Pyrosequencing Characterization of the Microbiota from Atlantic Intertidal Marine Sponges Reveals High Microbial Diversity and the Lack of Co-Occurrence Patterns

Sponges are ancient metazoans that host diverse and complex microbial communities. Sponge-associated microbial diversity has been studied from wide oceans across the globe, particularly in subtidal regions, but the microbial communities from intertidal sponges have remained mostly unexplored. Here we used pyrosequencing to characterize the microbial communities in 12 different co-occurring intertidal marine sponge species sampled from the Atlantic coast, revealing a total of 686 operational taxonomic units (OTUs) at 97% sequence similarity. Taxonomic assignment of 16S ribosomal RNA tag sequences estimated altogether 26 microbial groups, represented by bacterial (75.5%) and archaeal (22%) domains. Proteobacteria (43.4%) and Crenarchaeota (20.6%) were the most dominant microbial groups detected in all the 12 marine sponge species and ambient seawater. The Crenarchaeota microbes detected in three Atlantic Ocean sponges had a close similarity with Crenarchaeota from geographically separated subtidal Red Sea sponges. Our study showed that most of the microbial communities observed in sponges (73%) were also found in the surrounding ambient seawater suggesting possible environmental acquisition and/or horizontal transfer of microbes. Beyond the microbial diversity and community structure assessments (NMDS, ADONIS, ANOSIM), we explored the interactions between the microbial communities coexisting in sponges using the checkerboard score (C-score). Analyses of the microbial association pattern (co-occurrence) among intertidal sympatric sponges revealed the random association of microbes, favoring the hypothesis that the sponge-inhabiting microbes are recruited from the habitat mostly by chance or influenced by environmental factors to benefit the hosts.     

Metagenomic Investigation of Bacterial and Archaeal Diversity of Hammam Essalihine Hot Spring from Khenchela,Algeria

Abstract

Hot springs are natural environments where hot groundwater comes out from the earth. Exploring the microbial diversity present in hot springs is important first to determine the microorganisms able to proliferate there and to understand their role in biogeochemical cycles. In Algeria, research concerning microbial populations in those ecosystems is limited. This study describes bacterial and archaeal diversity of the ‘Hammam Essalihine’ hot spring in Khenchela province in north-east Algeria using a culture-independent approach. This is the first microbial diversity investigation in the ‘Hammam Essalihine’ hot spring using next-generation sequencing techniques to assess the species classification of thermophilic microorganisms. Genomic DNA was extracted from water samples and the V4–V5 region of 16S rRNA gene were amplified, sequenced, and analyzed. The average temperature of water varies from 68 to 70?°C. High-throughput sequencing analysis revealed the presence of 21 bacterial phyla, including an unknown phylum and distributed across 42 families and 39 genera. The majority of the sequences were observed to belong to the kingdom Bacteria. The bacterial community from this hot spring is dominated by Proteobacteria (41.52%), Chloroflexi (7.62%), and Bacteroidetes (7.62%), whereas the community of Archaea is scarcely present in the study site and the two identified operational taxonomic units (OTUs) are far from what is known in the GenBank database. The study shows several uncharacterized sequences, indicating that the water of ‘Hammam Essalihine’ hot spring contains undescribed microorganisms. This study is thought to add to the understanding of thermophile diversity and ecology of ‘Hammam Essalihine’ hot spring.     

Survey of Archaeal Diversity Reveals an Abundance of Halophilic Archaea in a Low-Salt, Sulfide- and Sulfur-Rich Spring

The archaeal community in a sulfide- and sulfur-rich spring with a stream water salinity of 0.7 to 1.0% in southwestern Oklahoma was studied by cloning and sequencing of 16S rRNA genes. Two clone libraries were constructed from sediments obtained at the hydrocarbon-exposed source of the spring and the microbial mats underlying the water flowing from the spring source. Analysis of 113 clones from the source library and 65 clones from the mat library revealed that the majority of clones belonged to the kingdom Euryarchaeota, while Crenarchaeota represented less than 10% of clones. Euryarchaeotal clones belonged to the orders Methanomicrobiales, Methanosarcinales, and Halobacteriales, as well as several previously described lineages with no pure-culture representatives. Those within the Halobacteriales represented 36% of the mat library and 4% of the source library. All cultivated members of this order are obligately aerobic halophiles. The majority of halobacterial clones encountered were not affiliated with any of the currently described genera of the family Halobacteriaceae. Measurement of the salinity at various locations at the spring, as well as along vertical gradients, revealed that soils adjacent to spring mats have a much higher salinity (NaCl concentrations as high as 32%) and a lower moisture content than the spring water, presumably due to evaporation. By use of a high-salt-plus-antibiotic medium, several halobacterial isolates were obtained from the microbial mats. Analysis of 16S rRNA genes indicated that all the isolates were members of the genus Haloferax. All isolates obtained grew at a wide range of salt concentrations, ranging from 6% to saturation, and all were able to reduce elemental sulfur to sulfide. We reason that the unexpected abundance of halophilic Archaea in such a low-salt, highly reduced environment could be explained by their relatively low salt requirement, which could be satisfied in specific locations of the shallow spring via evaporation, and their ability to grow under the prevalent anaerobic conditions in the spring, utilizing zero-valent sulfur compounds as electron acceptors. This study demonstrates that members of the Halobacteriales are not restricted to their typical high-salt habitats, and we propose a role for the Halobacteriales in sulfur reduction in natural ecosystems.     

Ultrastructure, Molecular Phylogenetics, and Chlorophyll a Content of Novel Cyanobacterial Symbionts in Temperate Sponges

Marine sponges often harbor photosynthetic symbionts that may enhance host metabolism and ecological success, yet little is known about the factors that structure the diversity, specificity, and nature of these relationships. Here, we characterized the cyanobacterial symbionts in two congeneric and sympatric host sponges that exhibit distinct habitat preferences correlated with irradiance: Ircinia fasciculata (higher irradiance) and Ircinia variabilis (lower irradiance). Symbiont composition was similar among hosts and dominated by the sponge-specific cyanobacterium Synechococcus spongiarum. Phylogenetic analyses of 16S-23S rRNA internal transcribed spacer (ITS) gene sequences revealed that Mediterranean Ircinia spp. host a specific, novel symbiont clade ("M") within the S. spongiarum species complex. A second, rare cyanobacterium related to the ascidian symbiont Synechocystis trididemni was observed in low abundance in I. fasciculata and likewise corresponded to a new symbiont clade. Symbiont communities in I. fasciculata exhibited nearly twice the chlorophyll a concentrations of I. variabilis. Further, S. spongiarum clade M symbionts in I. fasciculata exhibited dense intracellular aggregations of glycogen granules, a storage product of photosynthetic carbon assimilation rarely observed in I. variabilis symbionts. In both host sponges, S. spongiarum cells were observed interacting with host archeocytes, although the lower photosynthetic activity of Cyanobacteria in I. variabilis suggests less symbiont-derived nutritional benefit. The observed differences in clade M symbionts among sponge hosts suggest that ambient irradiance conditions dictate symbiont photosynthetic activity and consequently may mediate the nature of host-symbiont relationships. In addition, the plasticity exhibited by clade M symbionts may be an adaptive attribute that allows for flexibility in host-symbiont interactions across the seasonal fluctuations in light and temperature characteristic of temperate environments.     

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 β and δ-Proteobacteria performed this role in H. pigmentifera. We observed greater OTU diversity for C. cavernosa (H^ǀ 2.07) compared to H. pigmentifera (H^ǀ 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.     

Bacterial and Archaeal Diversity in Sediments of West Lake Bonney,McMurdo Dry Valleys,Antarctica

Bacterial and archaeal diversity was examined in a sediment core from Lake Bonney, Antarctica. Members of the Archaea showed both low abundance and diversity, whereas bacterial diversity was moderately high and some phyla were fairly abundant, even in geologically old samples. Microbial diversity correlated with sample texture and differed in silty and coarse samples.     

Bacterial and Archaeal Phylogenetic Diversity of a Cold Sulfur-Rich Spring on the Shoreline of Lake Erie,Michigan

Studies of sulfidic springs have provided new insights into microbial metabolism, groundwater biogeochemistry, and geologic processes. We investigated Great Sulphur Spring on the western shore of Lake Erie and evaluated the phylogenetic affiliations of 189 bacterial and 77 archaeal 16S rRNA gene sequences from three habitats: the spring origin (11-m depth), bacterial-algal mats on the spring pond surface, and whitish filamentous materials from the spring drain. Water from the spring origin water was cold, pH 6.3, and anoxic (H₂, 5.4 nM; CH₄, 2.70 μM) with concentrations of S²⁻ (0.03 mM), SO₄²⁻ (14.8 mM), Ca²⁺ (15.7 mM), and HCO₃⁻ (4.1 mM) similar to those in groundwater from the local aquifer. No archaeal and few bacterial sequences were >95% similar to sequences of cultivated organisms. Bacterial sequences were largely affiliated with sulfur-metabolizing or chemolithotrophic taxa in Beta-, Gamma-, Delta-, and Epsilonproteobacteria. Epsilonproteobacteria sequences similar to those obtained from other sulfidic environments and a new clade of Cyanobacteria sequences were particularly abundant (16% and 40%, respectively) in the spring origin clone library. Crenarchaeota sequences associated with archaeal-bacterial consortia in whitish filaments at a German sulfidic spring were detected only in a similar habitat at Great Sulphur Spring. This study expands the geographic distribution of many uncultured Archaea and Bacteria sequences to the Laurentian Great Lakes, indicates possible roles for epsilonproteobacteria in local aquifer chemistry and karst formation, documents new oscillatorioid Cyanobacteria lineages, and shows that uncultured, cold-adapted Crenarchaeota sequences may comprise a significant part of the microbial community of some sulfidic environments.Cold, sulfidic springs upwelling into caves (1, 16-19) or exposed at the land surface (14, 15, 31, 39, 47, 50, 51) have recently been shown to harbor unique microbial communities, reflective of the aqueous sulfur chemistry of the upwelling groundwater or of unique cave conditions. Within these spring and cave ecosystems, new and unique Epsilonproteobacteria 16S rRNA gene sequences associated with a limited number of cultured isolates that carry out oxidation of sulfur compounds have been discovered (7). The abundance of Epsilonproteobacteria sequences in these settings and associated biogeochemical research have led to new interest in the role of microbially mediated sulfuric acid speleogenesis as an important limestone dissolution process that may contribute to the development of karst features in limestone bedrock (19). Additionally, in streamlets from sulfidic springs, unique symbioses between uncultured Euryarchaeota, Crenarchaeota, and Epsilonproteobacteria spp. that grow in whitish, macroscopically visible filaments have been described (31, 51). Sulfur cycling was identified as a major means of energy production and maintenance of microbial communities in cold, saline, perennial springs emanating from permafrost in the Arctic (47). Studies of cold, sulfidic springs have therefore provided new insights into microbial metabolism, ecology, and evolution as well as groundwater biogeochemistry and geologic processes.All studies of sulfidic springs to date have focused on terrestrial landscapes typically associated with limestone (CaCO₃) bedrock. Limestone is one of several carbonate sedimentary rocks deposited by ancient seas, which may contain significant amounts of gypsum (CaSO₄·2H₂O) as well as pockets of hydrocarbon deposits, both a source of sulfur. Water that moves for long distances through such rocks evolves through sequential dissolution and precipitation reactions to a geochemistry that bears little resemblance to freshwater. SO₄²⁻ becomes available for microbial reduction to sulfide in aquifer zones where conditions are appropriate. Where spring waters rich in CaCO₃, CO₂, and sulfide emerge at the surface, carbonate deposition and microbially mediated sulfide oxidation occur. These processes result in tufa deposits and the whitish crusts often noted in sulfidic spring outflows (12, 13). Carbonate bedrock underlies large portions of the lower Laurentian Great Lakes. Caves in contact with lake water occur on islands in Lake Erie and along the Bruce Peninsula in Ontario, Canada. A cold, sulfidic spring is located in Ancaster, Ontario, about 5 km from the Lake Ontario shoreline (13). Recently, plumes of high-conductivity sulfidic groundwater, surrounded by whitish filamentous materials and variously colored microbial mats, were reported to occur at a 93-m depth in Lake Huron (2, 49). However, there have been few molecular surveys of Bacteria or Archaea in any Great Lakes environment, and no reports focusing on the molecular phylogenetic diversity of microorganisms associated with these Great Lakes sulfidic environments.Along the western shoreline of Lake Erie and within Monroe County, MI, sinkholes and springs are abundant in the Silurian-Devonian carbonate bedrock, and Ca²⁺ and Mg²⁺ with SO₄²⁻ or HCO₃⁻ dominate groundwater composition (43). In some areas of Monroe County, sulfide in groundwater prohibits its use as a drinking water source. Great Sulphur Spring (GSS) was first described by Sherzer in 1900 (53) and was named for its sulfide-rich water. The spring arises from Silurian-Devonian carbonate bedrock within 0.5 km of the Lake Erie shoreline and is a convenient location for accessing sulfide-rich groundwater and for exploring potential interactions between groundwater and lake water. As part of a larger study of nearshore groundwater interactions with Lake Erie (27) and to better understand the potential role of microorganisms in sulfur chemistry of nearshore groundwater, we evaluated the chemistry and bacterial and archaeal 16S rRNA gene diversity of GSS. Our study documents a unique microbial community for the Laurentian Great Lakes, comprised in large part of new lineages and uncultivated members of the Archaea, Deltaproteobacteria, Epsilonproteobacteria, and Cyanobacteria. These sequences suggest a microbial community structure driven by (possibly H₂S-based) carbon fixation and chemolithotrophy of reduced compounds such as H₂, H₂S, or reduced nitrogen compounds, all consistent with spring geochemistry.     

Thermodynamic Characterization of a Triheme Cytochrome Family from Geobacter sulfurreducens Reveals Mechanistic and Functional Diversity

A family of five periplasmic triheme cytochromes (PpcA-E) was identified in Geobacter sulfurreducens, where they play a crucial role by driving electron transfer from the cytoplasm to the cell exterior and assisting the reduction of extracellular acceptors. The thermodynamic characterization of PpcA using NMR and visible spectroscopies was previously achieved under experimental conditions identical to those used for the triheme cytochrome c₇ from Desulfuromonas acetoxidans. Under such conditions, attempts to obtain NMR data were complicated by the relatively fast intermolecular electron exchange. This work reports the detailed thermodynamic characterization of PpcB, PpcD, and PpcE under optimal experimental conditions. The thermodynamic characterization of PpcA was redone under these new conditions to allow a proper comparison of the redox properties with those of other members of this family. The heme reduction potentials of the four proteins are negative, differ from each other, and cover different functional ranges. These reduction potentials are strongly modulated by heme-heme interactions and by interactions with protonated groups (the redox-Bohr effect) establishing different cooperative networks for each protein, which indicates that they are designed to perform different functions in the cell. PpcA and PpcD appear to be optimized to interact with specific redox partners involving e⁻/H⁺ transfer via different mechanisms. Although no evidence of preferential electron transfer pathway or e⁻/H⁺ coupling was found for PpcB and PpcE, the difference in their working potential ranges suggests that they may also have different physiological redox partners. This is the first study, to our knowledge, to characterize homologous cytochromes from the same microorganism and provide evidence of their different mechanistic and functional properties. These findings provide an explanation for the coexistence of five periplasmic triheme cytochromes in G. sulfurreducens.     

Bacterial,Archaeal and Fungal Succession in the Forefield of a Receding Glacier

Glacier forefield chronosequences, initially composed of barren substrate after glacier retreat, are ideal locations to study primary microbial colonization and succession in a natural environment. We characterized the structure and composition of bacterial, archaeal and fungal communities in exposed rock substrates along the Damma glacier forefield in central Switzerland. Soil samples were taken along the forefield from sites ranging from fine granite sand devoid of vegetation near the glacier terminus to well-developed soils covered with vegetation. The microbial communities were studied with genetic profiling (T-RFLP) and sequencing of clone libraries. According to the T-RFLP profiles, bacteria showed a high Shannon diversity index (H) (ranging from 2.3 to 3.4) with no trend along the forefield. The major bacterial lineages were Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes and Cyanobacteria. An interesting finding was that Euryarchaeota were predominantly colonizing young soils and Crenarchaeota mainly mature soils. Fungi shifted from an Ascomycota-dominated community in young soils to a more Basidiomycota-dominated community in old soils. Redundancy analysis indicated that base saturation, pH, soil C and N contents and plant coverage, all related to soil age, correlated with the microbial succession along the forefield.     

Phylogenetic Diversity of Parabasalian Symbionts from Termites, Including the Phylogenetic Position of Pseudotrypanosoma and Trichonympha

ABSTRACT The phylogenetic diversity of parabasalian flagellates from termite hindguts has been examined by small subunit ribosomal RNA (rRNA) amplification and sequencing. Two species of particular interest, the giant trichomonad Pseudotrypanosoma giganteum and the hypermastigote Trichonympha magna, were isolated from the gut of Porotermes adamsoni by micropipetting. and the rRNA genes from these small populations amplified and sequenced. rRNA genes representing Hypermastigida and the Trichomonadida families Devescovinidae and Trichomonadidae. were also recovered by amplification from whole hindguts of three termites, P. adamsoni, Cryptotermes brevis , and Cryptotermes dudleyi. The parabasalian rRNA genes from C. brevis were found to comprise a unique and extremely heterogeneous lineage with no clear affinities to any known parabasalian rRNAs. In addition, one of the sequences isolated from P. Adamsoni was found to be similar to another uncharacterised rRNA gene from Reticulitermes flavipes. The phylogeny of all known parabasalian small subunit rRNAs was examined with these new sequences. We find many taxonomic groups to be supported by rRNA, but not all. We have found the root of parabasalia to be very difficult to discern accurately, but have nevertheless identified several possible positions.     

Diversity in a Cold Hot-Spot: DNA-Barcoding Reveals Patterns of Evolution among Antarctic Demosponges (Class Demospongiae,Phylum Porifera)

Background

The approximately 350 demosponge species that have been described from Antarctica represent a faunistic component distinct from that of neighboring regions. Sponges provide structure to the Antarctic benthos and refuge to other invertebrates, and can be dominant in some communities. Despite the importance of sponges in the Antarctic subtidal environment, sponge DNA barcodes are scarce but can provide insight into the evolutionary relationships of this unique biogeographic province.

Methodology/Principal Findings

We sequenced the standard barcoding COI region for a comprehensive selection of sponges collected during expeditions to the Ross Sea region in 2004 and 2008, and produced DNA-barcodes for 53 demosponge species covering about 60% of the species collected. The Antarctic sponge communities are phylogenetically diverse, matching the diversity of well-sampled sponge communities in the Lusitanic and Mediterranean marine provinces in the Temperate Northern Atlantic for which molecular data are readily available. Additionally, DNA-barcoding revealed levels of in situ molecular evolution comparable to those present among Caribbean sponges. DNA-barcoding using the Segregating Sites Algorithm correctly assigned approximately 54% of the barcoded species to the morphologically determined species.

Conclusion/Significance

A barcode library for Antarctic sponges was assembled and used to advance the systematic and evolutionary research of Antarctic sponges. We provide insights on the evolutionary forces shaping Antarctica''s diverse sponge communities, and a barcode library against which future sequence data from other regions or depth strata of Antarctica can be compared. The opportunity for rapid taxonomic identification of sponge collections for ecological research is now at the horizon.     

Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in Escherichia coli CV601 and Pseudomonas putida UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in several P. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriT probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5α by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobacter sp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.     

Inter- and Intraspecific Variations of Bacterial Communities Associated with Marine Sponges from San Juan Island,Washington

This study attempted to assess whether conspecific or congeneric sponges around San Juan Island, Washington, harbor specific bacterial communities. We used a combination of culture-independent DNA fingerprinting techniques (terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis [DGGE]) and culture-dependent approaches. The results indicated that the bacterial communities in the water column consisted of more diverse bacterial ribotypes than and were drastically different from those associated with the sponges. High levels of similarity in sponge-associated bacterial communities were found only in Myxilla incrustans and Haliclona rufescens, while the bacterial communities in Halichondria panicea varied substantially among sites. Certain terminal restriction fragments or DGGE bands were consistently obtained for different individuals of M. incrustans and H. rufescens collected from different sites, suggesting that there are stable or even specific associations of certain bacteria in these two sponges. However, no specific bacterial associations were found for H. panicea or for any one sponge genus. Sequencing of nine DGGE bands resulted in recovery of seven sequences that best matched the sequences of uncultured Proteobacteria. Three of these sequences fell into the sponge-specific sequence clusters previously suggested. An uncultured alphaproteobacterium and a culturable Bacillus sp. were found exclusively in all M. incrustans sponges, while an uncultured gammaproteobacterium was unique to H. rufescens. In contrast, the cultivation approach indicated that sponges contained a large proportion of Firmicutes, especially Bacillus, and revealed large variations in the culturable bacterial communities associated with congeneric and conspecific sponges. This study revealed sponge species-specific but not genus- or site-specific associations between sponges and bacterial communities and emphasized the importance of using a combination of techniques for studying microbial communities.Marine sponges (phylum Porifera) harbor a remarkable array of microorganisms, including bacteria (51, 54), unicellular algae (50), cyanobacteria (45, 48), dinoflagellates (14), zoochlorellae (58), and members of the domain Archaea (33). Of these microorganisms, bacteria are the most dominant group of microbial associates in sponges and can account for up to 40 to 50% of a sponge''s biomass (17). The density of bacteria can be up to 10⁸ to 10¹⁰ bacteria per g (wet weight) of sponge (18). The great abundance of bacteria in sponges caused workers to coin the term “bacteriosponges” (35) and has attracted much research interest in the role and specificity of the sponge-bacterium association.There are generally two pathways by which sponges may acquire their bacterial associates. The first pathway is filter feeding and selective retention of bacteria (44). Bacteria in the surrounding seawater can be captured by sponges when sponges filter the food particles out of the water column. The bacteria that resist digestion by sponge choanocytes and archaeocytes can survive and live inside the sponges. Sponge-associated bacterial communities acquired via this pathway are therefore heavily influenced by the type of bacteria in the water column. The second pathway is vertical transmission of bacterial associates from adult sponges to their progeny (10, 40, 49). Bacteria acquired through this pathway may exhibit specificity for genera or species of sponges due to coevolution (for a review, see reference 44).Sponge-bacterium symbioses are often mutualistic; while bacteria may benefit from the favorable nutritional conditions in sponges (18, 44), some bacterial associates may help their hosts eliminate metabolic waste (4), stabilize the sponge skeleton (36), and defend against pathogens, predators, or competitors via the production of bioactive secondary metabolites (5, 21, 39, 47). In addition, some cyanobacterial symbionts are a source of nutrients for their hosts because of their photosynthetic and nitrogen-fixing abilities (3, 57).There is a large body of knowledge concerning the possible roles of bacterial associates in sponges, but whether sponges harbor specific bacterial communities deserves more detailed study. Many previous investigations demonstrated that associated bacterial communities in certain species of sponges were highly similar and consistently different from the bacterial communities in the ambient environment (6, 22, 46). For instance, Hentschel et al. (16) showed that there were uniform microbial communities in the marine sponges Aplysina aerophoba, Rhopaloeides odorabile, and Theonella swinhoei from different geographic regions that were distinct from those in the water column or in sediments. In addition, Friedrich et al. (12) demonstrated that the composition of sponge-associated bacterial communities was resistant to environmental perturbations resulting from transplantation to different habitats. These studies suggest that there is a stable, specific, and perhaps mutualistic relationship between the two types of organisms (44). However, some bacterium-sponge symbioses do not appear to be consistent. For instance, Wichels et al. (56) demonstrated that the bacterial communities associated with the North Sea sponge Halichondria panicea varied substantially over time. Qian et al. (34) and Lee et al. (25) showed that the congeneric Callyspongia and Mycale sponges from different biogeographic regions had different bacterial associates. Although the discrepancies may be attributed to the methods employed in different studies, whether consistent sponge-bacterium associations occur in different species or genera of sponges remains unclear. Furthermore, most of the previous studies focused on only one sponge species or involved a few sponges from geographically separated regions. So far, there has been no large-scale study comparing the bacterial communities associated with sponges of different genera and species.In this study, we compared the bacterial communities associated with marine sponges around San Juan Island, Washington, in order to investigate the specificity of congeneric and conspecific sponge-associated bacterial communities. Most previous studies have relied on only one method to assess the bacterial communities, limiting the resolution of community assessments. In addition, different studies have used different approaches to address the same question, creating uncertainties and making generalizations difficult. Here, we employed both culture-independent and -dependent approaches to compensate for the limitations of different methods and to obtain a more reliable assessment of the associated bacterial communities. We used two DNA fingerprinting techniques, terminal restriction fragment length polymorphism (TRFLP) (27) and denaturing gradient gel electrophoresis (DGGE) (11). TRFLP is an effective, sensitive, high-throughput technique that differentiates bacterial community structures, while DGGE allows subsequent identification of bacteria of interest by excision and sequencing of specific bands. Both techniques have been successfully and widely used for characterization of bacterial communities in marine samples (13, 26, 28, 43, 56). In addition, bacteria associated with sponges were isolated using cultivation methods and identified by comparative analysis of 16S rRNA gene sequences. Phylogenetic affiliations of the isolates were determined and compared for different samples in order to study the specificity of the sponge-associated culturable bacterial communities.     

Phylogenetic Diversity and Characterization of Novel and Efficient Cellulase Producing Bacterial Isolates from Various Extreme Environments

A set of 300 bacterial strains isolated from various extreme environments were screened for the presence of cellulase activity on CMC agar plates. Phylogenetic analysis of the positive strain, based on 16S rRNA gene sequences indicated that the isolates were clustered within Firmicutes and Actinobacteria. A majority (17) of the isolates were identified as Bacillus, Paenibacillus, and Lysinibacillus sp., and the remaining three were identified as Arthobacter, Rhodococcus, and Bhargavaea cecembensis. Among the 20 positive isolates, 6 were evaluated for the production of cellulases on five different cellulosic substrates. Two isolates, B. cecembensis and Bacillus sp., based on maximum enzyme production on all cellulosic substrates, especially CMC and rice straw, were evaluated in terms of enzyme properties and kinetics. The enzymes of these two isolates are found to be active over broad range of pH and temperature. Such thermostable enzymes facilitate the development of efficient and cost-effective forms of the simultaneous saccharification and fermentation process converting lignocellulosic biomass into biofuels and value-added products.