Genomic Characteristics of the Toxic Bloom-Forming Cyanobacterium Microcystis aeruginosa NIES-102.

Microcystis aeruginosa, a bloom-forming cyanobacterium distributed mainly in freshwater environments, can be divided into at least 12 groups (A-K and X) based on multi-locus phylogenetic analyses. In this study, we characterized the genome of microcystin-producing M. aeruginosa NIES-102, assigned to group A, isolated from Lake Kasumigaura, Japan. The complete genome sequence of M. aeruginosa NIES-102 comprised a 5.87-Mbp circular chromosome containing 5,330 coding sequences. The genome was the largest among all sequenced genomes for the species. In a comparison with the genome of M. aeruginosa NIES-843, which belongs to the same group, the microcystin biosynthetic gene cluster and CRISPR-Cas locus were highly similar. A synteny analysis revealed small-scale rearrangements between the two genomes. Genes encoding transposases were more abundant in these two genomes than in other Microcystis genomes. Our results improve our understanding of structural genomic changes and adaptation to a changing environment in the species.

Complete Genome Sequence of Microcystis aeruginosa NIES-2481 and Common Genomic Features of Group G M. aeruginosa.

Microcystis aeruginosa is a freshwater bloom-forming cyanobacterium that is distributed worldwide. M. aeruginosa can be divided into at least 8 phylogenetic groups (A-G and X) at the intraspecific level. Here, we report the complete genome sequence of M. aeruginosa NIES-2481, which was isolated from Lake Kasumigaura, Japan, and is assigned to group G. The complete genome sequence of M. aeruginosa NIES-2481 comprises a 4.29-Mbp circular chromosome and a 147,539-bp plasmid; the circular chromosome and the plasmid contain 4,332 and 167 protein-coding genes, respectively. Comparative analysis with the complete genome of M. aeruginosa NIES-2549, which belongs to the same group with NIES-2481, showed that the genome size is the smallest level in previously sequenced M. aeruginosa strains, and the genomes do not contain a microcystin biosynthetic gene cluster in common. Synteny analysis revealed only small-scale rearrangements between the two genomes.

Comparison of the terrestrial cyanobacterium Leptolyngbya sp. NIES-2104 and the freshwater Leptolyngbya boryana PCC 6306 genomes.

The cyanobacterial genus Leptolyngbya is widely distributed throughout terrestrial environments and freshwater. Because environmental factors, such as oxygen level, available water content, and light intensity, vary between soil surface and water bodies, terrestrial Leptolyngbya should have genomic differences with freshwater species to adapt to a land habitat. To study the genomic features of Leptolyngbya species, we determined the complete genome sequence of the terrestrial strain Leptolyngbya sp. NIES-2104 and compared it with that of the near-complete sequence of the freshwater Leptolyngbya boryana PCC 6306. The greatest differences between these two strains were the presence or absence of a nitrogen fixation gene cluster for anaerobic nitrogen fixation and several genes for tetrapyrrole synthesis, which can operate under micro-oxic conditions. These differences might reflect differences in oxygen levels where these strains live. Both strains have the genes for trehalose biosynthesis, but only Leptolyngbya sp. NIES-2104 has genetic capacity to produce a mycosporine-like amino acid, mycosporine-glycine. Mycosporine-glycine has an antioxidant action, which may contribute to adaptation to terrestrial conditions. These features of the genomes yielded additional insights into the classification and physiological characteristics of these strains.

Complete Genome Sequence of Microcystis aeruginosa NIES-2549, a Bloom-Forming Cyanobacterium from Lake Kasumigaura, Japan.

Microcystis aeruginosa NIES-2549 is a freshwater bloom-forming cyanobacterium isolated from Lake Kasumigaura, Japan. We report the complete 4.29-Mbp genome sequence of NIES-2549 and its annotation and discuss the genetic diversity of M. aeruginosa strains. This is the third genome sequence of M. aeruginosa isolated from Lake Kasumigaura.

Murasaki: a fast, parallelizable algorithm to find anchors from multiple genomes.

BACKGROUND: With the number of available genome sequences increasing rapidly, the magnitude of sequence data required for multiple-genome analyses is a challenging problem. When large-scale rearrangements break the collinearity of gene orders among genomes, genome comparison algorithms must first identify sets of short well-conserved sequences present in each genome, termed anchors. Previously, anchor identification among multiple genomes has been achieved using pairwise alignment tools like BLASTZ through progressive alignment tools like TBA, but the computational requirements for sequence comparisons of multiple genomes quickly becomes a limiting factor as the number and scale of genomes grows. METHODOLOGY/PRINCIPAL FINDINGS: Our algorithm, named Murasaki, makes it possible to identify anchors within multiple large sequences on the scale of several hundred megabases in few minutes using a single CPU. Two advanced features of Murasaki are (1) adaptive hash function generation, which enables efficient use of arbitrary mismatch patterns (spaced seeds) and therefore the comparison of multiple mammalian genomes in a practical amount of computation time, and (2) parallelizable execution that decreases the required wall-clock and CPU times. Murasaki can perform a sensitive anchoring of eight mammalian genomes (human, chimp, rhesus, orangutan, mouse, rat, dog, and cow) in 21 hours CPU time (42 minutes wall time). This is the first single-pass in-core anchoring of multiple mammalian genomes. We evaluated Murasaki by comparing it with the genome alignment programs BLASTZ and TBA. We show that Murasaki can anchor multiple genomes in near linear time, compared to the quadratic time requirements of BLASTZ and TBA, while improving overall accuracy. CONCLUSIONS/SIGNIFICANCE: Murasaki provides an open source platform to take advantage of long patterns, cluster computing, and novel hash algorithms to produce accurate anchors across multiple genomes with computational efficiency significantly greater than existing methods. Murasaki is available under GPL at http://murasaki.sourceforge.net.

Whole genome assembly of a natto production strain Bacillus subtilis natto from very short read data.

BACKGROUND: Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and functions as a starter for the production of the traditional Japanese food "natto" made from soybeans. Although re-sequencing whole genomes of several laboratory domesticated B. subtilis 168 derivatives has already been attempted using short read sequencing data, the assembly of the whole genome sequence of a closely related strain, B. subtilis natto, from very short read data is more challenging, particularly with our aim to assemble one fully connected scaffold from short reads around 35 bp in length. RESULTS: We applied a comparative genome assembly method, which combines de novo assembly and reference guided assembly, to one of the B. subtilis natto strains. We successfully assembled 28 scaffolds and managed to avoid substantial fragmentation. Completion of the assembly through long PCR experiments resulted in one connected scaffold for B. subtilis natto. Based on the assembled genome sequence, our orthologous gene analysis between natto BEST195 and Marburg 168 revealed that 82.4% of 4375 predicted genes in BEST195 are one-to-one orthologous to genes in 168, with two genes in-paralog, 3.2% are deleted in 168, 14.3% are inserted in BEST195, and 5.9% of genes present in 168 are deleted in BEST195. The natto genome contains the same alleles in the promoter region of degQ and the coding region of swrAA as the wild strain, RO-FF-1. These are specific for gamma-PGA production ability, which is related to natto production. Further, the B. subtilis natto strain completely lacked a polyketide synthesis operon, disrupted the plipastatin production operon, and possesses previously unidentified transposases. CONCLUSIONS: The determination of the whole genome sequence of Bacillus subtilis natto provided detailed analyses of a set of genes related to natto production, demonstrating the number and locations of insertion sequences that B. subtilis natto harbors but B. subtilis 168 lacks. Multiple genome-level comparisons among five closely related Bacillus species were also carried out. The determined genome sequence of B. subtilis natto and gene annotations are available from the Natto genome browser http://natto-genome.org/.

Whole-genome tiling array analysis of Mycobacterium leprae RNA reveals high expression of pseudogenes and noncoding regions.

Whole-genome sequence analysis of Mycobacterium leprae has revealed a limited number of protein-coding genes, with half of the genome composed of pseudogenes and noncoding regions. We previously showed that some M. leprae pseudogenes are transcribed at high levels and that their expression levels change following infection. In order to clarify the RNA expression profile of the M. leprae genome, a tiling array in which overlapping 60-mer probes cover the entire 3.3-Mbp genome was designed. The array was hybridized with M. leprae RNA from the SHR/NCrj-rnu nude rat, and the results were compared to results from an open reading frame array and confirmed by reverse transcription-PCR. RNA expression was detected from genes, pseudogenes, and noncoding regions. The signal intensities obtained from noncoding regions were higher than those from pseudogenes. Expressed noncoding regions include the M. leprae unique repetitive sequence RLEP and other sequences without any homology to known functional noncoding RNAs. Although the biological functions of RNA transcribed from M. leprae pseudogenes and noncoding regions are not known, RNA expression analysis will provide insights into the bacteriological significance of the species. In addition, our study suggests that M. leprae will be a useful model organism for the study of the molecular mechanism underlying the creation of pseudogenes and the role of microRNAs derived from noncoding regions.

Accurate identification of orthologous segments among multiple genomes.

MOTIVATION: The accurate detection of orthologous segments (also referred to as syntenic segments) plays a key role in comparative genomics, as it is useful for inferring genome rearrangement scenarios and computing whole-genome alignments. Although a number of algorithms for detecting orthologous segments have been proposed, none of them contain a framework for optimizing their parameter values. METHODS: In the present study, we propose an algorithm, named OSfinder (Orthologous Segment finder), which uses a novel scoring scheme based on stochastic models. OSfinder takes as input the positions of short homologous regions (also referred to as anchors) and explicitly discriminates orthologous anchors from non-orthologous anchors by using Markov chain models which represent respective geometric distributions of lengths of orthologous and non-orthologous anchors. Such stochastic modeling makes it possible to optimize parameter values by maximizing the likelihood of the input dataset, and to automate the setting of the optimal parameter values. RESULTS: We validated the accuracies of orthology-mapping algorithms on the basis of their consistency with the orthology annotation of genes. Our evaluation tests using mammalian and bacterial genomes demonstrated that OSfinder shows higher accuracy than previous algorithms. AVAILABILITY: The OSfinder software was implemented as a C++ program. The software is freely available at http://osfinder.dna.bio.keio.ac.jp under the GNU General Public License. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

[Development of a large-scale comparative genome system and its application to the analysis of mycobacteria genomes].

As the number of whole genome sequences available continues to increase rapidly, the raw scale of the sequence data being used in analysis is the first hurdle for comparative genome analysis. When performing whole genome alignments, large-scale rearrangements make it necessary to first find out roughly which short well-conserved segments correspond to what other segments (termed anchors). Successful results have been achieved by adapting tools like BLAT and BLASTZ on a problem-to-problem basis, but the work required to perform a single alignment is considerable. Recently, new programs such as Mauve and Pattern-Hunter can handle slightly larger inputs, but the memory/time requirements for sequences like Human and Chimp X chromosomes are prohibitive for most computational environments. Our novel algorithm, which we have implemented in a program called Murasaki (available at http://murasaki.dna.bio.keio.ac.jp), makes it possible to identify anchors of multiple large sequences on the scale of several hundred megabases (e.g. three mammal chromosomes) in a matter of minutes. We also demonstrate an application of Murasaki to the comparative analysis of multiple mycobacteria genomes.