Identification of Restriction-Modification Systems of Bifidobacterium animalis subsp. lactis CNCM I-2494 by SMRT Sequencing and Associated Methylome Analysis

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.

Pangenome analysis of Bifidobacterium longum and site-directed mutagenesis through by-pass of restriction-modification systems

“…The negative impact of R-M systems on transformation efficiency of bifidobacteria has been demonstrated in B. breve UCC2003 [ 15 ], B. adolescentis ATCC 15703 [ 20 ] and B. bifidum S17 [ 53 ]. However, such an R-M barrier can be overcome by the appropriate pre-methylation of plasmid DNA [ 15 , 20 , 21 , 54 ].…”

“…In a recent study, data gained from methylome analysis led to a moderate improvement in the transformation efficiency of the strain Bifidobacterium animalis subsp. lactis CNCM I-2494 [ 21 ]. However, overcoming R-M systems of a given species is but one tool of the expanding bifidobacterial genetic tool box, which now includes conjugation-based methods [ 22 , 23 ], a temperature-sensitive plasmid [ 24 ] and a double-crossover, marker-less gene deletion system [ 25 ].…”

Next-generation sequencing as an approach to dairy starter selection

“…Combining whole genome sequencing and MTase motif analysis, the functions of one or more bacterially encoded R-Ms can be predicted which can then be confirmed using heterologous gene expression coupled with restriction endonuclease assays. This approach has been applied to both bacteria and bacteriophages alike (Murphy et al 2014 ; O'Connell-Motherway et al 2014 ), though it has not yet been applied to lactococcal or streptococcal strains.…”

A Transposon Mutagenesis System for Bifidobacterium longum subsp. longum Based on an IS 3 Family Insertion Sequence, IS Blo11

“…Although the mutagenesis system we describe was demonstrated to work in B. longum 105-A and NCC2705, genetically manipulatable strains, application to other Bifidobacterium species or strains may require optimization of transposase expression and of the transformation protocol (35). For example, premethylation by heterologous expression of Bifidobacterium methylases in the cloning host may be required to protect vectors against restriction enzymes and thereby enhance transformation efficiency (36)(37)(38). It should be noted that B. longum strains lacking ISBlo5 copies may be suitable for generation of a saturated transposon library, because the artificial transposon circle pBFS12 prefers an insertion into IS256 family ISBlo5 copies (Table 3).…”