Delayed gut microbiota development in high-risk for asthma infants is temporarily modifiable by Lactobacillus supplementation.

Gut microbiota dysbiosis and metabolic dysfunction in infancy precedes childhood atopy and asthma development. Here we examined gut microbiota maturation over the first year of life in infants at high risk for asthma (HR), and whether it is modifiable by early-life Lactobacillus supplementation. We performed a longitudinal comparison of stool samples collected from HR infants randomized to daily oral Lactobacillus rhamnosus GG (HRLGG) or placebo (HRP) for 6 months, and healthy (HC) infants. Meconium microbiota of HRP participants is distinct, follows a delayed developmental trajectory, and is primarily glycolytic and depleted of a range of anti-inflammatory lipids at 6 months of age. These deficits are partly rescued in HRLGG infants, but this effect was lost at 12 months of age, 6 months after cessation of supplementation. Thus we show that early-life gut microbial development is distinct, but plastic, in HR infants. Our findings offer a novel strategy for early-life preventative interventions.

Not All Particles Are Equal: The Selective Enrichment of Particle-Associated Bacteria from the Mediterranean Sea.

We have used two metagenomic approaches, direct sequencing of natural samples and sequencing after enrichment, to characterize communities of prokaryotes associated to particles. In the first approximation, different size filters (0.22 and 5 µm) were used to identify prokaryotic microbes of free-living and particle-attached bacterial communities in the Mediterranean water column. A subtractive metagenomic approach was used to characterize the dominant microbial groups in the large size fraction that were not present in the free-living one. They belonged mainly to Actinobacteria, Planctomycetes, Flavobacteria and Proteobacteria. In addition, marine microbial communities enriched by incubation with different kinds of particulate material have been studied by metagenomic assembly. Different particle kinds (diatomaceous earth, sand, chitin and cellulose) were colonized by very different communities of bacteria belonging to Roseobacter, Vibrio, Bacteriovorax, and Lacinutrix that were distant relatives of genomes already described from marine habitats. Besides, using assembly from deep metagenomic sequencing from the particle-specific enrichments we were able to determine a total of 20 groups of contigs (eight of them with >50% completeness) and reconstruct de novo five new genomes of novel species within marine clades (>79% completeness and <1.8% contamination). We also describe for the first time the genome of a marine Rhizobiales phage that seems to infect a broad range of Alphaproteobacteria and live in habitats as diverse as soil, marine sediment and water column. The metagenomic recruitment of the communities found by direct sequencing of the large size filter and by enrichment had nearly no overlap. These results indicate that these reconstructed genomes are part of the rare biosphere which exists at nominal levels under natural conditions.

Pseudorhizobium pelagicum gen. nov., sp. nov. isolated from a pelagic Mediterranean zone.

Two novel Alphaproteobacteria strains, R1-200B4(T) and R2-400B4, were isolated from the Mediterranean Sea off the coast of Alicante in Spain. The phylogenetic analysis of the 16S rRNA gene showed that they are related to members of Family Rhizobiaceae. The 16S rRNA gene sequence of strain R1-200B4(T) presents 97.0% and 95.6% similarity with respect to the type strains of the type species from genera Neorhizobium and Rhizobium, Neorhizobium galegae HAMBI 540(T) and Rhizobium leguminosarum USDA 2370(T), respectively. The remaining genera of family Rhizobiaceae showed similarities lower than 95%. The recA and atpD gene sequences of strain R1-200B4(T) showed, respectively, 90% and 88.6% similarity with respect to N. galegae HAMBI 540(T) and 87% and 86% with respect to R. leguminosarum USDA 2370(T). The calculated ANI values between the genomes of the strain R1-200B4(T) and those of N. galegae HAMBI 540(T) and R. leguminosarum 3841 are 75.9% and 74.0%, respectively. The major fatty acids are those from summed feature 8 (C18:1 ω6c/C18:1 ω7c) and the C16:0. Catalase and oxidase were positive. Nitrate reduction and aesculin hydrolysis were positive. Production of ß-galactosidase and urease was positive. The production of indol, arginine dehydrolase or gelatinase was negative. Growth was observed in presence of 7% NaCl. Therefore, based on the phylogenetic, chemotaxonomic and phenotypic data obtained in this study, we propose to classify the strains isolated in this study in a new genus named Pseudorhizobium gen. nov. and a new species named Pseudorhizobium pelagicum sp. nov. with the type strain R1-200B4(T) (=LMG 28314(T)=CECT 8629(T)).

Microbial transformation of the Deepwater Horizon oil spill-past, present, and future perspectives.

The Deepwater Horizon blowout, which occurred on April 20, 2010, resulted in an unprecedented oil spill. Despite a complex effort to cap the well, oil and gas spewed from the site until July 15, 2010. Although a large proportion of the hydrocarbons was depleted via natural processes and human intervention, a substantial portion of the oil remained unaccounted for and impacted multiple ecosystems throughout the Gulf of Mexico. The depth, duration and magnitude of this spill were unique, raising many questions and concerns regarding the fate of the hydrocarbons released. One major question was whether or not microbial communities would be capable of metabolizing the hydrocarbons, and if so, by what mechanisms and to what extent? In this review, we summarize the microbial response to the oil spill as described by studies performed during the past four years, providing an overview of the different responses associated with the water column, surface waters, deep-sea sediments, and coastal sands/sediments. Collectively, these studies provide evidence that the microbial response to the Deepwater Horizon oil spill was rapid and robust, displaying common attenuation mechanisms optimized for low molecular weight aliphatic and aromatic hydrocarbons. In contrast, the lack of evidence for the attenuation of more recalcitrant hydrocarbon components suggests that future work should focus on both the environmental impact and metabolic fate of recalcitrant compounds, such as oxygenated oil components.

RNA sequencing provides evidence for functional variability between naturally co-existing Alteromonas macleodii lineages.

BACKGROUND: Alteromonas macleodii is a ubiquitous gammaproteobacterium shown to play a biogeochemical role in marine environments. Two A. macleodii strains (AltDE and AltDE1) isolated from the same sample (i.e., the same place at the same time) show considerable genomic differences. In this study, we investigate the transcriptional response of these two strains to varying growth conditions in order to investigate differences in their ability to adapt to varying environmental parameters. RESULTS: RNA sequencing revealed transcriptional changes between all growth conditions examined (e.g., temperature and medium) as well as differences between the two A. macleodii strains within a given condition. The main inter-strain differences were more marked in the adaptation to grow on minimal medium with glucose and, even more so, under starvation. These differences suggested that AltDE1 may have an advantage over AltDE when glucose is the major carbon source, and co-culture experiments confirmed this advantage. Additional differences were observed between the two strains in the expression of ncRNAs and phage-related genes, as well as motility. CONCLUSIONS: This study shows that the genomic diversity observed in closely related strains of A. macleodii from a single environment result in different transcriptional responses to changing environmental parameters. This data provides additional support for the idea that greater diversity at the strain level of a microbial community could enhance the community's ability to adapt to environmental shifts.

Expanding the marine virosphere using metagenomics.

Viruses infecting prokaryotic cells (phages) are the most abundant entities of the biosphere and contain a largely uncharted wealth of genomic diversity. They play a critical role in the biology of their hosts and in ecosystem functioning at large. The classical approaches studying phages require isolation from a pure culture of the host. Direct sequencing approaches have been hampered by the small amounts of phage DNA present in most natural habitats and the difficulty in applying meta-omic approaches, such as annotation of small reads and assembly. Serendipitously, it has been discovered that cellular metagenomes of highly productive ocean waters (the deep chlorophyll maximum) contain significant amounts of viral DNA derived from cells undergoing the lytic cycle. We have taken advantage of this phenomenon to retrieve metagenomic fosmids containing viral DNA from a Mediterranean deep chlorophyll maximum sample. This method allowed description of complete genomes of 208 new marine phages. The diversity of these genomes was remarkable, contributing 21 genomic groups of tailed bacteriophages of which 10 are completely new. Sequence based methods have allowed host assignment to many of them. These predicted hosts represent a wide variety of important marine prokaryotic microbes like members of SAR11 and SAR116 clades, Cyanobacteria and also the newly described low GC Actinobacteria. A metavirome constructed from the same habitat showed that many of the new phage genomes were abundantly represented. Furthermore, other available metaviromes also indicated that some of the new phages are globally distributed in low to medium latitude ocean waters. The availability of many genomes from the same sample allows a direct approach to viral population genomics confirming the remarkable mosaicism of phage genomes.

A hybrid NRPS-PKS gene cluster related to the bleomycin family of antitumor antibiotics in Alteromonas macleodii strains.

Although numerous marine bacteria are known to produce antibiotics via hybrid NRPS-PKS gene clusters, none have been previously described in an Alteromonas species. In this study, we describe in detail a novel hybrid NRPS-PKS cluster identified in the plasmid of the Alteromonasmacleodii strain AltDE1 and analyze its relatedness to other similar gene clusters in a sequence-based characterization. This is a mobile cluster, flanked by transposase-like genes, that has even been found inserted into the chromosome of some Alteromonasmacleodii strains. The cluster contains separate genes for NRPS and PKS activity. The sole PKS gene appears to carry a novel acyltransferase domain, quite divergent from those currently characterized. The predicted specificities of the adenylation domains of the NRPS genes suggest that the final compound has a backbone very similar to bleomycin related compounds. However, the lack of genes involved in sugar biosynthesis indicates that the final product is not a glycopeptide. Even in the absence of these genes, the presence of the cluster appears to confer complete or partial resistance to phleomycin, which may be attributed to a bleomycin-resistance-like protein identified within the cluster. This also suggests that the compound still shares significant structural similarity to bleomycin. Moreover, transcriptomic evidence indicates that the NRPS-PKS cluster is expressed. Such sequence-based approaches will be crucial to fully explore and analyze the diversity and potential of secondary metabolite production, especially from increasingly important sources like marine microbes.

The Montastraea faveolata microbiome: ecological and temporal influences on a Caribbean reef-building coral in decline.

Coral-associated microbial communities, including protists, bacteria, archaea and viruses, are important components of the coral holobiont that influence the health of corals and coral reef ecosystems. Evidence suggests that the composition of these microbial communities is affected by numerous parameters; however, little is known about the confluence of these ecological and temporal effects. In this study, we used ribosomal RNA gene sequencing to identify the zooxanthellae, bacteria and archaea associated with healthy and yellow band diseased (YBD) colonies in the Media Luna reef of La Parguera, Puerto Rico, in order to examine the influence of YBD on the Montastraea faveolata microbiome. In addition, we evaluated the influence of season on the differences between healthy and YBD M. faveolata microbiomes by sampling from the same tagged colonies in both March and September of 2007. To the best of our knowledge, this is the first coral microbiome study to examine sequences from the zooxanthellar, bacterial and archaeal communities simultaneously from individual coral samples. Our results confirm differences in the M. faveolata zooxanthellar, bacterial and archaeal communities between healthy and YBD colonies in March; however, the September communities do not exhibit the same differences. Moreover, we provide evidence that the differences in the M. faveolata microbiomes between March and September are more significant than those observed between healthy and YBD. This data suggest that the entire coral microbiome, not just the bacterial community, is a dynamic environment where both disease and season play important roles.

Metagenomic analysis and metabolite profiling of deep-sea sediments from the Gulf of Mexico following the Deepwater Horizon oil spill.

Marine subsurface environments such as deep-sea sediments, house abundant and diverse microbial communities that are believed to influence large-scale geochemical processes. These processes include the biotransformation and mineralization of numerous petroleum constituents. Thus, microbial communities in the Gulf of Mexico are thought to be responsible for the intrinsic bioremediation of crude oil released by the Deepwater Horizon (DWH) oil spill. While hydrocarbon contamination is known to enrich for aerobic, oil-degrading bacteria in deep-seawater habitats, relatively little is known about the response of communities in deep-sea sediments, where low oxygen levels may hinder such a response. Here, we examined the hypothesis that increased hydrocarbon exposure results in an altered sediment microbial community structure that reflects the prospects for oil biodegradation under the prevailing conditions. We explore this hypothesis using metagenomic analysis and metabolite profiling of deep-sea sediment samples following the DWH oil spill. The presence of aerobic microbial communities and associated functional genes was consistent among all samples, whereas, a greater number of Deltaproteobacteria and anaerobic functional genes were found in sediments closest to the DWH blowout site. Metabolite profiling also revealed a greater number of putative metabolites in sediments surrounding the blowout zone relative to a background site located 127 km away. The mass spectral analysis of the putative metabolites revealed that alkylsuccinates remained below detection levels, but a homologous series of benzylsuccinates (with carbon chain lengths from 5 to 10) could be detected. Our findings suggest that increased exposure to hydrocarbons enriches for Deltaproteobacteria, which are known to be capable of anaerobic hydrocarbon metabolism. We also provide evidence for an active microbial community metabolizing aromatic hydrocarbons in deep-sea sediments of the Gulf of Mexico.

Intra- and intergenomic variation of ribosomal RNA operons in concurrent Alteromonas macleodii strains.

Biodiversity estimates based on ribosomal operon sequence diversity rely on the premise that a sequence is characteristic of a single specific taxon or operational taxonomic unit (OTU). Here, we have studied the sequence diversity of 14 ribosomal RNA operons (rrn) contained in the genomes of two isolates (five operons in each genome) and four metagenomic fosmids, all from the same seawater sample. Complete sequencing of the isolate genomes and the fosmids establish that they represent strains of the same species, Alteromonas macleodii, with average nucleotide identity (ANI) values >97 %. Nonetheless, we observed high levels of intragenomic heterogeneity (i.e., variability between operons of a single genome) affecting multiple regions of the 16S and 23S rRNA genes as well as the internally transcribed spacer 1 (ITS-1) region. Furthermore, the ribosomal operons exhibited intergenomic heterogeneity (i.e., variability between operons located in separate genomes) in each of these regions, compounding the variability. Our data reveal the extensive heterogeneity observed in natural populations of A. macleodii at a single point in time and support the idea that distinct lineages of A. macleodii exist in the deep Mediterranean. These findings highlight the potential of rRNA fingerprinting methods to misrepresent species diversity while simultaneously failing to recognize the ecological significance of individual strains.

Polyclonality of concurrent natural populations of Alteromonas macleodii.

We have analyzed a natural population of the marine bacterium, Alteromonas macleodii, from a single sample of seawater to evaluate the genomic diversity present. We performed full genome sequencing of four isolates and 161 metagenomic fosmid clones, all of which were assigned to A. macleodii by sequence similarity. Out of the four strain genomes, A. macleodii deep ecotype (AltDE1) represented a different genome, whereas AltDE2 and AltDE3 were identical to the previously described AltDE. Although the core genome (~80%) had an average nucleotide identity of 98.51%, both AltDE and AltDE1 contained flexible genomic islands (fGIs), that is, genomic islands present in both genomes in the same genomic context but having different gene content. Some of the fGIs encode cell surface receptors known to be phage recognition targets, such as the O-chain of the lipopolysaccharide, whereas others have genes involved in physiological traits (e.g., nutrient transport, degradation, and metal resistance) denoting microniche specialization. The presence in metagenomic fosmids of genomic fragments differing from the sequenced strain genomes, together with the presence of new fGIs, indicates that there are at least two more A. macleodii clones present. The availability of three or more sequences overlapping the same genomic region also allowed us to estimate the frequency and distribution of recombination events among these different clones, indicating that these clustered near the genomic islands. The results indicate that this natural A. macleodii population has multiple clones with a potential for different phage susceptibility and exploitation of resources, within a seemingly unstructured habitat.

Temperature regulation of virulence factors in the pathogen Vibrio coralliilyticus.

Sea surface temperatures (SST) are rising because of global climate change. As a result, pathogenic Vibrio species that infect humans and marine organisms during warmer summer months are of growing concern. Coral reefs, in particular, are already experiencing unprecedented degradation worldwide due in part to infectious disease outbreaks and bleaching episodes that are exacerbated by increasing SST. For example, Vibrio coralliilyticus, a globally distributed bacterium associated with multiple coral diseases, infects corals at temperatures above 27 °C. The mechanisms underlying this temperature-dependent pathogenicity, however, are unknown. In this study, we identify potential virulence mechanisms using whole genome sequencing of V. coralliilyticus ATCC (American Type Culture Collection) BAA-450. Furthermore, we demonstrate direct temperature regulation of numerous virulence factors using proteomic analysis and bioassays. Virulence factors involved in motility, host degradation, secretion, antimicrobial resistance and transcriptional regulation are upregulated at the higher virulent temperature of 27 °C, concurrent with phenotypic changes in motility, antibiotic resistance, hemolysis, cytotoxicity and bioluminescence. These results provide evidence that temperature regulates multiple virulence mechanisms in V. coralliilyticus, independent of abundance. The ecological and biological significance of this temperature-dependent virulence response is reinforced by climate change models that predict tropical SST to consistently exceed 27 °C during the spring, summer and fall seasons. We propose V. coralliilyticus as a model Gram-negative bacterium to study temperature-dependent pathogenicity in Vibrio-related diseases.

Antimicrobial resistance of the coral pathogen Vibrio coralliilyticus and Caribbean sister phylotypes isolated from a diseased octocoral.

Vibrio coralliilyticus is a global marine pathogen that has been found to cause disease in several marine organisms, including corals. This study is the first report of the isolation of V. coralliilyticus from a diseased Caribbean octocoral, Pseudopterogorgia americana. Five sister phylotypes were positively identified using 16S rRNA gene sequencing, recA probes specific for V. coralliilyticus, and rep-PCR fingerprinting. The antimicrobial resistance was compared between pathogenic strains of V. coralliilyticus and the Caribbean strains. First, the antimicrobial resistance of V. coralliilyticus-type strain ATCC BAA-450 was determined using an agar-overlay antimicrobial bioassay at 24 degrees C and 27 degrees C, temperatures which are relevant to its known temperature-dependent virulence. From 108 distinct bacteria isolated from P. americana, 12 inhibited the V. coralliilyticus-type strain at 24 degrees C and five at 27 degrees C. Next, the phenotypic comparison of two Caribbean phylotypes and three V. coralliilyticus reference strains against a subset of 30 bacteria demonstrated a similar resistance trend. At both temperatures, the reference strains were inhibited by three bacteria isolates, while the Caribbean strains were inhibited by four to nine bacteria. Additionally, V. coralliilyticus-type strain ATCC BAA-450 and one of the Caribbean strains were inhibited by a higher number of bacteria at 24 degrees C compared with 27 degrees C. Together, these results highlight that V. coralliilyticus strains have antimicrobial resistance to the majority of coral-associated bacteria tested, which may be temperature-dependent in some strains. Furthermore, all V. coralliilyticus strains tested showed multi-drug resistance to a range of 11-16 (out of 26) commercial antibiotics. This study establishes V. coralliilyticus in association with a Caribbean octocoral and demonstrates its resistance to the antimicrobial activity of coral-associated bacteria and to commercial antibiotics.

Microbial functional structure of Montastraea faveolata, an important Caribbean reef-building coral, differs between healthy and yellow-band diseased colonies.

A functional gene array (FGA), GeoChip 2.0, was used to assess the biogeochemical cycling potential of microbial communities associated with healthy and Caribbean yellow band diseased (YBD) Montastraea faveolata. Over 6700 genes were detected, providing evidence that the coral microbiome contains a diverse community of archaea, bacteria and fungi capable of fulfilling numerous functional niches. These included carbon, nitrogen and sulfur cycling, metal homeostasis and resistance, and xenobiotic contaminant degradation. A significant difference in functional structure was found between healthy and YBD M. faveolata colonies and those differences were specific to the physical niche examined. In the surface mucopolysaccharide layer (SML), only two of 31 functional categories investigated, cellulose degradation and nitrification, revealed significant differences, implying a very specific change in microbial functional potential. Coral tissue slurry, on the other hand, revealed significant changes in 10 of the 31 categories, suggesting a more generalized shift in functional potential involving various aspects of nutrient cycling, metal transformations and contaminant degradation. This study is the first broad screening of functional genes in coral-associated microbial communities and provides insights regarding their biogeochemical cycling capacity in healthy and diseased states.

N- and C-terminal domains of beta-catenin, respectively, are required to initiate and shape axon arbors of retinal ganglion cells in vivo.

We used deletion mutants to study beta-catenin function in axon arborization of retinal ganglion cells (RGCs) in live Xenopus laevis tadpoles. A deletion mutant betacatDeltaARM consists of the N- and C-terminal domains of wild-type beta-catenin that contain, respectively, alpha-catenin and postsynaptic density-95 (PSD-95)/discs large (Dlg)/zona occludens-1 (ZO-1) (PDZ) binding sites but lacks the central armadillo repeat region that binds cadherins and other proteins. Expression of DeltaARM in RGCs of live tadpoles perturbed axon arborization in two distinct ways: some RGC axons did not form arbors, whereas the remaining RGC axons formed arbors with abnormally long and tangled branches. Expression of the N- and C-terminal domains of beta-catenin separately in RGCs resulted in segregation of these two phenotypes. The axons of RGCs overexpressing the N-terminal domain of beta-catenin developed no or very few branches, whereas axons of RGCs overexpressing the C-terminal domain of beta-catenin formed arbors with long, tangled branches. Additional analysis revealed that the axons of RGCs that did not form arbors after overexpression of DeltaARM or the N-terminal domain of beta-catenin were frequently mistargeted within the tectum. These results suggest that interactions of the N-terminal domain of beta-catenin with alpha-catenin and of the C-terminal domain with PDZ domain-containing proteins are required, respectively, to initiate and shape axon arbors of RGCs in vivo.