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

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

Expanding the Marine Virosphere Using Metagenomics

<div><p>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, <i>Cyanobacteria</i> and also the newly described low GC <i>Actinobacteria</i>. 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.</p></div

Control of axonal branching and synapse formation by focal adhesion kinase

The formation of neuronal networks in the central nervous system (CNS) requires precise control of axonal branch development and stabilization. Here we show that cell-specific ablation of the murine gene Ptk2 (more commonly known as fak), encoding focal adhesion kinase (FAK), increases the number of axonal terminals and synapses formed by neurons in vivo. Consistent with this, fak mutant neurons also form greater numbers of axonal branches in culture because they have increased branch formation and reduced branch retraction. Expression of wild-type FAK, but not that of several FAK variants that prevent interactions with regulators of Rho family GTPases including the p190 Rho guanine nuclear exchange factor (p190RhoGEF), rescues the axonal arborization phenotype observed in fak mutant neurons. In addition, expression of a mutant p190RhoGEF that cannot associate with FAK results in a phenotype very similar to that of neurons lacking FAK. Thus, FAK functions as a negative regulator of axonal branching and synapse formation, and it seems to exert its actions, in part, through Rho family GTPases.This work was supported by a grant from the NIH and by the Howard Hughes Medical Institute (HHMI). B.R. was supported by a grant from the Ministerio de Educación y Cultura of Spain and by the HHMI. L.F.R. is an Investigator of the HHMI. B.R. is a Ramón y Cajal Investigator from Ministerio de Ciencia y Tecnología of Spain.Peer reviewe

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

General features of CGR groups and putative host assignment.

<p>CGR (Complete Genome Representative): contig representing a complete phage genome of a cluster of highly related contigs (>95% identity and 20% coverage in nucleotide sequence). The putative host taxon assigned to one or more CGRs in a group is shown. The last column shows the evidence for host assignment in brackets next to the host name. SS: putative host inferred by the high sequence similarity to known phages; AMG: Auxiliary metabolic gene; INT: putative host inferred by an exact match of a putative phage site-specific attachment site (attP) in an integrase carrying CGR to a host tRNA (host site-specific attachment site attB). The asterisk (*) in G13 indicates that host prediction was performed using a GF (genome fragment of an incomplete phage genome) and not a CGR.</p

Genomic comparison of novel, complete phage genomes (CGRs) with known tailed phages.

<p>An all-vs-all comparison of several reference tailed bacteriophage genomes with the 208 CGRs identified in this study was achieved by a clustering based on a sequence similarity derived metric (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003987#s4" target="_blank">Materials and Methods</a> for details). Branches are colored according to phage family classification (See color key top left). Branches representing unclassified phages are shown in black. The ICTV (International Committee on Taxonomy of Viruses) nomenclature of several phages is also shown for reference. In addition, color dots indicate positions of phages infecting important marine microbes (color key at the bottom). The CGRs in this study are represented by blue diamonds at the tip of the branches, and the CGR groups are highlighted in grey and labeled (G1–G21). For those groups where host prediction was possible for one or more CGRs, a taxonomic rank of the host and the organisms supporting the prediction, and the nature of the evidence supporting the assignment (<i>SS</i>: sequence similarity, <i>INT</i>: integrase/att) are shown. The asterisk (<b>*</b>) in G13 indicates that host prediction was performed using an incomplete genome and not a CGR.</p

Novel Autographivirinae phages.

<p>G7, G8 and G9 belonging to the subfamily Autographivirinae are compared to each other, and to the closest related reference phage genomes (boxed). CGR names are abbreviated, e.g. S45-C4 for uvMED-CGR-U-MedDCM-OCT-S45-C4 (for the complete names see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003987#pgen.1003987.s001" target="_blank">Data S1</a>). Size and GC% of the CGR/phage genomes are also indicated. Selected genes are uniformly colored and labeled. AA (amino acids) and NT (nucleotides) labels to the right indicate if the genome comparisons were made using TBLASTX or BLASTN. A color scale for the %identity (protein or nucleotide) is shown on the bottom right side. A 5 Kb length scale is also shown (bottom right). Some gene clusters are shown displaced and underlined in the graphic indicating that they have been moved to improve comparison across all genomes.</p