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The complete genome sequence of strain BB2000 reveals differences from the Proteus mirabilis reference strain

Nora L. Sullivan1, Alecia N. Septer, Andrew T. Fields, Larissa M. Wenren, and Karine A. Gibbs*

Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138
1

current address: W.M. Keck Science Department, 925 N. Mills Ave, Claremont

McKenna, Pitzer and Scripps Colleges, Claremont, CA 91711

*Corresponding author: Karine A. Gibbs, Ph.D. Department of Molecular and Cellular Biology Harvard University 16 Divinity Avenue Cambridge, MA 02138 Em: kagibbs@mcb.harvard.edu

Running title: Complete genome sequence of P. mirabilis BB2000

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Abstract We announce the complete genome for Proteus mirabilis strain BB2000, a model system for self recognition. This opportunistic pathogen contains a single, circular chromosome (3,846,754 base pairs). Comparisons between this genome and that of strain HI4320 reveal genetic variations corresponding to previously unknown physiological and self-recognition differences.

	
  

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The gut commensal bacterium Proteus mirabilis is the primary cause of urinary tract infections in patients with long-term indwelling catheters (1-4). Interestingly, migrating colonies of P. mirabilis cells can distinguish self from non-self: a visible boundary forms at the interface between two genetically distinct colonies, while two genetically identical populations merge together (5). The genetic determinants of this self-recognition behavior, first identified in P. mirabilis strain BB2000, included selfidentity genes containing numerous inter-strain nucleotide polymorphisms and suggested that additional genetic differences between strains are likely (6). To date, only the genome of P. mirabilis strain HI4320 (NCBI NC_010554) has been completed (7). Here we report a second closed genome, that of the genetically distinct strain, BB2000 (8). BB2000 genomic DNA was isolated and sequenced using standard protocols. Briefly, DNA was isolated from cells cultured in modified LB broth using phenol/chloroform extraction and ethanol (9). Beckman Coulter Genomics (Danvers, MA) performed initial library preparation and sequencing using the Roche 454 platform. Illumina sequencing was used to confirm the 454 data and resolve stretches of unknown nucleotides; genomic DNA libraries were prepared according to the Illumina Multiplexing Sample Preparation protocol and sequenced by Harvard FAS Systems Biology Core using an Illumina HiSeq 2000. Illumina reads were assembled onto the 454 genomic data using Galaxy software (10). Genome closure was accomplished by amplifying across gaps using polymerase chain reactions followed by Sanger sequencing performed by Genewiz Corporation (South Plainfield, NJ). The P. mirabilis BB2000 genome consists of a single chromosome (3,846,754 base pairs) with 38.6% G+C content. Potential coding sequences (CDSs) were identified

	
  

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using the xBase annotation service, which predicted CDS regions using Glimmer (11), and assigned predicted protein products based on a direct comparison to the P. mirabilis HI4320 genome (12-16). CDSs absent in the HI4320 genome were assigned “hypothetical protein” as the predicted product. Twenty-eight genes related to selfrecognition (6, 17) were annotated manually using blastx (12) and the HMMER web interface (18). Sequence assembly and annotation were completed using Artemis software (19). The BB2000 genome encodes 3,457 potential CDSs, of which 2,592 are assigned a putative function; the remaining 865 CDSs are classified as hypothetical proteins, with an additional 81 tRNA genes and 22 rRNA genes. Comparison of the BB2000 genome to that of strain HI4320 (7) revealed 93% similarity between the chromosomes. The CDSs unique to each genome include genes related to phage, toxin elements, and self recognition. The HI4320 genome encodes iron acquisition proteins that are absent in BB2000. Strain HI4320 also contains a plasmid (NCBI NC_010555.1) (7), and the HI4320 chromosome encodes a complete set of tra genes for conjugative transfer. No plasmid was identified in BB2000, nor does its genome encode tra genes or any HI4320 plasmid-encoded genes. Further analysis of variations between P. mirabilis isolates will advance our understanding of the genetic determinants of pathogenicity and self recognition. Nucleotide sequence accession number. The P. mirabilis BB2000 genome sequence has been deposited in GenBank under the accession number BankIt1590180 BB2000 CP004022.

ACKNOWLEDGMENTS

	
  

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Harvard University provided funding for this research.

The authors thank Beckman Coulter Genomics, the Harvard FAS Systems Biology Core, and the Harvard Research Computing Group for insightful advice during the genome construction.

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