ACCEPTED. Korean patient isolate in an effort to understand the prevalence, antibiotic resistance, and

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1 JB Accepts, published online ahead of print on June 00 J. Bacteriol. doi:./jb.00-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved The Complete Genome Sequence of Neisseria gonorrhoeae NCCP Gyung Tae Chung 1, Jeong Sik Yoo 1, Hee Bok Oh 1, Yeong Seon Lee 1, Sun Ho Cha, Sang Jun Kim, Cheon Kwon Yoo * Centers for Infectious Diseases, National Institute of Health, Korea Centers for Disease Control and Prevention, Seoul 1-01, Korea 1, GenoTech Corporation - Jang-Dong, Yuseong-Gu, Daejeon 0-, Korea, and Division of Biosafety Evaluation and Control, National Institute of Health, Korea Centers for Disease Control and Prevention, Seoul 1-01, Korea Neisseria gonorrhoeae is an obligate human pathogen that is the etiological agent of gonorrhea. We explored variations in genes of a multidrug-resistant N. gonorrhoeae Korean patient isolate in an effort to understand the prevalence, antibiotic resistance, and importance of horizontal gene transfer within this important, naturally competent organism. Here we report the complete annotated genome sequence of N. gonorrhoeae strain NCCP. Downloaded from on July 1, 01 by guest *Corresponding Author: Cheon Kwon Yoo ckyoo@nih.go.kr Tel: --0-1

2 Fax: Short title: Genome sequence of N. gonorrhoeae Keywords: N. gonorrhoeae, genome sequence Neisseria gonorrhoeae NCCP was isolated from a vaginal smear of a Korean patient. This strain fit the pattern of antimicrobial resistance that is prevalent in Korea, namely chromosome-mediated resistance to penicillin and tetracycline, and a ciprofloxacin MICof 1 mg/l. The complete genome sequence of NCCP was determined by whole-genome shotgun sequencing. Two genome libraries were generated by random shearing of genomic DNA; one was a fosmid library with mean insert size kb (CopyControl Fosmid library production kit, Epicentre, Madison, WI), and the other was a smaller insert library of 1~ kb. Automated DNA sequencing chromatograms were analyzed by the Phred/Phrap/Consed software package ( Gap closure and additional sequencing of low- Downloaded from on July 1, 01 by guest 1 coverage regions were accomplished via primer walking of gap-spanning clones and direct 1 sequencing of PCR products. In particular, the order of 1 contigs was predicted by 1 comparison with the genome sequence of N. gonorrhoeae FA0 (strain AE00) and

3 1 1 then confirmed by PCR. To solve problems with misassembled regions caused by repetitive sequences and to close remaining sequence gaps, we used long PCR along with six fosmid clones. Their relationships were reassembled manually based on location information for paired-end reads using Consed. The completed genome sequence had -fold sequence coverage with an error rate of 0.1 per,000 bases. Open reading frame (ORF) prediction and annotation were performed using GLIMMER () and BLAST. Functional assignment of genes was performed by searching translated ORFs against the COG () and KEGG () databases. The genome of NCCP consists of one circler chromosome (,,0 bp), encoding, predicted ORFs, and one plasmid (,1 bp) encoding 1 predicted ORFs. The estimated coding density over the entire genome is %, and the average G+C content is.%, values that are similar to those of strain FA0. The strain NCCP genome encodes trnas and four copies of 1S-S-S ribosomal RNA operons. Downloaded from on July 1, 01 by guest 1 Genome structure comparisons between NCCP and FA0 were performed using 1 the programs ACT () and MUMMER (). Genome colinearity between these strains is 1 interrupted by NCCP-specific and FA0-specific regions as well as by several

4 1 1 inversions and translocations. The strain NCCP genome is, bp larger than that of FA0, and the overall genome sequence identity is.%. This difference in genome size is caused by a gonococcal genetic island (GGI) in the NCCP. The GGI is present in 0% of gonococcal isolates and encodes a type IV secretion system (). The GGI of NCCP encodes 1 predicted ORFs (AY00) and is similar to the GGI of N. gonorrhoeae strain MSA ( kb). The GGI sequence similarity between these two strains is.%. As with other Neisseria sp., NCCP genome contains hundreds of repetitive sequence elements. We analyzed these repetitive elements using EMBOSS () and Nicolas method (1). The most abundant repeat type is the DNA uptake sequence ('-gccgtctgaa-'), comprising 1, copies throughout the genome. The next abundant repeat type is neisseria intergenic mosaic elements (RS: 1 copies, and drs: 1 copies). (). The NCCP genome also contains copies of Correia elements. (1) Downloaded from on July 1, 01 by guest 1

5 Nucleotide sequence accession number. The complete genome sequence of N. gonorrhoeae NCCP has been assigned GenBank accession number CP Funding for the sequencing project was provided by the National Institute of Health, Ministry of Health and Welfare, Republic of Korea. REFFERENCES 1. Buisine, N., C. M. Tang, and R. Chalmers. 00. Transposon-like Correia elements: structure, distribution and genetic exchange between pathogenic Neisseria sp. FEBS Letters. : -. Carver, T. J., K. M. Rutherford, M. Berriman, M. A. Rajandream, B. G. Barrell, and J. Parkhill. 00. ACT: the Artemis Comparison Tool. Bioinformatics. 1:-.. Delcher, A. L., D. Harmon, S. Kasif, O. White, and S. L. Salzberg. 1. Improved microbial gene identification with GLIMMER. Nucleic Acid Res. :-1.. Hamilton H. L., N. M. Dominguez, K. J. Schwartz, K. T. Hackett, and J. P. Dillard. 00. Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system. Molecular Microbiology. : -.. Kanehisa, M., S. Goto, S. Kawashima, Y. Okuno, and M. Hattori. 00. The KEGG resource for deciphering the genome. Nucleic Acid Res. :D-D0.. Kurtz S., A. Phillippy, A. L. Delcher, M. Smoot., M. Shumway, C. Antonescu, and S. L. Salzberg. 00. Versatile and open software for comparing large genomes. Genome Biology. :R1. Downloaded from on July 1, 01 by guest

6 . Liu, S. V., N. J. Saunders, A. Jeffries, and R. F. Rest. 00. Genome analysis and strain comparison of Correia repeats and Correia repeat-enclosed elements in pathogenic Neisseria. J. Bacteriol. 1:1-1.. Rice, P., I. Longden, and A. Bleasby EMBOSS: the European molecular biology open software suite. Trends Genet. 1: -. Tatusov, R. L., D. A. Natale, I. V. Garkavtsev, T. A. Tatusova, U. T. Shankavaram, B. S. Rao, B. Kiryutin, M. Y. Galperin, N. D. Fedorova, and E. V. Koonin The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acid Res. :-. Downloaded from on July 1, 01 by guest