Meningococcal Genetic Variation Mechanisms Viewed through Comparative Analysis of Serogroup C Strain FAM18 Stephen D. Bentley 1* , George S. Vernikos 1 , Lori A. S. Snyder 2 , Carol Churcher 1 , Claire Arrowsmith 1 , Tracey Chillingworth 1 , Ann Cronin 1 , Paul H. Davis 1 , Nancy E. Holroyd 1 , Kay Jagels 1 , Mark Maddison 1 , Sharon Moule 1 , Ester Rabbinowitsch 1 , Sarah Sharp 1 , Louise Unwin 1 , Sally Whitehead 1 , Michael A. Quail 1 , Mark Achtman 3 , Bart Barrell 1 , Nigel J. Saunders 2 , Julian Parkhill 1 1 Wellcome Trust Sanger Institute, Hinxton, United Kingdom, 2 Bacterial Pathogenesis and Functional Genomics Group, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom, 3 Molekulare Biologie, Max-Planck Institut fu ¨ r Infektionsbiologie, Berlin, Germany The bacterium Neisseria meningitidis is commonly found harmlessly colonising the mucosal surfaces of the human nasopharynx. Occasionally strains can invade host tissues causing septicaemia and meningitis, making the bacterium a major cause of morbidity and mortality in both the developed and developing world. The species is known to be diverse in many ways, as a product of its natural transformability and of a range of recombination and mutation-based systems. Previous work on pathogenic Neisseria has identified several mechanisms for the generation of diversity of surface structures, including phase variation based on slippage-like mechanisms and sequence conversion of expressed genes using information from silent loci. Comparison of the genome sequences of two N. meningitidis strains, serogroup B MC58 and serogroup A Z2491, suggested further mechanisms of variation, including C-terminal exchange in specific genes and enhanced localised recombination and variation related to repeat arrays. We have sequenced the genome of N. meningitidis strain FAM18, a representative of the ST-11/ET-37 complex, providing the first genome sequence for the disease-causing serogroup C meningococci; it has 1,976 predicted genes, of which 60 do not have orthologues in the previously sequenced serogroup A or B strains. Through genome comparison with Z2491 and MC58 we have further characterised specific mechanisms of genetic variation in N. meningitidis, describing specialised loci for generation of cell surface protein variants and measuring the association between noncoding repeat arrays and sequence variation in flanking genes. Here we provide a detailed view of novel genetic diversification mechanisms in N. meningitidis. Our analysis provides evidence for the hypothesis that the noncoding repeat arrays in neisserial genomes (neisserial intergenic mosaic elements) provide a crucial mechanism for the generation of surface antigen variants. Such variation will have an impact on the interaction with the host tissues, and understanding these mechanisms is important to aid our understanding of the intimate and complex relationship between the human nasopharynx and the meningococcus. Citation: Bentley SD, Vernikos GS, Snyder LAS, Churcher C, Arrowsmith C, et al. (2007) Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C Strain FAM18. PLoS Genet 3(2): e23. doi:10.1371/journal.pgen.0030023 Introduction N. meningitidis (the meningococcus) colonizes the non- ciliated columnar mucosal cells of the human nasopharynx as a harmless commensal organism and, as such, is carried by five to ten percent of the adult population [1,2]. Some strains are able to cross the mucosa into the bloodstream from where they can cause septicaemia or meningitis and, as a result, are a major cause of disease worldwide [2]. Several genetic loci have been associated with disease [3,4], but for most strains the mechanism of virulence is not well defined. The close interaction with the human host is reflected in enriched diversity and variability at the bacterial cell surface. There are 12 different polysaccharide capsules, which are the basis of serogrouping, some of which are virulence determinants [5– 7]. Vaccines targeted to the capsule types most commonly associated with disease have been successful, though capsule switching is a cause of concern [8]. Many meningococcal surface-exposed proteins and carbohydrates are also highly variable, creating a major challenge in the development of a universal meningococcal vaccine [9,10]. Current models of bacterial populations describe a spectrum of structures ranging from clonal, where lineages are derived from a common ancestor and horizontal genetic exchange plays no role, to nonclonal (or panmictic), where rates of horizontal genetic exchange are so high that genetic differences between isolates are effectively randomised and Editor: Claire M. Fraser-Liggett, The Institute for Genomic Research, United States of America Received September 8, 2006; Accepted December 21, 2006; Published February 16, 2007 A previous version of this article appeared as an Early Online Release on December 21, 2006 (doi:10.1371/journal.pgen.0030023.eor). Copyright: Ó 2007 Bentley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abbreviations: CDS, coding sequences; CREE, Correia repeat enclosed element; MDA meningococcal disease associated; NIME, neisserial intergenic mosaic element; RS element, repeat sequence element * To whom correspondence should be addressed. E-mail: sdb@sanger.ac.uk PLoS Genetics | www.plosgenetics.org February 2007 | Volume 3 | Issue 2 | e23 0230