Identification of Restriction-Modification Systems of Bifidobacterium animalis subsp. lactis CNCM I-2494 by SMRT Sequencing and Associated Methylome Analysis Mary O9Connell Motherway 1 , Debbie Watson 1 , Francesca Bottacini 1 , Tyson A. Clark 2 , Richard J. Roberts 3 , Jonas Korlach 2 , Peggy Garault 4 , Christian Chervaux 4 , Johan E. T. van Hylckama Vlieg 4 , Tamara Smokvina 4 , Douwe van Sinderen 1 * 1 Alimentary Pharmabiotic Centre and School of Microbiology, National University of Ireland, Cork, Ireland, 2 Pacific Biosciences, Menlo Park, California, United States of America, 3 New England Biolabs, Ipswich, Massachusetts, United States of America, 4 Danone Nutricia Research, Palaiseau, France Abstract Bifidobacterium animalis subsp. lactis CNCM I-2494 is a component of a commercialized fermented dairy product for which beneficial effects on health has been studied by clinical and preclinical trials. To date little is known about the molecular mechanisms that could explain the beneficial effects that bifidobacteria impart to the host. Restriction-modification (R-M) systems have been identified as key obstacles in the genetic accessibility of bifidobacteria, and circumventing these is a prerequisite to attaining a fundamental understanding of bifidobacterial attributes, including the genes that are responsible for health-promoting properties of this clinically and industrially important group of bacteria. The complete genome sequence of B. animalis subsp. lactis CNCM I-2494 is predicted to harbour the genetic determinants for two type II R-M systems, designated BanLI and BanLII. In order to investigate the functionality and specificity of these two putative R-M systems in B. animalis subsp. lactis CNCM I-2494, we employed PacBio SMRT sequencing with associated methylome analysis. In addition, the contribution of the identified R-M systems to the genetic accessibility of this strain was assessed. Citation: O9Connell Motherway M, Watson D, Bottacini F, Clark TA, Roberts RJ, et al. (2014) Identification of Restriction-Modification Systems of Bifidobacterium animalis subsp. lactis CNCM I-2494 by SMRT Sequencing and Associated Methylome Analysis. PLoS ONE 9(4): e94875. doi:10.1371/journal.pone.0094875 Editor: Tom Coenye, Ghent University, Belgium Received February 15, 2014; Accepted March 20, 2014; Published April 17, 2014 Copyright: ß 2014 O9Connell Motherway 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. Funding: This work was financially supported by Danone, a HRB postdoctoral fellow–Grant no. PDTM/20011/9 and by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan (grant nos. 07/CE/B1368 and SFI/12/RC/2273). The funder, Danone, funded the research and provided support in the form of salaries for authors PG, CC, JvHV and TS. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing Interests: We have the following interests: Peggy Garault, Christian Chervaux , Johan E. T. van Hylckama Vlieg and Tamara Smokvina are employed by Danone Nutricia Research, one of the funders of this study. Danone is selling the product containing the strain described in the submitted manuscript. Danone Nutricia Research financed part of this study. Tyson A. Clark and Jonas Korlach are employed by Pacific Biosciences and Richard J. Roberts by New England Biolabs. DvS, MOCM, DW, TS and PG are named inventors on pending patent application PCT/IB2013/002951 filed December 02 2013 "Genetic Transformation of Bifidobacteria". There are no further patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: d.vansinderen@ucc.ie Introduction Bifidobacteria are a common component of the microbiota of the gastrointestinal tract (GIT) of a broad range of hosts, and their presence is associated with a positive health status of the gut [1,2]. Compared to other micro-organisms of industrial and pharma- ceutical relevance, relatively little is known about the precise molecular mechanisms that explain how bifidobacteria confer beneficial effects, although scientific progress has been made in recent times [3,2]. A typical example of this is Bifidobacterium animalis subsp. lactis CNCM I-2494, which is a component of a fermented dairy product studied in several placebo-controlled clinical trials [4–6]. Veiga et al. [7] recently reported on the marked reduction in intestinal inflammation observed in a murine model of inflamma- tory bowel disease (IBD) upon administration of fermented milk containing B. animalis subsp. lactis CNCM I-2494, while Agostini et al [8] demonstrated that a dairy product containing this strain reduces visceral hypersensitivity associated with acute stress in a murine model. However, the molecular mechanisms of these observations remain unclear. Insights into bacterial modes of action may be obtained by comparative and functional genomic approaches. The last decade has seen a vast expansion of complete genome sequences obtained from various human, animal or insect-derived bifidobacterial species [9]. While these have contributed very significantly to advancing our knowledge on bifidobacterial genomics, genetics and metabolism, the availability of a genome sequence is merely a first step towards a better understanding of the beneficial attributes of a specific strain. The complete genome sequence of CNCM I- 2494 has been reported [10] and genetic determinants potentially involved in host interaction have been identified. However, demonstration of the role and functionality of these determinants demands the availability of suitable molecular tools. In this respect, the genus Bifidobacterium is renowned for being genetically recalcitrant and while numerous E. coli-bifidobacterial shuttle vectors have been created [11], the successful application of these vectors can be strain-dependent. At present the ability to access the chromosome to create mutants via site-specific homologous PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e94875