Search

Elevated seawater temperatures have contributed to the rise of coral disease mediated by bacterial pathogens, such as the globally distributed Vibrio coralliilyticus, which utilizes coral mucus as a chemical cue to locate stressed corals. However, the physiological events in the pathogens that follow their entry into the coral host environment remain unknown. Here, we present simultaneous measurements of the behavioral and transcriptional responses of V. coralliilyticus BAA-450 incubated in coral mucus. Video microscopy revealed a strong and rapid chemokinetic behavioral response by the pathogen, characterized by a two-fold increase in average swimming speed within 6 min of coral mucus exposure. RNA sequencing showed that this bacterial behavior was accompanied by an equally rapid differential expression of 53% of the genes in the V. coralliilyticus genome. Specifically, transcript abundance 10 min after mucus exposure showed upregulation of genes involved in quorum sensing, biofilm formation, and nutrient metabolism, and downregulation of flagella synthesis and chemotaxis genes. After 60 min, we observed upregulation of genes associated with virulence, including zinc metalloproteases responsible for causing coral tissue damage and algal symbiont photoinactivation, and secretion systems that may export toxins. Together, our results suggest that V. coralliilyticus employs a suite of behavioral and transcriptional responses to rapidly shift into a distinct infection mode within minutes of exposure to the coral microenvironment., The ISME Journal, 15 (12), ISSN:1751-7362, ISSN:1751-7370

Infectious diseases are one of the major drivers of coral reef decline worldwide. White plague-like disease (WPL) is a widespread disease with a complex etiology that infects several coral species, including the Brazilian endemic species Mussismilia braziliensis. Gene expression profiles of healthy and WPL-affected M. braziliensis were analyzed in winter and summer seasons. The de novo assembly of the M. braziliensis transcriptome from healthy and white plague samples produced a reference transcriptome containing 119,088 transcripts. WPL-diseased samples were characterized by repression of immune system and cellular defense processes. Autophagy and cellular adhesion transcripts were also repressed in WPL samples, suggesting exhaustion of the coral host defenses. Seasonal variation leads to plasticity in transcription with upregulation of intracellular signal transduction, apoptosis regulation, and oocyte development in the summer. Analysis of the active bacterial rRNA indicated that Pantoea bacteria were more abundant in WPL corals, while Tistlia, Fulvivirga, and Gammaproteobacteria Ga0077536 were more abundant in healthy samples. Cyanobacteria proliferation was also observed in WPL, mostly in the winter. These results indicate a scenario of dysbiosis in WPL-affected M. braziliensis, with the loss of potentially symbiotic bacteria and proliferation of opportunistic microbes after the start of the infection process.

Marine actinomycetes in the genus Salinispora fail to grow when seawater is replaced with deionized (DI) water in complex growth media. While bioinformatic analyses have led to the identification of a number of candidate marine adaptation genes, there is currently no experimental evidence to support the genetic basis for the osmotic requirements associated with this taxon. One hypothesis is that the lineage-specific loss of mscL is responsible for the failure of strains to grow in media prepared with DI water. The mscL gene encodes a conserved transmembrane protein that reduces turgor pressure under conditions of acute osmotic downshock. In the present study, the mscL gene from a Micromonospora strain capable of growth on media prepared with DI water was transformed into S. tropica strain CNB-440. The single-copy, chromosomal genetic complementation yielded a recombinant Salinispora mscL + strain that demonstrated an increased capacity to survive osmotic downshock. The enhanced survival of the S. tropica transformant provides experimental evidence that the loss of mscL is associated with the failure of Salinispora spp. to grow in low-osmotic-strength media.

The subsurface microbiology of an Athabasca oil sands reservoir in western Canada containing severely biodegraded oil was investigated by combining 16S rRNA gene- and polar lipid-based analyses of reservoir formation water with geochemical analyses of the crude oil and formation water. Biomass was filtered from formation water, DNA was extracted using two different methods, and 16S rRNA gene fragments were amplified with several different primer pairs prior to cloning and sequencing or community fingerprinting by denaturing gradient gel electrophoresis (DGGE). Similar results were obtained irrespective of the DNA extraction method or primers used. Archaeal libraries were dominated by Methanomicrobiales (410 of 414 total sequences formed a dominant phylotype affiliated with a Methanoregula sp.), consistent with the proposed dominant role of CO2-reducing methanogens in crude oil biodegradation. In two bacterial 16S rRNA clone libraries generated with different primer pairs, > 99% and 100% of the sequences were affiliated with Epsilonproteobacteria (n = 382 and 72 total clones respectively). This massive dominance of Epsilonproteobacteria sequences was again obtained in a third library (99% of sequences; n = 96 clones) using a third universal bacterial primer pair (inosine-341f and 1492r). Sequencing of bands from DGGE profiles and intact polar lipid analyses were in accordance with the bacterial clone library results. Epsilonproteobacterial OTUs were affiliated with Sulfuricurvum, Arcobacter and Sulfurospirillum spp. detected in other oil field habitats. The dominant organism revealed by the bacterial libraries (87% of all sequences) is a close relative of Sulfuricurvum kujiense – an organism capable of oxidizing reduced sulfur compounds in crude oil. Geochemical analysis of organic extracts from bitumen at different reservoir depths down to the oil water transition zone of these oil sands indicated active biodegradation of dibenzothiophenes, and stable sulfur isotope ratios for elemental sulfur and sulfate in formation waters were indicative of anaerobic oxidation of sulfur compounds. Microbial desulfurization of crude oil may be an important metabolism for Epsilonproteobacteria indigenous to oil reservoirs with elevated sulfur content and may explain their prevalence in formation waters from highly biodegraded petroleum systems.

Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus.

We have characterized the molecular and genomic diversity of the microbiota of the starlet sea anemone Nematostella vectensis, a cnidarian model for comparative developmental and functional biology and a year-round inhabitant of temperate salt marshes. Molecular phylogenetic analysis of 16S rRNA gene clone libraries revealed four ribotypes associated with N. vectensis at multiple locations and times. These associates include two novel ribotypes within the ε-Proteobacterial order Campylobacterales and the Spirochetes, respectively, each sharing 99% 16S rRNA identity with Endozoicomonas elysicola and Pseudomonas oleovorans, respectively. Species-specific PCR revealed that these populations persisted in N. vectensis asexually propagated under laboratory conditions. cDNA indicated expression of the Campylobacterales and Endozoicomonas 16S rRNA in anemones from Sippewissett Marsh, MA. A collection of bacteria from laboratory raised N. vectensis was dominated by isolates from P. oleovorans and Rhizobium radiobacter. Isolates from field-collected anemones revealed an association with Limnobacter and Stappia isolates. Genomic DNA sequencing was carried out on 10 cultured bacterial isolates representing field- and laboratory-associates, i.e., Limnobacter spp., Stappia spp., P. oleovorans and R. radiobacter. Genomes contained multiple genes identified as virulence (host-association) factors while S. stellulata and L. thiooxidans genomes revealed pathways for mixotrophic sulfur oxidation. A pilot metatranscriptome of laboratory-raised N. vectensis was compared to the isolate genomes and indicated expression of ORFs from L. thiooxidans with predicted functions of motility, nutrient scavenging (Fe and P), polyhydroxyalkanoate synthesis for carbon storage, and selective permeability (porins). We hypothesize that such activities may mediate acclimation and persistence of bacteria in a N. vectensis holobiont defined by both internal and external gradients of chemicals and nutrients in a dynamic coastal habitat., Massachusetts Institute of Technology. Department of Civil and Environmental Engineering, National Science Foundation (U.S.). Graduate Research Fellowship, National Research Foundation of Korea (Fellowship)

Cyanobacterial harmful algal blooms (cyanoHABs) appear to be increasing in frequency on a global scale. The Cyanobacteria in blooms can produce toxic secondary metabolites that make freshwater dangerous for drinking and recreation. To characterize microbial activities in a cyanoHAB, transcripts from a eutrophic freshwater reservoir in Singapore were sequenced for six samples collected over one day-night period. Transcripts from the Cyanobacterium Microcystis dominated all samples and were accompanied by at least 533 genera primarily from the Cyanobacteria, Proteobacteria, Bacteroidetes and Actinobacteria. Within the Microcystis population, abundant transcripts were from genes for buoyancy, photosynthesis and synthesis of the toxin microviridin, suggesting that these are necessary for competitive dominance in the Reservoir. During the day, Microcystis transcripts were enriched in photosynthesis and energy metabolism while at night enriched pathways included DNA replication and repair and toxin biosynthesis. Microcystis was the dominant source of transcripts from polyketide and non-ribosomal peptide synthase (PKS and NRPS, respectively) gene clusters. Unexpectedly, expression of all PKS/NRPS gene clusters, including for the toxins microcystin and aeruginosin, occurred throughout the day-night cycle. The most highly expressed PKS/NRPS gene cluster from Microcystis is not associated with any known product. The four most abundant phyla in the reservoir were enriched in different functions, including photosynthesis (Cyanobacteria), breakdown of complex organic molecules (Proteobacteria), glycan metabolism (Bacteroidetes) and breakdown of plant carbohydrates, such as cellobiose (Actinobacteria). These results provide the first estimate of secondary metabolite gene expression, functional partitioning and functional interplay in a freshwater cyanoHAB., Singapore. National Research Foundation (Singapore MIT Alliance for Research and Technology (SMART), Center for Environmental Sensing and Modeling (CENSAM) research program), National Science Foundation (U.S.) (Postdoctoral Research Fellowship in Biology, Grant No. DBI-1202865), National Institute of Environmental Health Sciences (NIEHS Grant P30-ES002109 to the MIT Center for Environmental Health Sciences), MIT International Science and Technology Initiatives (MISTI-Hayashi fund)

New bioinformatic tools are needed to analyze the growing volume of DNA sequence data. This is especially true in the case of secondary metabolite biosynthesis, where the highly repetitive nature of the associated genes creates major challenges for accurate sequence assembly and analysis. Here we introduce the web tool Natural Product Domain Seeker (NaPDoS), which provides an automated method to assess the secondary metabolite biosynthetic gene diversity and novelty of strains or environments. NaPDoS analyses are based on the phylogenetic relationships of sequence tags derived from polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, respectively. The sequence tags correspond to PKS-derived ketosynthase domains and NRPS-derived condensation domains and are compared to an internal database of experimentally characterized biosynthetic genes. NaPDoS provides a rapid mechanism to extract and classify ketosynthase and condensation domains from PCR products, genomes, and metagenomic datasets. Close database matches provide a mechanism to infer the generalized structures of secondary metabolites while new phylogenetic lineages provide targets for the discovery of new enzyme architectures or mechanisms of secondary metabolite assembly. Here we outline the main features of NaPDoS and test it on four draft genome sequences and two metagenomic datasets. The results provide a rapid method to assess secondary metabolite biosynthetic gene diversity and richness in organisms or environments and a mechanism to identify genes that may be associated with uncharacterized biochemistry.

The fish fauna of the Southern Ocean is dominated by species of the perciform suborder Notothenioidei, which constitute 46% of fish species and 90% of biomass. Notothenioids have undergone rapid morphological and ecological diversification and developed physiological adaptations to a cold, highly oxygenated environment. Microbes inhabiting animal intestines include those that perform essential nutritional functions, but notothenioid gut microbial communities have not been investigated using cultivation-independent approaches. We analyzed bacterial 16S rRNA gene sequences obtained from the intestinal tract of Notothenia coriiceps and Chaenocephalus aceratus, which differ in their pelagic distribution and feeding strategies. Both samples showed dominance of Gammaproteobacteria (mostly Vibrionaceae), as has been reported for temperate teleost species. Both samples showed low diversity relative to that reported for other fish microbiota studies, with C. aceratus containing fewer OTUs than N. coriiceps. Despite the small sample size of this preliminary study, our findings suggest that Antarctic notothenioids carry a gut microbiota similar in composition to that of temperate fish, but exhibiting lower species-level diversity. The omnivorous N. coriiceps individual exhibited greater diversity than the exclusively carnivorous C. aceratus individual, which may indicate that increasing herbivory in fish leads to gut microbe diversification, as found in mammals. Lastly, we detected members of taxa containing known microbial pathogens, which have not been previously reported in Antarctic notothenioid fish.