Many marine sponges harbor dense communities of microbes that aid in the chemical defense of thes... more Many marine sponges harbor dense communities of microbes that aid in the chemical defense of these nonmotile hosts. Metabolites that comprise this chemical arsenal can have pharmaceutically-relevant activities such as antibacterial, antiviral, antifungal and anticancer properties. Previous investigation of the Caribbean giant barrel sponge Xestospongia muta revealed a microbial community including novel Actinobacteria, a phylum well known for its production of antibiotic compounds. This novel assemblage was investigated for its ability to produce compounds that inhibit M. tuberculosis by using a bioinformatics approach. Microbial extracts were tested for their ability to inhibit growth of M. tb and genomes of the 11 strains that showed anti-M. tb activity including Micrococcus (n=2), Micromonospora (n=4), Streptomyces (n=3), and Brevibacterium spp. (n=2) were sequenced by using Illumina MiSeq. Three assembly algorithms/pipelines (SPAdes, A5-miseq and Shovill) were compared for their...
Coral reefs are the most biodiverse of all marine ecosystems. Bacteria are known to be abundant a... more Coral reefs are the most biodiverse of all marine ecosystems. Bacteria are known to be abundant and active in seawater around corals, inside coral tissues, and within their surface microlayer. Very little is known, however, about the structure, composition and maintenance of these bacterial communities. In the current study we characterize the culturable bacterial community within the mucus of healthy specimens of the Red Sea solitary coral Fungia scutaria. This was achieved using culture-based methods and molecular techniques for the identification of the bacterial isolates. More than 30% of the isolated bacteria were novel species and a new genus. The culturable heterotrophic bacterial community of the mucus of this coral is composed mainly of the bacterial groups Gammaproteobacteria, Alphaproteobacteria and of Actinobacteria. This study provides the first evidence of actinomycetes isolated from corals.
We report here the whole-genome sequencing results of two bacterial isolates, Aeromonas jandaei I... more We report here the whole-genome sequencing results of two bacterial isolates, Aeromonas jandaei IMET J and Cloacibacterium normanense IMET F, that inhibit (possibly due to denitrifying gene clusters) and promote (possibly due to an ammonification system), respectively, the growth of the microalgal strains Scenedesmus HTB1 and Chlorella vulgaris 1807.
Many fundamental advances in molecular biology have resulted from the study of interactions betwe... more Many fundamental advances in molecular biology have resulted from the study of interactions between bacteriophages and their bacterial hosts. These advances include confirmation that DNA is the carrier of genetic information (16), the discovery of messenger RNA as the intermediate between DNA and protein (15), and the discovery of restriction endonucleases (2), a prerequisite for the growth of genetic engineering and biotechnology. Several bacteriophages have been very intensively investigated at the molecular level, ...
Marine sponges are hosts to diverse and dense bacterial communities and thus provide a potential ... more 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.
Oleaginous microalgae are promising feedstock for biofuels, yet the genetic diversity, origin and... more 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.
Abstract : Funding was provided for U.S. participants in the 5th International Marine Biotechnolo... more Abstract : Funding was provided for U.S. participants in the 5th International Marine Biotechnology Conference held in Townsville, Australia from September 29 to October 5, 2000. Our funds were used for travel awards for U.S. participants and advanced participation by U.S. scientists in the important emerging area of marine biotechnology.
The antimalarial guided fractionation of the culture of marine Streptomyces sp. strain H668 led t... more The antimalarial guided fractionation of the culture of marine Streptomyces sp. strain H668 led to the isolation of a new polyether metabolite. The structure was determined by comprehensive NMR and MS assignments. This new metabolite showed in vitro antimalarial activity against both the chloroquine-susceptible (D6) and -resistant (W2) clones of Plasmodium falciparum, without cytotoxicity to normal cells (Vero) making it a promising first lead from this marine bacterium.
Copper is commonly used as an antifouling agent on ship hulls. Alteromonas spp. are early coloniz... more Copper is commonly used as an antifouling agent on ship hulls. Alteromonas spp. are early colonizers of copper-based antifouling paint, but their mechanism of tolerance is poorly understood. Sequencing of A. macleodii strains isolated from copper test materials for marine ships indicated the presence of multiple megaplasmids. Plasmids serve as key vectors in horizontal gene transfer and confer traits such as metal resistance, detoxification, ecological interaction, and antibiotic resistance. Bioinformatic analysis identified many metal resistance genes and genes associated with mobility. Understanding the molecular mechanisms and capacity for gene transfer within marine biofilms provides a platform for the development of novel antifouling solutions targeting genes involved in copper tolerance and biofilm formation.
<p>Bars indicate the mean number of pellets eaten (out of 10 pellets offered) for multiple ... more <p>Bars indicate the mean number of pellets eaten (out of 10 pellets offered) for multiple individuals of the given sponge at the given site. Error bars show standard error. In every case, 10/10 control pellets were consumed. The dotted line indicates a threshold for statistical significance as determined by a modified Fisher’s Exact Test; a sample is significantly deterrent (<i>p</i> < 0.05) if 6 or fewer pellets are consumed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174816#pone.0174816.ref044" target="_blank">44</a>]. The horizontal axis specifies species and growth form used to generate each extract: XDFL = <i>Xestospongia deweerdtae</i> free-living; XDA = <i>X</i>. <i>deweerdtae</i> associated; PL = <i>Plakortis deweerdtaephila</i>. The number of replicate individual sponges tested from each site is shown as a number overlaid on the bar. Tabulated data are archived in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174816#pone.0174816.s001" target="_blank">S1 Table</a>.</p
<p>The horizontal axis indicates polarity fractions, and different shades on bars represent... more <p>The horizontal axis indicates polarity fractions, and different shades on bars represent different concentrations of the extract fraction. Vertical axis and other details as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174816#pone.0174816.g001" target="_blank">Fig 1</a>.</p
cern in the world, as much of the population relies on groundwater as its major source of drinkin... more cern in the world, as much of the population relies on groundwater as its major source of drinking water as well as on soil as cultivable land. Heavy-metal contamination brings a potential health harzard that can cause metal toxicoses in animals and humans (Volesky and Holan, 1995). Trace elements such as cadmium, copper, and mercury are very toxic heavy metals and have been found in the human environment at increased concentrations, because a wide variety of industrial activities have accelerated the release of these metals at higher rates than natural geochemical cycling processes can tolerate (Nriagu and Pacyma, 1988). Automobile and leather factories, and sugar mills located in Tucumán, a northwestern state of Argentine, are potential sources of effluent contamination of aquifers and rivers. Salí is one of the most important rivers of Tucumán. The Salí River flows to the Río Hondo reservoir in northeast Argentina. This reservoir is a source of drinking water, irrigation and fish...
The culturability of Vibrio cholerae O1 serotype Inaba strain 569B was decreased by the addition ... more The culturability of Vibrio cholerae O1 serotype Inaba strain 569B was decreased by the addition of glucose to cell suspensions in starvation media. A similar effect was observed with sucrose, maltose, and fructose. We term this inhibitory effect glucose shock. It was not observed with arabinose or xylose or with carboxylates, such as acetate and pyruvate. No acidification of the medium occurred in the presence of these carbohydrates. Glucose shock was prevented by the addition of nitrogen or phosphorus sources. In the presence of phosphate, the bacterium produced formic acid from glucose. The phenomenon of glucose shock was also observed in V. cholerae O1 serotype Inaba strain RIMD 2203082 but not in strain RIMD 2203088 (O1 Inaba), IID 936 (O1 Ogawa), or RIMD 2214034 (non-O1). The culturability of Escherichia coli, Enterobacter aerogenes, and Listonella anguillarum did not decrease in starvation media with added glucose. Hence, the phenomenon should have ecological significance in ...
F1000 - Post-publication peer review of the biomedical literature, 2006
The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Ocu... more The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Oculina patagonica was determined by analyses of the 16S rRNA genes of isolated colonies and from a 16S rRNA clone library of extracted DNA. By SYBR gold staining, the numbers of bacteria in mucus and tissue samples were 6.2 ؋ 10 7 and 8.3 ؋ 10 8 /cm 2 of coral surface, respectively, 99.8% of which failed to produce colonies on Marine Agar. From analysis of mucus DNA, the most-abundant bacterium was Vibrio splendidus, representing 68% and 50% of the clones from the winter and summer, respectively. After removal of mucus from coral by centrifugation, analyses of DNA from the crushed tissue revealed a large diversity of bacteria, with Vibrio species representing less than 5% of the clones. The most-abundant culturable bacteria were a Pseudomonas sp. (8 to 14%) and two different ␣-proteobacteria (6 to 18%). Out of a total 1,088 16S rRNA genes sequenced, 400 different operational taxonomic units were identified (>99.5% identity). Of these, 295 were novel (<99% identical to any sequences in the GenBank database). This study provides a comprehensive database for future examinations of changes in the bacterial community during bleaching events.
F1000 - Post-publication peer review of the biomedical literature, 2011
The vast majority of bacterial species remain uncultured, and this severely limits the investigat... more The vast majority of bacterial species remain uncultured, and this severely limits the investigation of their physiology, metabolic capabilities, and role in the environment. High-throughput sequencing of RNA transcripts (RNA-seq) allows the investigation of the diverse physiologies from uncultured microorganisms in their natural habitat. Here, we report the use of RNAseq for characterizing the metatranscriptome of the simple gut microbiome from the medicinal leech Hirudo verbana and for utilizing this information to design a medium for cultivating members of the microbiome. Expression data suggested that a Rikenella-like bacterium, the most abundant but uncultured symbiont, forages on sulfated-and sialated-mucin glycans that are fermented, leading to the secretion of acetate. Histological stains were consistent with the presence of sulfated and sialated mucins along the crop epithelium. The second dominant symbiont, Aeromonas veronii, grows in two different microenvironments and is predicted to utilize either acetate or carbohydrates. Based on the metatranscriptome, a medium containing mucin was designed, which enabled the cultivation of the Rikenella-like bacterium. Metatranscriptomes shed light on microbial metabolism in situ and provide critical clues for directing the culturing of uncultured microorganisms. By choosing a condition under which the desired organism is rapidly proliferating and focusing on highly expressed genes encoding hydrolytic enzymes, binding proteins, and transporters, one can identify an organism's nutritional preferences and design a culture medium. IMPORTANCE The number of prokaryotes on the planet has been estimated to exceed 10 30 cells, and the overwhelming majority of them have evaded cultivation, making it difficult to investigate their ecological, medical, and industrial relevance. The application of transcriptomics based on high-throughput sequencing of RNA transcripts (RNA-seq) to microorganisms in their natural environment can provide investigators with insight into their physiologies under optimal growth conditions. We utilized RNAseq to learn more about the uncultured and cultured symbionts that comprise the relatively simple digestive-tract microbiome of the medicinal leech. The expression data revealed highly expressed hydrolytic enzymes and transporters that provided critical clues for the design of a culture medium enabling the isolation of the previously uncultured Rikenella-like symbiont. This directed culturing method will greatly aid efforts aimed at understanding uncultured microorganisms, including beneficial symbionts, pathogens, and ecologically relevant microorganisms, by facilitating genome sequencing, physiological characterization, and genetic manipulation of the previously uncultured microbes.
Pigment was produced by Escherichia coli cells carrying recombinant plasmids pNILl00, pNIL200 and... more Pigment was produced by Escherichia coli cells carrying recombinant plasmids pNILl00, pNIL200 and pNIL400 containing DNA from Rhodococcus sp. E. coli cells containing pNILlOO or pNIL200 (with DNA inserts from Rhodococcus sp. JLlO and Rhodococcus sp. ATCC 21 145 respectively) produced both blue and pink pigments, while cells containing pNIL400 (with a DNA insert from Rhodococcus sp. ATCC 21 145) produced only pink pigment. Colonies of E. coli(pNIL100) and E. coli(pNIL200) were dark blue, whereas E. coli(pNIL400) colonies were pink. No pigment was detected in Streptomyces griseus transformants containing pNILl00, pNIL200 or pNIL400. Restriction endonuclease mapping indicated that the cloned DNA fragments were different. The pigment gene(s) in pNIL200 producing both the blue and pink pigments were contained within a 2.8 kb DNA fragment. The pigments produced by E. coli transformants containing pNIL200 were characterized by visible and UV spectroscopy. No similar pigments were detected in Rhodococcus sp. ATCC 21 145. 0001-5019 0 1989 SGM Hill et al. (1 989) This study This study This study This study This study This study This study Vieira & Messing (1982) Vieira & Messing (1982) This study This study METHODS Bacteria, plasmids and growth conditions. The bacterial strains and plasmids used are listed in Table 1. Plasmids coding for pigment production were designated Pig+. E. coli cells were grown at 37 "C in Luria-Bertani (LB) medium (Maniatis et al., 1982). Rhodococcus strains were grown in LB medium at 30 "C. Rhodococcus sp. JLlO and Rhodococcus sp. ATCC 21 145 were previously classified as Nocardia corallina JLlO and Nocardia sp. ATCC 21 145 respectively. Developments in nocardioform taxonomy (Goodfellow & Alderson, 1977 ; Goodfellow & Cross, 1984) make it clear that both strains belong to the genus Rhodococcus not Nocardia. Rhodococcus sp. JL10, which forms pink to coral red colonies, was isolated from soil and harbours a 2-7 kb plasmid designated pKUlOO (Kirby & Usdin, 1985). Rhodococcus sp. ATCC 21 145 forms buff-coloured colonies and is described by Raymond (1971) in a patent for the production of hydroxyphenylketobutyric acids by the microbial oxidation of naphthalene. Preparation of DNA. Rhodococcus chromosomal DNA was prepared as described by Hopwood e f al. (1985) for Strepfomyces total DNA (procedure 1). The positive selection Streptomyces-E. coli shuttle vector pLR591 (Hill et al., 1989) was prepared from E. coli K51412 by the method of Ish-Horowicz & Burke (1981) and purified by CsCl equilibrium gradient centrifugation. This procedure was also used for the preparation of recombinant plasmids coding for pigment production. Restriction endonucleases (Boehringer-Mannheim) were used according to the conditions of Maniatis et al. (1982). Ligations were performed with T4 DNA ligase (Boehringer-Mannheim) according to the manufacturer's instructions, using the ligation buffer of King & Blakesley (1986). Construction of Rhodococcusgenomic libraries. Chromosomal DNA of Rhodococcus sp. JL 10 and Rhodococcus sp. ATCC 21 145 was partially digested with Sau3A endonuclease and DNA fragments of 5-10 kb were obtained by sucrose gradient centrifugation. The vector pLR591 was digested with BglII endonuclease ; vector and insert DNA were mixed in ratios between 1 : 1 and 50 : 1 at a final DNA concentration of 0.1-3 pg pl-l. Competent E. coli DK1 and E. coli LKl 11 cells were transformed by the method of Cohen et al. (1972) as modified by Dagert & Ehrlich (1979). E. coli transformants were expressed at 42 "C in LB medium with vigorous shaking for 1 h and plated on LB agar containing 100 pg ampicillin ml-l. The expression step ensures efficient killing of E. coli transformants containing pLR591 plasmids without inserts as the intact lethal EcoRI gene is efficiently expressed at 42 "C in these parental plasmids. Plasmids containing DNA inserts in the BglII cloning site within the EcoRI gene will not code for EcoRI endonuclease and cells containing recombinant plasmids will therefore survive (Hill et al., 1989). Southern blot hybridization. PstI endonuclease digested chromosomal DNA from Rhodococcus sp. JLlO and Rhodococcus sp. ATCC 21 145 was separated by gel electrophoresis (Maniatis et al., 1982), transferred to filters by blotting (Southern, 1975), and dried at 80 "C for 2 h under vacuum. Plasmids pNILlOO and pNIL200, nicktranslated with [cr-32P]dATP (Amersham), were used as hybridization probes. Blotted filters were prehybridized for 2 h at 65 "C in a solution containing 2 x SSC, 0.1 % (w/v) SDS, 1 x Denhardt's solution and 100 pg denatured herring sperm DNA ml-1 (1 x SSC is 0.15 M-NaC1, 0.015 M-trisodium citrate, pH 7.0; 1 x Denhardt's solution is
Marine sponges are an extremely rich and important source of natural products. Mariculture is one... more Marine sponges are an extremely rich and important source of natural products. Mariculture is one solution to the so-called "supply problem" that often hampers further studies and development of novel compounds from sponges. We report the extended culture (767 days) at sea in depths of 10 and 20 m of three sponge species: Negombata magnifica, Amphimedon chloros and Theonella swinhoei that produce latrunculin-B, halitoxin and swinholide-A, respectively. Since sponge-associated microorganisms may be the true producers of many of the natural products found in sponges and also be linked to the health of the sponges, we examined the stability of the bacterial communities in cultured versus wild sponges. Growth rate of the sponges (ranging from 308 to 61 and −19 (%)(year −1) in N. magnifica, A. chloros and T. swinhoei, respectively) differed significantly between species but not between the two depths at which the species were cultivated. Survivorship varied from 96% to 57%. During culture all species maintained the content of the desired natural product. Denaturing gradient gel electrophoresis analysis of the sponge-associated bacterial consortia revealed that differences existed between cultured and wild sponges in T. swinhoei and A. chloros but the communities remained quite stable OPEN ACCESS Mar. Drugs 2011, 9 2202 in N. magnifica. The cultivation technique for production of natural products was found to be most appropriate for N. magnifica, while for T. swinhoei and A. chloros it was less successful, because of poorer growth and survival rates and shifts in their bacterial consortia.
Many marine sponges harbor dense communities of microbes that aid in the chemical defense of thes... more Many marine sponges harbor dense communities of microbes that aid in the chemical defense of these nonmotile hosts. Metabolites that comprise this chemical arsenal can have pharmaceutically-relevant activities such as antibacterial, antiviral, antifungal and anticancer properties. Previous investigation of the Caribbean giant barrel sponge Xestospongia muta revealed a microbial community including novel Actinobacteria, a phylum well known for its production of antibiotic compounds. This novel assemblage was investigated for its ability to produce compounds that inhibit M. tuberculosis by using a bioinformatics approach. Microbial extracts were tested for their ability to inhibit growth of M. tb and genomes of the 11 strains that showed anti-M. tb activity including Micrococcus (n=2), Micromonospora (n=4), Streptomyces (n=3), and Brevibacterium spp. (n=2) were sequenced by using Illumina MiSeq. Three assembly algorithms/pipelines (SPAdes, A5-miseq and Shovill) were compared for their...
Coral reefs are the most biodiverse of all marine ecosystems. Bacteria are known to be abundant a... more Coral reefs are the most biodiverse of all marine ecosystems. Bacteria are known to be abundant and active in seawater around corals, inside coral tissues, and within their surface microlayer. Very little is known, however, about the structure, composition and maintenance of these bacterial communities. In the current study we characterize the culturable bacterial community within the mucus of healthy specimens of the Red Sea solitary coral Fungia scutaria. This was achieved using culture-based methods and molecular techniques for the identification of the bacterial isolates. More than 30% of the isolated bacteria were novel species and a new genus. The culturable heterotrophic bacterial community of the mucus of this coral is composed mainly of the bacterial groups Gammaproteobacteria, Alphaproteobacteria and of Actinobacteria. This study provides the first evidence of actinomycetes isolated from corals.
We report here the whole-genome sequencing results of two bacterial isolates, Aeromonas jandaei I... more We report here the whole-genome sequencing results of two bacterial isolates, Aeromonas jandaei IMET J and Cloacibacterium normanense IMET F, that inhibit (possibly due to denitrifying gene clusters) and promote (possibly due to an ammonification system), respectively, the growth of the microalgal strains Scenedesmus HTB1 and Chlorella vulgaris 1807.
Many fundamental advances in molecular biology have resulted from the study of interactions betwe... more Many fundamental advances in molecular biology have resulted from the study of interactions between bacteriophages and their bacterial hosts. These advances include confirmation that DNA is the carrier of genetic information (16), the discovery of messenger RNA as the intermediate between DNA and protein (15), and the discovery of restriction endonucleases (2), a prerequisite for the growth of genetic engineering and biotechnology. Several bacteriophages have been very intensively investigated at the molecular level, ...
Marine sponges are hosts to diverse and dense bacterial communities and thus provide a potential ... more 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 (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;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.
Oleaginous microalgae are promising feedstock for biofuels, yet the genetic diversity, origin and... more 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.
Abstract : Funding was provided for U.S. participants in the 5th International Marine Biotechnolo... more Abstract : Funding was provided for U.S. participants in the 5th International Marine Biotechnology Conference held in Townsville, Australia from September 29 to October 5, 2000. Our funds were used for travel awards for U.S. participants and advanced participation by U.S. scientists in the important emerging area of marine biotechnology.
The antimalarial guided fractionation of the culture of marine Streptomyces sp. strain H668 led t... more The antimalarial guided fractionation of the culture of marine Streptomyces sp. strain H668 led to the isolation of a new polyether metabolite. The structure was determined by comprehensive NMR and MS assignments. This new metabolite showed in vitro antimalarial activity against both the chloroquine-susceptible (D6) and -resistant (W2) clones of Plasmodium falciparum, without cytotoxicity to normal cells (Vero) making it a promising first lead from this marine bacterium.
Copper is commonly used as an antifouling agent on ship hulls. Alteromonas spp. are early coloniz... more Copper is commonly used as an antifouling agent on ship hulls. Alteromonas spp. are early colonizers of copper-based antifouling paint, but their mechanism of tolerance is poorly understood. Sequencing of A. macleodii strains isolated from copper test materials for marine ships indicated the presence of multiple megaplasmids. Plasmids serve as key vectors in horizontal gene transfer and confer traits such as metal resistance, detoxification, ecological interaction, and antibiotic resistance. Bioinformatic analysis identified many metal resistance genes and genes associated with mobility. Understanding the molecular mechanisms and capacity for gene transfer within marine biofilms provides a platform for the development of novel antifouling solutions targeting genes involved in copper tolerance and biofilm formation.
<p>Bars indicate the mean number of pellets eaten (out of 10 pellets offered) for multiple ... more <p>Bars indicate the mean number of pellets eaten (out of 10 pellets offered) for multiple individuals of the given sponge at the given site. Error bars show standard error. In every case, 10/10 control pellets were consumed. The dotted line indicates a threshold for statistical significance as determined by a modified Fisher’s Exact Test; a sample is significantly deterrent (<i>p</i> < 0.05) if 6 or fewer pellets are consumed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174816#pone.0174816.ref044" target="_blank">44</a>]. The horizontal axis specifies species and growth form used to generate each extract: XDFL = <i>Xestospongia deweerdtae</i> free-living; XDA = <i>X</i>. <i>deweerdtae</i> associated; PL = <i>Plakortis deweerdtaephila</i>. The number of replicate individual sponges tested from each site is shown as a number overlaid on the bar. Tabulated data are archived in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174816#pone.0174816.s001" target="_blank">S1 Table</a>.</p
<p>The horizontal axis indicates polarity fractions, and different shades on bars represent... more <p>The horizontal axis indicates polarity fractions, and different shades on bars represent different concentrations of the extract fraction. Vertical axis and other details as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174816#pone.0174816.g001" target="_blank">Fig 1</a>.</p
cern in the world, as much of the population relies on groundwater as its major source of drinkin... more cern in the world, as much of the population relies on groundwater as its major source of drinking water as well as on soil as cultivable land. Heavy-metal contamination brings a potential health harzard that can cause metal toxicoses in animals and humans (Volesky and Holan, 1995). Trace elements such as cadmium, copper, and mercury are very toxic heavy metals and have been found in the human environment at increased concentrations, because a wide variety of industrial activities have accelerated the release of these metals at higher rates than natural geochemical cycling processes can tolerate (Nriagu and Pacyma, 1988). Automobile and leather factories, and sugar mills located in Tucumán, a northwestern state of Argentine, are potential sources of effluent contamination of aquifers and rivers. Salí is one of the most important rivers of Tucumán. The Salí River flows to the Río Hondo reservoir in northeast Argentina. This reservoir is a source of drinking water, irrigation and fish...
The culturability of Vibrio cholerae O1 serotype Inaba strain 569B was decreased by the addition ... more The culturability of Vibrio cholerae O1 serotype Inaba strain 569B was decreased by the addition of glucose to cell suspensions in starvation media. A similar effect was observed with sucrose, maltose, and fructose. We term this inhibitory effect glucose shock. It was not observed with arabinose or xylose or with carboxylates, such as acetate and pyruvate. No acidification of the medium occurred in the presence of these carbohydrates. Glucose shock was prevented by the addition of nitrogen or phosphorus sources. In the presence of phosphate, the bacterium produced formic acid from glucose. The phenomenon of glucose shock was also observed in V. cholerae O1 serotype Inaba strain RIMD 2203082 but not in strain RIMD 2203088 (O1 Inaba), IID 936 (O1 Ogawa), or RIMD 2214034 (non-O1). The culturability of Escherichia coli, Enterobacter aerogenes, and Listonella anguillarum did not decrease in starvation media with added glucose. Hence, the phenomenon should have ecological significance in ...
F1000 - Post-publication peer review of the biomedical literature, 2006
The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Ocu... more The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Oculina patagonica was determined by analyses of the 16S rRNA genes of isolated colonies and from a 16S rRNA clone library of extracted DNA. By SYBR gold staining, the numbers of bacteria in mucus and tissue samples were 6.2 ؋ 10 7 and 8.3 ؋ 10 8 /cm 2 of coral surface, respectively, 99.8% of which failed to produce colonies on Marine Agar. From analysis of mucus DNA, the most-abundant bacterium was Vibrio splendidus, representing 68% and 50% of the clones from the winter and summer, respectively. After removal of mucus from coral by centrifugation, analyses of DNA from the crushed tissue revealed a large diversity of bacteria, with Vibrio species representing less than 5% of the clones. The most-abundant culturable bacteria were a Pseudomonas sp. (8 to 14%) and two different ␣-proteobacteria (6 to 18%). Out of a total 1,088 16S rRNA genes sequenced, 400 different operational taxonomic units were identified (>99.5% identity). Of these, 295 were novel (<99% identical to any sequences in the GenBank database). This study provides a comprehensive database for future examinations of changes in the bacterial community during bleaching events.
F1000 - Post-publication peer review of the biomedical literature, 2011
The vast majority of bacterial species remain uncultured, and this severely limits the investigat... more The vast majority of bacterial species remain uncultured, and this severely limits the investigation of their physiology, metabolic capabilities, and role in the environment. High-throughput sequencing of RNA transcripts (RNA-seq) allows the investigation of the diverse physiologies from uncultured microorganisms in their natural habitat. Here, we report the use of RNAseq for characterizing the metatranscriptome of the simple gut microbiome from the medicinal leech Hirudo verbana and for utilizing this information to design a medium for cultivating members of the microbiome. Expression data suggested that a Rikenella-like bacterium, the most abundant but uncultured symbiont, forages on sulfated-and sialated-mucin glycans that are fermented, leading to the secretion of acetate. Histological stains were consistent with the presence of sulfated and sialated mucins along the crop epithelium. The second dominant symbiont, Aeromonas veronii, grows in two different microenvironments and is predicted to utilize either acetate or carbohydrates. Based on the metatranscriptome, a medium containing mucin was designed, which enabled the cultivation of the Rikenella-like bacterium. Metatranscriptomes shed light on microbial metabolism in situ and provide critical clues for directing the culturing of uncultured microorganisms. By choosing a condition under which the desired organism is rapidly proliferating and focusing on highly expressed genes encoding hydrolytic enzymes, binding proteins, and transporters, one can identify an organism's nutritional preferences and design a culture medium. IMPORTANCE The number of prokaryotes on the planet has been estimated to exceed 10 30 cells, and the overwhelming majority of them have evaded cultivation, making it difficult to investigate their ecological, medical, and industrial relevance. The application of transcriptomics based on high-throughput sequencing of RNA transcripts (RNA-seq) to microorganisms in their natural environment can provide investigators with insight into their physiologies under optimal growth conditions. We utilized RNAseq to learn more about the uncultured and cultured symbionts that comprise the relatively simple digestive-tract microbiome of the medicinal leech. The expression data revealed highly expressed hydrolytic enzymes and transporters that provided critical clues for the design of a culture medium enabling the isolation of the previously uncultured Rikenella-like symbiont. This directed culturing method will greatly aid efforts aimed at understanding uncultured microorganisms, including beneficial symbionts, pathogens, and ecologically relevant microorganisms, by facilitating genome sequencing, physiological characterization, and genetic manipulation of the previously uncultured microbes.
Pigment was produced by Escherichia coli cells carrying recombinant plasmids pNILl00, pNIL200 and... more Pigment was produced by Escherichia coli cells carrying recombinant plasmids pNILl00, pNIL200 and pNIL400 containing DNA from Rhodococcus sp. E. coli cells containing pNILlOO or pNIL200 (with DNA inserts from Rhodococcus sp. JLlO and Rhodococcus sp. ATCC 21 145 respectively) produced both blue and pink pigments, while cells containing pNIL400 (with a DNA insert from Rhodococcus sp. ATCC 21 145) produced only pink pigment. Colonies of E. coli(pNIL100) and E. coli(pNIL200) were dark blue, whereas E. coli(pNIL400) colonies were pink. No pigment was detected in Streptomyces griseus transformants containing pNILl00, pNIL200 or pNIL400. Restriction endonuclease mapping indicated that the cloned DNA fragments were different. The pigment gene(s) in pNIL200 producing both the blue and pink pigments were contained within a 2.8 kb DNA fragment. The pigments produced by E. coli transformants containing pNIL200 were characterized by visible and UV spectroscopy. No similar pigments were detected in Rhodococcus sp. ATCC 21 145. 0001-5019 0 1989 SGM Hill et al. (1 989) This study This study This study This study This study This study This study Vieira & Messing (1982) Vieira & Messing (1982) This study This study METHODS Bacteria, plasmids and growth conditions. The bacterial strains and plasmids used are listed in Table 1. Plasmids coding for pigment production were designated Pig+. E. coli cells were grown at 37 "C in Luria-Bertani (LB) medium (Maniatis et al., 1982). Rhodococcus strains were grown in LB medium at 30 "C. Rhodococcus sp. JLlO and Rhodococcus sp. ATCC 21 145 were previously classified as Nocardia corallina JLlO and Nocardia sp. ATCC 21 145 respectively. Developments in nocardioform taxonomy (Goodfellow & Alderson, 1977 ; Goodfellow & Cross, 1984) make it clear that both strains belong to the genus Rhodococcus not Nocardia. Rhodococcus sp. JL10, which forms pink to coral red colonies, was isolated from soil and harbours a 2-7 kb plasmid designated pKUlOO (Kirby & Usdin, 1985). Rhodococcus sp. ATCC 21 145 forms buff-coloured colonies and is described by Raymond (1971) in a patent for the production of hydroxyphenylketobutyric acids by the microbial oxidation of naphthalene. Preparation of DNA. Rhodococcus chromosomal DNA was prepared as described by Hopwood e f al. (1985) for Strepfomyces total DNA (procedure 1). The positive selection Streptomyces-E. coli shuttle vector pLR591 (Hill et al., 1989) was prepared from E. coli K51412 by the method of Ish-Horowicz & Burke (1981) and purified by CsCl equilibrium gradient centrifugation. This procedure was also used for the preparation of recombinant plasmids coding for pigment production. Restriction endonucleases (Boehringer-Mannheim) were used according to the conditions of Maniatis et al. (1982). Ligations were performed with T4 DNA ligase (Boehringer-Mannheim) according to the manufacturer's instructions, using the ligation buffer of King & Blakesley (1986). Construction of Rhodococcusgenomic libraries. Chromosomal DNA of Rhodococcus sp. JL 10 and Rhodococcus sp. ATCC 21 145 was partially digested with Sau3A endonuclease and DNA fragments of 5-10 kb were obtained by sucrose gradient centrifugation. The vector pLR591 was digested with BglII endonuclease ; vector and insert DNA were mixed in ratios between 1 : 1 and 50 : 1 at a final DNA concentration of 0.1-3 pg pl-l. Competent E. coli DK1 and E. coli LKl 11 cells were transformed by the method of Cohen et al. (1972) as modified by Dagert & Ehrlich (1979). E. coli transformants were expressed at 42 "C in LB medium with vigorous shaking for 1 h and plated on LB agar containing 100 pg ampicillin ml-l. The expression step ensures efficient killing of E. coli transformants containing pLR591 plasmids without inserts as the intact lethal EcoRI gene is efficiently expressed at 42 "C in these parental plasmids. Plasmids containing DNA inserts in the BglII cloning site within the EcoRI gene will not code for EcoRI endonuclease and cells containing recombinant plasmids will therefore survive (Hill et al., 1989). Southern blot hybridization. PstI endonuclease digested chromosomal DNA from Rhodococcus sp. JLlO and Rhodococcus sp. ATCC 21 145 was separated by gel electrophoresis (Maniatis et al., 1982), transferred to filters by blotting (Southern, 1975), and dried at 80 "C for 2 h under vacuum. Plasmids pNILlOO and pNIL200, nicktranslated with [cr-32P]dATP (Amersham), were used as hybridization probes. Blotted filters were prehybridized for 2 h at 65 "C in a solution containing 2 x SSC, 0.1 % (w/v) SDS, 1 x Denhardt's solution and 100 pg denatured herring sperm DNA ml-1 (1 x SSC is 0.15 M-NaC1, 0.015 M-trisodium citrate, pH 7.0; 1 x Denhardt's solution is
Marine sponges are an extremely rich and important source of natural products. Mariculture is one... more Marine sponges are an extremely rich and important source of natural products. Mariculture is one solution to the so-called "supply problem" that often hampers further studies and development of novel compounds from sponges. We report the extended culture (767 days) at sea in depths of 10 and 20 m of three sponge species: Negombata magnifica, Amphimedon chloros and Theonella swinhoei that produce latrunculin-B, halitoxin and swinholide-A, respectively. Since sponge-associated microorganisms may be the true producers of many of the natural products found in sponges and also be linked to the health of the sponges, we examined the stability of the bacterial communities in cultured versus wild sponges. Growth rate of the sponges (ranging from 308 to 61 and −19 (%)(year −1) in N. magnifica, A. chloros and T. swinhoei, respectively) differed significantly between species but not between the two depths at which the species were cultivated. Survivorship varied from 96% to 57%. During culture all species maintained the content of the desired natural product. Denaturing gradient gel electrophoresis analysis of the sponge-associated bacterial consortia revealed that differences existed between cultured and wild sponges in T. swinhoei and A. chloros but the communities remained quite stable OPEN ACCESS Mar. Drugs 2011, 9 2202 in N. magnifica. The cultivation technique for production of natural products was found to be most appropriate for N. magnifica, while for T. swinhoei and A. chloros it was less successful, because of poorer growth and survival rates and shifts in their bacterial consortia.
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