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1.  Replicating phages in the epidermal mucosa of the eel (Anguilla anguilla) 
In this work, we used the eel (Anguilla anguilla) as an animal model to test the hypothesis of Barr et al. (2013a,b) about the putative role of the epidermal mucosa as a phage enrichment layer. To this end, we analyzed the microbial content of the skin mucus of wild and farmed eels by using a metagenomic approach. We found a great abundance of replicating phage genomes (concatemers) in all the samples. They were assembled in four complete genomes of three Myovirus and one Podovirus. We also found evidences that ΦKZ and Podovirus phages could be part of the resident microbiota associated to the eel mucosal surface and persist on them over the time. Moreover, the viral abundance estimated by epiflorescent counts and by metagenomic recruitment from eel mucosa was higher than that of the surrounding water. Taken together, our results support the hypothesis that claims a possible role of phages in the animal mucus as agents controlling bacterial populations, including pathogenic species, providing a kind of innate immunity.
doi:10.3389/fmicb.2015.00003
PMCID: PMC4310352
metagenomics; phage; eel; mucosa; immunity
2.  From Metagenomics to Pure Culture: Isolation and Characterization of the Moderately Halophilic Bacterium Spiribacter salinus gen. nov., sp. nov. 
Applied and Environmental Microbiology  2014;80(13):3850-3857.
Recent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dominated by both Euryarchaeota and halophilic bacteria, with a gammaproteobacterium closely related to the genera Alkalilimnicola and Arhodomonas being one of the most predominant microorganisms, making up to 15% of the total prokaryotic population. Here we used several strategies and culture media in order to isolate this organism in pure culture. We report the isolation and taxonomic characterization of this new, never before cultured microorganism, designated M19-40T, isolated from a saltern located in Isla Cristina, Spain, using a medium with a mixture of 15% salts, yeast extract, and pyruvic acid as the carbon source. Morphologically small curved cells (young cultures) with a tendency to form long spiral cells in older cultures were observed in pure cultures. The organism is a Gram-negative, nonmotile bacterium that is strictly aerobic, non-endospore forming, heterotrophic, and moderately halophilic, and it is able to grow at 10 to 25% (wt/vol) NaCl, with optimal growth occurring at 15% (wt/vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that strain M19-40T has a low similarity with other previously described bacteria and shows the closest phylogenetic similarity with species of the genera Alkalilimnicola (94.9 to 94.5%), Alkalispirillum (94.3%), and Arhodomonas (93.9%) within the family Ectothiorhodospiraceae. The phenotypic, genotypic, and chemotaxonomic features of this new bacterium showed that it constitutes a new genus and species, for which the name Spiribacter salinus gen. nov., sp. nov., is proposed, with strain M19-40T (= CECT 8282T = IBRC-M 10768T = LMG 27464T) being the type strain.
doi:10.1128/AEM.00430-14
PMCID: PMC4054224  PMID: 24747894
3.  Genome Sequence of “Thalassospira australica” NP3b2T Isolated from St. Kilda Beach, Tasman Sea 
Genome Announcements  2014;2(6):e01139-14.
Here, we present the draft genome of “Thalassospira australica” NP3b2T, a potential poly(ethylene terephthalate) (PET) plastic biodegrader. This genomic information will enhance information on the genetic basis of metabolic pathways for the degradation of PET plastic.
doi:10.1128/genomeA.01139-14
PMCID: PMC4241657  PMID: 25395631
4.  Metagenomics of the Mucosal Microbiota of European Eels 
Genome Announcements  2014;2(6):e01132-14.
European eels are an economically important and threatened species that are prone to rapid collapse in farm conditions. Using metagenomics, we show that the eel mucosal microbiota has specific features distinguishing it from the surrounding aquatic community. This is a first step in dissecting the resident microbiota of this critical barrier that may have implications for maintenance of healthy eel populations.
doi:10.1128/genomeA.01132-14
PMCID: PMC4223461  PMID: 25377710
5.  RNA sequencing provides evidence for functional variability between naturally co-existing Alteromonas macleodii lineages 
BMC Genomics  2014;15(1):938.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-938) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-938
PMCID: PMC4223743  PMID: 25344729
Alteromonas macleodii; Genomic diversity; RNA-seq; Transcriptomics; Genomic islands; CRISPR
6.  Metagenomics of Ancient Fermentation Pits Used for the Production of Chinese Strong-Aroma Liquor 
Genome Announcements  2014;2(5):e01045-14.
The complex microbiota of pit mud of solid-state fermentation reactors used for the production of Chinese liquor is responsible for producing one of the oldest distillates in the world. We apply a deep-sequencing approach to characterize the microbiota from pits that have been in use for up to 440 years.
doi:10.1128/genomeA.01045-14
PMCID: PMC4208321  PMID: 25342677
7.  Genomes of Alteromonas australica, a world apart 
BMC Genomics  2014;15(1):483.
Background
Alteromonas is a genus of marine bacteria that is very easy to isolate and grow in the laboratory. There are genomes available of the species Alteromonas macleodii from different locations around the world and an Alteromonas sp. isolated from a sediment in Korea. We have analyzed the genomes of two strains classified by 16S rRNA (>99% similarity) as the recently described species Alteromonas australica, and isolated from opposite ends of the world; A. australica DE170 was isolated in the South Adriatic (Mediterranean) at 1000 m depth while A. australica H17T was isolated from a sea water sample collected in St Kilda Beach, Tasman Sea.
Results
Although these two strains belong to a clearly different species from A. macleodii, the overall synteny is well preserved and the flexible genomic islands seem to code for equivalent functions and be located at similar positions. Actually the genomes of all the Alteromonas species known to date seem to preserve synteny quite well with the only exception of the sediment isolate SN2. Among the specific metabolic features found for the A. australica isolates there is the degradation of xylan and production of cellulose as extracellular polymeric substance by DE170 or the potential ethanol/methanol degradation by H17T.
Conclusions
The genomes of the two A. australica isolates are not more different than those of strains of A. macleodii isolated from the same sample. Actually the recruitment from metagenomes indicates that all the available genomes are found in most tropical-temperate marine samples analyzed and that they live in consortia of several species and multiple clones within each. Overall the hydrolytic activities of the Alteromonas genus as a whole are impressive and fit with its known capabilities to exploit sudden inputs of organic matter in their environment.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-483) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-483
PMCID: PMC4119200  PMID: 24942065
Alteromonas australica; Biogeography; Alteromonas; Genomic Island; Population genomics; Integron
8.  Pangenome Evidence for Extensive Interdomain Horizontal Transfer Affecting Lineage Core and Shell Genes in Uncultured Planktonic Thaumarchaeota and Euryarchaeota 
Genome Biology and Evolution  2014;6(7):1549-1563.
Horizontal gene transfer (HGT) is an important force in evolution, which may lead, among other things, to the adaptation to new environments by the import of new metabolic functions. Recent studies based on phylogenetic analyses of a few genome fragments containing archaeal 16S rRNA genes and fosmid-end sequences from deep-sea metagenomic libraries have suggested that marine planktonic archaea could be affected by high HGT frequency. Likewise, a composite genome of an uncultured marine euryarchaeote showed high levels of gene sequence similarity to bacterial genes. In this work, we ask whether HGT is frequent and widespread in genomes of these marine archaea, and whether HGT is an ancient and/or recurrent phenomenon. To answer these questions, we sequenced 997 fosmid archaeal clones from metagenomic libraries of deep-Mediterranean waters (1,000 and 3,000 m depth) and built comprehensive pangenomes for planktonic Thaumarchaeota (Group I archaea) and Euryarchaeota belonging to the uncultured Groups II and III Euryarchaeota (GII/III-Euryarchaeota). Comparison with available reference genomes of Thaumarchaeota and a composite marine surface euryarchaeote genome allowed us to define sets of core, lineage-specific core, and shell gene ortholog clusters for the two archaeal lineages. Molecular phylogenetic analyses of all gene clusters showed that 23.9% of marine Thaumarchaeota genes and 29.7% of GII/III-Euryarchaeota genes had been horizontally acquired from bacteria. HGT is not only extensive and directional but also ongoing, with high HGT levels in lineage-specific core (ancient transfers) and shell (recent transfers) genes. Many of the acquired genes are related to metabolism and membrane biogenesis, suggesting an adaptive value for life in cold, oligotrophic oceans. We hypothesize that the acquisition of an important amount of foreign genes by the ancestors of these archaeal groups significantly contributed to their divergence and ecological success.
doi:10.1093/gbe/evu127
PMCID: PMC4122925  PMID: 24923324
horizontal gene transfer; Thaumarchaeota; Euryarchaeota; ammonia-oxidizing archaea; uncultured archaea
9.  Homologous recombination is involved in the diversity of replacement flexible genomic islands in aquatic prokaryotes 
Frontiers in Genetics  2014;5:147.
Different strains of the same prokaryotic species, even very similar ones, vary in large regions of their genomes. This flexible genome represents a huge reservoir of diversity that allows prokaryotes to exploit their environment efficiently. Most of the flexible genome is concentrated in genomic islands, some of which are present in all the strains and coding for similar functions but containing different genes. These replacement genomic islands are typically involved in exposed cellular structures, and their diversity has been connected to their recognition as targets by prokaryotic viruses (phages). We have compared genomes of closely related aquatic microbes from different origins and found examples of recent replacement of some of these flexible genomic islands. In all cases, that include Gram positive and negative bacteria and one archaeon, the replaced regions boundaries contain tell-tale peaks of increased, mostly synonymous, nucleotide substitutions. They tended to be sharper at the boundary closest to the origin of replication of the island. We will present the hypothesis that replacement flexible genomic islands are often exchanged by homologous recombination between different clonal frames. These recombination events are possibly selected due to the immediate reward provided by a change in the phage sensitivity spectrum.
doi:10.3389/fgene.2014.00147
PMCID: PMC4033161  PMID: 24904647
homologous recombination; SNP; genomic island; aquatic bacteria; phage predation; genomic diversity
10.  Comparison of prokaryotic community structure from Mediterranean and Atlantic saltern concentrator ponds by a metagenomic approach 
We analyzed the prokaryotic community structure of a saltern pond with 21% total salts located in Isla Cristina, Huelva, Southwest Spain, close to the Atlantic ocean coast. For this purpose, we constructed a metagenome (designated as IC21) obtained by pyrosequencing consisting of 486 Mb with an average read length of 397 bp and compared it with other metagenomic datasets obtained from ponds with 19, 33, and 37% total salts acquired from Santa Pola marine saltern, located in Alicante, East Spain, on the Mediterranean coast. Although the salinity in IC21 is closer to the pond with 19% total salts from Santa Pola saltern (designated as SS19), IC21 is more similar at higher taxonomic levels to the pond with 33% total salts from Santa Pola saltern (designated as SS33), since both are predominated by the phylum Euryarchaeota. However, there are significant differences at lower taxonomic levels where most sequences were related to the genus Halorubrum in IC21 and to Haloquadratum in SS33. Within the Bacteroidetes, the genus Psychroflexus is the most abundant in IC21 while Salinibacter dominates in SS33. Sequences related to bacteriorhodopsins and halorhodopsins correlate with the abundance of Haloquadratum in Santa Pola SS19 to SS33 and of Halorubrum in Isla Cristina IC21 dataset, respectively. Differences in composition might be attributed to local ecological conditions since IC21 showed a decrease in the number of sequences related to the synthesis of compatible solutes and in the utilization of phosphonate.
doi:10.3389/fmicb.2014.00196
PMCID: PMC4021199  PMID: 24847316
metagenomics; haloarchaea; halophilic bacteria; saltern; prokaryotic diversity
11.  Tales from a thousand and one phages 
Bacteriophage  2014;4: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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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