JB Accepts, published online ahead of print on 8 July 2011 J. Bacteriol. doi:10.1128/jb.05596-11 Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Genome Announcements 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Complete genome sequence of Clostridium acetobutylicum DSM 1731, a solvent producing strain with multi-replicon genome architecture Guanhui Bao 1,2, Runjiang Wang 1,2, Yan Zhu 1,2, Hongjun Dong 1,2, Shaoming Mao 1, Yanping Zhang 1, Zugen Chen 4, Yin Li 1*, Yanhe Ma 1,4 1 Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 2 Graduate School of Chinese Academy of Sciences, Beijing, China 3 Department of Human Genetics, School of Medicine, University of California, Los Angeles, CA 90095 4 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China *Corresponding author: Mailing address for Yin Li: Institute of Microbiology, Chinese Academy of Sciences, No.1 West Beichen Road, Chaoyang District, Beijing 100101, China. Phone: 86-10-64807485. Fax: 86-10-64807485. E-mail: yli@im.ac.cn. Abstract Clostridium acetobutylicum is an important microorganism for solvent production. We report the complete genome sequence of C. acetobutylicum DSM 1731, a genome
25 26 27 with multi-replicon architecture. Comparison with the sequenced type strain C. acetobutylicum ATCC 824, the genome of strain DSM1731 harbors a 1.7 kb insertion and a novel 11.1 kb plasmid, which might be acquired during evolution. 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Acetone butanol ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest industrial fermentations ever known, and historically ranked the second next to the ethanol fermentation by yeast. C. acetobutylicum is a low-gc-content, gram-positive, spore-forming and obligate anaerobe (2). Recent omics studies including genomics (4), transcriptomics (12, 13), proteomics (9, 10) and metabolomics (1) have greatly improved our understandings to the physiology of this species. Here we report the complete genome sequence of Clostridium acetobutylicum DSM 1731, which was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The genome was sequenced at the Department of Human Genetics in University of California Los Angeles, utilizing the Solexa technology according to Illumina s recommendations. A total of 6,043,147 reads, counting up to 217,553,292 bases were obtained, providing a 52X coverage. Assembly was performed by AMOScmp (5) and Velvet (14), using comparative genome assembly and de novo assembly. The gaps were filled by sequencing polymerase chain reaction (PCR) fragments using ABI 3730. The complete genome of C. acetobutylicum DSM 1731 consists of a single, circle chromosome of 3,942,462 bp (30.9% GC content), two plasmids: psmba (191,996 bp, 30.9% GC content) and psmbb (11,123 bp, 25.9% GC content).
50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 Putative protein coding sequences were identified by GeneMark.hmm (8) and Glimmer (3). trnascan-se (7) was used to predict transfer RNA genes and RNAmmer 1.2 server (6) was employed to detect rrna genes. BLASTP were applied to perform annotation, followed by manual curation. The chromosome of C. acetobutylicum DSM 1731 contains 3,765 predicated protein-coding genes, 11 rrna operons and 73 trna genes, while the two plasmids contains 176 (psmba) and 9 (psmbb) protein coding genes, respectively. Functional classification of all proteins was conducted using NCBI clusters of orthologous groups (COG), where 3072, 126, and 2 COG classified genes are located on the chromosome, psmba, and psmbb, respectively. Comparing with the genome sequence of C. acetobutylicum strain ATCC 824 (11) and EA 2018 (4), the most remarkable feature of strain DSM 1731 is the presence of a 11.1 kb plasmid, psmbb, which has not been previously reported in this species. In addition, 7 out of the 9 predicted genes of psmbb are hypothetical proteins, suggesting it plays an unknown role in clostridial physiology. Besides, 345 single nucleotide variations (SNVs) were identified between the chromosomes of strains DSM 1731 and ATCC 824. The largest insertion is 1,753 bp in the position of 1,278,525-1,280,278, located within a hypothetical protein gene. Consistent with strain EA 2018, the size of the megaplamid in strain DSM 1731 (psmba) is 4 bp smaller than that of strain ATCC824. Nucleotide sequence accession numbers The complete genome sequence of Clostridium acetobutylicum DSM 1731 has been deposited in GenBank under the accession numbers between CP002660 and
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