Russell T. Hill

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile

ABSTRACT Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of theActinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria,Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.

Characterization of a Culturable Alphaproteobacterial Symbiont Common to Many Marine Sponges and Evidence for Vertical Transmission via Sponge Larvae

ABSTRACT A closely related group of alphaproteobacteria were found to be present in seven genera of marine sponges from several locations and were shown to be transferred between sponge generations through the larvae in one of these sponges. Isolates of the alphaproteobacterium were cultured from the sponges Axinella corrugata, Mycale laxissima, Monanchora unguifera, and Niphates digitalis from Key Largo, Florida; Didiscus oxeata and Monanchora unguifera from Discovery Bay, Jamaica; an Acanthostronglyophora sp. from Manado, Indonesia; and Microciona prolifera from the Cheasapeake Bay in Maryland. Isolates were very similar to each other on the basis of 16S rRNA gene sequence (>99% identity) and are closely related to Pseudovibrio denitrificans. The bacterium was never isolated from surrounding water samples and was cultured from larvae of M. laxissima, indicating that it is a vertically transmitted symbiont in this sponge. Denaturing gradient gel electrophoresis, 16S rRNA gene clone library analysis, and fluorescent in situ hybridization with probes specific to the alphaproteobacterium confirmed the presence of this bacterium in the M. laxissima larvae. The alphaproteobacterium was densely associated with the larvae rather than being evenly distributed throughout the mesohyl. This is the first report of the successful culture of a bacterial symbiont of a sponge that is transferred through the gametes.

Novel actinobacteria from marine sponges.

Actinobacteria exclusively within the sub-class Acidimicrobidae were shown by 16S rDNA community analysis to be major components of the bacterial community associated with two sponge species in the genus Xestospongia. Four groups of Actinobacteria were identified in Xestospongia spp., with three of these four groups being found in both Xestospongia muta from Key Largo, Florida and Xestospongia testudinaria from Manado, Indonesia. This suggests that these groups are true symbionts in these sponges and may play a common role in both the Pacific and Atlantic sponge species. The fourth group was found only in X. testudinaria and was a novel assemblage distantly related to any previously sequenced actinobacterial clones. The only actinobacteria that were obtained in initial culturing attempts were Gordonia, Micrococcus and Brachybacterium spp., none of which were represented in the clone libraries. The closest cultured actinobacteria to all the Acidimicrobidae clones from Xestospongia spp. are ‘Microthrix parvicella’ and Acidimicrobium spp. Xestospongia spp. can now be targeted as source material from which to culture novel Acidimicrobidae to investigate their potential as producers of bioactive compounds. Isolation of sponge-associated Acidimicrobidae will also make it possible to elucidate their role as sponge symbionts.

Diversity and expression of nitrogen fixation genes in bacterial symbionts of marine sponges

Marine sponges contain complex assemblages of bacterial symbionts, the roles of which remain largely unknown. We identified diverse bacterial nifH genes within sponges and found that nifH genes are expressed in sponges. This is the first demonstration of the expression of any protein-coding bacterial gene within a sponge. Two sponges Ircinia strobilina and Mycale laxissima were collected from Key Largo, Florida and had delta(15)N values of c. 0-1 per thousand and 3-4 per thousand respectively. The potential for nitrogen fixation by symbionts was assessed by amplification of nifH genes. Diverse nifH genes affiliated with Proteobacteria and Cyanobacteria were detected, and expression of nifH genes affiliated with those from cyanobacteria was detected. The nifH genes from surrounding seawater were similar to those of Trichodesmium and clearly different from the cyanobacterial nifH genes detected in the two sponges. This study advances understanding of the role of bacterial symbionts in sponges and suggests that provision of fixed nitrogen is a means whereby symbionts benefit sponges in nutrient-limited reef environments. Nitrogen fixation by sponge symbionts is possibly an important source of new nitrogen to the reef environment that heretofore has been neglected and warrants further investigation.

Diversity of aerobic and anaerobic ammonia-oxidizing bacteria in marine sponges

Aerobic ammonia-oxidizing bacteria (AAOB) are known to have an important function in the marine nitrogen cycle. Anaerobic ammonium oxidation (anammox) carried out by some members of Planctomycetales is also an important process in marine ecosystems. Ammonia-monooxygenase gene (amoA) fragments were amplified to investigate the potential for nitrification and the diversity of the AAOB in two marine sponges Ircinia strobilina and Mycale laxissima. All of the AmoA sequences obtained from the two sponges clustered with the AmoA sequences of the Betaproteobacteria Nitrosospira spp. To investigate the anaerobic ammonia-oxidizing bacteria (AnAOB) in sponges, 16S rRNA gene fragments of Planctomycetales and anammox bacteria were also amplified with specific primers, and clone libraries were constructed. The Planctomycetales diversity detected in the two sponges was different. The Planctomycetales community in M. laxissima was affiliated with Pirellula, Planctomyces and anammox bacteria, while all of the I. strobilina Planctomycetales clones were solely affiliated with the candidate phylum ‘Poribacteria’. Interestingly, sequences related to anammox genera were recovered only from M. laxissima. This is the first report of anammox bacteria in marine sponges. It is intriguing to find AAOB and AnAOB in M. laxissima, but the nature of their interaction with the sponge host and with each other remains unclear. This work further supports the potential of sponge-associated microorganisms for nitrification and sheds light on anammox as a new aspect of the nitrogen cycle in marine sponges.

New drugs from marine microbes: the tide is turning

This is a mini-review demonstrating that investigation of the genomics of marine microbes from all three domains has the potential to revolutionize the search for secondary metabolites originally thought to be the product of marine invertebrates. The basis for the review was a symposium at the 2005 Annual Meeting of the SIM covering some aspects of the potential for marine microbes to be the primary producers of such metabolites. The work reported at that symposium has been integrated into a fuller discussion of current published literature on the subject with examples drawn from bacteria, cyanophytes and fungi.

Nannochloropsis Genomes Reveal Evolution of Microalgal Oleaginous Traits

Oleaginous microalgae are promising feedstock for biofuels, yet the genetic diversity, origin and evolution of oleaginous traits remain largely unknown. Here we present a detailed phylogenomic analysis of five oleaginous Nannochloropsis species (a total of six strains) and one time-series transcriptome dataset for triacylglycerol (TAG) synthesis on one representative strain. Despite small genome sizes, high coding potential and relative paucity of mobile elements, the genomes feature small cores of ca. 2,700 protein-coding genes and a large pan-genome of >38,000 genes. The six genomes share key oleaginous traits, such as the enrichment of selected lipid biosynthesis genes and certain glycoside hydrolase genes that potentially shift carbon flux from chrysolaminaran to TAG synthesis. The eleven type II diacylglycerol acyltransferase genes (DGAT-2) in every strain, each expressed during TAG synthesis, likely originated from three ancient genomes, including the secondary endosymbiosis host and the engulfed green and red algae. Horizontal gene transfers were inferred in most lipid synthesis nodes with expanded gene doses and many glycoside hydrolase genes. Thus multiple genome pooling and horizontal genetic exchange, together with selective inheritance of lipid synthesis genes and species-specific gene loss, have led to the enormous genetic apparatus for oleaginousness and the wide genomic divergence among present-day Nannochloropsis. These findings have important implications in the screening and genetic engineering of microalgae for biofuels.

Enrichment, Isolation, and Phylogenetic Identification of Polycyclic Aromatic Hydrocarbon-Degrading Bacteria from Elizabeth River Sediments

ABSTRACT The diversity of indigenous bacteria in sediments from several sites in the Elizabeth River (Virginia) able to degrade multiple polycyclic aromatic hydrocarbons (PAHs) was investigated by the use of classical selective enrichment and molecular analyses. Enrichment cultures containing naphthalene, phenanthrene, fluoranthene, or pyrene as a sole carbon and energy source were monitored by denaturing gradient gel electrophoresis (DGGE) to detect changes in the bacterial-community profile during enrichment and to determine whether the representative strains present were successfully cultured. The DGGE profiles of the final enrichments grown solely on naphthalene and pyrene showed no clear relationship with the site from which the inoculum was obtained. The enrichments grown solely on pyrene for two sample sites had >80% similarity, which suggests that common pyrene-degrading strains may be present in these sediments. The final enrichments grown on fluoranthene and phenanthrene remained diverse by site, suggesting that these strains may be influenced by environmental conditions. One hundred and one isolates were obtained, comprising representatives of the actinomycetes and alpha-, beta-, and gammaproteobacteria, including seven novel isolates with 16S rRNA gene sequences less than 98% similar to known strains. The ability to degrade multiple PAHs was demonstrated by mineralization of 14C-labeled substrate and growth in pure culture. This supports our hypothesis that a high diversity of bacterial strains with the ability to degrade multiple PAHs can be confirmed by the combined use of classical selective enrichment and molecular analyses. This large collection of diverse PAH-degrading strains provides a valuable resource for studies on mechanisms of PAH degradation and bioremediation.

Diversity and quorum-sensing signal production of Proteobacteria associated with marine sponges

Marine sponges are hosts to diverse and dense bacterial communities and thus provide a potential environment for quorum sensing. Quorum sensing, a key factor in cell-cell communication and bacterial colonization of higher animals, might be involved in the symbiotic interactions between bacteria and their sponge hosts. Given that marine Proteobacteria are known to produce N-acyl homoserine lactone (AHL) signal molecules, we tested the production of AHLs by Alpha- and Gammaproteobacteria isolated from marine sponges Mycale laxissima and Ircinia strobilina and the surrounding water column. We used three different AHL biodetection systems in diffusion assays: Chromobacterium violaceum, Agrobacterium tumefaciens and Sinorhizobium meliloti with optimal sensitivity to short-chain (C4-C6), moderate-chain (C8-C12) and long-chain (>or= C14) AHLs respectively. Thirteen of 23 isolates from M. laxissima and five of 25 isolates from I. strobilina were found to produce AHLs. Signals were detected from two of eight proteobacterial strains from the water column. Thin-layer chromatographic assays based on the A. tumefaciens reporter system were utilized to determine the AHL profiles of the positive isolates. The types and amounts of AHLs synthesized varied considerably among the strains. Small ribosomal rRNA gene sequencing revealed that the AHL-producing alphaproteobacterial isolates were mainly from the Silicibacter-Ruegeria subgroup of the Roseobacter clade. Two-dimensional gel electrophoresis (2DGE)-based proteomic analyses were congruent with phylogenetic relationships but provided higher resolution to differentiate these closely related AHL-producing strains.

The expanding role of marine microbes in pharmaceutical development

Marine microbes have received growing attention as sources of bioactive metabolites and offer a unique opportunity to both increase the number of marine natural products in clinical trials as well as expedite their development. This review focuses specifically on those molecules currently in the clinical pipeline that are established or highly likely to be produced by bacteria based on expanding circumstantial evidence. We also include an example of how compounds from harmful algal blooms may yield both tools for measuring environmental change as well as leads for pharmaceutical development. An example of the karlotoxin class of compounds isolated from the dinoflagellate Karlodinium veneficum reveals a significant environmental impact in the form of massive fish kills, but also provides opportunities to construct new molecules for the control of cancer and serum cholesterol assisted by tools associated with rational drug design.