Molecular Understanding of the Symbiotic Relationships between Sponge and Bacteria{dagger}

Werner E. G. Müller,* Vladislav A. Grebenjuk, Narsinh L. Thakur, Archana N. Thakur, Renato Batel, Anatoli Krasko, Isabel M. Müller, and Hans J. Breter

Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität Mainz, D-55099 Mainz, Germany

Received 15 August 2003/ Accepted 14 January 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sponges (phylum Porifera), known to be the richest producers among the metazoans of bioactive secondary metabolites, are assumed to live in a symbiotic relationship with microorganisms, especially bacteria. Until now, the molecular basis of the mutual symbiosis, the exchange of metabolites for the benefit of the other partner, has not been understood. We show with the demosponge Suberites domuncula as a model that the sponge expresses under optimal aeration conditions the enzyme tyrosinase, which synthesizes diphenols from monophenolic compounds. The cDNA isolated was used as a probe to determine the steady-state level of gene expression. The gene expression level parallels the level of specific activity in sponge tissue, indicating that without aeration the tyrosinase level drops drastically; this effect is reversible. The SB2 bacterium isolated from the sponge surface grew well in M9 minimal salt medium supplemented with the dihydroxylated aromatic compound protocatechuate; this carbon source supported growth more than did glucose. From the SB2 bacterium the protocatechuate gene cluster was cloned and sequenced. This cluster comprises all genes coding for enzymes involved in the conversion of protocatechuate to acetyl coenzyme A. Expression is strongly induced if the bacteria are cultivated on M9-protocatechuate medium; the genes pcaQ (encoding the putative transcriptional activator of the pca operon) and pcaDC were used for quantitative PCR analyses. We conclude that metabolites, in this case diphenols, which might be produced by the sponge S. domuncula are utilized by the sponge surface-associated bacterium for energy generation. This rationale will help to further uncover the symbiotic pathways between sponges and their associated "nonculturable" microorganisms; our approach is flanked by the establishment of an EST (expressed sequence tags) database in our laboratory.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sponges (phylum Porifera) are among the metazoan organisms the richest source of bioactive compounds (18, 49). It is established that their secondary metabolites display strong and selective inhibitory activities against distinct pathways in higher metazoans (50), such as inhibition of tyrosine kinases (e.g., aeroplysinin) (29), angiogenesis (aeroplysinin) (44), and replication of viruses, e.g. herpes simplex viruses (arabinofuranosyl adenine) (38) or retroviruses (e.g., avarol) (24, 48). Sponges, as marine sessile filter feeders processing around 2 tons of water daily per kg of tissue (59), are exposed to immense loads of bacteria (56) and viruses (3), as well as to eukaryotic grazers and invaders. Sponges resist these attackers by interfering with metabolic pathways which remained conserved from the lowest metazoan phylum, the Porifera, to humans (36, 37). Lately, the pharmacological properties of the secondary metabolites, especially those from sponges, have gained importance with regard to biotechnology and their potential application in human therapy (6, 43).

It has been suggested that many of the bioactive metabolites are produced by microorganisms which are associated, either commensally or symbiotically, with the eukaryotic host (43). This assumption can be validated only by experimental demonstration of the origin of the secondary metabolites. Experimental evidence, through analysis by PCR of ribosomal DNA (rDNA), has been provided indicating that sponges live in a symbiotic relationship with distinct bacteria (1). However, cultivation of these microorganisms in vitro is usually not successful, most probably due to inappropriate cultivation conditions. In the sponge Halichondria panicea the bacteria require sponge lectins for their growth (39).

The marine environment is rich in inorganic components like phosphorus, potassium, magnesium, sodium, calcium, and to some extent iron (27), but it is poor in amino acids and proteins as well as in carbon sources. Sponges possess the {gamma}-glutamyl cycle, which allows them to accumulate N-containing organic compounds from the environment (31). With respect to the requirement for carbon sources some bacteria differ in their needs from those of their host. One hydrocarbon transformation which produces starting substrates for oxidative catabolism is the protocatechuate/catechol/ß-ketoadipate pathway (21). In bacteria most aromatic compounds are first converted to catechol or protocatechuate, whose aromatic ring is subsequently cleaved with formation of succinate, pyruvate, and/or acetyl coenzyme A (acetyl-CoA), which in turn can enter, for example, the citric acid cycle. Important for our research focus is also the pathway which results in the formation of building blocks, e.g. malonyl-CoA for the synthesis of polyketides. Also in sponges these units are subsequently utilized by bacteria to produce polyketides (40), e.g., swinholide from the lithistid sponge Theonella swinhoei (4).

In a previous study we presented evidence that some bacteria growing on the surface of sponges, e.g. on Suberites domuncula, produce bioactive compounds to protect their host against other bacterial invaders (57). S. domuncula can easily be kept under controlled aquarium conditions. To design a suitable growth medium for such microorganisms, we examined whether on the surface of this sponge bacteria are present which require as carbon sources oxidation products of aromatic compounds, e.g., protocatechuate and catechol, that are produced by the sponge host. We especially studied the enzyme tyrosinase (EC 1.1.18.1), which had been described in 1903 (10). It turned out to be highly active in S. domuncula, and therefore we identified and sequenced the tyrosinase from this sponge. Tyrosinases catalyze hydroxylation of monophenols or oxidation of o-diphenols to o-quinones or both with consumption of molecular oxygen (13; reviewed in reference 35). In lower-animal phyla tyrosinase is involved in immune responses via the production of melanin (53, 63).

The present study focused on a probable correlation between the expression of tyrosinase in the sponge host (formation of dihydroxylated compounds) and the growth dependence of sponge-associated bacterial strain(s) on such metabolites. Indeed, the surface bacteria from S. domuncula, termed SB2 ({alpha}-proteobacterium MBIC 3368), were found to grow on inorganic medium supplemented with dihydroxybenzoate (protocatechuate). We subsequently isolated and sequenced the underlying gene cluster for the protocatechuate branch of the ß-ketoadipate pathway.

Since oxygen supply is crucial for sponge metabolism (20), we determined in a final set of experiments the abundance of bacteria on the surface of S. domuncula in relation to oxygen supply We found a close correlation between the level of expression of tyrosinase and {delta}-aminolevulinic acid dehydratase (ALAD), an enzyme which catalyzes the synthesis of porphobilinogen from two molecules of {delta}-aminolevulinic acid (25), and the oxygen supply to the sponges. The expression of both enzymes was strongly downregulated in animals kept at low oxygen; in parallel, the enzyme activity of tyrosinase was reduced too. Eventually, animals living under low oxygen conditions almost completely lost the fauna of SB2 bacteria on their surfaces. These data suggest that the SB2 bacteria grow on dihydroxylated aromatic compounds produced very likely by the sponge host.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and enzymes.
The sources of chemicals and enzymes used were given previously (28, 30, 64). All other chemicals were obtained from Fa. Karl Roth GmbH (Karlsruhe, Germany) or Roche (Mannheim, Germany).

Sponges.
Live specimens of S. domuncula (Porifera, Demospongiae, Hadromerida) were collected by scuba diving near Rovinj (Croatia) from depths between 15 and 35 m. The sponges were brought to Mainz (Germany) in seawater (at 17 to 20°C) and kept there in 103-liter tanks at 17°C before use in the experiments. Under normal conditions, the animals are kept in the aquarium under controlled aeration; air is added to the aquarium water through a fleece filter compartment (34). The quality of the marine water is controlled, e.g. for ammonia content. The sponges were kept in aquaria in Mainz for more than 4 months prior to their use (34).

In one series of experiments the animals were kept in a 5-liter beaker, filled with 4 liters of seawater, without aeration for 1 to 3 days. Tissue samples (1 cm3) were taken from the exopinacoderm region of the specimens and used for (i) determination of tyrosinase activity, (ii) determination of the expression levels of three selected genes, or (iii) PCR analysis to monitor the abundance of the SB2 bacteria. Previously this strain was found to be a member of the {alpha}-proteobacteria (57). The sponge specimens were subsequently transferred back to the aerated aquarium and kept for three more days.

Determination of tyrosinase.
Tissue from control or treated animals, ablated from the exopinacoderm area (except the osculum region), was extracted with 3 volumes of K-phosphate buffer (pH 6.5; supplemented with 0.1 M NaCl). The homogenate was centrifuged (20,000 x g, 10 min, 4°C), and the supernatant was used for the experiments. Aliquots were tested for enzymatic activity in the phosphate buffer as described previously (14), using 1 mM (final concentration) L-tyrosine as the substrate. Oxidation of tyrosine was followed at 280 nm, and the conversion from tyrosine to L-DOPA [3,4-(dihydroxyphenyl)alanine] was quantitated using an {varepsilon} for this conversion of 1,440 liters M–1 cm–1. One unit of activity was defined as a {Delta}E of 0.001/min (pH 6.5; 35°C) and correlated with the amount of protein present in the assay. The protein concentration was determined by application of the Micro BCA protein assay (Pierce, Rockford, Ill.).

Four sponge specimens were used for the experiments. Tissue samples of 0.1 to 0.3 g were cut off at day 0. The specimens were then transferred into seawater without aeration for 1 to 3 days; after this time the same specimens were kept under optimal aeration for an additional 3 days (total incubation period of 6 days).

S. domuncula tyrosinase-related protein cDNA.
The complete sponge cDNA, encoding the tyrosinase-related protein (TYRP_SUBDO), termed SDTYRP, was isolated from the S. domuncula cDNA library (30) by PCR (2). A degenerate forward primer directed against the characteristic amino acid motif found within the putative copper-binding site B in mammalian tyrosinases (33) was used in conjunction with the 3'-end vector-specific primer 5'-AAC/T-GAC/T-CCI-ATA/C/T-TTC/T-TTA/G-CTI-CAC/T-CAC/T-3' (I = inosine). This motif is located within human tyrosinase (NP_000363; 35) from amino acid 382 (aa382) to aa390. The PCR was carried out with an initial denaturation at 95°C for 3 min, followed by 30 amplification cycles at 95°C for 30 s, 60°C for 45 s, and 74°C for 1.5 min and a final extension step at 62°C for 10 min. The reaction mixture was as described earlier (63). A fragment of ~500 bp was used to isolate the cDNA from the library (2). The clone, SDTYRP, was sequenced using an automatic DNA sequenator (Li-Cor 4200).

S. domuncula ALAD cDNA.
In a recent study a genomic contig including the allograft inflammatory factor gene was identified and isolated from a genomic library of S. domuncula (7). Genomic fragments (~200 to 600 bp) were used to screen for the cDNA of the ALAD in the S. domuncula library, following described protocols (2, 62). The 1,246-nucleotide (nt) cDNA (SDALAD) was isolated and sequenced (34).

Sequence analysis.
Sequences were analyzed using the computer programs BLAST (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) and FASTA (http://www.ncbi.nlm.nih.gov/BLAST/fasta.html). Multiple alignments were performed with CLUSTAL W version 1.6 (58). Phylogenetic trees were constructed on the basis of amino acid sequence alignments by neighbor joining, as implemented in the Neighbor program from the PHYLIP package (19). The distance matrices were calculated using the Dayhoff PAM matrix model as described previously (11). The degree of support for internal branches was further assessed by bootstrapping (19). The graphic presentation was prepared with GeneDoc, version 1.1.004 (K. B. Nicholas and H. B. Nicholas, Jr. [http://www.psc.edu/biomed/genedoc/gddl.htm]).

Northern blotting.
For this series of experiments the animals were kept with or without oxygen, as indicated. RNA was extracted from liquid nitrogen-pulverized sponge tissue with TRIzol reagent (Gibco BRL, Grand Island, N.Y.). Then 5 µg of total RNA was electrophoresed through a 1% formaldehyde-agarose gel and blotted onto a Hybond-N+ nylon membrane following the manufacturer's instructions (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) (63). Hybridization was performed with ~0.5-kb parts of the SDTYRP, SDALAD, or SDTUB cDNA. The cDNA of the housekeeping gene ß-tubulin, SDTUB, of S. domuncula was used as an internal standard. The probes were labeled with the PCR DIG (digoxigenin) probe synthesis kit according to the manufacturer's instructions (Roche). After a washing, DIG-labeled nucleic acid was detected with anti-DIG Fab fragments (conjugated to alkaline phosphatase; dilution of 1:10,000) and visualized by a chemiluminescence technique using CDP, the chemiluminescence substrate alkaline phosphatase, according to the instructions of the manufacturer (Roche) as described previously (55).

Isolation, culture, and growth studies of sponge-associated bacterium.
The bacterium SB2 was isolated from the sponge (S. domuncula) surface and cultured as described before (57). Initially, these bacteria were cultured on B1 medium (0.25% peptone, 0.15% yeast extract, 0.15% glycerol, 1.6% agar in seawater). The growth (at 21°C) of this bacterium was investigated in M9 minimal medium (47) supplemented with either 4 mM Na-benzoate, Na-protocatechuate, or glucose. An overnight liquid culture of SB2 was used to inoculate (1:10) M9 minimal medium with different carbon sources. The optical densities at 600 nm of bacterial cultures were recorded at different time intervals up to 48 h. The growth curve was plotted.

The phylogenetic identification of the SB2 isolate, based on rDNA sequencing, was performed as described previously (23). The sequences obtained were aligned using the ABI Prism Auto assembler version 2.1 software (Perkin-Elmer, Foster City, Calif.) and entered into the BLAST (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) and ARB 16S rDNA sequence (www.arb.home.de) databases.

In the studies to determine the expression of the protocatechuate gene cluster, the SB2 bacterium was grown for 3 h at room temperature (25 to 27°C) with shaking. The culture consisted of either 4 mM benzoate, 4 mM Na-protocatechuate, or 10 mM glucose. For the real-time quantitative PCR (Q-PCR) experiments the SB2 bacteria were grown in a peptone-containing medium (see above) until an optical density at 600 nm of 0.5 U was reached. Then the cells were collected by centrifugation and resuspended in 7 ml of M9 minimal medium supplemented with the respective carbon source. After 3 h of shaking the RNA was isolated from 2 ml of the culture.

Bacterial extract and testing for bioactivity.
The organic extract was prepared as follows. The bacterial isolate (SB2) was inoculated into a conical flask (1-liter capacity) with 500 ml of culture broth: peptone (2.5 g), K2HPO4 (0.1 g), yeast extract (1.25 g), glucose (0.5 g), MgSO4 (0.1 g), seawater (250 ml), and distilled water (250 ml) (the pH was adjusted to 7.2 to 7.5) (15). The flask was placed onto a shaker (100 rpm, 30°C, 3 days). After addition of 150 ml of n-butanol, the mixture was kept at 40°C for 24 h, stirred for 20 min, and centrifuged and the butanol layer was evaporated. Dry residue was stored below 5°C until further use.

Antibacterial and antifungal activities were tested by using the standard paper disk diffusion method against gram-positive and gram-negative bacteria and yeasts (57). A bacterial extract (1 mg/disk) was applied to sterile paper disks (6-mm diameter). The solvent was evaporated before the disks were placed onto agar plates that had been seeded with bacterial reference strains. The assay was carried out in triplicate. The diameters of the inhibition zones (diameter of inhibition zone minus diameter of disk) were measured in millimeters after incubation at 30°C for 24 h. Control disks (soaked with solvent only) were prepared in the same manner. Medium inhibition is defined as an inhibition zone of approximately 10 mm, and strong inhibition is an inhibition zone of 11 to 15 mm.

Cytotoxicity assay.
A bacterial extract was tested for cytotoxicity against human cervix HeLa S3 cells (ATCC [American Type Culture Collection, Manassas, Va.] CCL 2.2) and PC12 cells (ATCC CRL 1721) as described earlier (8). The PC12 cells were grown in Dulbecco's modified Eagle's medium (supplemented with 10% horse serum and 5% fetal calf serum), and HeLa cells were grown in RPMI (with 10% fetal calf serum). The cells were seeded into 96-well plates at a concentration of 1.4 x 104 cells cm–2 and incubated at 37°C. After incubation for 24 h the bacterial extract was added at different concentrations to the wells containing cells. The final volume in each well was 200 µl. Then the plates were incubated at 37°C for 72 h. Cell viability was determined using the MTT (methylthiazolyldiphenyl-tetrazolium bromide) colorimetric assay (52). The plates were read at 595 nm using an enzyme-linked immunosorbent assay plate reader (Bio-Rad 3550, equipped with the program NCIMR IIIB) after overnight incubation at 37°C. The 50% effective doses were determined by logit regression (46).

Isolation and identification of the protocatechuate (pca) gene cluster from SB2 bacteria.
The genomic library was constructed from SB2 in the CopyControl pCC1FOS cosmid vector, using the CopyControl fosmid library production kit (Epicentre, Madison, Wis.) following the manufacturer's instructions. Briefly, SB2 DNA was sheared mechanically by being passed through a yellow pipette tip, end repaired, and size fractionated by pulsed-field gel electrophoresis using a 1% low-melting-point agarose gel. For visualization, crystal violet (GeneCraft, Münster, Germany) was included in the gel and electrophoresis buffer. A 40-kb fraction was excised and recovered with GELase (Epicentre). Fractionated DNA was ligated into a predigested vector at room temperature for 2 h (Fast-Link ligation system; Epicentre) and packaged with MaxPlax Lambda Packaging Extract (Epicentre). An aliquot of packaging reaction mixtures was plated onto the EPI300 plating strain for library titer estimation. Library titer was estimated as 1 x 107 CFU/ml or 4 x 107 CFU/µg of DNA. To check the insert size, 10 clones were randomly isolated and digested with BamHI and EcoRI. Digests were resolved on a 15-cm-long 0.8% agarose-TAE gel. By this procedure the average insert size was determined to be 40 kb.

Plasmid and cosmid clones were routinely grown overnight in Luria-Bertani medium (pH 7.5) supplied with appropriate antibiotics at 37°C with vigorous shaking. For the induction of the CopyControl cosmid clones to high copy number, overnight cultures were inoculated (in a ratio of 1:5) into fresh Luria-Bertani medium supplemented with Induction Solution (Epicentre) and grown for an additional 5 h at 37°C. Plasmid and cosmid DNAs were isolated by using the High Pure plasmid isolation kit (Roche). The average yield was 5 µg of cosmid DNA per 1 ml of "induced" bacterial culture.

We are primarily interested in those "nonculturable" bacteria which comprise polyketide synthase (PKS) clusters; degenerate primers around the active site of ß-ketoacyl synthases (PROSITE identification number PDOC00529) were used to identify the PKS gene cluster(s) in the genome of SB2 (40). The fragment obtained (insert size, 739 bp) was used to screen the genomic library. The probe was labeled with the PCR DIG Labeling Mix Plus kit (Roche). Cosmid colonies were lifted on nylon filters (Duralon-UV membranes; Stratagene, La Jolla, Calif.) and hybridized according to the colony screening by hybridization procedure (2). Briefly, hybridization was done at 30°C in EasyHyb solution (Roche) overnight. The next day, filters were washed in 2x SSC (1x SSC is 15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS), followed by 1x SSC-0.1% SDS and then 0.5x SSC-0.1% SDS for 20 min at room temperature; finally the filters were washed with 0.25x SSC-0.1% SDS at 50°C for 30 min. The DIG-labeled probe was detected with an anti-DIG-alkaline phosphatase conjugate (Roche) by a colorimetric method according to the manufacturer's instructions. More then 20 colonies produced a strong signal; 12 of the colonies were selected for DNA isolation. Selected clones were digested with BamHI, EcoRI, and BamHI/EcoRI, resolved on 20-cm-long 1% agarose-TAE gels, and subjected to hybridization by Southern analysis (2) with the same probe (PKS probe, 739 bp) under the conditions described above. Sequencing was performed by application of the Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing kit with 7-deaza-dGTP (Amersham) and primers labeled with IRD-700 or IRD-800. Sequencing ladders were resolved in SequaGel XR gels (National Diagnostics, Atlanta, Ga.) on an automatic DNA sequenator (Li-Cor 4200). Initially, cosmid clones were directly sequenced from both ends. Selected clones were subcloned partially with EcoRI and BamHI into the pBluescript SK plasmid vector (Stratagene). Subclones were end sequenced. Most sequencing data were obtained from EZ::TN<KAN-2> insertion clones (Epicentre) which were prepared according to the manufacturer's instructions. These clones were sequenced bidirectionally. Gap regions were covered by sequencing with specific primers.

For data analysis, compilation of the sequence was done with the Lasergene software package (DNASTAR, Inc, Madison, Wis.). DNA and translated protein sequence homology searches were done primarily with BLASTX and PLASTP programs from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST/) and also from the ProDom website (http://protein.toulouse.inra.fr/prodom/2002.1/html/home.php). For genes and potential promoter prediction, we used the FGENESB-Pattern/Markov chain-based bacterial operon and gene prediction program from the SoftBerry website (Softberry, Inc., Mount Kisco, N.Y.).

A 20-kb PKS cluster was cloned and sequenced. To identify the regions upstream as well as downstream, more than 10 kb was additionally sequenced in each direction. The upstream region was found to comprise the gene cluster encoding the catabolic enzymes of the protocatechuate branch of the 3-oxoadipate pathway. The part of the sequence of the open reading frame (ORF) flanking dctP, encoding a protein similar to the periplasmic membrane protein DctP (Agrobacterium tumefaciens; EMBL/GenBank accession number AAG27615.1), and the PKS cluster comprises the protocatechuate cluster and is addressed here.

Real-time Q-PCR analysis of the protocatechuate gene cluster.
The technique of real-time Q-PCR was applied to assess the expression of the protocatechuate gene cluster as described previously (60). Cells were harvested from 2 ml of bacterial cultures by centrifugation (10,000 x g; 30 s), and RNA was isolated with TRIzol reagent (Invitrogen) following the manufacturer's instructions with one exception: TRIzol was preheated to 90°C. The RNA concentration was estimated by UV spectrophotometry.

For cDNA synthesis, 5 µg of RNA was treated with 1 U of RNase-free DNase (Roche) in 30 µl of reverse transcription buffer (50 mM Tris/HCl [pH 8.3], 30 mM KCl, 8 mM MgCl2) at 37°C for 15 min. Then EDTA was added to a final concentration of 5 mM and DNase was heat inactivated at 65°C for 10 min. The reaction volume was brought to 60 µl with reverse transcription buffer supplemented with 10 mM dithiothreitol, 0.5 mM deoxynucleoside triphosphates, a 1 µM concentration of random nanomers, and 4 U of RNase inhibitor (MB Enzymes, Münster, Germany); 50 U of MMLV-H reverse transcriptase (MB Enzymes) was added and incubated at 37°C for 45 min. After incubation, EDTA was added to a final concentration of 4 mM and the reverse transcriptase was heat inactivated at 65°C for 10 min.

For amplification of the pcaQ gene within this cluster, 2 µl of the cDNA was used as the template in at least three replicates. The following primers were used: forward primer, 5'-CTGCGAGGGGATGGTTCTTGTC-3'; and reverse primer, 5'-AGCCAGCCGTCACCCGTACTATTC-3'. The product size was 450 bp, and the annealing temperature was 65°C. In parallel, the pcaDC gene was amplified by using the forward primer 5'-TCGTTGGTTCTCATCGGGCTTTCA-3' and the reverse primer 5'-TACGCCGTGTCTGCATGCCTTGTT-3'; the product size was 401 bp, and the annealing temperature was 64°C. Real-time Q-PCR was carried out with an iCycler (Bio-Rad, München; Germany) as described previously (22). Each reaction mixture (volume of 25 µl) contained 1 µl of cDNA (starting with approximately 200 ng of reverse-transcribed total RNA in several dilutions), 12.5 µl of 2x SYBR Green PCR Master Mix (ABI, Werrington, United Kingdom), and primers at a final concentration of 330 nM. The PCR conditions were initial denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 20 s, annealing for 20 s, and extension at 72°C for 1 min; the annealing temperature was 65°C (64°C) during the first 5 cycles, 62°C during cycles 6 to 10, 60°C during cycles 11 to 20, and 58°C during cycles 21 to 40. The amount of fluorescence, correlating with the amount of product, was determined during the elongation phase within each amplification cycle. By this procedure the log phase of amplification could be detected. The iCycler software was used to calculate the relative fluorescence units and to define that cycle at which the sample attained the threshold level of fluorescence. A plot, the threshold cycle versus log10 copy number for the PCR standard and probes, was established. One reference cosmid, comprising the PKS cluster, was added as a standard. The relative number of transcripts in the samples was calculated with the iCycler software, applying the standard curve data. The expression levels of pcaQ and pcaDC in each series of experiments, using protocatechuate or Na-benzoate as a carbon source, are correlated with the transcript number measured in the medium containing glucose (this value was set to 100%).

Determination of the abundance of the SB2 bacteria on the surfaces of the animals.
To determine the number of bacteria growing on the surfaces of the sponge specimens semiquantitatively, tissue samples of 3-mm thickness from the surface were used to isolate DNA as described previously (40). Then the DNA samples were used in the PCR assay using the gene-specific primers from the pcaDC gene of the protocatechuate cluster. The PCR conditions were as described above using the forward primer 5'-TCGTTGGTTCTCATCGGGCTTTCA-3' and the reverse primer 5'-TACGCCGTGTCTGCATGCCTTGTT-3'.

Nucleotide sequence accession numbers.
The cDNA sequences have been deposited (EMBL/GenBank) for the tyrosinase-like protein from S. domuncula under the accession number AJ574915 and for the ALAD under accession number AJ575745. The protocatechuate gene cluster from the sponge-associated bacterium SB2, including the genes dctP, pcaD/pcaC, pcaDE, pcaH, pcaG, pcaI, pcaJ, pcaF, and pcaB, were deposited under accession number AJ577848.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tyrosinase activity in S. domuncula in relation to oxygen.
In order to assess whether the sponge S. domuncula contains tyrosinase, tissue was extracted and assayed for enzymatic activity. The results show that extracts from controls (specimens kept under optimal aeration) contain tyrosinase with a specific activity of 2.8 U/mg of protein. If the same specimens are transferred to containers with seawater but without aeration, the activity drops after 1 day to 0.9 U/mg and after 3 days to 0.05 U/mg. However, when transferred back to the seawater milieu under optimal aeration, the sponges again express an increasing tyrosinase activity: after one day, 0.6 U/mg, and after 6 days, 2.3 U/mg (Fig. 1).



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  		FIG. 1. Determination of tyrosinase activity in tissue from S. domuncula after incubation of the animals in the absence of aeration (minus) for 1 to 3 days or under optimal aeration in the aquarium for the following 3 days (days 4 to 6). Five animals were taken for the experiments, and tissue samples were taken from each specimen at the indicated time point. The enzyme activity was determined as described in Materials and Methods. Five parallel experiments were performed; the mean values and standard deviations are given. Con, control.

 
S. domuncula tyrosinase-related protein cDNA.
The complete cDNA encoding the sponge tyrosinase-related protein was obtained by the PCR cloning technique. As described in Materials and Methods a pair of degenerate primers was designed for the conserved putative copper-binding site B in mammalian tyrosinases. The cDNA, named SDTYRP, shows that the enzyme is a member of the tyrosinase family. The SDTYRP cDNA comprises 1,448 nt [without the poly(A) tail]; the size determined by Northern blotting was found to be 1.5 kb, indicating that the full-length cDNA was isolated. The ORF, ranging from nt 40 to 42 (nt40-42) to nt1813-1815 (stop), encodes a polypeptide of 591 aa. The calculated size of the protein is 65,501 Da; the polypeptide is termed TYRP_SUBDO (Fig. 2A). It is most similar to the vertebrate tyrosinase/monophenol oxidase (EC 1.14.18.1) family with an "expect value" (E) (degree of similarity between each pair of proteins after alignment) of e–58 (http://www.isrec.isb-sib.ch/cgi-bin/PFSCAN_form_parser). It should be mentioned that the instability index was computed to be 42, indicating that this protein is metabolically unstable (PC/GENE, Data Banks CD-ROM, release 14.0; IntelliGenetics, Inc., Mountain View, Calif.).



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  		FIG. 2. S. domuncula tyrosinase-related protein and ALAD. (A) The tyrosinase-related protein (TYRP_SUBDO) is aligned with the human tyrosinase (TYR_HUMAN; NP_000363) (32). Amino acids similar or identical between the two sequences are shaded in black. The amino acid residues characteristic for the two copper binding sites (COP-A and COP-B) are marked. The conserved cysteine (C), glycine (G), and proline (P) residues are indicated. (B) Phylogenetic tree constructed from the two tyrosinase sequences together with the prophenol oxidase A3 from D. melanogaster (PPOA3_DROME; BAB43866), the common central domain of tyrosinase from C. elegans (TYRP_CAEEL; NP_499836), the Vps70p hypothetical protein from S. cerevisiae (Vps70_YEAST; NP_012660), and the expressed protein At1g from A. thaliana (At1g_ARATH; NP_566814). After alignment the tree was built and rooted using the plant sequence as an outgroup. The scale bar indicates an evolutionary distance of 0.1 aa substitution per position in the sequence. (C) The sponge ALAD enzyme (ALAD_SUBDO) is compared with the same enzyme from humans (ALAD; NP_000022) (26), the D. melanogaster hypothetical protein CG10335-PA (CG10335_DROME; NP_648564), the only distantly related enzyme, IMP dehydrogenase from C. elegans (IMPDH_CAEEL; T32709), the chain A, Schiff-base complex of ALAD from S. cerevisiae (ALAD_YEAST; 1YLV_A) (16), and the porphobilinogen synthase (ALAD) from A. thaliana (ALAD_ARATH; NP_177132). A tree was constructed after the sequences were aligned; the plant sequence was used as the outgroup.

 
Comparing the sponge TYRP_SUBDO with the related proteins, two characteristic domains, the copper-binding sites A and B (33), can be delimited; in S. domuncula they range from aa213 to aa230 for copper-binding site A and aa409 to aa421 for copper-binding site B (Fig. 2A). Within these sequences the consensus patterns read H-x (4)-F-P-x-W-H-R-x (2)-M-x (3)-E (site A) and D-P-x-F-x (7)-H-H-x (3)-D (site B).

Phylogenetic analysis of S. domuncula tyrosinase-related protein.
The sponge tyrosinase-related protein is most similar to the human tyrosinase (32) with a similarity/identity score of 43%/27%. Less related are the two other metazoan sequences, from deuterostomians, compared here: the Drosophila melanogaster prophenol oxidase A3 (BAB43866), 24%/13%; and the Caenorhabditis elegans tyrosinase (NP_499836), 25%/11%. Only distantly related are the Vps70p hypothetical protein from Saccharomyces cerevisiae (NP_012660) and the expressed At1g protein from Arabidopsis thaliana (NP_566814) both with scores of <20%/<10%. After alignment the tree was built and rooted using the plant sequence as an outgroup. Based on this calculation it becomes evident that the sponge protein groups together with the human tyrosinase in one branch while the two other metazoan sequences form a separate one (Fig. 2B).

ALAD.
The nucleotide sequence SDALAD comprises one ORF which spans 329 amino acids. The deduced polypeptide, the putative ALAD, termed ALAD_SUBDO, has an Mr of 35,964 and displays the characteristic domains found in other proteins of this family, e.g. the ALAD active site and the putative metal-binding sites B and C; details are given elsewhere (34). A rooted phylogenetic tree was constructed (Fig. 2C) which shows that sponge ALAD shares highest sequence similarity with the human ALAD, with 80% similar amino acids. Lower is the sequence similarity of sponge ALAD to the related sequences from D. melanogaster and C. elegans and to the S. cerevisiae and A. thaliana proteins.

Level of expression of the tyrosinase and ALAD genes in S. domuncula tissue in relation to aeration.
Three cDNA probes were applied to determine their expression levels in S. domuncula tissue, cDNAs for the housekeeping gene ß-tubulin (transcript size, 1.6 kb), tyrosinase (1.5 kb), and ALAD (1.3 kb); all cDNAs had been isolated from S. domuncula. Under the conditions used all three genes were found to be highly expressed in S. domuncula tissue. The expression level of the ß-tubulin gene remained unchanged under different aeration conditions (Fig. 3). However, in the tissue of a specimen which lived for 3 days without additional aeration the steady-state levels of the tyrosinase and ALAD genes dropped drastically, being more pronounced for the ALAD gene. To determine if the reduction of the expression of the two genes, for tyrosinase and ALAD, is reversible, the same animal was transferred back to the aquarium and was kept again under optimal aeration conditions. The Northern blot studies showed that the steady-state expression level of the two genes reverted to normal after 3 days (Fig. 3). Three parallel experiments with different specimens were performed; they were all found to respond to the same extent (results of the Northern blot experiments for the two last specimens are not shown here).



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  		FIG. 3. Expression of tyrosinase and ALAD gene tissue from S. domuncula (Northern blotting). RNA was extracted from control animals which had been kept under optimal aeration in the aquarium (C), from animals which remained for 3 days in the absence of additional aeration in the beaker (day 1-3; –O2), or from the same specimens which were subsequently transferred back to the aerated aquarium for three more days (day 4-6; +O2). RNA was extracted, and 5 µg of total RNA per slot was size separated; after blot transfer hybridization was performed either with the tyrosinase (TYRP), ALAD (ALAD), or ß-tubulin (TUB) probe, all isolated from S. domuncula.

 
The S. domuncula surface-associated bacterium SB2.
The growth of the SB2 bacterium was studied in M9 minimal medium in the presence of 4 mM Na-benzoate, 4 mM Na-protocatechuate, or 10 mM glucose. As expected, the bacterium did not grow in the absence of any carbon source and it showed slight growth in the presence of Na-benzoate. However, this bacterium grew well by utilizing Na-protocatechuate or glucose as a carbon source and reached a stationary phase during 12 to 24 h. Interestingly, Na-protocatechuate was even more supportive for the growth of this bacterium than glucose (Fig. 4).



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  		FIG. 4. Growth curve of the sponge surface-associated bacterium SB2 in M9 minimal medium supplemented with 4 mM protocatechuate (Pca), 10 mM glucose (Glc), or 4 mM Na-benzoate (Ben); exponential phase, 3 to12 h; stationary phase, 12 to 24 h; decline, after 24 h. OD600, optical density at 600 nm.

 
The bacterium SB2 was identified following the rDNA sequencing approach. The 398-nt 16S rDNA sequence is 98.5% similar to a sequence from the {alpha}-Proteobacterium MBIC3368 (gene for 16S rRNA, partial sequence; GenBank accession number AF 218241).

Production of bioactive compounds by the surface-associated bacterium SB2.
Antibacterial activity was tested by using the paper disk assay as described in Materials and Methods. An amount of 1 mg of bacterial extract per disk was applied and transferred onto the agar surface covered with both gram-positive (Staphylococcus) and gram-negative (Escherichia coli) strains. The results show that only weak inhibition was displayed by the extract in the assays with Staphylococcus aureus WT, S. aureus 118, and S. aureus A134 while a strong inhibition was measured in the assays with S. epidermidis 40. The extract was inactive against E. coli DH5{alpha}. The cytostatic activity of the extract was tested against two tumor cell lines, HeLa S3 and PC12 cells. The growth inhibitory activity of the extract was low, as can be deduced from the ED50 values (HeLa S3 cells, >100 µg/ml; PC12 cells, 25 µg/ml).

The protocatechuate (pca) gene cluster from the bacterium SB2.
In our approach to elucidate the PKS clusters in symbiotic bacteria from sponges the genome of the bacterium SB2 was analyzed. PKS sequences were found and sequenced, as described in Materials and Methods.

However, upstream from the PKS cluster we found a 13,000-bp DNA operon, the protocatechuate (pca) gene cluster, and isolated it from the bacterium SB2. The ORFs were predicted by applying the programs listed in Materials and Methods. The general orientation of the predicted genes is directed by those downstream from pcaDC. A schematic outline of the organization of the gene cluster is given in Fig. 5.



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  		FIG. 5. Structure of the gene cluster encoding the protocatechuate enzymes, which catabolize protocatechuate, isolated from the bacterium SB2. The gene arrangement is given above. The borders of the genes are schematically presented below; the numbering of the nucleotides refer to the ORF for the respective deduced polypeptide (the size in number of amino acids is given within the boxes). The arrows indicate the direction of transcription. The classification of the deduced enzymes are taken from reference 9. The regions within the cluster which had been selected for the Q-PCR are indicated.

 
The first ORF, flanking the pca contig, spans nt194 to nt1225 and encodes 344 aa (transcription in opposite direction). The product shows highest similarity (48%) to the periplasmic membrane protein DctP (A. tumefaciens). The following ORF, likewise in the opposite direction, encodes the putative transcriptional activator of the pca operon; it spans nt1474 to nt2412 and encodes 313 aa, sharing 70% similarity with the LysR family protein (Sinorhizobium meliloti; GenBank accession number CAC49892.1); the gene is also termed pcaQ (9). The coding sequence for the potential oxoadipate enol-lactonase (PcaD) follows from nt2496 to nt3266; the product comprises 267 aa and has 66% similarity to the beta-ketoadipate enol-lactone hydrolase protein (S. meliloti; CAC49891.1). The coding sequence for PcaD is immediately flanked downstream by that for the putative PcaC, the 4-carboxymuconolactone decarboxylase, from nt3267 to nt3696. The deduced 143 aa have 55% similarity to the 3-oxoadipate enol-lactone hydrolase/4-carboxymuconolactone decarboxylase of Rhodococcus opacus (AAC38246.1). In gram-negative bacteria PcaC and PcaD are fused to one protein, PcaL (17). The PcaCD coding sequence is followed by that for the protocatechuate 3,4-dioxygenase beta-chain, nt3697 to nt4437. The 247-aa PcaH shares 81% similarity with the putative protocatechuate 3,4-dioxygenase beta-chain protein from S. meliloti (CAC49889.1). The adjacent sequence, ranging from nt4439 to nt5050, encodes the protocatechuate 3,4-dioxygenase alpha chain, comprising 612 aa (PcaG) and sharing 82% similarity with the protocatechuate 3,4-dioxygenase alpha chain from Mesorhizobium loti (BAB53362.1). Next, an ORF, nt5060 to nt5910, follows which putatively encodes the acetate CoA-transferase alpha subunit (PcaI), 88% similar to the acetoacetyl-CoA transferase beta subunit from M. loti (BAB50894.1). The interacting acetoacetyl-CoA transferase beta subunit (PcaJ) follows, encoded by nt5911 to nt6702. This deduced 264-aa protein shares 83% similarity with the putative CoA-transferase subunit B protein from Sinorhizobium meliloti (CAC49900.1). Then, the sequence for the putative beta-ketoadipyl CoA thiolase (PcaF) follows within the nt6702-to-nt7901 fragment; 86% of the amino acids are similar to those of the beta-ketoadipyl CoA thiolase from M. loti (BAB50893.1). Finally, an ORF follows from nt8137 to nt8973 which encodes the putative 3-carboxy-cis,cis-muconate cycloisomerase (PcaB), with 66% similarity to the 3-carboxy-cis,cis-muconate cycloisomerase from A. tumefaciens (AAK88905.1).

Expression of the protocatechuate (pca) gene cluster in relation to the carbon source.
The surface-associated bacteria were cultivated in M9 minimal salt medium supplemented with 4 mM Na-benzoate, 4 mM Na-protocatechuate, or 10 mM glucose. After reverse transcription, the expression of the protocatechuate cluster in the sponge surface-associated bacterium SB2 was determined by Q-PCR as described in Materials and Methods. Two genes were selected to determine the levels of expression, pcaQ, encoding the putative transcriptional activator, and pcaDC, encoding the decarboxylase oxoadipate enol-lactonase/beta-ketoadipate enol-lactone hydrolase.

As shown in Fig. 6, the upregulation of the expression of the gene encoding the activator, pcaQ, is twofold stronger in bacteria grown in Na-protocatechuate than in bacteria grown in glucose. In comparison, the level of expression of the gene encoding the enzyme, pcaDC, is sevenfold higher in Na-protocatechuate medium than in glucose medium.



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  		FIG. 6. Expression level of the protocatechuate cluster in the sponge surface-associated bacterium SB2. The level of expression of the cluster was determined by Q-PCR as described in Materials and Methods. RNA was from the bacterium cultured in M9 minimal salt medium supplemented with Na-benzoate (Ben), protocatechuate (Pca), or glucose (Glc). RNA was extracted and reverse transcribed, and the resulting levels of cDNA for two genes from the protocatechuate cluster, pcaQ (left) and pcaC (right), were determined. Two different dilutions of cDNA in the assays were analyzed, a 5-fold dilution (black bars) and a 25-fold dilution (gray bars). The level of expression is correlated with the level of the respective transcripts measured in bacteria grown in the presence of glucose.

 
Abundance of the SB2 bacteria on the surfaces of the animals.
The amount of SB2 bacteria on the surfaces of the sponge specimens kept for 3 days without aeration was assessed; subsequently the same specimens were maintained for three more days under optimal aeration, and the amount of SB2 bacteria was assessed again. Then DNA was extracted and PCR analyzed with gene-specific primers for the protocatechuate cluster, the pcaDC gene. The signal of the fragment, which can be readily amplified in DNA from control animals, was almost absent in DNA obtained from a specimen kept without aeration. However, if the same animal was transferred back to optimal aeration conditions, the signal was as high as that in the controls (data not shown).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The term symbiosis was introduced by De Bary (12), who defined this phenomenon as the "cohabitation of non-related organisms" that can be classified either as "antagonistic," destructive for each of the partners, or as "mutual," with shared benefits for the symbionts involved. The consequence of a mutual symbiosis is also the mutual dependence which is reflected by adaptation of the metabolisms of the two partners (12). The extreme form of a mutual symbiotic relationship is the dependence of one of the partners on metabolites which are formed by the second partner, which itself cannot utilize them. In other words, one partner is utilizing a final metabolite of the other to meet its needs for energy supply. The optimal strategy for metabolite supply in a symbiotic relationship would be an exchange of organic compounds which are toxic for nonwanted partners and beneficial only for the wanted symbiont. In a recent study we reported that the compound quinolinic acid is produced by the sponge S. domuncula during the process of apoptosis, thus attracting a eukaryotic epibiont, the mollusk Bittium sp., which removes the dying and rejected tissue; for other metazoans quinolinic acid is a neurotoxic agent (51). Now, we present experimental observations, based on molecular biological data, which indicate that the sponge host S. domuncula produces hydroxylated aromatic compounds which can be used by epibiotic bacteria as a carbon source.

In most organisms, including bacteria, aromatic compounds from the hydroxylated lignin or catechin/catechol/protocatechuate/shikimate class cannot be metabolized or are even toxic (42). Some of the compounds can however be degraded or metabolized by the two branches of the ß-ketoadipate pathway, forming metabolites which are used in the tricarboxylic acid cycle (54).

As outlined above (see reference 57), sponges use bacteria living either in the interior of the tissue, where some of them are even encapsulated in specific cells (5), or on the surface of the sponge host for the production of bioactive compounds. The establishment of a bacterial community on the surface of the sponge also provides the advantage that potential toxic products synthesized by them can be readily cleared and released into the surrounding water. To the best of our knowledge there has been, until now, no report which hints at the nature of an obligatory metabolite for sponge surface-associated bacteria which is produced by the sponge.

In the present study we investigated the growth of SB2 bacteria in M9 minimal medium supplemented with an organic carbon source like Na-benzoate or protocatechuate in comparison to that in medium supplemented with glucose. The results were interesting with the medium having Na-protocatechuate as a carbon source. As the genes involved in the degradation pathways of this aromatic compound were identified in SB2, it became easier to formulate the medium for its growth. This approach highlights the importance of molecular biological studies with potential bacteria in order to formulate the appropriate medium for their growth.

This investigation gains additional importance since the bacterium SB2 is a producer of bioactive compounds. Furthermore, this bacterium was found to be associated with several different sponges. Webster and Hill (61) reported that the {alpha}-Proteobacterium MBIC3368 strain NW001 dominates the culturable microbial community of the Australian sponge Rhopaloeides odorabile. The {alpha}-Proteobacterium MBIC3368 strain SB89 was also recovered from the Mediterranean sponge Aplysina aerophoba, which displays antimicrobial activity against various gram-positive and gram-negative bacteria (23).

The question of organic nutritional sources required by this bacterial strain to grow on the surface of the sponge can hardly be answered directly. It appears impossible to feed the sponge with radiolabeled precursors and follow their fate up to the bacterium, because sponges filter unusually large amounts of water through their canal system; in some sponges the filtration rate reaches 0.002 to 0.84 cm3 per s per cm3 of sponge tissue (41). An alternative approach is to expose the sponge to different environmental conditions and to determine the production of potential metabolites for the surface-associated bacteria. One enzyme of the sponge S. domuncula, which is described here on cDNA level, is the tyrosinase; it converts monophenolic compounds to diphenols (13). The cDNA of the S. domuncula tyrosinase was cloned, and it was found to be highly similar to the mammalian tyrosinases.

The activity of tyrosinase in S. domuncula strongly depends on the cultivation condition of the sponge. Under optimal aeration conditions the specific expression of this enzyme is high, and it undergoes drastic reduction under low oxygen supply. This effect is reversible. To confirm this change on the gene transcription level, Northern blot studies were conducted. It was found that the steady-state expression level drops to almost zero after the transfer of the animals to a nonaerated environment; again this change is reversible. In parallel with the changes in tyrosinase gene expression, the expression of a second gene involved in oxygen supply/consumption responds to aeration. One key enzyme in the synthesis of heme is ALAD. This enzyme forms porphobilinogen from {delta}-aminolevulinic acid (25) in the synthesis of the oxygen-transporting porphyrins (45). The induction of gene expression was recently shown to be highly dependent on oxygen supply in primmorphs, a specific cell culture system in sponges (34). Based on these observations with the two genes, encoding oxygen-metabolizing (tyrosinase) and transporting (ALAD) enzymes, combined with the finding that the expression of the housekeeping gene remains unchanged, it must be concluded that oxygen plays a crucial role in the control of expression of ALAD as well as of tyrosinase.

In a subsequent series of experiments, a gene cluster from the bacterium SB2 was cloned and sequenced which codes for the enzymes of the protocatechuate cluster. These enzymes convert protocatechuate to succinyl-CoA and acetyl-CoA (9). The cluster contains, besides the gene for the regulator/activator of this cluster/pathway, pcaQ, also the genes for dioxygenase (pcaH and pcaG), cycloisomerase (pcaB), decarboxylase (pcaC), hydrolase (pcaD), transferase (pcaI and pcaJ), and finally the thiolase (pcaF). The arrangement of the genes within this cluster varies among different bacterial taxa (see reference 9). The arrangement of the protocatechuate cluster found in SB2 is most similar to that described for A. tumefaciens.

We demonstrated that the metabolite protocatechuate, formed by the sponge tyrosinase, causes a strong upregulation of the transcription of the protocatechuate cluster in the SB2 bacterium. The increase is even stronger than the one seen for glucose. Almost no induction is observed for the aromatic compound benzoate. The positive correlation between expression of the tyrosinase gene and the activity of tyrosinase with the abundance of this bacterium on the surface of the sponge can be considered the first compelling experimental evidence that the regulation of the bacterial fauna in sponges is controlled by oxygen. As SB2 is found to be associated with different sponge species, future studies will investigate the biochemical basis of the specificity of this association between bacteria and sponges.

The understanding of the molecular basis of the symbiotic relationship between sponges and their associated microorganisms is crucial for the optimization of the culture conditions for symbiotic bacteria; most of them are considered to be nonculturable in free media. This knowledge will be a prerequisite for the successful exploitation of resources, which are considered to be most diverse with respect to the spectrum of bioactive secondary metabolites. The principle of the symbiotic relationship, the production of diphenols via the tyrosinase of sponges, and their (potential) transfer to the surface-associated bacteria will contribute to the further elucidation of other pathways likewise involved in the sponge-microorganism relationship. Our EST (expressed sequence tags) database for the sponge S. domuncula comprises more than 15,000 sequences, which will help to elucidate those pathways.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Mü 348/14), the Bundesministerium für Bildung und Forschung (project: Center of Excellence BIOTECmarin), the European Commission (project: SPONGE), and the International Human Frontier Science Program (RG-333/96-M).


   FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität Mainz, Duesbergweg 6, 55099 Mainz, Germany. Phone: 6131-3925910. Fax: 6131-3925243. E-mail: wmueller{at}mail.uni-mainz.de. Back

{dagger} This paper is dedicated to Prof. Axel Zeeck (Göttingen, Germany) on the occasion of his 65th birthday. Back


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, April 2004, p. 2332-2341, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2332-2341.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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