Eubacterium limosum KIST612 is an anaerobic acetogenic bacterium that uses CO as the sole carbon/energy source and produces acetate, butyrate, and ethanol. To evaluate its potential as a syngas microbial catalyst, we have sequenced the complete 4.3-Mb genome of E. limosum KIST612.Synthesis gas (syngas) (H 2 , CO 2 , and CO) has been highlighted for use as a potential feedstock for the production of biofuels and valuable chemicals (9, 16). Eubacterium limosum KIST612 isolated from an anaerobic digester has been considered a microbial syngas catalyst due to its rapid growth under high CO pressure (Ͼ1 atm) and production of acetate and butyrate and ethanol from CO (5-7). To understand its physiological properties (e.g., a high tolerance to CO and production of ethanol) and provide metabolic engineering principles, we attempted to obtain the complete genome sequence information for this microorganism.The genome of E. limosum KIST612 was sequenced by a combination of Illumina Genome Analyzer IIx (GAIIx) and Roche 454 GS FLX (454 GS FLX) platforms. We obtained two libraries of 643,326 single-end (SE) reads and 291,735 pairedend (PE) reads containing 3-kb inserts from 454 GS FLX. The third genomic library of 35,235,888 PE reads containing 400-bp inserts was obtained from GAIIx. To combine these three libraries (454 GS FLX SE and PE and GAIIx PE) into a single procedure, we first assembled GAIIx PE reads into 296 contigs (4,635,997 bases) by the ABySS 1.20 assembler (15) and split into overlapping ϳ1.5-kb fake reads (45,221 reads). We merged these fake reads with 454 SE and PE reads (total 935,061 reads) and assembled into 9 scaffolds (34 contigs) by the Newbler gsAssembler 2.3 (454 Life Sciences, Branford, CT). We determined the actual order of 9 scaffolds in a single contig with a series of PCRs based on a permutation table of scaffolds. The genome was finished by filling gaps with sequencing and primer walking of PCR products using an ABI 3730 capillary sequencer (Applied Biosystems, CA).The complete genome of E. limosum KIST612 consisted of 4,276,902 bp in a single circular chromosome with an average GϩC content of 47.5%. Approximately 91% of the nucleotides were predicted as 4,516 protein-coding regions by the union of Glimmer (8), GeneMarkS (3), and Prodigal (10). The predicted proteins were annotated by BLAST (1) and the RAST server (2). Seventy-eight percent (3,541) of the open reading frames were annotated with known proteins. Five copies of the 16S-23S-5S rRNA operon and a separate 5S rRNA locus were predicted by RNAmmer 1.2 (12), and the 58 tRNA genes were identified by tRNAscan-SE 1.23 (13).Metabolic pathway analysis revealed that E. limosum KIST612 uses the Wood-Ljungdahl pathway to fix CO (or CO 2 ) and converts it into acetyl coenzyme A (acetyl-CoA), like other syngas-utilizing acetogens such as Moorella thermoacetica (14), Clostridium ljungdahlii (11), and Clostridium carboxidivorans strain P7 T (4). E. limosum KIST612 also contains 10 genes annotated as subunits of hydrogenases that may provide reducing...

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Verrucosispora maris AB-18-032 is a marine actinomycete that produces atrop-abyssomicin C and proximicin A, both of which have novel structures and modes of action. In order to understand the biosynthesis of these compounds, to identify further biosynthetic potential, and to facilitate rational improvement of secondary metabolite titers, we have sequenced the complete 6.7-Mb genome of Verrucosispora maris AB-18-032.Verrucosispora maris AB-18-032 was isolated from a deep marine sediment sample collected from the East Sea (Sea of Japan) (3, 9). It produces unique polycyclic type 1 polyketide antibiotics, namely, the abyssomicins, which are the first known natural-product inhibitors of the para-aminobenzoic acid biosynthetic pathway (6, 7). In particular, atrop-abyssomicin C is active against Gram-positive bacteria, including Bacillus subtilis, methicillin-resistant Staphylococcus aureus and vancomycin-resistant S. aureus (6,7,9), and Mycobacterium tuberculosis (5). The strain also produces proximicin A, a furan analogue of netropsin with novel antitumor properties (4, 10). Here, we announce the genome sequence of Verrucosispora maris AB-18-032, the first member of this genus to be sequenced, which should provide fundamental understanding of the biosynthesis of these antibiotics and facilitate bioengineering and biodiscovery efforts involving this strain.The complete genome of Verrucosispora maris AB-18-032 was sequenced using Roche 454 genome sequencer (GS) FLX (9.4-, 9.9-, 6.7-, and 6.8-times coverage of single-end reads and 13.3-times coverage of paired-end reads having 3-kb inserts) and Solexa (Illumina genome analyzer [GA], 43-times coverage with ϳ350-bp inserts and paired-end reads) sequencing technology.To combine the GA library with 454 GS FLX libraries, reads were assembled into 1,730 contigs using the ABySS 1.20 assembler (11), and the contigs were shredded into 15,379 fake reads. The fake reads and 454 GS FLX reads were assembled into 5 scaffolds (116 contigs) by using a combination of Newbler gsAssembler 2.3 (454 Life Sciences, Branford, CT) and CABOG assembler (8). The actual order of the scaffolds was determined by a series of PCRs based on a permutation table, and gaps were filled by primer-walking PCR followed by sequencing using an ABI 3730 system (Applied Biosystems, CA). Finally, Polisher software (Alla Lapidus, unpublished data) was used with the Illumina data to correct for homopolymers in the 454 reads and increase consensus quality. Annotation was performed by the RAST server (2) and the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) (http://www.ncbi.nlm.nih.gov/genomes/static /Pipeline.html), and both annotations were scrutinized by manual inspection based on BLAST searches (1).The genome of Verrucosispora maris AB-18-032 has a single circular chromosome of 6,673,976 bp with a GC content of 70.9%. A circular plasmid of 58,295 bp with a GC content of 70.3%, pVMKU, was also identified. The chromosome has 5,947 coding sequences (CDS), 51 tRNA genes, and 9 rRNA genes, and the pla...

HER2-positive luminal B breast cancer (BC), a subset of the luminal B subtype, is ER-positive and HER2-positive BC which is approximately 10% of all BC. However, HER2-positive luminal B BC has received less attention and is less represented in previous molecular analyses than other subtypes. Hence, it is important to elucidate the molecular biology of HER2-positive luminal B BC to stratify patients in a way that allows them to receive their respective optimal treatment. We performed molecular profiling using targeted next-generation sequencing on 94 HER2-positive luminal B BC to identify its molecular characteristics. A total of 134 somatic nonsynonymous mutations, including 131 nonsynonymous single nucleotide variants and three coding insertions/deletions were identified in 30 genes of 75 samples. PIK3CA was most frequently mutated (38/94, 40.4%), followed by TP53 (31/94, 33.0%), and others were detected at lower frequencies. Recurrent germline mutations of MLH1 V384D were found in 13.8% (13/94), with a significantly high TP53 mutations rate. The frequency of MLH1 V384D germline mutation in individuals with HER2-positive luminal B BC was significantly higher than that observed in the controls. All 13 cases were classified as microsatellite stable tumors. Tumor mutation burdens (TMB) were not significantly different between MLH1 V384D carrier and wild type. The concordant results of microsatellite instability (MSI) and TMB suggest that the haploinsufficiency of MLH1 plays a role as a tumor predisposition factor rather than a direct oncogenic driver. Our study identified, for the first time, that MLH1 V384D germline variant is frequently detected in HER2-positive luminal B BC. MLH1 V384D germline variant may not only contribute to gastrointestinal cancer predisposition but may also contribute to BC in East Asians.