Two Strains of Crocosphaera watsonii with Highly Conserved Genomes are Distinguished by Strain-Specific Features

Unicellular nitrogen-fixing cyanobacteria are important components of marine phytoplankton. Although non-nitrogen-fixing marine phytoplankton generally exhibit high gene sequence and genomic diversity, gene sequences of natural populations and isolated strains of Crocosphaera watsonii, one of the two most abundant open ocean unicellular cyanobacteria groups, have been shown to be 98–100% identical. The low sequence diversity in Crocosphaera is a dramatic contrast to sympatric species of Prochlorococcus and Synechococcus, and raises the question of how genome differences can explain observed phenotypic diversity among Crocosphaera strains. Here we show, through whole genome comparisons of two phenotypically different strains, that there are strain-specific sequences in each genome, and numerous genome rearrangements, despite exceptionally low sequence diversity in shared genomic regions. Some of the strain-specific sequences encode functions that explain observed phenotypic differences, such as exopolysaccharide biosynthesis. The pattern of strain-specific sequences distributed throughout the genomes, along with rearrangements in shared sequences is evidence of significant genetic mobility that may be attributed to the hundreds of transposase genes found in both strains. Furthermore, such genetic mobility appears to be the main mechanism of strain divergence in Crocosphaera which do not accumulate DNA microheterogeneity over the vast majority of their genomes. The strain-specific sequences found in this study provide tools for future physiological studies, as well as genetic markers to help determine the relative abundance of phenotypes in natural populations

Approaching the uncultured endosymbiont of Riftia pachyptila by physiological proteomics

Author Posting. © The Authors, 2006. This is the author's version of the work. It is posted here by permission of AAAS for personal use, not for redistribution. The definitive version was published in Science 315 (2007): 247-250, doi:10.1126/science.1132913.The bacterial endosymbiont of the deep-sea tube worm Riftia pachyptila has never been successfully cultivated outside its host. In the absence of cultivation data we have taken a proteomic approach based on the metagenome sequence to study the metabolism of this peculiar microorganism in detail. As one result, we found that three major sulfide oxidation proteins constitute ~12% of the total cytosolic proteome, highlighting the essential role of these enzymes for the symbiont’s energy metabolism. Unexpectedly, the symbiont uses the reductive tricarboxylic acid (TCA) cycle in addition to the previously identified Calvin cycle for CO2 fixation.This work was supported by the DFG, grant Schw595/3-1. Other funding sources were: NSF (OCE 04-52333) and NASA Astrobiology Institute (NNA04CC04A) for SMS, MH: postdoctoral scholarship from WHOI, HF: Academic Senate (RF811S and RE518S)

Mechanisms of Thermal Adaptation Revealed From the Genomes of the Antarctic Archaea Methanogenium frigidum and Methanococcoides burtonii

We generated draft genome sequences for two cold-adapted Archaea, Methanogenium frigidum and Methanococcoides burtonii, to identify genotypic characteristics that distinguish them from Archaea with a higher optimal growth temperature (OGT). Comparative genomics revealed trends in amino acid and tRNA composition, and structural features of proteins. Proteins from the cold-adapted Archaea are characterized by a higher content of noncharged polar amino acids, particularly Gin and Thr and a lower content of hydrophobic amino acids, particularly Leu. Sequence data from nine methanogen genomes (OGT 15degrees-98degreesC) were used to generate IIII modeled protein structures. Analysis of the models from the cold-adapted Archaea showed a strong tendency in the solvent-accessible area for more Gin, Thr, and hydrophobic residues and fewer charged residues. A cold shock domain (CSD) protein (CspA homolog) was identified in M. frigidum, two hypothetical proteins with CSD-folds in M. burtonii, and a unique winged helix DNA-binding domain protein in M. burtonii. This suggests that these types of nucleic acid binding proteins have a critical role in cold-adapted Archaea. Structural analysis of tRNA sequences from the Archaea indicated that GC content is the major factor influencing tRNA stability in hyperthermophiles, but not in the psychrophiles, mesophiles or moderate thermophiles. Below an OGT of 60degreesC, the GC content in tRNA was largely unchanged, indicating that any requirement for flexibility of tRNA in psychrophiles is mediated by other means. This is the first time that comparisons have been performed with genome data from Archaea spanning the growth temperature extremes. from psychrophiles to hyperthermophile

Comparative genomics and natural distributions of phenotypically distinct strains of the nitrogen-fixing cyanobacterium Crocosphaera watsonii

Crocosphaera watsonii is an ecologically important marine unicellular diazotrophic cyanobacterium. It is often abundant in oligotrophic ocean regions where it provides fixed nitrogen to nutrient-limited phytoplankton communities. Previous genetic studies have observed genetic rearrangements but very little sequence variation among natural populations or cultivated strains of Crocosphaera. Those strains exhibit two phenotypes (large- and small-cell) with characteristics that suggest different ecological roles and niches. Prior to this work, the genetic basis for the phenotypic differences was unknown, and molecular methods for enumerating natural C. watsonii could not differentiate between phenotypes. To address those unanswered scientific questions, these studies compared the genomes of six C. watsonii strains, three of each phenotype, which were isolated over large spatial and temporal distances. A large portion of those genome sequences were shared among all strains with nearly 100% nucleotide identity. However, there were also genes that were specific to each strain, and others were specific to each phenotype, including some which could explain phenotypic differences (e.g. EPS biosynthesis). Relative to small-cell strains, large-cell strains had larger genomes and additional genetic capabilities, including possibly increased adaptations to iron and phosphorus limitation. Clustering based on genome sequences and content showed that strains with a common phenotype were evolutionarily most closely related, regardless of their time and location of isolation. Surprisingly, the genome of the C. watsonii type-strain, WH8501, was quite unusual, even compared to those with the same phenotype, suggesting it may not be appropriately representative of the species. To investigate distributions of Crocosphaera types in the marine environment, molecular assays were developed, based on phenotype-specific genes, and applied to samples from the North and South Pacific. In those samples, small-cells dominated in the upper 75 m where abundance of both types was much greater, while large-cells dominated in samples with lower counts between 100 m and 175 m. There was also more evidence that large-cells form aggregates in the N. Pacific. Future studies will be important to determine which of the initial C. watsonii patterns described here can be generalized, both in genomes, and in natural distributions of the two types

Comparative genomics and natural distributions of phenotypically distinct strains of the nitrogen-fixing cyanobacterium Crocosphaera watsonii

Crocosphaera watsonii is an ecologically important marine unicellular diazotrophic cyanobacterium. It is often abundant in oligotrophic ocean regions where it provides fixed nitrogen to nutrient-limited phytoplankton communities. Previous genetic studies have observed genetic rearrangements but very little sequence variation among natural populations or cultivated strains of Crocosphaera. Those strains exhibit two phenotypes (large- and small-cell) with characteristics that suggest different ecological roles and niches. Prior to this work, the genetic basis for the phenotypic differences was unknown, and molecular methods for enumerating natural C. watsonii could not differentiate between phenotypes. To address those unanswered scientific questions, these studies compared the genomes of six C. watsonii strains, three of each phenotype, which were isolated over large spatial and temporal distances. A large portion of those genome sequences were shared among all strains with nearly 100% nucleotide identity. However, there were also genes that were specific to each strain, and others were specific to each phenotype, including some which could explain phenotypic differences (e.g. EPS biosynthesis). Relative to small-cell strains, large-cell strains had larger genomes and additional genetic capabilities, including possibly increased adaptations to iron and phosphorus limitation. Clustering based on genome sequences and content showed that strains with a common phenotype were evolutionarily most closely related, regardless of their time and location of isolation. Surprisingly, the genome of the C. watsonii type-strain, WH8501, was quite unusual, even compared to those with the same phenotype, suggesting it may not be appropriately representative of the species. To investigate distributions of Crocosphaera types in the marine environment, molecular assays were developed, based on phenotype-specific genes, and applied to samples from the North and South Pacific. In those samples, small-cells dominated in the upper 75 m where abundance of both types was much greater, while large-cells dominated in samples with lower counts between 100 m and 175 m. There was also more evidence that large-cells form aggregates in the N. Pacific. Future studies will be important to determine which of the initial C. watsonii patterns described here can be generalized, both in genomes, and in natural distributions of the two types

Coupling FACS and genomic methods for the characterization of uncultivated symbionts.

Symbioses between microbes are likely widespread and functionally relevant in diverse biological systems; however, they are difficult to discover. Most microbes remain uncultivated, symbioses can be relatively rare or dynamic, and intercellular connections can be delicate. Thus, traditional methods such as microscopy are inadequate for efficient discovery and precise characterization of novel interactions, their metabolic basis, and the species involved. High-throughput metagenomic sequencing of entire microbial communities has revolutionized the field of microbial ecology; however, genomic signals from symbionts can get buried in sequences from abundant organisms and evidence for direct links between microbial species cannot be gained from bulk samples. Thus, a specialized approach to the characterization of symbioses between naturally occurring microbes is required. This chapter presents methods for combining fluorescence-activated cell sorting to isolate and separate uncultivated symbionts with molecular biology techniques for DNA amplification in order to characterize uncultivated symbionts through genomic and metagenomic techniques

Whole genome comparison of six Crocosphaera watsonii strains with differing phenotypes.

Crocosphaera watsonii, a unicellular nitrogen-fixing cyanobacterium found in oligotrophic oceans, is important in marine carbon and nitrogen cycles. Isolates of C. watsonii can be separated into at least two phenotypes with environmentally important differences, indicating possibly distinct ecological roles and niches. To better understand the evolutionary history and variation in metabolic capabilities among strains and phenotypes, this study compared the genomes of six C. watsonii strains, three from each phenotypic group, which had been isolated over several decades from multiple ocean basins. While a substantial portion of each genome was nearly identical to sequences in the other strains, a few regions were identified as specific to each strain and phenotype, some of which help explain observed phenotypic features. Overall, the small-cell type strains had smaller genomes and a relative loss of genetic capabilities, while the large-cell type strains were characterized by larger genomes, some genetic redundancy, and potentially increased adaptations to iron and phosphorus limitation. As such, strains with shared phenotypes were evolutionarily more closely related than those with the opposite phenotype, regardless of isolation location or date. Unexpectedly, the genome of the type-strain for the species, C. watsonii WH8501, was quite unusual even among strains with a shared phenotype, indicating it may not be an ideal representative of the species. The genome sequences and analyses reported in this study will be important for future investigations of the proposed differences in adaptation of the two phenotypes to nutrient limitation, and to identify phenotype-specific distributions in natural Crocosphaera populations