Nonribosomal peptide synthetase (NRPS) adenylation (A) domain genes were investigated by polymerase chain reaction for 109 bacteria isolated from four South China Sea sponges, Stelletta tenuis, Halichondria rugosa, Dysidea avara, and Craniella australiensis. Meanwhile, the antimicrobial bioassay of bacteria with NRPS genes were carried out to confirm the screening of NRPS genes. Fifteen bacteria were found to contain NRPS genes and grouped into two phyla Firmicutes (13 of 15) and Proteobacteria (two of 15) according to 16S rDNA sequences. Based on the phylogenetic analysis of the conserved A domain amino acid sequences, most of the NRPS fragments (11 of 15) showed below 70% similarity to their closest relatives suggesting the novelty of these NRPS genes. All of the 15 bacteria with NRPS genes have antimicrobial activities, with most of them exhibiting activity against multiple indicators including fungi and gram-positive and gramnegative bacteria. The different antimicrobial spectra indicate the chemical diversity of biologically active metabolites of sponge-associated bacteria and the possible role of bacterial symbionts in the host's antimicrobial chemical defense. Phylogenetic analysis based on the representative NRPS genes shows high diversity of marine NRPS genes. The combined molecular technique and bioassay strategy will be useful to obtain sponge-associated bacteria with the potential to synthesize bioactive compounds.

The gene cloning, purification, properties, kinetics, and antifungal activity of chitinase from marine Streptomyces sp. DA11 associated with South China sponge Craniella australiensis were investigated. Alignment analysis of the amino acid sequence deduced from the cloned conserved 451 bp DNA sequence shows the chitinase belongs to ChiC type with 80% similarity to chitinase C precursor from Streptomyces peucetius. Through purification by 80% ammonium sulfate, affinity binding to chitin and diethylaminoethyl-cellulose anion-exchange chromatography, 6.15-fold total purification with a specific activity of 2.95 Umg-1 was achieved. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a molecular weight of approximately 34 kDa and antifungal activities were observed against Aspergillus niger and Candida albicans. The optimal pH, temperature, and salinity for chitinase activity were 8.0, 50℃, and 45g‰ psu, respectively, which may contribute to special application of this marine microbe-derived chitinase compared with terrestrial chitinases. The chitinase activity was increased by Mn2+, Cu2+, and Mg2+, while strongly inhibited by Fe2+ and Ba2+. Meanwhile, SDS, ethyleneglycoltetraacetic acid, urea, and ethylenediaminetetraacetic acid were found to have significantly inhibitory effect on chitinase activity. With colloidal chitin as substrates instead of powder chitin, higher Vmax (0.82 mg product/min•mg protein) and lower Km (0.019 mg/ml) values were achieved. The sponge’s microbial symbiont with chitinase activity may contribute to chitin degradation and antifungal defense. To our knowledge, it was the first time to study sponge-associated microbial chitinase.

Marine animals and plants such as sponges, sea squirts, corals, worms and algae host diverse and abundant symbiotic microorganisms. Marine microbial symbionts are possible the true producers or take part in the biosynthesis of some bioactive marine natural products isolated from the marine organism hosts. Investigation of the pharmaceutical metabolites may reveal the biosynthesis mechanisms of related natural products and solve the current problem of supply limitation in marine drug development. This paper reviews the advances in diversity revelation, biological activity and related pharmaceutical metabolites, and functional genes of marine microbial symbionts from the China Sea.

In this paper, statistical Plackett-Burman design and Box-Behnken Response Surface Methodology were applied to optimize the medium components to improve the chitinase activity of Streptomyces sp. DA11 associated with South China Sea sponge Craniella australiensis. Firstly, galactose and peptone were found to be the suitable carbon and nitrogen sources for the growth and chitinase activity by single factor Seriatim-Factorial test. Secondly, galactose, colloidal chitin and MgSO4 7H2O were proved to have remarkable effects on chitinase activity. Finally, an optimal medium was obtained by Box-Behnken methodology, which consisted of 5.00g/L galactose, 2.62g/L colloidal chitin, 0.10g/L MgSO4 7H2O and 12.5g/L peptone, 1.5g/L PO4 3 (KH2PO4 0.45g/L, K2HPO4 1.05g/L), 12.5g/L powder chitin, 0.03g/L FeSO4 and 0.03g/L ZnSO4 7H2O with artificial sea water (ASW). With this optimal medium, both the chitinase activity and cell growth were remarkably enhanced. The chitinase activity of 1559.2U/g cell dry weight (36.43U/mL) and the maximum cell dry weight of 23.3g/L were reached after incubation of 72 h, which were 39.2-fold and 2.6-fold higher than that of the basic medium, respectively. The results suggest that Streptomyces sp. DA11 isolated from marine sponge might be a potential strain for the production of chitinolytic enzymes. To our knowledge, this is the first report of the statistical optimization of chitinase production from marine sponge-associated Streptomyces.

Culture-independent 16S rDNA-DGGE fingerprinting and phylogenetic analysis were used to reveal the community structure and diversity of the predominant bacteria associated with the four sponges Stelletta tenui, Halichrondria, Dysidea avara, and Craniella australiensis from the South China Sea for the first time. Sponge total community DNA extracted with a direct grinding disruption based method was used successfully after series dilution for 16S rDNA PCR amplification, which simplifies the current procedure and results in good DGGE banding profiles. 16S rDNA-V3 fragments from 42 individual DGGE bands were sequenced and the detailed corresponding bacteria were found in sponges for the fist time based on BLAST results. The sponge-associated bacteria are sponge host-specific because each of the tested four sponges from the same geographical location has different predominant bacterial diversity. Proteobacteria, e.g. a, h and g subdivisions, make up the majority of the predominant bacteria in sponges and are perhaps in close symbiotic relationship with sponges. Though similar bacteria with close phylogenetic relationships were found among different sponges, the sponge-associated predominant bacterial community structures differ. Sponge C. australiensis has the greatest bacterial diversity, with the four bacteria phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria, followed by the sponge D. avara with the two phyla Proteobacteria and Bacteroidetes, and the sponges S. tenui and Halichrondria with the phylum Proteobacteria. DGGE fingerprint-based analysis should ideally be integrated with band cloning and sequencing, phylogenetic analysis and molecular techniques to obtain precise results in terms of the microbial community and diversity.