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 Crocosphaerawatsonii, 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.

Hydrogen cycling by the unicellular marine diazotroph Crocosphaera watsonii strain WH8501.

The hydrogen (H2) cycle associated with the dinitrogen (N2) fixation process was studied in laboratory cultures of the marine cyanobacterium Crocosphaera watsonii. The rates of H2 production and acetylene (C2H2) reduction were continuously measured over the diel cycle with simultaneous measurements of fast repetition rate fluorometry and dissolved oxygen. The maximum rate of H2 production was coincident with the maximum rates of C2H2 reduction. Theoretical stoichiometry for N2 fixation predicts an equimolar ratio of H2 produced to N2 fixed. However, the maximum rate of net H2 production observed was 0.09 nmol H2 µg chlorophyll a (chl a)⁻¹ h⁻¹) compared to the N2 fixation rate of 5.5 nmol N2 µg chl a⁻¹ h⁻¹, with an H2 production/N2 fixation ratio of 0.02. The 50-fold discrepancy between expected and observed rates of H2 production was hypothesized to be a result of H2 reassimilation by uptake hydrogenase. This was confirmed by the addition of carbon monoxide (CO), a potent inhibitor of hydrogenase, which increased net H2 production rates ∼40-fold to a maximum rate of 3.5 nmol H2 µg chl a⁻¹ h⁻¹. We conclude that the reassimilation of H2 by C. watsonii is highly efficient (> 98%) and hypothesize that the tight coupling between H2 production and consumption is a consequence of fixing N2 at nighttime using a finite pool of respiratory carbon and electrons acquired from daytime solar energy capture. The H2 cycle provides unique insight into N2 fixation and associated metabolic processes in C. watsonii.

Genome-wide analysis of diel gene expression in the unicellular N(2)-fixing cyanobacterium Crocosphaera watsonii WH 8501.

The unicellular cyanobacterium Crocosphaera watsonii is an important nitrogen fixer in oligotrophic tropical and subtropical oceans. Metabolic, energy and cellular processes in cyanobacteria are regulated by the circadian mechanism, and/or follow the rhythmicity of light-dark cycles. The temporal separation of metabolic processes is especially essential for nitrogen fixation because of inactivation of the nitrogenase by oxygen. Using a microarray approach, we analyzed gene expression in cultures of Crocosphaera watsonii WH 8501 (C. watsonii) over a 24-h period and compared the whole-genome transcription with that in Cyanothece sp. ATCC 51142 (Cyanothece), a unicellular diazotroph that inhabits coastal marine waters. Similar to Cyanothece, regulation at the transcriptional level in C. watsonii was observed for all major metabolic and energy processes including photosynthesis, carbohydrate and amino acid metabolisms, respiration, and nitrogen fixation. Increased transcript abundance for iron acquisition genes by the end of the day appeared to be a general pattern in the unicellular diazotrophs. In contrast, genes for some ABC transporters (for example, phosphorus acquisition), DNA replication, and some genes encoding hypothetical proteins were differentially expressed in C. watsonii only. Overall, C. watsonii showed a higher percentage of genes with light-dark cycling patterns than Cyanothece, which may reflect the habitats preferences of the two cyanobacteria. This study represents the first whole-genome expression profiling in cultivated Crocosphaera, and the results will be useful in determining the basal physiology and ecology of the endemic Crocosphaera populations.

Seasonal Expression of the Picocyanobacterial Phosphonate Transporter Gene phnD in the Sargasso Sea.

In phosphorus-limited marine environments, picocyanobacteria (Synechococcus and Prochlorococcus spp.) can hydrolyze naturally occurring phosphonates as a P source. Utilization of 2-aminoethylphosphonate (2-AEP) is dependent on expression of the phn genes, encoding functions required for uptake, and C-P bond cleavage. Prior work has indicated that expression of picocyanobacterial phnD, encoding the phosphonate binding protein of the phosphonate ABC transporter, is a proxy for the assimilation of phosphonates in natural assemblages of Synechococcus spp. and Prochlorococcus spp (Ilikchyan et al., 2009). In this study, we expand this work to assess seasonal phnD expression in the Sargasso Sea. By RT-PCR, our data confirm that phnD expression is constitutive for the Prochlorococcus spp. detected, but in Synechococcus spp. phnD transcription follows patterns of phosphorus availability in the mixed layer. Specifically, our data suggest that phnD is repressed in the spring when P is bioavailable following deep winter mixing. In the fall, phnD expression follows a depth-dependent pattern reflecting depleted P at the surface following summertime drawdown, and elevated P at depth.

Detection and expression of the phosphonate transporter gene phnD in marine and freshwater picocyanobacteria.

We describe a PCR-based assay designed to detect expression of the phosphonate assimilation gene phnD from picocyanobacteria. The phnD gene encodes the phosphonate binding protein of the ABC-type phosphonate transporter, present in many of the picocyanobacterial genome sequences. Detection of phnD expression can indicate a capacity of picoplankton to utilize phosphonates, a refractory form of phosphorus that can represent 25% of the high-molecular-weight dissolved organic phosphorus pool in marine systems. Primer sets were designed to specifically amplify phnD sequences from marine and freshwater Synechococcus spp., Prochlorococcus spp. and environmental samples from the ocean and Laurentian Great Lakes. Quantitative RT-PCR from cultured marine Synechococcus sp. strain WH8102 and freshwater Synechococcus sp. ARC-21 demonstrated induction of phnD expression in P-deficient media, suggesting that phn genes are regulated coordinately with genes under phoRB control. Last, RT-PCR of environmental RNA samples from the Sargasso Sea and Pacific Ocean detected phnD expression from the endemic picocyanobacterial population. Synechococcus spp. phnD expression yielded a depth-dependent pattern following gradients of P bioavailability. By contrast, the Prochlorococcus spp. primers revealed that in all samples tested, phnD expression was constitutive. The method described herein will allow future studies aimed at understanding the utilization of naturally occurring phoshonates in the ocean as well as monitoring the acquisition of synthetic phosphonate herbicides (e.g. glyphosate) by picocyanobacteria in freshwaters.

The Alternative Splicing Mutation Database: a hub for investigations of alternative splicing using mutational evidence.

BACKGROUND: Some mutations in the internal regions of exons occur within splicing enhancers and silencers, influencing the pattern of alternative splicing in the corresponding genes. To understand how these sequence changes affect splicing, we created a database of these mutations. FINDINGS: The Alternative Splicing Mutation Database (ASMD) serves as a repository for all exonic mutations not associated with splicing junctions that measurably change the pattern of alternative splicing. In this initial published release (version 1.2), only human sequences are present, but the ASMD will grow to include other organisms, (see Availability and requirements section for the ASMD web address).This relational database allows users to investigate connections between mutations and features of the surrounding sequences, including flanking sequences, RNA secondary structures and strengths of splice junctions. Splicing effects of the mutations are quantified by the relative presence of alternative mRNA isoforms with and without a given mutation. This measure is further categorized by the accuracy of the experimental methods employed. The database currently contains 170 mutations in 66 exons, yet these numbers increase regularly.We developed an algorithm to derive a table of oligonucleotide Splicing Potential (SP) values from the ASMD dataset. We present the SP concept and tools in detail in our corresponding article. CONCLUSION: The current data set demonstrates that mutations affecting splicing are located throughout exons and might be enriched within local RNA secondary structures. Exons from the ASMD have below average splicing junction strength scores, but the difference is small and is judged not to be significant.

Calculation of splicing potential from the Alternative Splicing Mutation Database.

BACKGROUND: The Alternative Splicing Mutation Database (ASMD) presents a collection of all known mutations inside human exons which affect splicing enhancers and silencers and cause changes in the alternative splicing pattern of the corresponding genes. FINDINGS: An algorithm was developed to derive a Splicing Potential (SP) table from the ASMD information. This table characterizes the influence of each oligonucleotide on the splicing effectiveness of the exon containing it. If the SP value for an oligonucleotide is positive, it promotes exon retention, while negative SP values mean the sequence favors exon skipping. The merit of the SP approach is the ability to separate splicing signals from a wide range of sequence motifs enriched in exonic sequences that are attributed to protein-coding properties and/or translation efficiency. Due to its direct derivation from observed splice site selection, SP has an advantage over other computational approaches for predicting alternative splicing. CONCLUSION: We show that a vast majority of known exonic splicing enhancers have highly positive cumulative SP values, while known splicing silencers have core motifs with strongly negative cumulative SP values. Our approach allows for computation of the cumulative SP value of any sequence segment and, thus, gives researchers the ability to measure the possible contribution of any sequence to the pattern of splicing.