Pyrosequencing revealed shifts of prokaryotic communities between healthy and disease-like tissues of the Red Sea sponge Crella cyathophora.

Sponge diseases have been widely reported, yet the causal factors and major pathogenic microbes remain elusive. In this study, two individuals of the sponge Crella cyathophora in total that showed similar disease-like characteristics were collected from two different locations along the Red Sea coast separated by more than 30 kilometers. The disease-like parts of the two individuals were both covered by green surfaces, and the body size was much smaller compared with adjacent healthy regions. Here, using high-throughput pyrosequencing technology, we investigated the prokaryotic communities in healthy and disease-like sponge tissues as well as adjacent seawater. Microbes in healthy tissues belonged mainly to the Proteobacteria, Cyanobacteria and Bacteroidetes, and were much more diverse at the phylum level than reported previously. Interestingly, the disease-like tissues from the two sponge individuals underwent shifts of prokaryotic communities and were both enriched with a novel clade affiliated with the phylum Verrucomicrobia, implying its intimate connection with the disease-like Red Sea sponge C. cyathophora. Enrichment of the phylum Verrucomicrobia was also considered to be correlated with the presence of algae assemblages forming the green surface of the disease-like sponge tissues. This finding represents an interesting case of sponge disease and is valuable for further study.

Pyrosequencing reveals the microbial communities in the Red Sea sponge Carteriospongia foliascens and their impressive shifts in abnormal tissues.

Abnormality and disease in sponges have been widely reported, yet how sponge-associated microbes respond correspondingly remains inconclusive. Here, individuals of the sponge Carteriospongia foliascens under abnormal status were collected from the Rabigh Bay along the Red Sea coast. Microbial communities in both healthy and abnormal sponge tissues and adjacent seawater were compared to check the influences of these abnormalities on sponge-associated microbes. In healthy tissues, we revealed low microbial diversity with less than 100 operational taxonomic units (OTUs) per sample. Cyanobacteria, affiliated mainly with the sponge-specific species "Candidatus Synechococcus spongiarum," were the dominant bacteria, followed by Bacteroidetes and Proteobacteria. Intraspecies dynamics of microbial communities in healthy tissues were observed among sponge individuals, and potential anoxygenic phototrophic bacteria were found. In comparison with healthy tissues and the adjacent seawater, abnormal tissues showed dramatic increase in microbial diversity and decrease in the abundance of sponge-specific microbial clusters. The dominated cyanobacterial species Candidatus Synechococcus spongiarum decreased and shifted to unspecific cyanobacterial clades. OTUs that showed high similarity to sequences derived from diseased corals, such as Leptolyngbya sp., were found to be abundant in abnormal tissues. Heterotrophic Planctomycetes were also specifically enriched in abnormal tissues. Overall, we revealed the microbial communities of the cyanobacteria-rich sponge, C. foliascens, and their impressive shifts under abnormality.

Toward understanding the dynamics of microbial communities in an estuarine system.

Community assembly theories such as species sorting theory provide a framework for understanding the structures and dynamics of local communities. The effect of theoretical mechanisms can vary with the scales of observation and effects of specific environmental factors. Based on 16S rRNA gene tag pyrosequencing, different structures and temporal succession patterns were discovered between the surface sediments and bottom water microbial communities in the Pearl River Estuary (PRE). The microbial communities in the surface sediment samples were more diverse than those in the bottom water samples, and several genera were specific for the water or sediment communities. Moreover, water temperature was identified as the main variable driving community dynamics and the microbial communities in the sediment showed a greater temporal change. We speculate that nutrient-based species sorting and bacterial plasticity to the temperature contribute to the variations observed between sediment and water communities in the PRE. This study provides a more comprehensive understanding of the microbial community structures in a highly dynamic estuarine system and sheds light on the applicability of ecological theoretical mechanisms.

Microbial sulfur cycle in two hydrothermal chimneys on the Southwest Indian Ridge.

UNLABELLED: Sulfur is an important element in sustaining microbial communities present in hydrothermal vents. Sulfur oxidation has been extensively studied due to its importance in chemosynthetic pathways in hydrothermal fields; however, less is known about sulfate reduction. Here, the metagenomes of hydrothermal chimneys located on the ultraslow-spreading Southwest Indian Ridge (SWIR) were pyrosequenced to elucidate the associated microbial sulfur cycle. A taxonomic summary of known genes revealed a few dominant bacteria that participated in the microbial sulfur cycle, particularly sulfate-reducing Deltaproteobacteria. The metagenomes studied contained highly abundant genes related to sulfur oxidation and reduction. Several carbon metabolic pathways, in particular the Calvin-Benson-Bassham pathway and the reductive tricarboxylic acid cycles for CO2 fixation, were identified in sulfur-oxidizing autotrophic bacteria. In contrast, highly abundant genes related to the oxidation of short-chain alkanes were grouped with sulfate-reducing bacteria, suggesting an important role for short-chain alkanes in the sulfur cycle. Furthermore, sulfur-oxidizing bacteria were associated with enrichment for genes involved in the denitrification pathway, while sulfate-reducing bacteria displayed enrichment for genes responsible for hydrogen utilization. In conclusion, this study provides insights regarding major microbial metabolic activities that are driven by the sulfur cycle in low-temperature hydrothermal chimneys present on an ultraslow midocean ridge. IMPORTANCE: There have been limited studies on chimney sulfides located at ultraslow-spreading ridges. The analysis of metagenomes of hydrothermal chimneys on the ultraslow-spreading Southwest Indian Ridge suggests the presence of a microbial sulfur cycle. The sulfur cycle should be centralized within a microbial community that displays enrichment for sulfur metabolism-related genes. The present study elucidated a significant role of the microbial sulfur cycle in sustaining an entire microbial community in low-temperature hydrothermal chimneys on an ultraslow spreading midocean ridge, which has characteristics distinct from those of other types of hydrothermal fields.

In situ environment rather than substrate type dictates microbial community structure of biofilms in a cold seep system.

Using microscopic and molecular techniques combined with computational analysis, this study examined the structure and composition of microbial communities in biofilms that formed on different artificial substrates in a brine pool and on a seep vent of a cold seep in the Red Sea to test our hypothesis that initiation of the biofilm formation and spreading mode of microbial structures differs between the cold seep and the other aquatic environments. Biofilms on different substrates at two deployment sites differed morphologically, with the vent biofilms having higher microbial abundance and better structural features than the pool biofilms. Microbes in the pool biofilms were more taxonomically diverse and mainly composed of various sulfate-reducing bacteria whereas the vent biofilms were exclusively dominated by sulfur-oxidizing Thiomicrospira. These results suggest that the redox environments at the deployment sites might have exerted a strong selection on microbes in the biofilms at two sites whereas the types of substrates had limited effects on the biofilm development.

Molecular techniques revealed highly diverse microbial communities in natural marine biofilms on polystyrene dishes for invertebrate larval settlement.

Biofilm microbial communities play an important role in the larval settlement response of marine invertebrates. However, the underlying mechanism has yet to be resolved, mainly because of the uncertainties in characterizing members in the communities using traditional 16S rRNA gene-based molecular methods and in identifying the chemical signals involved. In this study, pyrosequencing was used to characterize the bacterial communities in intertidal and subtidal marine biofilms developed during two seasons. We revealed highly diverse biofilm bacterial communities that varied with season and tidal level. Over 3,000 operational taxonomic units with estimates of up to 8,000 species were recovered in a biofilm sample, which is by far the highest number recorded in subtropical marine biofilms. Nineteen phyla were found, of which Cyanobacteria and Proteobacteria were the most dominant one in the intertidal and subtidal biofilms, respectively. Apart from these, Actinobacteria, Bacteroidetes, and Planctomycetes were the major groups recovered in both intertidal and subtidal biofilms, although their relative abundance varied among samples. Full-length 16S rRNA gene clone libraries were constructed for the four biofilm samples and showed similar bacterial compositions at the phylum level to those revealed by pyrosequencing. Laboratory assays confirmed that cyrids of the barnacle Balanus amphitrite preferred to settle on the intertidal rather than subtidal biofilms. This preference was independent of the biofilm bacterial density or biomass but was probably related to the biofilm community structure, particularly, the Proteobacterial and Cyanobacterial groups.

Effect of polybrominated diphenyl ether (PBDE) treatment on the composition and function of the bacterial community in the sponge Haliclona cymaeformis.

Marine sponges play important roles in benthic environments and are sensitive to environmental stresses. Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants since the 1970s and are cytotoxic and genotoxic to organisms. In the present study, we studied the short-period effect of PBDE-47 (2,2',4,4'-tetrabromodiphenyl ether) treatment on the community structure and functional gene composition of the bacterial community inhabiting the marine sponge Haliclona cymaeformis. Our results showed that the bacterial community shifted from an autotrophic bacteria-dominated community to a heterotrophic bacteria-dominated community in response to PBDE-47 in a time- and concentration-dependent manner. A potentially symbiotic sulfur-oxidizing bacterium (SOB) was dominant (>80% in abundance) in the untreated sponge. However, exposure to a high concentration (1 µg/L) of PBDE-47 caused a substantial decrease in the potential symbiont and an enrichment of heterotrophic bacteria like Clostridium. A metagenomic analysis showed a selective effect of the high concentration treatment on the functional gene composition of the enriched heterotrophic bacteria, revealing an enrichment for the functions responsible for DNA repair, multidrug efflux pumping, and bacterial chemotaxis and motility. This study demonstrated that PBDE-47 induced a shift in the composition of the community and functional genes in the sponge-associated bacterial community, revealing the selective effect of PBDE-47 treatment on the functions of the bacterial community in the microenvironment of the sponge.

Adaptation of intertidal biofilm communities is driven by metal ion and oxidative stresses.

Marine organisms in intertidal zones are subjected to periodical fluctuations and wave activities. To understand how microbes in intertidal biofilms adapt to the stresses, the microbial metagenomes of biofilms from intertidal and subtidal zones were compared. The genes responsible for resistance to metal ion and oxidative stresses were enriched in both 6-day and 12-day intertidal biofilms, including genes associated with secondary metabolism, inorganic ion transport and metabolism, signal transduction and extracellular polymeric substance metabolism. In addition, these genes were more enriched in 12-day than 6-day intertidal biofilms. We hypothesize that a complex signaling network is used for stress tolerance and propose a model illustrating the relationships between these functions and environmental metal ion concentrations and oxidative stresses. These findings show that bacteria use diverse mechanisms to adapt to intertidal zones and indicate that the community structures of intertidal biofilms are modulated by metal ion and oxidative stresses.

Artifactual pyrosequencing reads in multiple-displacement-amplified sediment metagenomes from the Red Sea.

The Multiple Displacement Amplification (MDA) protocol is reported to introduce different artifacts into DNA samples with impurities. In this study, we report an artifactual effect of MDA with sediment DNA samples from a deep-sea brine basin in the Red Sea. In the metagenomes, we showed the presence of abundant artifactual 454 pyrosequencing reads over sizes of 50 to 220 bp. Gene fragments translocated from neighboring gene regions were identified in these reads. Occasionally, the translocation occurred between the gene fragments from different species. Reads containing these gene fragments could form a strong stem-loop structure. More than 60% of the artifactual reads could fit the structural models. MDA amplification is probably responsible for the massive generation of the artifactual reads with the secondary structure in the metagenomes. Possible sources of the translocations and structures are discussed.

Autotrophic microbe metagenomes and metabolic pathways differentiate adjacent Red Sea brine pools.

In the Red Sea, two neighboring deep-sea brine pools, Atlantis II and Discovery, have been studied extensively, and the results have shown that the temperature and concentrations of metal and methane in Atlantis II have increased over the past decades. Therefore, we investigated changes in the microbial community and metabolic pathways. Here, we compared the metagenomes of the two pools to each other and to those of deep-sea water samples. Archaea were generally absent in the Atlantis II metagenome; Bacteria in the metagenome were typically heterotrophic and depended on aromatic compounds and other extracellular organic carbon compounds as indicated by enrichment of the related metabolic pathways. In contrast, autotrophic Archaea capable of CO2 fixation and methane oxidation were identified in Discovery but not in Atlantis II. Our results suggest that hydrothermal conditions and metal precipitation in the Atlantis II pool have resulted in elimination of the autotrophic community and methanogens.

Profundibacterium mesophilum gen. nov., sp. nov., a novel member in the family Rhodobacteraceae isolated from deep-sea sediment in the Red Sea, Saudi Arabia.

A slow-growing, strictly aerobic, Gram-negative, coccus bacterial strain, designated KAUST100406-0324(T), was isolated from sea-floor sediment collected from the Red Sea, Saudi Arabia. The catalase- and oxidase-positive strain was non-sporulating and only slightly halophilic. Optimum growth occurred at 20-25 °C and at pH values ranging from 7.0 to 8.0. The major cellular fatty acids of the strain were unsaturated C18 : 1ω6c and/or C18 : 1ω7c, C18 : 1ω7c 11-methyl and C16 : 1ω7c and/or C16 : 1ω6c. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine and two unidentified phospholipids. Ubiquinone 10 was the predominant lipoquinone. The DNA G+C content of strain KAUST100406-0324(T) was 64.0 mol%. Phylogenetic analysis of 16S rRNA gene sequences revealed that the novel strain belonged to the family Rhodobacteraceae of the class Alphaproteobacteria but formed a distinct evolutionary lineage from other bacterial species with validly published names. The 16S rRNA gene sequence of the novel strain was distantly related, but formed a monophyletic cluster with, those of bacteria from two moderately halophilic genera, Hwanghaeicola and Maribius. The similarity of the sequence between the novel strain KAUST100406-0324(T) and the type strains Hwanghaeicola aestuarii Y26(T) (accession number FJ230842), Maribius pelagius B5-6(T) (DQ514326) and Maribius salinus CL-SP27(T) (AY906863) were 94.5 %, 95.2 % and 95.3 %, respectively. Based on the physiological, phylogenetic and chemotaxonomic characteristics presented in this study, we propose that this strain represents a novel species of a new genus in the family Rhodobacteraceae, for which the name of Profundibacterium mesophilum gen. nov., sp. nov. was proposed, with KAUST100406-0324(T) ( = JCM 17872(T)  = NRRL B-59665(T)) as the type strain.

Deep sequencing of Myxilla (Ectyomyxilla) methanophila, an epibiotic sponge on cold-seep tubeworms, reveals methylotrophic, thiotrophic, and putative hydrocarbon-degrading microbial associations.

The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ(13)C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge.

Spatial and species variations in bacterial communities associated with corals from the Red Sea as revealed by pyrosequencing.

Microbial associations with corals are common and are most likely symbiotic, although their diversity and relationships with environmental factors and host species remain unclear. In this study, we adopted a 16S rRNA gene tag-pyrosequencing technique to investigate the bacterial communities associated with three stony Scleractinea and two soft Octocorallia corals from three locations in the Red Sea. Our results revealed highly diverse bacterial communities in the Red Sea corals, with more than 600 ribotypes detected and up to 1,000 species estimated from a single coral species. Altogether, 21 bacterial phyla were recovered from the corals, of which Gammaproteobacteria was the most dominant group, and Chloroflexi, Chlamydiae, and the candidate phylum WS3 were reported in corals for the first time. The associated bacterial communities varied greatly with location, where environmental conditions differed significantly. Corals from disturbed areas appeared to share more similar bacterial communities, but larger variations in community structures were observed between different coral species from pristine waters. Ordination methods identified salinity and depth as the most influential parameters affecting the abundance of Vibrio, Pseudoalteromonas, Serratia, Stenotrophomonas, Pseudomonas, and Achromobacter in the corals. On the other hand, bacteria such as Chloracidobacterium and Endozoicomonas were more sensitive to the coral species, suggesting that the host species type may be influential in the associated bacterial community, as well. The combined influences of the coral host and environmental factors on the associated microbial communities are discussed. This study represents the first comparative study using tag-pyrosequencing technology to investigate the bacterial communities in Red Sea corals.

The regulatory role of the NO/cGMP signal transduction cascade during larval attachment and metamorphosis of the barnacle Balanus (=Amphibalanus) amphitrite.

The barnacle Balanus amphitrite is among the most dominant fouling species on intertidal rocky shores in tropical and subtropical areas and is thus a target organism in antifouling research. After being released from adults, the swimming nauplius undertakes six molting cycles and then transforms into a cyprid. Using paired antennules, a competent cyprid actively explores and selects a suitable substratum for attachment and metamorphosis (collectively known as settlement). This selection process involves the reception of exogenous signals and subsequent endogenous signal transduction. To investigate the involvement of nitric oxide (NO) and cyclic GMP (cGMP) during larval settlement of B. amphitrite, we examined the effects of an NO donor and an NO scavenger, two nitric oxide synthase (NOS) inhibitors and a soluble guanylyl cyclase (sGC) inhibitor on settling cyprids. We found that the NO donor sodium nitroprusside (SNP) inhibited larval settlement in a dose-dependent manner. In contrast, both the NO scavenger carboxy-PTIO and the NOS inhibitors aminoguanidine hemisulfate (AGH) and S-methylisothiourea sulfate (SMIS) significantly accelerated larval settlement. Suppression of the downstream guanylyl cyclase (GC) activity using a GC-selective inhibitor ODQ could also significantly accelerate larval settlement. Interestingly, the settlement inhibition effects of SNP could be attenuated by ODQ at all concentrations tested. In the developmental expression profiling of NOS and sGC, the lowest expression of both genes was detected in the cyprid stage, a crucial stage for the larval decision to attach and metamorphose. In summary, we concluded that NO regulates larval settlement via mediating downstream cGMP signaling.

Marinobacter xestospongiae sp. nov., isolated from the marine sponge Xestospongia testudinaria collected from the Red Sea.

A Gram-negative, catalase- and oxidase-positive, non-sporulating, rod-shaped and slightly halophilic bacterial strain, designated UST090418-1611(T), was isolated from the marine sponge Xestospongia testudinaria collected from the Red Sea coast of Saudi Arabia. Phylogenetic trees based on the 16S rRNA gene sequence placed strain UST090418-1611(T) in the family Alteromonadaceae with the closest relationship to the genus Marinobacter. The 16S rRNA gene sequence similarity between the strain and the type strains of recognized Marinobacter species ranged from 92.9 to 98.3%. Although strain UST090418-1611(T) shared high 16S rRNA gene sequence similarity with Marinobacter mobilis CN46(T), M. zhejiangensis CN74(T) and M. sediminum R65(T) (98.3, 97.4 and 97.3%, respectively), the relatedness of the strain to these three strains in DNA-DNA hybridization was only 58, 56 and 33%, respectively, supporting the novelty of the strain. In contrast to most strains in the genus Marinobacter, strain UST090418-1611(T) tolerated only 6% (w/v) NaCl, and optimal growth occurred at 2.0% (w/v) NaCl, pH 7.0-8.0 and 28-36 °C. The predominant cellular fatty acids were C(12:0) 3-OH, C(16:0), C(12:0) and summed feature 3 (C(16:1)ω6c and/or C(16:1)ω7c). The genomic DNA G+C content was 57.1 mol%. Based on the physiological, phylogenetic and chemotaxonomic characteristics presented in this study, we suggest that the strain represents a novel species in the genus Marinobacter, for which the name Marinobacter xestospongiae sp. nov. is proposed, with UST090418-1611(T) ( = JCM 17469(T)  = NRRL B-59512(T)) as the type strain.

Erythrobacter pelagi sp. nov., a member of the family Erythrobacteraceae isolated from the Red Sea.

A novel Gram-negative, aerobic, catalase- and oxidase-positive, non-sporulating, non-motile, rod-shaped bacterium, designated strain UST081027-248(T), was isolated from seawater of the Red Sea. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain UST081027-248(T) fell within the genus Erythrobacter. Levels of 16S rRNA gene sequence similarity between the novel strain and the type strains of Erythrobacter species ranged from 95.3 % (with Erythrobacter gangjinensis) to 98.2 % (with Erythrobacter citreus). However, levels of DNA-DNA relatedness between strain UST081027-248(T) and the type strains of closely related species were below 70 %. Optimal growth of the isolate occurred in the presence of 2.0 % NaCl, at pH 8.0-9.0 and at 28-36 °C. The isolate did not produce bacteriochlorophyll a. The predominant cellular fatty acids were C(17:1)ω6c, summed feature 8 (C(18:1)ω6c and/or C(18:1)ω7c) and C(15:0) 2-OH. The genomic DNA G+C content of strain UST081027-248(T) was 60.4 mol%. Phenotypic properties and phylogenetic distinctiveness clearly indicated that strain UST081027-248(T) represents a novel species of the genus Erythrobacter, for which the name Erythrobacter pelagi sp. nov. is proposed. The type strain is UST081027-248(T) ( = JCM 17468(T) = NRRL 59511(T)).

Sponges and sediments as monitoring tools of metal contamination in the eastern coast of the Red Sea, Saudi Arabia.

Sediments and sponges were collected from various locations along the eastern coast of the Red Sea, the Kingdom of Saudi Arabia. Total concentrations of Cd, Zn, Ag, Cu, Pb, As and Hg in the sediments were measured. Metal contamination was not significant in most of the studied sites and only one site was moderately polluted by Zn, Cu, and Pb. Sponges accumulated specific metals readily even though the metal exposure was low in the ambient environment. Contrasting interspecies differences in metal accumulation patterns were observed among the nine collected species of sponges. Significant positive correlations were found between the metal concentrations in the two species of sponges collected from the same sites. The strong ability to accumulate specific metals and the diversity of sponges that live in the Red Sea coastal areas make them a promising biomonitor of metal contamination in the areas.

Hydrothermally generated aromatic compounds are consumed by bacteria colonizing in Atlantis II Deep of the Red Sea.

Hydrothermal ecosystems have a wide distribution on Earth and many can be found in the basin of the Red Sea. Production of aromatic compounds occurs in a temperature window of ∼60-150 °C by utilizing organic debris. In the past 50 years, the temperature of the Atlantis II Deep brine pool in the Red Sea has increased from 56 to 68 °C, whereas the temperature at the nearby Discovery Deep brine pool has remained relatively stable at about 44 °C. In this report, we confirmed the presence of aromatic compounds in the Atlantis II brine pool as expected. The presence of the aromatic compounds might have disturbed the microbes in the Atlantis II. To show shifted microbial communities and their metabolisms, we sequenced the metagenomes of the microbes from both brine pools. Classification based on metareads and the 16S rRNA gene sequences from clones showed a strong divergence of dominant bacterial species between the pools. Bacteria capable of aromatic degradation were present in the Atlantis II brine pool. A comparison of the metabolic pathways showed that several aromatic degradation pathways were significantly enriched in the Atlantis II brine pool, suggesting the presence of aromatic compounds. Pathways utilizing metabolites derived from aromatic degradation were also significantly affected. In the Discovery brine pool, the most abundant genes from the microbes were related to sugar metabolism pathways and DNA synthesis and repair, suggesting a different strategy for the utilization of carbon and energy sources between the Discovery brine pool and the Atlantis II brine pool.

Diversity and quantity of ammonia-oxidizing Archaea and Bacteria in sediment of the Pearl River Estuary, China.

The diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the sediment of the Pearl River Estuary were investigated by cloning and quantitative real-time polymerase chain reaction (qPCR). From one sediment sample S16, 36 AOA OTUs (3% cutoff) were obtained from three clone libraries constructed using three primer sets for amoA gene. Among the 36 OTUs, six were shared by all three clone libraries, two appeared in two clone libraries, and the other 28 were only recovered in one of the libraries. For AOB, only seven OTUs (based on 16S rRNA gene) and eight OTUs (based on amoA gene) were obtained, showing lower diversity than AOA. The qPCR results revealed that AOA amoA gene copy numbers ranged from 9.6 × 10(6) to 5.1 × 10(7) copies per gram of sediment and AOB amoA gene ranged from 9.5 × 10(4) to 6.2 × 10(5) copies per gram of sediment, indicating that the dominant ammonia-oxidizing microorganisms in the sediment of the Pearl River Estuary were AOA. The terminal restriction fragment length polymorphism results showed that the relative abundance of AOB species in the sediment samples of different salinity were significantly different, indicating that salinity might be a key factor shaping the AOB community composition.

Pyrosequencing reveals highly diverse and species-specific microbial communities in sponges from the Red Sea.

Marine sponges are associated with a remarkable array of microorganisms. Using a tag pyrosequencing technology, this study was the first to investigate in depth the microbial communities associated with three Red Sea sponges, Hyrtios erectus, Stylissa carteri and Xestospongia testudinaria. We revealed highly diverse sponge-associated bacterial communities with up to 1000 microbial operational taxonomic units (OTUs) and richness estimates of up to 2000 species. Altogether, 26 bacterial phyla were detected from the Red Sea sponges, 11 of which were absent from the surrounding sea water and 4 were recorded in sponges for the first time. Up to 100 OTUs with richness estimates of up to 300 archaeal species were revealed from a single sponge species. This is by far the highest archaeal diversity ever recorded for sponges. A non-negligible proportion of unclassified reads was observed in sponges. Our results demonstrated that the sponge-associated microbial communities remained highly consistent in the same sponge species from different locations, although they varied at different degrees among different sponge species. A significant proportion of the tag sequences from the sponges could be assigned to one of the sponge-specific clusters previously defined. In addition, the sponge-associated microbial communities were consistently divergent from those present in the surrounding sea water. Our results suggest that the Red Sea sponges possess highly sponge-specific or even sponge-species-specific microbial communities that are resistant to environmental disturbance, and much of their microbial diversity remains to be explored.