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Sponges (Porifera) contain many peptide-specialized metabolites with potent biological activities and significant roles in shaping marine ecology. It is well established that symbiotic bacteria produce bioactive "sponge" peptides, both on the ribosome (RiPPs) and nonribosomally. Here, we demonstrate that sponges themselves also produce many bioactive macrocyclic peptides, such as phakellistatins and related proline-rich macrocyclic peptides (PRMPs). Using the Stylissa carteri sponge transcriptome, methods were developed to find sequences encoding 46 distinct RiPP-type core peptides, of which ten encoded previously identified PRMP sequences. With this basis set, the genome and transcriptome of the sponge Axinella corrugata was interrogated to find 35 PRMP precursor peptides encoding 31 unique core peptide sequences. At least 11 of these produced cyclic peptides that were present in the sponge and could be characterized by mass spectrometry, including stylissamides A-D and seven previously undescribed compounds. Precursor peptides were encoded in the A. corrugata genome, confirm-ing their animal origin. The peptides contained signal peptide sequences and highly repetitive recognition sequence-core peptide elements with up to 25 PRMP copies in a single precursor. In comparison to sponges without PRMPs, PRMP sponges are incredibly enriched in potentially secreted polypeptides, with >23,000 individual signal peptide encoding genes found in a single transcriptome. The similarities between PRMP biosynthetic genes and neuropeptides in terms of their biosynthetic logic suggest a fundamental biology linked to circular peptides, possibly indicating a widespread and underappreciated diversity of signaling peptide post-translational modifications across the animal kingdom. [ABSTRACT FROM AUTHOR]

Class Demospongiae is the largest in the phylum Porifera (Sponges) and encompasses nearly 8,000 accepted species in three subclasses: Keratosa, Verongimorpha, and Heteroscleromorpha. Subclass Heteroscleromorpha contains ∼90% of demosponge species and is subdivided into 17 orders. The higher level classification of demosponges underwent major revision as the result of nearly three decades of molecular studies. However, because most of the previous molecular work only utilized partial data from a small number of nuclear and mitochondrial (mt) genes, this classification scheme needs to be tested by larger datasets. Here we compiled a mt dataset for 136 demosponge species—including 64 complete or nearly complete and six partial mt-genome sequences determined or assembled for this study—and used it to test phylogenetic relationships among Demospongiae in general and Heteroscleromorpha in particular. We also investigated the phylogenetic position of Myceliospongia araneosa, a highly unusual demosponge without spicules and spongin fibers, currently classified as Demospongiae incertae sedis, for which molecular data were not available. Our results support the previously inferred sister-group relationship between Heteroscleromorpha and Keratosa + Verongimorpha and suggest five main clades within Heteroscleromorpha: Clade C0 composed of order Haplosclerida; Clade C1 composed of Scopalinida, Sphaerocladina, and Spongillida; Clade C2 composed of Axinellida, Biemnida, Bubarida; Clade C3 composed of Tetractinellida; and Clade C4 composed of Agelasida, Clionaida, Desmacellida, Merliida, Suberitida, Poecilosclerida, Polymastiida, and Tethyida. The inferred relationships among these clades were (C0(C1(C2(C3+C4)))). Analysis of molecular data from M. araneosa placed it in the C3 clade as a sister taxon to the highly skeletonized tetractinellids Microscleroderma sp. and Leiodermatium sp. Molecular clock analysis dated divergences among the major clades in Heteroscleromorpha from the Cambrian to the Early Silurian, the origins of most heteroscleromorph orders in the middle Paleozoic, and the most basal splits within these orders around the Paleozoic to Mesozoic transition. Overall, the results of this study are mostly congruent with the accepted classification of Heteroscleromorpha, but add temporal perspective and new resolution to phylogenetic relationships within this subclass. [ABSTRACT FROM AUTHOR]

Sponges are recognized as a diverse and abundant component of mesophotic and deep-sea ecosystems worldwide. In Flower Garden Banks National Marine Sanctuary region within the northwestern Gulf of Mexico, sponges thrive among diverse biological and geological habitats between 16–200+ m deep (i.e., coral reefs and communities, algal nodules, and coralline algae reefs, mesophotic reefs, patch reefs, scarps, ridges, soft substrate, and rocky outcrops). A synoptic guide is presented, developed by studying common sponge species in the region, through direct sampling and in-situ photographic records. A total of 64 species is included: 60 are Demospongiae (14 orders), two are Hexactinellida (one order), and two are Homoscleromorpha (one order). Thirty-four taxa are identified to species and 13 were identified to have affinity with, but were not identical to, a known species. Fifteen taxa could only be identified to genus level, and the species remain as uncertain (incerta sedis), with the potential to represent new species or variants of known species. One specimen received only a family assignation. This study extends geographic or mesophotic occurrence data for eleven known species and includes several potentially new species. This work improves our knowledge of Gulf of Mexico sponge biodiversity and highlights the importance of the region for scientists and resource managers. [ABSTRACT FROM AUTHOR]

Cryptosporidium sp. are apicomplexan parasites that cause significant morbidity and possible mortality in humans and valuable livestock. There are no drugs on the market that are effective in the population most severely affected by this parasite. This study is the first high-throughput screen for potent anti-Cryptosporidium natural products sourced from a unique marine compound library. The Harbor Branch Oceanographic Institute at Florida Atlantic University has a collection of diverse marine organisms some of which have been subjected to medium pressure liquid chromatography to create an enriched fraction library. Numerous active compounds have been discovered from this library, but it has not been tested against Cryptosporidium parvum. A high-throughput in vitro growth inhibition assay was used to test 3764 fractions in the library, leading to the identification of 23 fractions that potently inhibited the growth of Cryptosporidium parvum. Bioassay guided fractionation of active fractions from a deep-sea sponge, Leiodermatium sp., resulted in the purification of leiodolide A, the major active compound in the organism. Leiodolide A displayed specific anti-Cryptosporidium activity at a half maximal effective concentration of 103.5 nM with selectivity indexes (SI) of 45.1, 11.9, 19.6 and 14.3 for human ileocecal colorectal adenocarcinoma cells (HCT-8), human hepatocellular carcinoma cells (Hep G2), human neuroblastoma cells (SH-SY5Y) and green monkey kidney cells (Vero), respectively. The unique structure of leiodolide A provides a valuable drug scaffold on which to develop new anti-Cryptosporidium compounds and supports the importance of screening natural product libraries for new chemical scaffolds. [ABSTRACT FROM AUTHOR]

A deep-water sponge of the Spongosorites genus yields a unique bis(indole) alkaloid, dragmacidin d. Spectroscopic analysis establishes its formula as C25H21BrN7O2. Dragmacidin d is unique among related sponge metabolites. It is the first bis(indole) alkaloid found with a C-4 alkyl group at the ring, the first dragmacidin with a2-aminoimidazole function and the first to have the 2(1H)-pyrazinone spacer. More important, dragmacidin d exhibits a wide range of antimicrobial activity, including inhibition of E. coli, B. subtilis and P. aeruginosa. It is also effective against the feline leukemia virus and human lung tumor cell lines.

The Caribbean marine sponge Discodermia sp. yields polydiscamide A, a discodermin depsipeptide that inhibits the growth of the human lung cancer A549. Spectroscopic, chemical degradation and analogue studies reveal a structure composed of 13 amino acids, including a unique 3-methylisoleucine. The side-chain phenylalanine is replaced by p-bromophenylalanine. The lactone ring has five instead of six amino acids. Valine and proline replace the leucine, threonine and sarcosine in the ring. Except for asparagine, which exhibits a D configuration, the common amino acids show the same absolute configuration as in other discodermins.

Background. Among all present demosponges, lithistids represent a polyphyletic group with exceptionally well-preserved fossils dating back to the Cambrian. Knowledge of their recent diversity, particularly in the Tropical Western Atlantic Ocean (TWA) where they are common in deep waters, is scarce making any comparison between present and past major 'lithistid' faunas difficult. In addition, the lack of sufficient molecular and morphological data hamper any predictions on phylogenetic relationships or phylodiversity from this region. The Harbor Branch Oceanographic Institute (HBOI, Fort Pierce, Florida) holds the largest collection of TWA lithistid sponges worldwide, however, the majority remain to be taxonomically identified and revised. Principal Findings. In this study we provide sequences of 249 lithistid demosponges using two independent molecular markers (28S rDNA (C1-D2) and cox1 mtDNA). In addition, a morphological documentation of 70 lithistid specimens is provided in the database of the Sponge Barcoding Project (SBP). This integrated dataset represents the largest and most comprehensive of the TWA lithistids to date. The phylogenetic diversity of 'lithistid' demosponges in the Bahamas and Jamaica are high in comparison to other TWA regions; Theonellidae and Corallistidae dominate the fauna, while Neopeltidae and Macandrewiidae are rare. A proposed tetractinellid suborder, one undescribed genus and several undescribed species are recognized and the Pacific 'lithistid' genera, Herengeria and Awhiowhio, are reported from the TWA for the first time. The higher-taxa relationships of desma-bearing tetractinellids are discussed and topics for revision suggested. Conclusion. This first integrative approach of TWA 'lithistid' demosponges contributes to a better understanding of their phylogenetic affinities, diversity and bathymetric distribution patterns within the TWA. As in the Pacific, the TWA 'lithistid' demosponges dominate deep-water habitats. Deeper taxonomic investigations will undoubtedly contribute to a better comparison between present major 'lithistid' faunas and their fossil record in the Mesozoic. [ABSTRACT FROM AUTHOR]

Vetulina Schmidt, 1879 (Demospongiae, Sphaerocladina, Vetulinidae) currently constitutes the only living representative of a once diverse Mesozoic group. Molecular data place Vetulina as a sister taxon to freshwater sponges (Spongillida) despite different skeletal composition. To date, only three extant species of this desma-bearing 'rock sponge' have been described from the Caribbean and Indian Ocean, all with similar growth forms and spiculation, but different desma and surface details. Comparison of these genetically very similar species was not possible until the present study. The distribution of Vetulina is taken to be a consequence of the closure of the Tethyan Seaway in the Early Miocene, suggesting a more widely distributed population with its origin in the Tethys Sea. To support this hypothesis in a molecular palaeobiological framework, we first increased the taxon sampling by describing and sequencing two new species of Vetulina from the Bahamas and Philippines and report Vetulina stalactites from nine additional locations in the Tropical Western Atlantic. A robust, dated phylogeny was calculated from the combined dataset and amended by five representative fossils. Our results point to an Eocene origin for Vetulina, even before the closure of the Tethyan Seaway in the Miocene, supporting the hypothesis that Vetulina presents a relict fauna with its origin in the Tethys Sea. [ABSTRACT FROM AUTHOR]

The Galápagos Islands, positioned in the confluence of warm and coldwater currents in the Eastern Pacific, is well known for the high degree of endemism of its marine invertebrate fauna. This fauna has been studied extensively in recent years: the echinoderms, corals and other benthic cnidarians, but little is known about the deep- and shallow-water sponge faunas. To date, only 70 sponge species have been described from the Galápagos Islands, 37 of which are endemic. Of these 70 species, only one shallow-water species of desma-bearing Tetractinellida (Demospongiae), Corallistes isabela, has been reported. In 1995, Harbor Branch Oceanographic Institution, Florida, led an expedition around the Galápagos archipelago, focussed on the collection of deep-water Porifera. Here, we describe seven new species and provide DNA barcodes for the tetractinellids from these collections. Phylogenetic relationships of these new species are discussed and compared with other material from the Caribbean, the Central and West Pacific Oceans. The new species represent five genera (Craniella, and desma-bearing Tetractinellida Neophrissospongia, Corallistes, Racodiscula and Scleritoderma). Phylogenetic reconstructions combining independent markers (mtDNA and rDNA) support the generic affiliation of these new species and confirm the separation of Eastern Pacific species from Caribbean and Central to West Pacific species. [ABSTRACT FROM AUTHOR]

Although sponges are important components of benthic ecosystems of the Caribbean Sea, their diversity remained poorly investigated in the Lesser Antilles. By organizing a training course in Martinique, we wanted both to promote taxonomy and to provide a first inventory of the sponge diversity on this island. The course was like a naturalist expedition, with a field laboratory and a classroom nearby. Early-career scientists and environmental managers were trained in sponge taxonomy. We gathered unpublished data and conducted an inventory at 13 coastal sites. We explored only shallow water habitats (0–30 m), such as mangroves, reefs or rocky bottoms and underwater caves. According to this study, the sponge fauna of Martinique is currently represented by a minimum of 191 species, 134 of which we could assign species names. One third of the remaining non-identified sponge species we consider to be new to science. Martinique appears very remarkable because of its littoral marine fauna harboring sponge aggregations with high biomass and species diversity dominating over coral species. In mangroves, sponges cover about 10% of the surface of subtidal roots. Several submarine caves are true reservoirs of hidden and insufficiently described sponge diversity. Thanks to this new collaborative effort, the Eastern Caribbean has gained a significant increase of knowledge, with sponge diversity of this area potentially representing 40% of the total in the Caribbean Sea. We thus demonstrated the importance of developing exploratory and educational research in areas historically devoid of biodiversity inventories and systematics studies. Finally, we believe in the necessity to consider not only the number of species but their distribution in space to evaluate their putative contribution to ecosystem services and our willingness to preserve them. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial introns intermit coding regions of genes and feature characteristic secondary structures and splicing mechanisms. In metazoans, mitochondrial introns have only been detected in sponges, cnidarians, placozoans and one annelid species. Within demosponges, group I and group II introns are present in six families. Based on different insertion sites within the cox1 gene and secondary structures, four types of group I and two types of group II introns are known, which can harbor up to three encoding homing endonuclease genes (HEG) of the LAGLIDADG family (group I) and/or reverse transcriptase (group II). However, only little is known about sponge intron mobility, transmission, and origin due to the lack of a comprehensive dataset. We analyzed the largest dataset on sponge mitochondrial group I introns to date: 95 specimens, from 11 different sponge genera which provided novel insights into the evolution of group I introns. Results: For the first time group I introns were detected in four genera of the sponge family Scleritodermidae (Scleritoderma, Microscleroderma, Aciculites, Setidium). We demonstrated that group I introns in sponges aggregate in the most conserved regions of cox1. We showed that co-occurrence of two introns in cox1 is unique among metazoans, but not uncommon in sponges. However, this combination always associates an active intron with a degenerating one. Earlier hypotheses of HGT were confirmed and for the first time VGT and secondary losses of introns conclusively demonstrated. Conclusion: This study validates the subclass Spirophorina (Tetractinellida) as an intron hotspot in sponges. Our analyses confirm that most sponge group I introns probably originated from fungi. DNA barcoding is discussed and the application of alternative primers suggested. [ABSTRACT FROM AUTHOR]

A deep-water sponge of the genus Spongosorites has yielded a bis-indole alkaloid which we have named dragmacidin G. Dragmacidin G was first reported by us in the patent literature and has recently been reported by Hitora et al. from a sponge of the genus Lipastrotheya. Dragmacidin G is the first in this series of compounds to have a pyrazine ring linking the two indole rings. It also has a rare N-(2-mercaptoethyl)-guanidine side chain. Dragmacidin G shows a broad spectrum of biological activity including inhibition of methicillin-resistant Staphylococcus aureus, Mycobacterium tuberculosis, Plasmodium falciparum, and a panel of pancreatic cancer cell lines. [ABSTRACT FROM AUTHOR]

Most lithistids occur worldwide in deep-water environments, but can be found in some places in shallow water. They are not well known in the tropical western Atlantic, despite the fact that they were first described in the late 1800s. We report here two species of poorly known theonellid demosponges (Astrophorina), Discodermia dissoluta and Theonella atlantica, and one new species, Theonella wrightae, from the north-west, south-west and south Florida shelf. There is considerable variability in habitus, colour and spiculation in this species. If samples are taken randomly from different specimens and from functionally and structurally different locations on the sponge, the specimens could be mistakenly identified or individuals of the same species could be described as different species. This report increases the number of lithistid sponges reported from the tropical western Atlantic region to 30 species, but our unpublished data suggest a much higher number of species present in this region. It is also the second report of the relatively deep-water (81 m) occurrence of D. dissoluta that is mostly known from depths as shallow as 10–30 m. [ABSTRACT FROM PUBLISHER]

Background: Demosponges are challenging for phylogenetic systematics because of their plastic and relatively simple morphologies and many deep divergences between major clades. To improve understanding of the phylogenetic relationships within Demospongiae, we sequenced and analyzed seven nuclear housekeeping genes involved in a variety of cellular functions from a diverse group of sponges. Methodology/Principal Findings: We generated data from each of the four sponge classes (i.e., Calcarea, Demospongiae, Hexactinellida, and Homoscleromorpha), but focused on family-level relationships within demosponges. With data for 21 newly sampled families, our Maximum Likelihood and Bayesian-based approaches recovered previously phylogenetically defined taxa: Keratosap, Myxospongiaep, Spongillidap, Haploscleromorphap (the marine haplosclerids) and Democlaviap. We found conflicting results concerning the relationships of Keratosap and Myxospongiaep to the remaining demosponges, but our results strongly supported a clade of Haploscleromorphap +Spongillidap +Democlaviap. In contrast to hypotheses based on mitochondrial genome and ribosomal data, nuclear housekeeping gene data suggested that freshwater sponges (Spongillidap) are sister to Haploscleromorphap rather than part of Democlaviap. Within Keratosap, we found equivocal results as to the monophyly of Dictyoceratida. Within Myxospongiaep , Chondrosida and Verongida were monophyletic. A well- supported clade within Democlaviap, Tetractinellidap , composed of all sampled members of Astrophorina and Spirophorina (including the only lithistid in our analysis), was consistently revealed as the sister group to all other members of Democlaviap. Within Tetractinellidap, we did not recover monophyletic Astrophorina or Spirophorina. Our results also reaffirmed the monophyly of order Poecilosclerida (excluding Desmacellidae and Raspailiidae), and polyphyly of Hadromerida and Halichondrida. Conclusions/Significance: These results, using an independent nuclear gene set, confirmed many hypotheses based on ribosomal and/or mitochondrial genes, and they also identified clades with low statistical support or clades that conflicted with traditional morphological classification. Our results will serve as a basis for future exploration of these outstanding questions using more taxon- and gene-rich datasets. [ABSTRACT FROM AUTHOR]

Lithistid sponges are known to produce a diverse array of compounds ranging from polyketides, cyclic and linear peptides, alkaloids, pigments, lipids, and sterols. A majority of these structurally complex compounds have very potent and interesting biological activities. It has been a decade since a thorough review has been published that summarizes the literature on the natural products reported from this amazing sponge order. This review provides an update on the current taxonomic classification of the Lithistida, describes structures and biological activities of 131 new natural products, and discusses highlights from the total syntheses of 16 compounds from marine sponges of the Order Lithistida providing a compilation of the literature since the last review published in 2002. [ABSTRACT FROM AUTHOR]

The lithistid sponge Discodermia dissoluta (family Theonellidae), is found in deep-waters throughout the Caribbean sea and is the source of discodermolide, a natural product with potential anticancer properties, and other secondary metabolites. As with other sponges, large numbers of microbes are harbored in the sponge mesohyl. The microbial population of the sponge mesohyl shows an abundance of large filamentous microbes. Fractionation of the dissociated sponge allowed enrichment of this microbe, which was then identified by analysis of the 16S rRNA genes. Its identity was confirmed through the use of fluorescent in situ hybridization. These studies have allowed the identification of this eubacterial microbe as belonging to the genus Entotheonella.The ISME Journal (2008) 2, 335–339; doi:10.1038/ismej.2007.91; published online 7 February 2008 [ABSTRACT FROM AUTHOR]

Marine biotechnology is an applied science, the goal of which is to develop goods and services from marine organisms and processes. The new wave of marine biotechnology research began in the early 1980s and includes some significant success stories. A new drug to manage pain is commercially available, and a new cancer drug has been recommended for approval, the first from a fish-eating snail and the second from a mangrove tunicate. Enzymes from hydrothermal vent microbes are routinely used in PCR reactions, and marine-derived molecular probes are helping understand the molecular basis of disease processes. Advances in aquaculture biotechnology have resulted in more efficient production of finfish and shellfish for human consumption, and polyunsaturated fatty acids from marine microalgae are used as nutritional supplements for adults and infants. Rapid diagnostic tools have been developed to monitor toxins in the environment and in seafood, and genetic fingerprinting techniques are helping to control illegal trade of threatened marine species. In the future, multidisciplinary programs in oceans and human health should focus not only on microbial pathogens and harmful algal bloom toxins but also on discovery of new chemicals to prevent or treat human diseases. And the development of biological and biochemical sensors to detect pathogens, contaminants, and toxins and to monitor human and environmental health indicators in the marine environment should be a very high priority in the establishment of U.S. coastal ocean observing systems. [ABSTRACT FROM AUTHOR]

Abstract: Creatine kinase (CK) is a member of a family of phosphoryl transfer enzymes called phosphagen (guanidino) kinases which play a central role in cellular energy homeostasis. There are three CK isoform gene groups, each coding for proteins targeted to different intracellular compartments — cytoplasmic (CytCK), mitochondrial (MtCK) and flagellar (FlgCK). The former two CKs are either dimeric or octameric while FlgCKs are contiguous trimers consisting of three fused, complete CK domains. Conventional wisdom supports the view that CKs evolved from a cytoplasmic, monomeric ancestral protein closely related to a phosphagen kinase homologue, arginine kinase (AK). Recently, it has been shown that a demosponge (Phylum Porifera) expresses a true MtCK and two dimeric, protoflagellar CKs (protoflgCK) with great similarity to FlgCKs. To further probe the early evolution of CK, we have obtained additional sequences for Mt- and protoflgCKs from two more demosponges and from three hexactinellid (glass) sponges as well as an MtCK sequence from a basal metazoan cnidarian. Phylogenetic analyses using Maximum Likelihood (ML) of these new CK sequences with other CKs and phosphagen kinases yielded a consensus tree containing an assemblage of MtCKs and a supercluster consisting of protoflg-, Flg- and CytCKs. The MtCKs appear basal in the tree topology consistent with prior results. Within the protoflg-, Flg- and CytCK supercluster, the protoflgCKs appear to be allied to the domains of the FlgCKs, although the support is not robust. PCR amplification of genomic DNA and sequencing of the genes for Mt- and protoflgCK from the demosponge Suberites fuscus showed that the sponge MtCK shares four–five common intron:exon boundaries with invertebrate, protochordate and vertebrate MtCKs supporting a common ancestry and the extreme conservation of intron:exon organization in MtCK genes. The protoflgCK gene organization was highly divergent in relation to other CK genes but shares a common intron:exon boundary with domain 2 of the gene for the FlgCK from the tunicate Ciona intestinalis, providing support for the linkage of the protoflgCKs with the FlgCKs. Our results show that the two, major CK gene lineages are present in arguably the oldest, extant metazoan group, the hexactinellid sponges, indicating that these two genes are ancient and confirming prior work that the MtCK gene is likely basal and ancestral. [Copyright &y& Elsevier]

The article focuses on the study of deep-water Lophelia Pertusa coral reefs' habitat and fauna off the southeastern U.S. Several dives with human occupied submersible were done to document the habitat, benthic fauna and fish diversity of some of the poorly known deep water reefs. 58 fish species and 142 benthic invertebrae taxa were discovered. According to the study, habitats are ecologically diverse vulnerable to physical destruction and irreplaceable resources.

Abstract: Bacteria, microalgae and yeast less than 10 μm in size are the primary food source of sponges but their relative contribution to somatic growth is poorly understood. In a laboratory study, the sponge Halichondria melanadocia was fed for 6 weeks a diet consisting solely of four bacterial strains, or a mixed diet consisting of bacteria, microalgae and yeast. Both diets were fed at three concentrations, based on the natural concentration (NC) of particles available to sponges: 1/5, 1 and 5NC. Mean final size of H. melanadocia was 40% greater on a mixed diet than on the bacteria diet, probably because of the greater supply of carbon and other essential nutrients in microalgae and yeast. Cell concentration also significantly affected the growth of H. melanadocia, with greatest growth for sponges fed at the highest cell concentration. The estimated carbon requirement for H. melanadocia to meet metabolic costs was 0.356 mg C l−1 or 103 μg C h−1 gDW−1. Many H. melanadocia appeared to be optimizing their surface area for food uptake. [Copyright &y& Elsevier]

Cites a study which surveyed deep-water sinkholes and bioherms of south Florida. Description and characteristics of deep-water reefs; Species of fish and macrobenthic communities found in deep-water sinkholes and bioherms; Prevalence of deep-water, high-relief bioherms off the southeastern U.S.

Abstract: A taxonomic survey was conducted to determine the microbial diversity held within the Harbor Branch Oceanographic Marine Microbial Culture Collection (HBMMCC). The collection consists of approximately 17,000 microbial isolates, with 11,000 from a depth of greater than 150ft seawater. A total of 2273 heterotrophic bacterial isolates were inventoried using the DNA fingerprinting technique amplified rDNA restriction analysis on approximately 750–800 base pairs (bp) encompassing hypervariable regions in the 5′ portion of the small subunit (SSU) 16S rRNA gene. Restriction fragment length polymorphism patterns obtained from restriction digests with RsaI, HaeIII, and HhaI were used to infer taxonomic similarity. SSU 16S rDNA fragments were sequenced from a total of 356 isolates for more definitive taxonomic analysis. Sequence results show that this subset of the HBMMCC contains 224 different phylotypes from six major bacterial clades (Proteobacteria (Alpha, Beta, Gamma), Cytophaga, Flavobacteria, and Bacteroides (CFB), Gram+ high GC content, Gram+ low GC content). The 2273 microorganisms surveyed encompass 834 α-Proteobacteria (representing 60 different phylotypes), 25 β-Proteobacteria (3 phylotypes), 767 γ-Proteobacteria (77 phylotypes), 122 CFB (17 phylotypes), 327 Gram+ high GC content (43 phylotypes), and 198 Gram+ low GC content isolates (24 phylotypes). Notably, 11 phylotypes were ⩽93% similar to the closest sequence match in the GenBank database even after sequencing a larger portion of the 16S rRNA gene (∼1400bp), indicating the likely discovery of novel microbial taxa. Furthermore, previously reported “uncultured” microbes, such as sponge-specific isolates, are part of the HBMMCC. The results of this research will be available online as a searchable taxonomic database (www.hboi.edu/dbmr/dbmr_hbmmd.html). [Copyright &y& Elsevier]