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1.  Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics 
PLoS ONE  2014;9(4):e93827.
This study explored the short-term planktonic microbial community structure and resilience in Lake Lanier (GA, USA) while simultaneously evaluating the technical aspects of identifying taxa via 16S rRNA gene amplicon and metagenomic sequence data. 16S rRNA gene amplicons generated from four temporally discrete samples were sequenced with 454 GS-FLX-Ti yielding ∼40,000 rRNA gene sequences from each sample and representing ∼300 observed OTUs. Replicates obtained from the same biological sample clustered together but several biases were observed, linked to either the PCR or sequencing-preparation steps. In comparisons with companion whole-community shotgun metagenome datasets, the estimated number of OTUs at each timepoint was concordant, but 1.5 times and ∼10 times as many phyla and genera, respectively, were identified in the metagenomes. Our analyses showed that the 16S rRNA gene captures broad shifts in community diversity over time, but with limited resolution and lower sensitivity compared to metagenomic data. We also identified OTUs that showed marked shifts in abundance over four close timepoints separated by perturbations and tracked these taxa in the metagenome vs. 16S rRNA amplicon data. A strong summer storm had less of an effect on community composition than did seasonal mixing, which revealed a distinct succession of organisms. This study provides insights into freshwater microbial communities and advances the approaches for assessing community diversity and dynamics in situ.
doi:10.1371/journal.pone.0093827
PMCID: PMC3979728  PMID: 24714158
2.  Tales from a thousand and one phages 
Bacteriophage  2014;4(1):e28265.
The sequencing of marine metagenomic fosmids led to the discovery of several new complete phage genomes. Among the 21 major sequence groups, 10 totally novel groups of marine phages could be identified. Some of these represent the first phages infecting large marine prokaryotic phyla, such as the Verrucomicrobia and the recently described Ca. Actinomarinales. Coming from a single deep photic zone sample the diversity of phages found is astonishing, and the comparison with a metavirome from the same location indicates that only 2% of the real diversity was recovered. In addition to this large macro-diversity, rich micro-diversity was also found, affecting host-recognition modules, mirroring the variation of cell surface components in their host marine microbes.
doi:10.4161/bact.28265
PMCID: PMC3945994  PMID: 24616837
metagenomics; metavirome; marine phages; deep chlorophyll maximum; constant-diversity; red-queen; phage evolution; pan-selectome; pan-genome
3.  Evidence for metaviromic islands in marine phages 
Metagenomic islands (MGIs) have been defined as genomic regions in prokaryotic genomes that under-recruit from metagenomes where most of the same genome recruits at close to 100% identity over most of its length. The presence of MGIs in prokaryotes has been associated to the diversity of concurrent lineages that vary at this level to disperse the predatory pressure of phages that, reciprocally, maintain high clonal diversity in the population and improve ecosystem performance. This was proposed as a Constant-Diversity (C-D) model. Here we have investigated the regions of phage genomes under-recruiting in a metavirome constructed with a sample from the same habitat where they were retrieved. Some of the genes found to under-recruit are involved in host recognition as would be expected from the C-D model. Furthermore, the recruitment of intragenic regions known to be involved in molecular recognition also had a significant under-recruitment compared to the rest of the gene. However, other genes apparently disconnected from the recognition process under-recruited often, specifically the terminases involved in packaging of the phage genome in the capsid and a few others. In addition, some highly related phage genomes (at nucleotide sequence level) had no metaviromic islands (MVIs). We speculate that the latter might be generalist phages with broad infection range that do not require clone specific lineages.
doi:10.3389/fmicb.2014.00027
PMCID: PMC3909814  PMID: 24550898
population genomics; phages; metaviromes; host recognition; marine phages; virome
4.  A Gene Island with Two Possible Configurations Is Involved in Chromatic Acclimation in Marine Synechococcus 
PLoS ONE  2013;8(12):e84459.
Synechococcus, the second most abundant oxygenic phototroph in the marine environment, harbors the largest pigment diversity known within a single genus of cyanobacteria, allowing it to exploit a wide range of light niches. Some strains are capable of Type IV chromatic acclimation (CA4), a process by which cells can match the phycobilin content of their phycobilisomes to the ambient light quality. Here, we performed extensive genomic comparisons to explore the diversity of this process within the marine Synechococcus radiation. A specific gene island was identified in all CA4-performing strains, containing two genes (fciA/b) coding for possible transcriptional regulators and one gene coding for a phycobilin lyase. However, two distinct configurations of this cluster were observed, depending on the lineage. CA4-A islands contain the mpeZ gene, encoding a recently characterized phycoerythrobilin lyase-isomerase, and a third, small, possible regulator called fciC. In CA4-B islands, the lyase gene encodes an uncharacterized relative of MpeZ, called MpeW. While mpeZ is expressed more in blue light than green light, this is the reverse for mpeW, although only small phenotypic differences were found among chromatic acclimaters possessing either CA4 island type. This study provides novel insights into understanding both diversity and evolution of the CA4 process.
doi:10.1371/journal.pone.0084459
PMCID: PMC3877281  PMID: 24391958
5.  Expanding the Marine Virosphere Using Metagenomics 
PLoS Genetics  2013;9(12):e1003987.
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.
Author Summary
Prokaryotic species contain extremely large gene pools (pan-genome) the study of which has been constrained by the difficulties in getting enough cultivated representatives of most of them. The situation of their viruses, also known as phages, that provide part of this genomic diversity and preserve it, is even worse. Here we have found a way to bypass the limitation imposed by pure culture to retrieve phage genomes. We obtained large insert clones (fosmids) from natural communities that are undergoing active viral attack. This has allowed us to triple the number of genomes of marine phages and could be similarly applied to other habitats, shedding light into the biology of the most numerous and least known biological entities on the planet. They exhibit a remarkable degree of variation at one single geographic site but some seem also to be prevalent worldwide. Their frequent mosaicism indicates a high level of promiscuity that goes beyond the already remarkable hybrid nature of prokaryotic genomes.
doi:10.1371/journal.pgen.1003987
PMCID: PMC3861242  PMID: 24348267
6.  Metagenome Sequencing of Prokaryotic Microbiota from Two Hypersaline Ponds of a Marine Saltern in Santa Pola, Spain 
Genome Announcements  2013;1(6):e00933-13.
Marine salterns are composed of several shallow ponds with a salinity gradient, from seawater to salt saturation, with gradually changing microbial populations. Here, we report the metagenome sequencing of the prokaryotic microbiota of two ponds with 13% and 33% salinity from a saltern in Santa Pola, Spain.
doi:10.1128/genomeA.00933-13
PMCID: PMC3828313  PMID: 24233589
7.  Genomes of “Spiribacter”, a streamlined, successful halophilic bacterium 
BMC Genomics  2013;14:787.
Background
Thalassosaline waters produced by the concentration of seawater are widespread and common extreme aquatic habitats. Their salinity varies from that of sea water (ca. 3.5%) to saturation for NaCl (ca. 37%). Obviously the microbiota varies dramatically throughout this range. Recent metagenomic analysis of intermediate salinity waters (19%) indicated the presence of an abundant and yet undescribed gamma-proteobacterium. Two strains belonging to this group have been isolated from saltern ponds of intermediate salinity in two Spanish salterns and were named “Spiribacter”.
Results
The genomes of two isolates of “Spiribacter” have been fully sequenced and assembled. The analysis of metagenomic datasets indicates that microbes of this genus are widespread worldwide in medium salinity habitats representing the first ecologically defined moderate halophile. The genomes indicate that the two isolates belong to different species within the same genus. Both genomes are streamlined with high coding densities, have few regulatory mechanisms and no motility or chemotactic behavior. Metabolically they are heterotrophs with a subgroup II xanthorhodopsin as an additional energy source when light is available.
Conclusions
This is the first bacterium that has been proven by culture independent approaches to be prevalent in hypersaline habitats of intermediate salinity (half a way between the sea and NaCl saturation). Predictions from the proteome and analysis of transporter genes, together with a complete ectoine biosynthesis gene cluster are consistent with these microbes having the salt-out-organic-compatible solutes type of osmoregulation. All these features are also consistent with a well-adapted fully planktonic microbe while other halophiles with more complex genomes such as Salinibacter ruber might have particle associated microniches.
doi:10.1186/1471-2164-14-787
PMCID: PMC3832224  PMID: 24225341
Halophilic bacteria; Xanthorhodopsin; Hypersaline; Saltern; Spiribacter; Moderate halophile
8.  Architecture and Gene Repertoire of the Flexible Genome of the Extreme Acidophile Acidithiobacillus caldus 
PLoS ONE  2013;8(11):e78237.
Background
Acidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the Acidithiobacillales. As such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. In this study, we explore the genomic diversity of A. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome.
Principal Findings
Comparative sequence analysis of A. caldus ATCC 51756 and SM-1 indicate that, despite sharing a conserved and highly syntenic genomic core, both strains have unique gene complements encompassing nearly 20% of their respective genomes. The differential gene complement of each strain is distributed between the chromosomal compartment, one megaplasmid and a variable number of smaller plasmids, and is directly associated to a diverse pool of mobile genetic elements (MGE). These include integrative conjugative and mobilizable elements, genomic islands and insertion sequences. Some of the accessory functions associated to these MGEs have been linked previously to the flexible gene pool in microorganisms inhabiting completely different econiches. Yet, others had not been unambiguously mapped to the flexible gene pool prior to this report and clearly reflect strain-specific adaption to local environmental conditions.
Significance
For many years, and because of DNA instability at low pH and recurrent failure to genetically transform acidophilic bacteria, gene transfer in acidic environments was considered negligible. Findings presented herein imply that a more or less conserved pool of actively excising MGEs occurs in the A. caldus population and point to a greater frequency of gene exchange in this econiche than previously recognized. Also, the data suggest that these elements endow the species with capacities to withstand the diverse abiotic and biotic stresses of natural environments, in particular those associated with its extreme econiche.
doi:10.1371/journal.pone.0078237
PMCID: PMC3826726  PMID: 24250794
9.  Tropical Aquatic Archaea Show Environment-Specific Community Composition 
PLoS ONE  2013;8(9):e76321.
The Archaea domain is ubiquitously distributed and extremely diverse, however, environmental factors that shape archaeal community structure are not well known. Aquatic environments, including the water column and sediments harbor many new uncultured archaeal species from which metabolic and ecological roles remain elusive. Some environments are especially neglected in terms of archaeal diversity, as is the case of pristine tropical areas. Here we investigate the archaeal composition in marine and freshwater systems from Ilha Grande, a South Atlantic tropical environment. All sampled habitats showed high archaeal diversity. No OTUs were shared between freshwater, marine and mangrove sediment samples, yet these environments are interconnected and geographically close, indicating environment-specific community structuring. Group II Euryarchaeota was the main clade in marine samples, while the new putative phylum Thaumarchaeota and LDS/RCV Euryarchaeota dominated freshwaters. Group III Euryarchaeota, a rare clade, was also retrieved in reasonable abundance in marine samples. The archaeal community from mangrove sediments was composed mainly by members of mesophilic Crenarchaeota and by a distinct clade forming a sister-group to Crenarchaeota and Thaumarchaeota. Our results show strong environment-specific community structuring in tropical aquatic Archaea, as previously seen for Bacteria.
doi:10.1371/journal.pone.0076321
PMCID: PMC3783403  PMID: 24086729
10.  A Hybrid NRPS-PKS Gene Cluster Related to the Bleomycin Family of Antitumor Antibiotics in Alteromonas macleodii Strains 
PLoS ONE  2013;8(9):e76021.
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.
doi:10.1371/journal.pone.0076021
PMCID: PMC3777966  PMID: 24069455
11.  Microbial Life in a Fjord: Metagenomic Analysis of a Microbial Mat in Chilean Patagonia 
PLoS ONE  2013;8(8):e71952.
The current study describes the taxonomic and functional composition of metagenomic sequences obtained from a filamentous microbial mat isolated from the Comau fjord, located in the northernmost part of the Chilean Patagonia. The taxonomic composition of the microbial community showed a high proportion of members of the Gammaproteobacteria, including a high number of sequences that were recruited to the genomes of Moritella marina MP-1 and Colwelliapsycherythraea 34H, suggesting the presence of populations related to these two psychrophilic bacterial species. Functional analysis of the community indicated a high proportion of genes coding for the transport and metabolism of amino acids, as well as in energy production. Among the energy production functions, we found protein-coding genes for sulfate and nitrate reduction, both processes associated with Gammaproteobacteria-related sequences. This report provides the first examination of the taxonomic composition and genetic diversity associated with these conspicuous microbial mat communities and provides a framework for future microbial studies in the Comau fjord.
doi:10.1371/journal.pone.0071952
PMCID: PMC3756073  PMID: 24015199
12.  Metagenomics uncovers a new group of low GC and ultra-small marine Actinobacteria 
Scientific Reports  2013;3:2471.
We describe a deep-branching lineage of marine Actinobacteria with very low GC content (33%) and the smallest free living cells described yet (cell volume ca. 0.013 μm3), even smaller than the cosmopolitan marine photoheterotroph, ‘Candidatus Pelagibacter ubique'. These microbes are highly related to 16S rRNA sequences retrieved by PCR from the Pacific and Atlantic oceans 20 years ago. Metagenomic fosmids allowed a virtual genome reconstruction that also indicated very small genomes below 1 Mb. A new kind of rhodopsin was detected indicating a photoheterotrophic lifestyle. They are estimated to be ~4% of the total numbers of cells found at the site studied (the Mediterranean deep chlorophyll maximum) and similar numbers were estimated in all tropical and temperate photic zone metagenomes available. Their geographic distribution mirrors that of picocyanobacteria and there appears to be an association between these microbial groups. A new sub-class, ‘Candidatus Actinomarinidae' is proposed to designate these microbes.
doi:10.1038/srep02471
PMCID: PMC3747508  PMID: 23959135
13.  Reconstruction of Novel Cyanobacterial Siphovirus Genomes from Mediterranean Metagenomic Fosmids 
Cellular metagenomes are primarily used for investigating microbial community structure and function. However, cloned fosmids from such metagenomes capture phage genome fragments that can be used as a source of phage genomes. We show that fosmid cloning from cellular metagenomes and sequencing at a high coverage is a credible alternative to constructing metaviriomes and allows capturing and assembling novel, complete phage genomes. It is likely that phages recovered from cellular metagenomes are those replicating within cells during sample collection and represent “active” phages, naturally amplifying their genomic DNA and increasing chances for cloning. We describe five sets of siphoviral contigs (MEDS1, MEDS2, MEDS3, MEDS4, and MEDS5), obtained by sequencing fosmids from the cellular metagenome of the deep chlorophyll maximum in the Mediterranean. Three of these represent complete siphoviral genomes and two represent partial ones. This is the first set of phage genomes assembled directly from cellular metagenomic fosmid libraries. They exhibit low sequence similarities to one another and to known siphoviruses but are remarkably similar in overall genome architecture. We present evidence suggesting they infect picocyanobacteria, likely Synechococcus. Four of these sets also define a novel branch in the phylogenetic tree of phage large subunit terminases. Moreover, some of these siphoviral groups are globally distributed and abundant in the oceans, comparable to some known myoviruses and podoviruses. This suggests that, as more siphoviral genomes become available, we will be better able to assess the abundance and influence of this diverse and polyphyletic group in the marine habitat.
doi:10.1128/AEM.02742-12
PMCID: PMC3553755  PMID: 23160125
14.  Complete Genome Sequence of the Copiotrophic Marine Bacterium Alteromonas macleodii Strain ATCC 27126T 
Journal of Bacteriology  2012;194(24):6998.
The genome of Alteromonas macleodii strain ATCC 27126T has been resequenced and closed into a single contig. We describe here the genome of this important and globally distributed marine bacterium.
doi:10.1128/JB.01565-12
PMCID: PMC3510622  PMID: 23209244
15.  Genomic Diversity of “Deep Ecotype” Alteromonas macleodii Isolates: Evidence for Pan-Mediterranean Clonal Frames 
Genome Biology and Evolution  2013;5(6):1220-1232.
We have compared genomes of Alteromonas macleodii “deep ecotype” isolates from two deep Mediterranean sites and two surface samples from the Aegean and the English Channel. A total of nine different genomes were analyzed. They belong to five clonal frames (CFs) that differ among them by approximately 30,000 single-nucleotide polymorphisms (SNPs) over their core genomes. Two of the CFs contain three strains each with nearly identical genomes (∼100 SNPs over the core genome). One of the CFs had representatives that were isolated from samples taken more than 1,000 km away, 2,500 m deeper, and 5 years apart. These data mark the longest proven persistence of a CF in nature (outside of clinical settings). We have found evidence for frequent recombination events between or within CFs and even with the distantly related A. macleodii surface ecotype. The different CFs had different flexible genomic islands. They can be classified into two groups; one type is additive, that is, containing different numbers of gene cassettes, and is very variable in short time periods (they often varied even within a single CF). The other type was more stable and produced the complete replacement of a genomic fragment by another with different genes. Although this type was more conserved within each CF, we found examples of recombination among distantly related CFs including English Channel and Mediterranean isolates.
doi:10.1093/gbe/evt089
PMCID: PMC3698932  PMID: 23729633
Alteromonas macleodii; SNPs; microevolution; recombination; horizontal gene transfer
16.  Assembly of a Marine Viral Metagenome after Physical Fractionation 
PLoS ONE  2013;8(4):e60604.
Metagenomic analyses of marine viruses generate an overview of viral genes present in a sample, but the percentage of the resulting sequence fragments that can be reassembled is low and the phenotype of the virus from which a given sequence derives is usually unknown. In this study, we employed physical fractionation to characterize the morphological and genomic traits of a subset of uncultivated viruses from a natural marine assemblage. Viruses from Kāne‘ohe Bay, Hawai‘i were fractionated by equilibrium buoyant density centrifugation in a cesium chloride (CsCl) gradient, and one fraction from the CsCl gradient was then further fractionated by strong anion-exchange chromatography. One of the fractions resulting from this two-dimensional separation appeared to be dominated by only a few virus types based on genome sizes and morphology. Sequences generated from a shotgun clone library of the viruses in this fraction were assembled into significantly more numerous contigs than have been generated with previous metagenomic investigations of whole DNA viral assemblages with comparable sequencing effort. Analysis of the longer contigs (up to 6.5 kb) assembled from our metagenome allowed us to assess gene arrangement in this subset of marine viruses. Our results demonstrate the potential for physical fractionation to facilitate sequence assembly from viral metagenomes and permit linking of morphological and genomic data for uncultivated viruses.
doi:10.1371/journal.pone.0060604
PMCID: PMC3620275  PMID: 23580170
17.  Novel group of podovirus infecting the marine bacterium Alteromonas macleodii 
Bacteriophage  2013;3(2):e24766.
Four novel, closely related podoviruses, which displayed lytic activity against the gamma-proteobacterium Alteromonas macleodii, have been isolated and sequenced. Alterophages AltAD45-P1 to P4 were obtained from water recovered near a fish farm in the Mediterranean Sea. Their morphology indicates that they belong to the Podoviridae. Their linear and dsDNA genomes are 100–104 kb in size, remarkably larger than any other described podovirus. The four AltAD45-phages share 99% nucleotide sequence identity over 97% of their ORFs, although an insertion was found in AltAD45-P1 and P2 and some regions were slightly more divergent. Despite the high overall sequence similarity among these four phages, the group with the insertion and the group without it, have different host ranges against the A. macleodii strains tested. The AltAD45-P1 to P4 phages have genes for DNA replication and transcription as well as structural genes, which are similar to the N4-like Podoviridae genus that is widespread in proteobacteria. However, in terms of their genomic structure, AltAD45-P1 to P4 differ from that of the N4-like phages. Some distinguishing features include the lack of a large virion encapsidated RNA polymerase gene, very well conserved among all the previously described N4-like phages, a single-stranded DNA binding protein and different tail protein genes. We conclude that the AltAD45 phages characterized in this study constitute a new genus within the Podoviridae.
doi:10.4161/bact.24766
PMCID: PMC3821669  PMID: 24228219
Alteromonas macleodii; Podoviridae; N4-like virus; lytic phage; marine phages
18.  Draft Genome of Spiribacter salinus M19-40, an Abundant Gammaproteobacterium in Aquatic Hypersaline Environments 
Genome Announcements  2013;1(1):e00179-12.
We have previously used a de novo metagenomic assembly approach to describe the presence of an abundant gammaproteobacterium comprising nearly 15% of the microbial community in an intermediate salinity solar saltern pond. We have obtained this microbe in pure culture and describe the genome sequencing of the halophilic photoheterotrophic microbe, Spiribacter salinus M19-40.
doi:10.1128/genomeA.00179-12
PMCID: PMC3569344  PMID: 23409269
19.  Insights into Dynamics of Mobile Genetic Elements in Hyperthermophilic Environments from Five New Thermococcus Plasmids 
PLoS ONE  2013;8(1):e49044.
Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.
doi:10.1371/journal.pone.0049044
PMCID: PMC3543421  PMID: 23326305
20.  Polyclonality of Concurrent Natural Populations of Alteromonas macleodii 
Genome Biology and Evolution  2012;4(12):1360-1374.
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.
doi:10.1093/gbe/evs112
PMCID: PMC3542563  PMID: 23212172
Alteromonas macleodii; metagenome; population genomics; genomic island; constant-diversity; phage
21.  Combined Analyses of the ITS Loci and the Corresponding 16S rRNA Genes Reveal High Micro- and Macrodiversity of SAR11 Populations in the Red Sea 
PLoS ONE  2012;7(11):e50274.
Bacteria belonging to the SAR11 clade are among the most abundant prokaryotes in the pelagic zone of the ocean. 16S rRNA gene-based analyses indicate that they constitute up to 60% of the bacterioplankton community in the surface waters of the Red Sea. This extremely oligotrophic water body is further characterized by an epipelagic zone, which has a temperature above 24°C throughout the year, and a remarkable uniform temperature (∼22°C) and salinity (∼41 psu) from the mixed layer (∼200 m) to the bottom at over 2000 m depth. Despite these conditions that set it apart from other marine environments, the microbiology of this ecosystem is still vastly understudied. Prompted by the limited phylogenetic resolution of the 16S rRNA gene, we extended our previous study by sequencing the internal transcribed spacer (ITS) region of SAR11 in different depths of the Red Sea’s water column together with the respective 16S fragment. The overall diversity captured by the ITS loci was ten times higher than that of the corresponding 16S rRNA genes. Moreover, species estimates based on the ITS showed a highly diverse population of SAR11 in the mixed layer that became diminished in deep isothermal waters, which was in contrast to results of the related 16S rRNA genes. While the 16S rRNA gene-based sequences clustered into three phylogenetic subgroups, the related ITS fragments fell into several phylotypes that showed clear depth-dependent shifts in relative abundances. Blast-based analyses not only documented the observed vertical partitioning and universal co-occurrence of specific phylotypes in five other distinct oceanic provinces, but also highlighted the influence of ecosystem-specific traits (e.g., temperature, nutrient availability, and concentration of dissolved oxygen) on the population dynamics of this ubiquitous marine bacterium.
doi:10.1371/journal.pone.0050274
PMCID: PMC3502338  PMID: 23185592
22.  Ribosomal Multi-Operon Diversity: An Original Perspective on the Genus Aeromonas 
PLoS ONE  2012;7(9):e46268.
16S rRNA gene (rrs) is considered of low taxonomic interest in the genus Aeromonas. Here, 195 Aeromonas strains belonging to populations structured by multilocus phylogeny were studied using an original approach that considered Ribosomal Multi-Operon Diversity. This approach associated pulsed-field gel electrophoresis (PFGE) to assess rrn operon number and distribution across the chromosome and PCR-temporal temperature gel electrophoresis (TTGE) to assess rrs V3 region heterogeneity. Aeromonads harbored 8 to 11 rrn operons, 10 operons being observed in more than 92% of the strains. Intraspecific variability was low or nul except for A. salmonicida and A. aquariorum suggesting that large chromosomic rearrangements might occur in these two species while being extremely rarely encountered in the evolution of other taxa. rrn operon number at 8 as well as PFGE patterns were shown valuable for taxonomic purpose allowing resolution of species complexes. PCR-TTGE revealed a high rate of strains (41.5%) displaying intragenomic rrs heterogeneity. Strains isolated from human samples more frequently displayed intragenomic heterogeneity than strains recovered from non-human and environmental specimens. Intraspecific variability ranged from 0 to 76.5% of the strains. The observation of species-specific TTGE bands, the recovery of identical V3 regions in different species and the variability of intragenomic heterogeneity (1–13 divergent nucleotides) supported the occurrence of mutations and horizontal transfer in aeromonad rrs evolution. Altogether, the presence of a high number of rrn operon, the high proportion of strains harboring divergent rrs V3 region and the previously demonstrated high level of genetic diversity argued in favor of highly adaptative capabilities of aeromonads. Outstanding features observed for A. caviae supported the ongoing process of adaptation to a specialized niche represented by the gut, previously hypothesized. 16S rRNA gene is an informative marker in the genus Aeromonas for both evolutionary and polyphasic taxonomic studies provided that multi-operon fingerprinting approaches are used.
doi:10.1371/journal.pone.0046268
PMCID: PMC3459834  PMID: 23032081
23.  Genomes of surface isolates of Alteromonas macleodii: the life of a widespread marine opportunistic copiotroph 
Scientific Reports  2012;2:696.
Alteromonas macleodii is a marine gammaproteobacterium with widespread distribution in temperate or tropical waters. We describe three genomes of isolates from surface waters around Europe (Atlantic, Mediterranean and Black Sea) and compare them with a previously described deep Mediterranean isolate (AltDE) that belongs to a widely divergent clade. The surface isolates are quite similar, the most divergent being the Black Sea (BS11) isolate. The genomes contain several genomic islands with different gene content. The recruitment of very similar genomic fragments from metagenomes in different locations indicates that the surface clade is globally abundant with little effect of geography, even the AltDE and the BS11 genomes recruiting from surface samples in open ocean locations. The finding of CRISPR protospacers of AltDE in a lysogenic phage in the Atlantic (English Channel) isolate illustrates a flow of genetic material among these clades and a remarkably wide distribution of this phage.
doi:10.1038/srep00696
PMCID: PMC3458243  PMID: 23019517
24.  Is the pan-genome also a pan-selectome? 
F1000Research  2012;1:16.
The comparative genomics of prokaryotes has shown the presence of conserved regions containing highly similar genes (the 'core genome') and other regions that vary in gene content (the ‘flexible’ regions). A significant part of the latter is involved in surface structures that are phage recognition targets. Another sizeable part provides for differences in niche exploitation. Metagenomic data indicates that natural populations of prokaryotes are composed of assemblages of clonal lineages or "meta-clones" that share a core of genes but contain a high diversity by varying the flexible component. This meta-clonal diversity is maintained by a collection of phages that equalize the populations by preventing any individual clonal lineage from hoarding common resources. Thus, this polyclonal assemblage and the phages preying upon them constitute natural selection units.
doi:10.12688/f1000research.1-16.v1
PMCID: PMC3782348  PMID: 24358823
25.  Conservation of Gene Cassettes among Diverse Viruses of the Human Gut 
PLoS ONE  2012;7(8):e42342.
Viruses are a crucial component of the human microbiome, but large population sizes, high sequence diversity, and high frequencies of novel genes have hindered genomic analysis by high-throughput sequencing. Here we investigate approaches to metagenomic assembly to probe genome structure in a sample of 5.6 Gb of gut viral DNA sequence from six individuals. Tests showed that a new pipeline based on DeBruijn graph assembly yielded longer contigs that were able to recruit more reads than the equivalent non-optimized, single-pass approach. To characterize gene content, the database of viral RefSeq proteins was compared to the assembled viral contigs, generating a bipartite graph with functional cassettes linking together viral contigs, which revealed a high degree of connectivity between diverse genomes involving multiple genes of the same functional class. In a second step, open reading frames were grouped by their co-occurrence on contigs in a database-independent manner, revealing conserved cassettes of co-oriented ORFs. These methods reveal that free-living bacteriophages, while usually dissimilar at the nucleotide level, often have significant similarity at the level of encoded amino acid motifs, gene order, and gene orientation. These findings thus connect contemporary metagenomic analysis with classical studies of bacteriophage genomic cassettes. Software is available at https://sourceforge.net/projects/optitdba/.
doi:10.1371/journal.pone.0042342
PMCID: PMC3416800  PMID: 22900013

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