Although two-dimensional electrophoresis (2D-electrophoresis) has been used to analyze bacterial protein polymorphisms and to distinguish between closely related pathogenic organisms [24–26], 2D-electrophoresis has not been used to compare bifidobacteria. In this study, our objective was to compare three human B. longum isolates with the model sequenced strain B. longum NCC2705 at the chromosome AZD8055 cost and proteome

levels. Pulse-field gel electrophoresis (PFGE) revealed a high degree of heterogeneity. Moreover, the isolates showed different patterns in terms of their cytoplasmic proteins that may reveal correlations with specific phenotypic differences of the B. longum strains. Our results show that this approach is a valuable tool for exploring the natural diversity and the various capabilities of bifidobacteria strains. Results and Discussion Epigenetic Reader Domain inhibitor In the present study, we chose B. longum NCC2705 as the reference strain because (i) B. longum is one of three species used as probiotics; (ii) the entire genome sequence is available, allowing protein identification using a public database [16]; (iii)

a proteome reference map had been established for this strain [19]. Three B. longum human isolates with known biological effects were compared to this reference strain. In an animal model, B. longum BS89 has a protective role against necrotizing enterocolitis via a sharp decrease of clostridia unless [27]. The two other isolates show differences in their abilities to stimulate the intestinal immune system in gnotobiotic mice by inducing either T-helper 2 (B. longum BS64) or T-helper 1 cytokines (B. longum BS49) [28]. Genotype comparison using PFGE We first compared the four strains at the genome level using PFGE [29]. XbaI macro-restriction analysis of genomic DNA from B. longum strains NCC2705, BS49, BS64 and BS89 generated clear and easy-to-interpret PFGE patterns (Figure 1). The four strains exhibited a high degree of genomic heterogeneity and low intraspecies relatedness: BS89, BS49 and BS64 shared 57.9, 29.3 and 20.9% identity, respectively, with NCC2705 macrorestriction patterns. Such genetic variability is consistent with the comparative genomic analysis

of B. longum strains NCC2705 and DJO10A, which showed substantial loss of genome regions, probably due to multiple phage insertion sites [18, 30]. Considering the various biological effects and genomic heterogeneity of the isolates, one might speculate that this heterogeneity could be related to functional differences that could be identified using proteomic analysis. Figure 1 Comparison of B. longum genomic DNA XbaI macrorestriction patterns using pulsed field gel electrophoresis (PFGE) genotyping. Comparison of cytosolic protein patterns of the B. longum strains We next used 2D-electrophoresis to analyze the cytosolic protein content of these four strains. Spot differences between the three human isolates, BS89, BS49 and BS64, and B.