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1.  Integrating metagenomic and amplicon databases to resolve the phylogenetic and ecological diversity of the Chlamydiae 
The ISME Journal  2013;8(1):115-125.
In the era of metagenomics and amplicon sequencing, comprehensive analyses of available sequence data remain a challenge. Here we describe an approach exploiting metagenomic and amplicon data sets from public databases to elucidate phylogenetic diversity of defined microbial taxa. We investigated the phylum Chlamydiae whose known members are obligate intracellular bacteria that represent important pathogens of humans and animals, as well as symbionts of protists. Despite their medical relevance, our knowledge about chlamydial diversity is still scarce. Most of the nine known families are represented by only a few isolates, while previous clone library-based surveys suggested the existence of yet uncharacterized members of this phylum. Here we identified more than 22 000 high quality, non-redundant chlamydial 16S rRNA gene sequences in diverse databases, as well as 1900 putative chlamydial protein-encoding genes. Even when applying the most conservative approach, clustering of chlamydial 16S rRNA gene sequences into operational taxonomic units revealed an unexpectedly high species, genus and family-level diversity within the Chlamydiae, including 181 putative families. These in silico findings were verified experimentally in one Antarctic sample, which contained a high diversity of novel Chlamydiae. In our analysis, the Rhabdochlamydiaceae, whose known members infect arthropods, represents the most diverse and species-rich chlamydial family, followed by the protist-associated Parachlamydiaceae, and a putative new family (PCF8) with unknown host specificity. Available information on the origin of metagenomic samples indicated that marine environments contain the majority of the newly discovered chlamydial lineages, highlighting this environment as an important chlamydial reservoir.
doi:10.1038/ismej.2013.142
PMCID: PMC3869019  PMID: 23949660
16S rRNA; next-generation sequencing; amplicon sequencing; metagenomics
2.  Metagenomic insights into strategies of carbon conservation and unusual sulfur biogeochemistry in a hypersaline Antarctic lake 
The ISME Journal  2013;7(10):1944-1961.
Organic Lake is a shallow, marine-derived hypersaline lake in the Vestfold Hills, Antarctica that has the highest reported concentration of dimethylsulfide (DMS) in a natural body of water. To determine the composition and functional potential of the microbial community and learn about the unusual sulfur chemistry in Organic Lake, shotgun metagenomics was performed on size-fractionated samples collected along a depth profile. Eucaryal phytoflagellates were the main photosynthetic organisms. Bacteria were dominated by the globally distributed heterotrophic taxa Marinobacter, Roseovarius and Psychroflexus. The dominance of heterotrophic degradation, coupled with low fixation potential, indicates possible net carbon loss. However, abundant marker genes for aerobic anoxygenic phototrophy, sulfur oxidation, rhodopsins and CO oxidation were also linked to the dominant heterotrophic bacteria, and indicate the use of photo- and lithoheterotrophy as mechanisms for conserving organic carbon. Similarly, a high genetic potential for the recycling of nitrogen compounds likely functions to retain fixed nitrogen in the lake. Dimethylsulfoniopropionate (DMSP) lyase genes were abundant, indicating that DMSP is a significant carbon and energy source. Unlike marine environments, DMSP demethylases were less abundant, indicating that DMSP cleavage is the likely source of high DMS concentration. DMSP cleavage, carbon mixotrophy (photoheterotrophy and lithoheterotrophy) and nitrogen remineralization by dominant Organic Lake bacteria are potentially important adaptations to nutrient constraints. In particular, carbon mixotrophy relieves the extent of carbon oxidation for energy production, allowing more carbon to be used for biosynthetic processes. The study sheds light on how the microbial community has adapted to this unique Antarctic lake environment.
doi:10.1038/ismej.2013.69
PMCID: PMC3965305  PMID: 23619305
metagenomics; Organic Lake; Antarctic microbial ecology; nutrient cycles; dimethylsulfide
4.  Ecotype Diversity and Conversion in Photobacterium profundum Strains 
PLoS ONE  2014;9(5):e96953.
Photobacterium profundum is a cosmopolitan marine bacterium capable of growth at low temperature and high hydrostatic pressure. Multiple strains of P. profundum have been isolated from different depths of the ocean and display remarkable differences in their physiological responses to pressure. The genome sequence of the deep-sea piezopsychrophilic strain Photobacterium profundum SS9 has provided some clues regarding the genetic features required for growth in the deep sea. The sequenced genome of Photobacterium profundum strain 3TCK, a non-piezophilic strain isolated from a shallow-water environment, is now available and its analysis expands the identification of unique genomic features that correlate to environmental differences and define the Hutchinsonian niche of each strain. These differences range from variations in gene content to specific gene sequences under positive selection. Genome plasticity between Photobacterium bathytypes was investigated when strain 3TCK-specific genes involved in photorepair were introduced to SS9, demonstrating that horizontal gene transfer can provide a mechanism for rapid colonisation of new environments.
doi:10.1371/journal.pone.0096953
PMCID: PMC4019646  PMID: 24824441
5.  A metaproteomic assessment of winter and summer bacterioplankton from Antarctic Peninsula coastal surface waters 
The ISME Journal  2012;6(10):1883-1900.
A metaproteomic survey of surface coastal waters near Palmer Station on the Antarctic Peninsula, West Antarctica, was performed, revealing marked differences in the functional capacity of summer and winter communities of bacterioplankton. Proteins from Flavobacteria were more abundant in the summer metaproteome, whereas winter was characterized by proteins from ammonia-oxidizing Marine Group I Crenarchaeota. Proteins prevalent in both seasons were from SAR11 and Rhodobacterales clades of Alphaproteobacteria, as well as many lineages of Gammaproteobacteria. The metaproteome data were used to elucidate the main metabolic and energy generation pathways and transport processes occurring at the microbial level in each season. In summer, autotrophic carbon assimilation appears to be driven by oxygenic photoautotrophy, consistent with high light availability and intensity. In contrast, during the dark polar winter, the metaproteome supported the occurrence of chemolithoautotrophy via the 3-hydroxypropionate/4-hydroxybutyrate cycle and the reverse tricarboxylic acid cycle of ammonia-oxidizing archaea and nitrite-oxidizing bacteria, respectively. Proteins involved in nitrification were also detected in the metaproteome. Taurine appears to be an important source of carbon and nitrogen for heterotrophs (especially SAR11), with transporters and enzymes for taurine uptake and degradation abundant in the metaproteome. Divergent heterotrophic strategies for Alphaproteobacteria and Flavobacteria were indicated by the metaproteome data, with Alphaproteobacteria capturing (by high-affinity transport) and processing labile solutes, and Flavobacteria expressing outer membrane receptors for particle adhesion to facilitate the exploitation of non-labile substrates. TonB-dependent receptors from Gammaproteobacteria and Flavobacteria (particularly in summer) were abundant, indicating that scavenging of substrates was likely an important strategy for these clades of Southern Ocean bacteria. This study provides the first insight into differences in functional processes occurring between summer and winter microbial communities in coastal Antarctic waters, and particularly highlights the important role that ‘dark' carbon fixation has in winter.
doi:10.1038/ismej.2012.28
PMCID: PMC3446797  PMID: 22534610
marine microorganisms; metaproteomics; antarctic microbiology; southern ocean microbiology
6.  Global biogeography of SAR11 marine bacteria 
Metagenomic samples from oceans around the globe were used to examine the biogeography of the dominant marine heterotrophic bacterial clade, SAR11. Analysis uncovers evidence of adaptive radiation in response to environmental parameters, particularly temperature.
By generating 37 new Antarctic metagenomes and analysing the internal transcribed spacer (ITS) regions of the SAR11 clade in a total of 128 surface marine metagenomes, we identified phylotype distributions that strongly correlated with temperature and latitude.By assembling SAR11 genomes from Antarctic metagenome data, we identified specific genes, biases in gene functions and signatures of positive selection in the genomes of the polar SAR11—genomic signatures of adaptive radiation.Our data demonstrate the importance of adaptive radiation in an organism's ability to proliferate throughout the world's oceans, and describe genomic traits characteristic of different phylotypes in specific marine biomes.These bacteria are important marine heterotrophs and have a fundamental role in oceanic nutrient cycling. These findings, therefore, have important implications for our ability to predict how changes in ocean temperature may affect bacterial ecology.
The ubiquitous SAR11 bacterial clade is the most abundant type of organism in the world's oceans, but the reasons for its success are not fully elucidated. We analysed 128 surface marine metagenomes, including 37 new Antarctic metagenomes. The large size of the data set enabled internal transcribed spacer (ITS) regions to be obtained from the Southern polar region, enabling the first global characterization of the distribution of SAR11, from waters spanning temperatures −2 to 30°C. Our data show a stable co-occurrence of phylotypes within both ‘tropical' (>20°C) and ‘polar' (<10°C) biomes, highlighting ecological niche differentiation between major SAR11 subgroups. All phylotypes display transitions in abundance that are strongly correlated with temperature and latitude. By assembling SAR11 genomes from Antarctic metagenome data, we identified specific genes, biases in gene functions and signatures of positive selection in the genomes of the polar SAR11—genomic signatures of adaptive radiation. Our data demonstrate the importance of adaptive radiation in the organism's ability to proliferate throughout the world's oceans, and describe genomic traits characteristic of different phylotypes in specific marine biomes.
doi:10.1038/msb.2012.28
PMCID: PMC3421443  PMID: 22806143
adaptive radiation; Antarctica; metagenome; Pelagibacter; phylotype distribution
7.  An assessment of actinobacterial diversity in the marine environment 
Antonie Van Leeuwenhoek  2008;94(1):51-62.
The 16S rRNA gene sequence diversity within the Phylum Actinobacteria was assessed from four sources: PCR-generated V6 sequence tags derived from seawater samples, metagenomic data from the Global Ocean Sampling (GOS) expedition, marine-derived sequences maintained in the Ribosomal Database Project (RDP), and select cultured strains for which sequence data is not yet available in the RDP. This meta-analysis revealed remarkable levels of phylogenetic diversity and confirms the existence of major, deeply rooted, and as of yet uncharacterized lineages within the phylum. A dramatic incongruence among cultured strains and those detected using culture-independent techniques was also revealed. Redundancy among the actinobacteria detected using culture-independent techniques suggests that greater sequence coverage or improved DNA extraction efficiencies may be required to detect the rare phylotypes that can be readily cultured from marine samples. Conversely, new strategies need to be developed for the cultivation of frequently observed but yet to be cultured marine actinobacteria.
doi:10.1007/s10482-008-9239-x
PMCID: PMC3375478  PMID: 18500568
Marine actinobacteria; Bacterial diversity; Metagenomics; Actinomycetes
8.  An integrative study of a meromictic lake ecosystem in Antarctica 
The ISME journal  2010;5(5):879-895.
In nature, the complexity and structure of microbial communities varies widely, ranging from a few species to thousands of species, and from highly structured to highly unstructured communities. Here, we describe the identity and functional capacity of microbial populations within distinct layers of a pristine, marine-derived, meromictic (stratified) lake (Ace Lake) in Antarctica. Nine million open reading frames were analyzed, representing microbial samples taken from six depths of the lake size fractionated on sequential 3.0, 0.8 and 0.1 μm filters, and including metaproteome data from matching 0.1 μm filters. We determine how the interactions of members of this highly structured and moderately complex community define the biogeochemical fluxes throughout the entire lake. Our view is that the health of this delicate ecosystem is dictated by the effects of the polar light cycle on the dominant role of green sulfur bacteria in primary production and nutrient cycling, and the influence of viruses/phage and phage resistance on the cooperation between members of the microbial community right throughout the lake. To test our assertions, and develop a framework applicable to other microbially driven ecosystems, we developed a mathematical model that describes how cooperation within a microbial system is impacted by periodic fluctuations in environmental parameters on key populations of microorganisms. Our study reveals a mutualistic structure within the microbial community throughout the lake that has arisen as the result of mechanistic interactions between the physico-chemical parameters and the selection of individual members of the community. By exhaustively describing and modelling interactions in Ace Lake, we have developed an approach that may be applicable to learning how environmental perturbations affect the microbial dynamics in more complex aquatic systems.
doi:10.1038/ismej.2010.185
PMCID: PMC3105772  PMID: 21124488
metagenomics/metaproteomics; Antarctic meromictic lake; green sulfur bacteria; virus/phage; nutrient cycle; Lotka–Volterra predator–prey model
9.  Importance of Proteins Controlling Initiation of DNA Replication in the Growth of the High-Pressure-Loving Bacterium Photobacterium profundum SS9▿  
Journal of Bacteriology  2009;191(20):6383-6393.
The molecular mechanism(s) by which deep-sea bacteria grow optimally under high hydrostatic pressure at low temperatures is poorly understood. To gain further insight into the mechanism(s), a previous study screened transposon mutant libraries of the deep-sea bacterium Photobacterium profundum SS9 and identified mutants which exhibited alterations in growth at high pressure relative to that of the parent strain. Two of these mutants, FL23 (PBPRA3229::mini-Tn10) and FL28 (PBPRA1039::mini-Tn10), were found to have high-pressure sensitivity and enhanced-growth phenotypes, respectively. The PBPRA3229 and PBPRA1039 genes encode proteins which are highly similar to Escherichia coli DiaA, a positive regulator, and SeqA, a negative regulator, respectively, of the initiation of DNA replication. In this study, we investigated the hypothesis that PBPRA3229 and PBPRA1039 encode DiaA and SeqA homologs, respectively. Consistent with this, we determined that the plasmid-carried PBPRA3229 and PBPRA1039 genes restored synchrony to the initiation of DNA replication in E. coli mutants lacking DiaA and SeqA, respectively. Additionally, PBPRA3229 restored the cold sensitivity phenotype of an E. coli dnaA(Cs) diaA double mutant whereas PBPRA1039 suppressed the cold sensitivity phenotype of an E. coli dnaA(Cs) single mutant. Taken together, these findings show that the genes disrupted in FL23 and FL28 encode DiaA and SeqA homologs, respectively. Consequently, our findings add support to a model whereby high pressure affects the initiation of DNA replication in P. profundum SS9 and either the presence of a positive regulator (DiaA) or the removal of a negative regulator (SeqA) promotes growth under these conditions.
doi:10.1128/JB.00576-09
PMCID: PMC2753030  PMID: 19700526
10.  The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ †  
Applied and Environmental Microbiology  2008;74(20):6298-6305.
Motility is a critical function needed for nutrient acquisition, biofilm formation, and the avoidance of harmful chemicals and predators. Flagellar motility is one of the most pressure-sensitive cellular processes in mesophilic bacteria; therefore, it is ecologically relevant to determine how deep-sea microbes have adapted their motility systems for functionality at depth. In this study, the motility of the deep-sea piezophilic bacterium Photobacterium profundum SS9 was investigated and compared with that of the related shallow-water piezosensitive strain Photobacterium profundum 3TCK, as well as that of the well-studied piezosensitive bacterium Escherichia coli. The SS9 genome contains two flagellar gene clusters: a polar flagellum gene cluster (PF) and a putative lateral flagellum gene cluster (LF). In-frame deletions were constructed in the two flagellin genes located within the PF cluster (flaA and flaC), the one flagellin gene located within the LF cluster (flaB), a component of a putative sodium-driven flagellar motor (motA2), and a component of a putative proton-driven flagellar motor (motA1). SS9 PF flaA, flaC, and motA2 mutants were defective in motility under all conditions tested. In contrast, the flaB and motA1 mutants were defective only under conditions of high pressure and high viscosity. flaB and motA1 gene expression was strongly induced by elevated pressure plus increased viscosity. Direct swimming velocity measurements were obtained using a high-pressure microscopic chamber, where increases in pressure resulted in a striking decrease in swimming velocity for E. coli and a gradual reduction for 3TCK which proceeded up to 120 MPa, while SS9 increased swimming velocity at 30 MPa and maintained motility up to a maximum pressure of 150 MPa. Our results indicate that P. profundum SS9 possesses two distinct flagellar systems, both of which have acquired dramatic adaptations for optimal functionality under high-pressure conditions.
doi:10.1128/AEM.01316-08
PMCID: PMC2570297  PMID: 18723648
11.  Large-Scale Transposon Mutagenesis of Photobacterium profundum SS9 Reveals New Genetic Loci Important for Growth at Low Temperature and High Pressure▿  
Journal of Bacteriology  2007;190(5):1699-1709.
Microorganisms adapted to piezopsychrophilic growth dominate the majority of the biosphere that is at relatively constant low temperatures and high pressures, but the genetic bases for the adaptations are largely unknown. Here we report the use of transposon mutagenesis with the deep-sea bacterium Photobacterium profundum strain SS9 to isolate dozens of mutant strains whose growth is impaired at low temperature and/or whose growth is altered as a function of hydrostatic pressure. In many cases the gene mutation-growth phenotype relationship was verified by complementation analysis. The largest fraction of loci associated with temperature sensitivity were involved in the biosynthesis of the cell envelope, in particular the biosynthesis of extracellular polysaccharide. The largest fraction of loci associated with pressure sensitivity were involved in chromosomal structure and function. Genes for ribosome assembly and function were found to be important for both low-temperature and high-pressure growth. Likewise, both adaptation to temperature and adaptation to pressure were affected by mutations in a number of sensory and regulatory loci, suggesting the importance of signal transduction mechanisms in adaptation to either physical parameter. These analyses were the first global analyses of genes conditionally required for low-temperature or high-pressure growth in a deep-sea microorganism.
doi:10.1128/JB.01176-07
PMCID: PMC2258685  PMID: 18156275
12.  The Unique 16S rRNA Genes of Piezophiles Reflect both Phylogeny and Adaptation▿ †  
In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few γ-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions.
doi:10.1128/AEM.01726-06
PMCID: PMC1800765  PMID: 17158629
13.  Laterally transferred elements and high pressure adaptation in Photobacterium profundum strains 
BMC Genomics  2005;6:122.
Background
Oceans cover approximately 70% of the Earth's surface with an average depth of 3800 m and a pressure of 38 MPa, thus a large part of the biosphere is occupied by high pressure environments. Piezophilic (pressure-loving) organisms are adapted to deep-sea life and grow optimally at pressures higher than 0.1 MPa. To better understand high pressure adaptation from a genomic point of view three different Photobacterium profundum strains were compared. Using the sequenced piezophile P. profundum strain SS9 as a reference, microarray technology was used to identify the genomic regions missing in two other strains: a pressure adapted strain (named DSJ4) and a pressure-sensitive strain (named 3TCK). Finally, the transcriptome of SS9 grown under different pressure (28 MPa; 45 MPa) and temperature (4°C; 16°C) conditions was analyzed taking into consideration the differentially expressed genes belonging to the flexible gene pool.
Results
These studies indicated the presence of a large flexible gene pool in SS9 characterized by various horizontally acquired elements. This was verified by extensive analysis of GC content, codon usage and genomic signature of the SS9 genome. 171 open reading frames (ORFs) were found to be specifically absent or highly divergent in the piezosensitive strain, but present in the two piezophilic strains. Among these genes, six were found to also be up-regulated by high pressure.
Conclusion
These data provide information on horizontal gene flow in the deep sea, provide additional details of P. profundum genome expression patterns and suggest genes which could perform critical functions for abyssal survival, including perhaps high pressure growth.
doi:10.1186/1471-2164-6-122
PMCID: PMC1239915  PMID: 16162277

Results 1-13 (13)