Genome sequence of Enterobacter mori type strain LMG 25706, a

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1 JB Accepts, published online ahead of print on 20 May 2011 J. Bacteriol. doi: /jb Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Genome sequence of Enterobacter mori type strain LMG 25706, a pathogenic bacterium of Morus alba L Bo Zhu 1, Guo-Qing Zhang 1, Miao-Miao Lou 1, Wen-Xiao Tian 1, Bin Li 1, Xue-Ping Zhou 1, Guo-Feng Wang 2, He Liu 1, Guan-Lin Xie 1, Gu-Lei Jin 3 1 State Key Laboratory of Rice Biology and Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Biotechnology, Zhejiang University, Hangzhou , China 2 Institute of Environmental and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, Danzhou , China 3 Institute of Bioinformatics, Zhejiang University, Hangzhou , China Corresponding author: Guan-Lin Xie : glxie@zju.edu.cn; Phone: Gu-Lei Jin: guleijin@zju.edu.cn; Phone: Key words: Genome sequencing; Enterobacter mori; Morus alba L; Pathogenic bacterium

2 Abstract Enterobacter mori is a plant pathogenic enterobacterium responsible of the bacterial wilt of Morus alba L. Here, we present the draft genome sequence of the type strain LMG To the best of our knowledge, this is the first genome sequence of a pathogenic bacterium in the Enterobacter genus

3 Mulberry (Morus alba L.) is an important economic plant widely grown in Asia, Africa and Europe. Mulberry leaves are ecologically important as the sole nutrition resource of the silkworm Bombyx mori, the cocoon of which is used to make silk. China produces about 75% of the world s raw silk, which is valued nearly $2 billion (2). In summer of 2006, a severe bacterial wilt was noted in mulberry orchards of Hangzhou, Zhejiang province of China. Our laboratory isolated the pathogen, and identified it as Enterobacter spp. (6-7, 10). Later, DNA-DNA hybridization, fatty acids analysis and phylogenetic analysis suggested that it was a new species in Enterobacter genus (9). We nominated it as Enterobacter mori (named after the mulberry genus Morus) and deposited the reference strain R18-2 at Belgian Co-ordinated Collections of Micro-organisms (BCCM) as the type strain LMG T (9). The genomic DNA, isolated using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) was whole-genome sequenced by using Illumina GA (Solexa). This resulted in 15,593,018 high quality filtered reads of 80 bp average read length and coverage equivalent to about 200 times. Quality filtered reads were assembled in silico with Velvet program (8). Based on the reference genome of Enterobacter cloacae subsp. cloacae ATCC T (4), a draft genome of LMG was completed. By subsequent PCR and re-sequencing, 84 genome gaps were closed, but remaining 23 scaffolds (N 50 of approximately 280kb with the largest scaffold size of 982.8kb). The E. mori LMG T genome has a 4,960,223 bases circular chromosome.

4 A total of 4,732 coding sequences (CDSs) were predicted using GLIMMER (5). Putative functions of encoding genes were automatically identified using GenDB annotation engine (3). The chromosome has three rrna operons and 63 trnas. Furthermore, 89.3% of the ORFs have orthologs in the reference strain E. cloacae subsp. cloacae ATCC (BLASTP < 1e-5), but 457 ORFs were not found in the released genomes of Enterobacter genus, of these, 124 ORFs did not give hits in current public databases. Although the genome of E. mori LMG lacks type III secration system (TTSS), which has been proved to be an important virulence associated system in Gram-negative pathogenic bacteria (1), it still possesses virulence properties recognized to be important in infection. At least 66 genes were found to be potentially involved in different secretion systems (Type I, Type II, Type VI, Sec-SRP and TaT). They form diverse gene clusters with high degree of syntery to the reference genome. Interestingly, several genes which were not found in reference genome have high similarity orthologs in Erwinia amylovora, a serious phytobacterium. The results suggest the differences between E. mori and E. cloacae subsp. cloacae. Overall, the genome sequence of E. mori LMG provides a foundation for both basic and agricultural applied researches. Nucleotide sequence accession numbers This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AEXB The version described in this paper is the first version, AEXB

5 Acknowledgements This work was supported by the Special Fund for Agro-scientific Research in the Public Interest ( ; ), the National Natural Science Foundation of China ( ), and Zhejiang Agri. Dep. (Ji-fa ). Reference 1. Cheng, L. W., and O. Schneewind Type III machines of Gram-negative bacteria: delivering the goods. Trends Microbiol. 8: Li, B., T. Su, X. L. Chen, B. P. Liu, B. Zhu, Y. Fang, W. Qiu, and G. L. Xie Effect of chitosan solution on the bacterial septicemia disease of Bombyx mori (Lepidoptera: Bombycidae) caused by Serratia marcescens. Appl. Entomol. Zool. 45: Meyer, F., A. Goesmann, A. C. McHardy, D. Bartels, T. Bekel, J. Clausen, J. Kalinowski, B. Linke, O. Rupp, R. Giegerich, and A. Puhler GenDB - an open source genome annotation system for prokaryote genomes. Nucleic Acids Res. 31: Ren, Y., Z. M. Zhou, X. Guo, Y. Y. Li, L. Feng, and L. Wang Complete genome sequence of Enterobacter cloacae subsp cloacae type strain ATCC J. Bacteriol. 192: Salzberg, S. L., A. L. Delcher, S. Kasif, and O. White Microbial gene identification using interpolated Markov models. Nucleic Acids Res. 26: Wang, G. F., K. Praphat, G. L. Xie, B. Zhu, B. Li, B. Liu, and Q. Zhou Bacterial wilt of mulberry (Morus alba) caused by Enterobacter cloacae in China. Plant Dis. 92: Wang, G. F., G. L. Xie, B. Zhu, J. S. Huang, B. Liu, P. Kawicha, L. Benyon, and Y. P. Duan Identification and characterization of the Enterobacter complex causing mulberry (Morus alba) wilt disease in China. Eur. J. Plant Pathol. 126: Zerbino, D. R., and E. Birney Velvet: Algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18: Zhu, B., M. Lou, G. Xie, G. Wang, Q. Zhou, F. Wang, Y. Fang, T. Su, B. Li, and Y. Duan Enterobacter mori sp. nov., a novel Enterobacter species associated with bacterial wilt on Morus alba L. Int. J. Syst. Evol. Microbiol. In press. (Online, January 7, 2011 as doi: /ijs ) 10. Zhu, B., G. F. Wang, G. L. Xie, Q. Zhou, M. Zhao, K. Praphat, B. Li, and W. X. Tian Enterobacter spp.: A new evidence causing bacterial wilt on mulberry. Sci. China C Life Sci. 53: