Characterization of Extracellular Protease from the Haloarcheon Halococcus sp. Strain GUGFAWS-3 (MF425611).

Halococcus agarilyticus GUGFAWS-3 (MF425611) was isolated from a marine white sponge of Haliclona sp., inhabiting the rocks in the intertidal region of Anjuna, Goa, India. Uniquely, the microbe simultaneously produces two halo-extremozymes in 25% NaCl, namely protease and lipase at 49.5 ± 0.4 and 3.67 ± 0.02 (U mL-1), respectively. The protease is constitutively produced in starch mineral salts medium with consistent 4 ± 1.0 mm zone of enzyme production, regardless of the non-availability of protein as substrate. The ethanol precipitated enzyme on dialysis and Sephadex G-200 gel filtration chromatography was partially purified to 12.26-fold and was active between 20 and 80 °C, 0-5 M NaCl, and pH 3-13. Optimum activity, however, was at 70 °C, 3 M NaCl, and pH 7. The enzyme was thermo stable at 70 °C with 50.26 ± 2.40% of relative enzyme activity at 75 min. Furthermore, it was stable in the presence of polar and non-polar organic solvents, detergents, and hydrocarbons. Several metal cations enhanced its activity in the order of Ca2+ > Ni2+ > Fe3+ > Co2+ > Mg2+ > Cu2+ > Mn2+. Dependence of enzyme on cysteine; serine, and metal ions was confirmed by ß-mercaptoethanol; PMSF and EDTA, respectively which induced its partial inhibition. Additionally, protease inhibited in vitro biofilm formation in Staphylococcus aureus. Conclusively, the production of a neutral halo-thermophilic protease is reported for the first time in the genus Halococcus.

Haloferax sulfurifontis GUMFAZ2 producing xylanase-free cellulase retrieved from Haliclona sp. inhabiting rocky shore of Anjuna, Goa-India.

Salt stable cellulases are implicated in detritic food webs of marine invertebrates for their role in the degradation of cellulosic material. A haloarchaeon, Haloferax sulfurifontis GUMFAZ2 producing cellulase was successfully isolated from marine Haliclona sp., a sponge inhabiting the rocky intertidal region of Anjuna, Goa. The culture produced extracellular xylanase-free cellulase with a maximum activity of 11.7 U/ml, using carboxymethylcellulose-Na (CMC-Na), as a sole source of carbon in 3.5 M NaCl containing medium, pH 7 at 40°C and produced cellobiose and glucose, detectable by thin-layer chromatography. Nondenaturing polyacrylamide gel electrophoresis of the crude enzyme, revealed a single protein band of 19.6 kDa which on zymographic analysis exhibited cellulase activity while corresponding sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a molecular weight of 46 kDa. Unlike conventional cellulases, this enzyme is active in presence of 5 M NaCl and does not have accompanying xylanase activity, hence can be considered as xylanase-free cellulase. Such enzymes from haloarchaea offer great potential for biotechnological application because of their stability at high salinity and is therefore worth pursuing.

Localized production of defence chemicals by intracellular symbionts of Haliclona sponges.

Marine sponges often house small-molecule-producing symbionts extracellularly in their mesohyl, providing the host with a means of chemical defence against predation and microbial infection. Here, we report an intriguing case of chemically mediated symbiosis between the renieramycin-containing sponge Haliclona sp. and its herein discovered renieramycin-producing symbiont Candidatus Endohaliclona renieramycinifaciens. Remarkably, Ca. E. renieramycinifaciens has undergone extreme genome reduction where it has lost almost all necessary elements for free living while maintaining a complex, multi-copy plasmid-encoded biosynthetic gene cluster for renieramycin biosynthesis. In return, the sponge houses Ca. E. renieramycinifaciens in previously uncharacterized cellular reservoirs (chemobacteriocytes), where it can acquire nutrients from the host and avoid bacterial competition. This relationship is highly specific to a single clade of Haliclona sponges. Our study reveals intracellular symbionts as an understudied source for defence chemicals in the oldest-living metazoans and paves the way towards discovering similar systems in other marine sponges.

Stable and Enriched Cenarchaeum symbiosum and Uncultured Betaproteobacteria HF1 in the Microbiome of the Mediterranean Sponge Haliclona fulva (Demospongiae: Haplosclerida).

Sponges harbor characteristic microbiomes derived from symbiotic relationships shaping their lifestyle and survival. Haliclona fulva is encrusting marine sponge species dwelling in coralligenous accretions or semidark caves of the Mediterranean Sea and the near Atlantic Ocean. In this work, we characterized the abundance and core microbial community composition found in specimens of H. fulva by means of electron microscopy and 16S amplicon Illumina sequencing. We provide evidence of its low microbial abundance (LMA) nature. We found that the H. fulva core microbiome is dominated by sequences belonging to the orders Nitrosomonadales and Cenarchaeales. Seventy percent of the reads assigned to these phylotypes grouped in a very small number of high-frequency operational taxonomic units, representing niche-specific species Cenarchaeum symbiosum and uncultured Betaproteobacteria HF1, a new eubacterial ribotype variant found in H. fulva. The microbial composition of H. fulva is quite distinct from those reported in sponge species of the same Haliclona genus. We also detected evidence of an excretion/capturing loop between these abundant microorganisms and planktonic microbes by analyzing shifts in seawater planktonic microbial content exposed to healthy sponge specimens maintained in aquaria. Our results suggest that horizontal transmission is very likely the main mechanism for symbionts' acquisition by H. fulva. So far, this is the first shallow water sponge species harboring such a specific and predominant assemblage composed of these eubacterial and archaeal ribotypes. Our data suggests that this symbiotic relationship is very stable over time, indicating that the identified core microbial symbionts may play key roles in the holobiont functioning.

Parendozoicomonas haliclonae gen. nov. sp. nov. isolated from a marine sponge of the genus Haliclona and description of the family Endozoicomonadaceae fam. nov. comprising the genera Endozoicomonas, Parendozoicomonas, and Kistimonas.

Two Gram-stain-negative, facultative anaerobic, motile, rod-shaped strains, S-B4-1UT and JOB-63a, forming small whitish transparent colonies on marine agar, were isolated from a sponge of the genus Haliclona. The strains shared 99.7% 16S rRNA gene sequence identity and a DNA-DNA hybridization value of 100%, but were differentiated by genomic fingerprinting using rep-PCRs. 16S rRNA gene sequence phylogeny placed the strains as a sister branch to the monophyletic genus Endozoicomonas (Oceanospirillales; Gammaproteobacteria) with 92.3-94.3% 16S rRNA gene sequence similarity to Endozoicomonas spp., 91.9 and 92.1% to Candidatus Endonucleobacter bathymodiolin, and 91.9 to 92.1% to the type strains of Kistimonas spp. Core genome based phylogeny of strain S-B4-1UT confirmed the phylogenetic placement. Major fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c) and 8 (C18:1 ω7c/C18:1 ω6c) followed by C10:0 3-OH, C16:0, and C18:0. The G+C content was 50.1-51.4mol%. The peptidoglycan diamino acid of strain S-B4-1UT was meso-diaminopimelic acid, the predominant polyamine spermidine, the major respiratory quinone ubiquinone Q-9; phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine were major polar lipids. Based on the clear phylogenetic distinction, the genus Parendozoicomonas gen. nov. is proposed, with Parendozoicomonas haliclonae sp. nov. as type species and strain S-B4-1UT (=CCM 8713T=DSM 103671T=LMG 29769T) as type strain and JOB-63a as a second strain of the species. Based on the 16S rRNA gene sequence phylogeny of the Oceanospirillales within the Gammaproteobacteria, the Endozoicomonaceae fam. nov. is proposed including the genera Endozoicomonas, Parendozoicomonas, and Kistimonas as well as the Candidatus genus Endonucleobacter.

Proposal of Litorimonas haliclonae sp. nov., isolated from a marine sponge of the genus Haliclona.

A bright-orange-pigmented, Gram-stain-negative, motile, and rod-shaped bacterium, strain MAA42T, was isolated from a marine sponge of the genus Haliclona, which is in long-time culture in a marine aquarium system at the Justus Liebig University Giessen, Germany. The strain grew at 4-34 °C (optimum 28 °C), in the presence of 0.5-9.5 % (w/v) NaCl (optimum 3.5 %) and at pH 4.5-10.0 (optimum pH 7.5). Strain MAA42T shared the highest 16S rRNA gene sequence similarity (98.1 %) with the type strain of Litorimonas taeanensis. Sequence similarities to all other closely related type strains were below 97 %. DNA-DNA hybridization of strain MAA42T with L. taeanensis DSM 22008T resulted in values of 4.7 % (reciprocal 17.7 %). Major cellular fatty acids of strain MAA42T were C18 : 1ω7c (66.2 %), C18 : 1 2-OH (17.4 %), and C18 : 0 (14.1 %). Spermidine was predominant in the polyamine pattern, and ubiquinone Q-10 was the major respiratory quinone. The polar lipid profile contained the major compounds phosphatidylglycerol, monoglycosyldiglyceride, three unidentified phospholipids, and one unidentified glycolipid. Glucuronopyranosyldiglyceride was present as a minor compound. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The genomic DNA G+C content was 52.8 mol%. Based on the genotypic, chemotaxonomic, and phenotypic analyses, strain MAA42T represents a novel species of the genus Litorimonas, for which the name Litorimonas haliclonae is proposed. The type strain is MAA42T (=CCM 8709T=CIP 111178T=LMG 29765T).

Winogradskyella haliclonae sp. nov., isolated from a marine sponge of the genus Haliclona.

A yellow-pigmented, Gram-stain-negative, motile and rod-shaped bacterium, strain M1A16T, was isolated from the internal tissue of a sponge of the genus Haliclona, which was long-term cultured in the CEMarin aquaria system at Justus Liebig University of Giessen. The strain grew well at 20-32 °C (optimum 25 °C), in the presence of 0-6 % NaCl (optimum 3 %), and at pH 5.5-9.0 (optimum pH 7.0-8.0). Phylogenetic analysis based on its 16S rRNA gene sequence placed the strain within the monophyletic cluster of the genus Winogradskyella with highest sequence similarity to Winogradskyella jejuensis CP32T (98.3 % 16S rRNA gene sequence similarity). Sequence similarities to all other type strains were 98.0 % or less. DNA-DNA hybridization of strain M1A16T with W. jejuensis CP32T resulted in hybridization values of 44.1 % (reciprocal 68.1 %). Major cellular fatty acids of strain M1A16T were iso-C15 : 1 G (18.1 %), iso-C15 : 0 (13.7 %), C16 : 1ω7c (12.9 %), iso-C17 : 0 3-OH (10.6 %) and iso-C16 : 0 3-OH (10.2 %). The overall polyamine content was very low with major components being cadaverine, spermidine and sym-homospermidine. The major quinone was menaquinone MK-6. The polar lipid profile contained predominantly phosphatidylethanolamine, two unidentified aminolipids and two unidentified lipids devoid of a detectable functional group. The genomic DNA G+C content was 32.7 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic analyses, strain M1A16T represents a novel species of the genus Winogradskyella, for which the name Winogradskyella haliclonae sp. nov. is proposed. The type strain is M1A16T (=DSM 103138T=CCM 8681T=LMG 29588T=CIP 111091T).

Antibacterial activity and mutagenesis of sponge-associated Pseudomonas fluorescens H41.

Marine sponges (phylum Porifera) are well known to harbour a complex and diverse bacterial community. Some of these sponge-associated bacteria have been shown to be the real producers of secondary metabolites with a wide range of activities from antimicrobials to anticancer agents. Previously, we revealed that the strain Pseudomonas fluorescens H41 isolated from the sponge Haliclona sp. (collected at the coast of Rio de Janeiro, Brazil) showed a strong antimicrobial activity against clinical and marine bacteria. Thus, in this study the genes involved in the antimicrobial activity of P. fluorescens H41 were identified. To this end, a library of mutants was generated via miniTnphoA3 transposon mutagenesis and the resulting clones were characterized for their antimicrobial activity. It was demonstrated that genes involved in the biosynthesis of the pyoverdine siderophore are related to the inhibitory activity of P. fluorescens H41. Therefore, this strain might play an important role in the biocontrol of the host sponge.

Bacterial Diversity Associated with Cinachyra cavernosa and Haliclona pigmentifera, Cohabiting Sponges in the Coral Reef Ecosystem of Gulf of Mannar, Southeast Coast of India.

Sponges are abundant, diverse and functionally important organisms of coral reef ecosystems. Sponge-associated microorganisms have been receiving greater attention because of their significant contribution to sponge biomass, biogeochemical cycles and biotechnological potentials. However, our understanding of the sponge microbiome is limited to a few species of sponges from restricted geographical locations. Here, we report for the first time the bacterial diversity of two cohabiting sponges, viz. Cinachyra cavernosa and Haliclona pigmentifera, as well as that in the ambient water from the coral reef ecosystems of the Gulf of Mannar, located along the southeast coast of India. Two hundred and fifty two clones in the 16S rRNA gene library of these sponges were grouped into eight distinct phyla, of which four belonged to the core group that are associated only with sponges. Phylogenetic analysis of the core bacteria showed close affinity to other sponge-associated bacteria from different geographical locations. γ-Proteobacteria, Chloroflexi, Planctomycetes and Deferribacter were the core groups in C. cavernosa while ß and δ-Proteobacteria performed this role in H. pigmentifera. We observed greater OTU diversity for C. cavernosa (Hǀ 2.07) compared to H. pigmentifera (Hǀ 1.97). UniFrac analysis confirmed the difference in bacterial diversity of the two sponge species and also between the sponges and the reef water (p<0.001). The results of our study restate the existence of a host driven force in shaping the sponge microbiome.

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

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 sea water via denaturing gradient gel electrophoresis (DGGE) indicates that it harbours 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 OF THE STUDY: 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.

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

UNLABELLED: Marine sponges are the most primitive metazoan and host symbiotic microorganisms. They are crucial components of the marine ecological system and play an essential role in pelagic processes. Copper pollution is currently a widespread problem and poses a threat to marine organisms. Here, we examined the effects of copper treatment on the composition of the sponge-associated bacterial community and the genetic features that facilitate the survival of enriched bacteria under copper stress. The 16S rRNA gene sequencing results showed that the sponge Haliclona cymaeformis harbored symbiotic sulfur-oxidizing Ectothiorhodospiraceae and photosynthetic Cyanobacteria as dominant species. However, these autotrophic bacteria decreased substantially after treatment with a high copper concentration, which enriched for a heterotrophic-bacterium-dominated community. Metagenomic comparison revealed a varied profile of functional genes and enriched functions, including bacterial motility and chemotaxis, extracellular polysaccharide and capsule synthesis, virulence-associated genes, and genes involved in cell signaling and regulation, suggesting short-period mechanisms of the enriched bacterial community for surviving copper stress in the microenvironment of the sponge. Microscopic observation and comparison revealed dynamic bacterial aggregation within the matrix and lysis of sponge cells. The bacteriophage community was also enriched, and the complete genome of a dominant phage was determined, implying that a lytic phage cycle was stimulated by the high copper concentration. This study demonstrated a copper-induced shift in the composition of functional genes of the sponge-associated bacterial community, revealing the selective effect of copper treatment on the functions of the bacterial community in the microenvironment of the sponge. IMPORTANCE: This study determined the bacterial community structure of the common sponge Haliclona cymaeformis and examined the effect of copper treatment on the community structure and functional gene composition, revealing that copper treatment had a selective effect on the functions of the bacterial community in the sponge. These findings suggest that copper pollution has an ecological impact on the sponge symbiont. The analysis showed that the untreated sponges hosted symbiotic autotrophic bacteria as dominant species, and the high-concentration copper treatment enriched for a heterotrophic bacterial community with enrichment for genes important for bacterial motility, supplementary cellular components, signaling and regulation, and virulence. Microscopic observation showed obvious bacterial aggregation and a reduction of sponge cell numbers in treated sponges, which suggested the formation of aggregates to reduce the copper concentration. The enrichment for functions of directional bacterial movement and supplementary cellular components and the formation of bacterial aggregates and phage enrichment are novel findings in sponge studies.

Metabolomic profiling and genomic study of a marine sponge-associated Streptomyces sp.

Metabolomics and genomics are two complementary platforms for analyzing an organism as they provide information on the phenotype and genotype, respectively. These two techniques were applied in the dereplication and identification of bioactive compounds from a Streptomyces sp. (SM8) isolated from the sponge Haliclona simulans from Irish waters. Streptomyces strain SM8 extracts showed antibacterial and antifungal activity. NMR analysis of the active fractions proved that hydroxylated saturated fatty acids were the major components present in the antibacterial fractions. Antimycin compounds were initially putatively identified in the antifungal fractions using LC-Orbitrap. Their presence was later confirmed by comparison to a standard. Genomic analysis of Streptomyces sp. SM8 revealed the presence of multiple secondary metabolism gene clusters, including a gene cluster for the biosynthesis of the antifungal antimycin family of compounds. The antimycin gene cluster of Streptomyces sp. SM8 was inactivated by disruption of the antimycin biosynthesis gene antC. Extracts from this mutant strain showed loss of antimycin production and significantly less antifungal activity than the wild-type strain. Three butenolides, 4,10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (1), 4,11-dihydroxy-10-methyl-dodec-2-en-1,4-olide (2), and 4-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide (3) that had previously been reported from marine Streptomyces species were also isolated from SM8. Comparison of the extracts of Streptomyces strain SM8 and its host sponge, H. simulans, using LC-Orbitrap revealed the presence of metabolites common to both extracts, providing direct evidence linking sponge metabolites to a specific microbial symbiont.

Subtilomycin: a new lantibiotic from Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans.

Bacteriocins are attracting increased attention as an alternative to classic antibiotics in the fight against infectious disease and multidrug resistant pathogens. Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans displays a broad spectrum antimicrobial activity, which includes Gram-positive and Gram-negative pathogens, as well as several pathogenic Candida species. This activity is in part associated with a newly identified lantibiotic, herein named as subtilomycin. The proposed biosynthetic cluster is composed of six genes, including protein-coding genes for LanB-like dehydratase and LanC-like cyclase modification enzymes, characteristic of the class I lantibiotics. The subtilomycin biosynthetic cluster in B. subtilis strain MMA7 is found in place of the sporulation killing factor (skf) operon, reported in many B. subtilis isolates and involved in a bacterial cannibalistic behaviour intended to delay sporulation. The presence of the subtilomycin biosynthetic cluster appears to be widespread amongst B. subtilis strains isolated from different shallow and deep water marine sponges. Subtilomycin possesses several desirable industrial and pharmaceutical physicochemical properties, including activity over a wide pH range, thermal resistance and water solubility. Additionally, the production of the lantibiotic subtilomycin could be a desirable property should B. subtilis strain MMA7 be employed as a probiotic in aquaculture applications.

Evaluation of differential protein expression in Haliclona aquarius and sponge-associated microorganisms under cadmium stress.

A comparative proteomic approach was used to assess differentially expressed proteins in marine sponges after 36 h of exposure to cadmium (Cd). After separation performed by 2-D polyacrylamide gel electrophoresis, 46 protein spots indicated differential expression, and 17 of these proteins were identified by electrospray ionization quadrupole time-of-flight mass spectrometry. From the proteins identified, 76% were attributed to sponge-associated microorganisms (fungi and bacteria), and 24% were attributed to Haliclona aquarius. Some of the proteins that were identified may be related to cell proliferation and differentiation or processes of oxidative stress repair and energy procurement. An integrated evaluation based on spot expression levels and the postulated functions of these proteins allowed a more accurate evaluation of the stress caused to the sponge holobiont system by cadmium exposure. This study could provide new insights into the use of a proteomic approach in the marine sponge to assess the effects of Cd pollution in a marine environment.

Diversity and bioactive potential of endospore-forming bacteria cultured from the marine sponge Haliclona simulans.

AIMS: Despite the frequent isolation of endospore-formers from marine sponges, little is known about the diversity and characterization of individual isolates. The main aims of this study were to isolate and characterize the spore-forming bacteria from the marine sponge Haliclona simulans and to examine their potential as a source for bioactive compounds. METHODS AND RESULTS: A bank of presumptive aerobic spore-forming bacteria was isolated from the marine sponge H. simulans. These represented c. 1% of the total culturable bacterial population. A subgroup of thirty isolates was characterized using morphological, phenotypical and phylogenetic analysis. A large diversity of endospore-forming bacteria was present, with the thirty isolates being distributed through a variety of Bacillus and Paenibacillus species. These included ubiquitous species, such as B. subtilis, B. pumilus, B. licheniformis and B. cereus group, as well as species that are typically associated with marine habitats, such as B. aquimaris, B. algicola and B. hwajinpoensis. Two strains carried the aiiA gene that encodes a lactonase known to be able to disrupt quorum-sensing mechanisms, and various isolates demonstrated protease activity and antimicrobial activity against different pathogenic indicator strains, including Clostridium perfringens, Bacillus cereus and Listeria monocytogenes. CONCLUSIONS: The marine sponge H. simulans harbours a diverse collection of endospore-forming bacteria, which produce proteases and antibiotics. This diversity appears to be overlooked by culture-dependent and culture-independent methods that do not specifically target sporeformers. SIGNIFICANCE AND IMPACT OF STUDY: Marine sponges are an as yet largely untapped and poorly understood source of endospore-forming bacterial diversity with potential biotechnological, biopharmaceutical and probiotic applications. These results also indicate the importance of combining different methodologies for the comprehensive characterization of complex microbial populations such as those found in marine sponges.

In vitro antiplasmodial activity of bacterium RJAUTHB 14 associated with marine sponge Haliclona Grant against Plasmodium falciparum.

Malaria is the most important parasitic disease, leading to annual death of about one million people, and the Plasmodium falciparum develops resistance to well-established antimalarial drugs. The newest antiplasmodial drug from a marine microorganism helps in addressing this problem. In the present study, Haliclona Grant were collected and subjected for enumeration and isolation of associated bacteria. The count of bacterial isolates was maximum in November 2007 (18 × 10(4) colony-forming units (CFU) g(-1), and the average count was maximum during the monsoon season (117 × 10(3) CFU g(-1)). Thirty-three morphologically different bacterial isolates were isolated from Haliclona Grant, and the extracellular ethyl acetate extracts were screened for antiplasmodial activity against P. falciparum. The antiplasmodial activity of bacterium RJAUTHB 14 (11.98 µg[Symbol: see text]ml(-1)) is highly comparable with the positive control chloroquine (IC(50) 19.59 µg[Symbol: see text]ml(-1)), but the other 21 bacterial extracts showed an IC(50) value of more than 100 µg[Symbol: see text]ml(-1). Statistical analysis reveals that significant in vitro antiplasmodial activity (P < 0.05) was observed between the concentrations and time of exposure. The chemical injury to erythrocytes showed no morphological changes in erythrocytes by the ethyl acetate extract of bacterial isolates after 48 h of incubation. The in vitro antiplasmodial activity might be due to the presence of reducing sugars and alkaloids in the ethyl acetate extracts of bacterium RJAUTHB 14. The 16S rRNA gene partial sequence of bacterium RJAUTHB 14 is deposited in NCBI (GenBank accession no. GU269569). It is concluded from the present study that the ethyl acetate extracts of bacterium RJAUTHB 14 possess lead compounds for the development of antiplasmodial drugs.

Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona (gellius) sp.

Three methods were examined to cultivate bacteria associated with the marine sponge Haliclona (gellius) sp.: agar plate cultures, liquid cultures, and floating filter cultures. A variety of oligotrophic media were employed, including media with aqueous and organic sponge extracts, bacterial signal molecules, and siderophores. More than 3,900 isolates were analyzed, and 205 operational taxonomic units (OTUs) were identified. Media containing low concentrations of mucin or a mixture of peptone and starch were most successful for the isolation of diversity, while the commonly used marine broth did not result in a high diversity among isolates. The addition of antibiotics generally led to a reduced diversity on plates but yielded different bacteria than other media. In addition, diversity patterns of isolates from agar plates, liquid cultures, and floating filters were significantly different. Almost 89% of all isolates were Alphaproteobacteria; however, members of phyla that are less commonly encountered in cultivation studies, such as Planctomycetes, Verrucomicrobia, and Deltaproteobacteria, were isolated as well. The sponge-associated bacteria were categorized into three different groups. The first group represented OTUs that were also obtained in a clone library from previously analyzed sponge tissue (group 1). Furthermore, we distinguished OTUs that were obtained from sponge tissue (in a previous study) but not from sponge isolates (group 2), and there were also OTUs that were not obtained from sponge tissue but were obtained from sponge isolates (group 3). The 17 OTUs categorized into group 1 represented 10 to 14% of all bacterial OTUs that were present in a large clone library previously generated from Haliclona (gellius) sp. sponge tissue, which is higher than previously reported cultivability scores for sponge-associated bacteria. Six of these 17 OTUs were not obtained from agar plates, which underlines that the use of multiple cultivation methods is worthwhile to increase the diversity of the cultivable microorganisms from sponges.

Streptomyces associated with a marine sponge Haliclona sp.; biosynthetic genes for secondary metabolites and products.

Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genus Streptomyces. Presence of this genus in a marine sponge, Haliclona sp., was investigated using culture-dependent and -independent techniques. 16S rRNA gene clone library analysis showed the presence of diverse Streptomyces in the sponge sample. In addition to the dominant genus Streptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genus Streptomyces were also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR-43 exhibited cytotoxic activity against cancer cell lines. JBIR-34 and -35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains.

Tetracycline resistance-encoding plasmid from Bacillus sp. strain #24, isolated from the marine sponge Haliclona simulans.

Knowledge of the nature of resistance determinants in natural habitats is fundamental to increasing our understanding of the development of antibiotic resistance in clinical settings. Here we provide the first report of a tetracycline resistance-encoding plasmid, pBHS24, from a marine sponge-associated bacterium, Bacillus sp. strain #24, isolated from Haliclona simulans.

Tetracenoquinocin and 5-iminoaranciamycin from a sponge-derived Streptomyces sp. Sp080513GE-26.

Two new anthracyclines, tetracenoquinocin (1) and 5-iminoaranciamycin (2), together with the known compounds aranciamycin (3) and antibiotic SM 173B were isolated from the culture of Streptomyces sp. Sp080513GE-26 associated with a marine sponge, Haliclona sp. The structures of 1 and 2 were established on the basis of extensive NMR and MS analyses along with (13)C-labeling experiments. The compounds 1-3 were evaluated for cytotoxicity against two cancer cell lines.