A Bifidobacterium mixed-species microarray for high resolution discrimination between intestinal bifidobacteria

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Abstract

A genomic DNA-based microarray was constructed containing over 6000 randomly cloned genomic fragments of approximately 1–2 kb from six mammalian intestinal Bifidobacterium spp. including B. adolescentis, B. animalis, B. bifidum, B. catenulatum, B. longum and B. pseudolongum. This Bifidobacterium Mixed-Species (BMS) microarray was used to differentiate between type strains and isolates belonging to a set of nine Bifidobacterium spp. Hierarchical clustering of genomic hybridization data confirmed the grouping of the Bifidobacterium spp. according to the 16S rRNA-based phylogenetic clusters. In addition, these genomic hybridization experiments revealed high homology between the type-strain B. animalis subsp. lactis LMG18314 and B. animalis subsp. animalis LMG10508 (79%) as well as between the type strains B. longum biotype longum LMG13197 and B. longum biotype infantis LMG8811 (72%) — nevertheless, discrimination between these species was possible due to the high resolution output of the BMS-array. In addition, it was shown that the BMS-array could be used for assigning unknown Bifidobacterium isolates to a species group. Finally, a set of 54 diagnostic clones for Bifidobacterium identification was selected and sequenced to advance the understanding of the species-related differences. Remarkably, a large fraction (31%) of these was predicted to encode proteins that belong to the bifidobacterial glycobiome and another 11% had functional homology with genes involved in the protection against foreign DNA. Overall, the BMS-microarray is a high-resolution diagnostic tool that is able to facilitate the detection of strain- and species-specific characteristics of bifidobacteria.

Introduction

Bifidobacterium species are known to be among the first and most dominant gut inhabitants in our early life (Favier et al., 2002, Xu and Gordon, 2003, Vaughan et al., 2005). Their main habitat is the intestinal tract of humans and other animals. Bifidobacteria are reported to derive their specific ecological success from their capacity to metabolize complex carbohydrates (Ventura et al., 2007b). This characteristic forms the basis for various claims on the prebiotic effect of dietary oligosaccharides that selectively stimulate growth of bifidobacteria in the human colon (for a review see Gibson et al., 2005) (Gibson et al., 2005). However, there is only limited information on the effect of bifidobacteria on human health (Guarner and Malagelada, 2003, Klijn et al., 2005, Boesten and De Vos, 2008) in spite of the fact that some Bifidobacterium strains are also marketed as probiotics (Juntunen et al., 2001, Parvez et al., 2006, Mohan et al., 2006).

The Bifidobacterium genus presently includes 34 species that are characterized by specific host-relations and can be grouped into six phylogenetic clusters (Ventura et al., 2006). Two of these contain Bifidobacterium spp. found in the human intestinal tract, including the B. adolescentis and B. longum clusters. Another relevant intestinal species is B. bifidum, which only includes human isolates but does not belong to any of the clusters but has a separate branch (Ventura et al., 2006). A fourth one, the B. pseudolongum cluster, is of interest in relation to the human gut because among others it contains B. animalis subsp. lactis that is isolated from fermented milk and is widely marketed as a human probiotic (Wall et al., 2007). As a consequence, this species is increasingly found in human fecal samples that are also known to contain B. adolescentis, B. catenulatum, B. longum, and B. bifidum (Matsuki et al., 1999). Although, fecal samples of healthy infants are significantly different (Matsuki et al., 1999) and apparently affected by the health status (Salminen et al., 2005, Marchesi and Shanahan, 2007), the Bifidobacterium spp. composition in the intestinal tract of healthy adults is stable over time (Zoetendal et al., 1998, Matsuki et al., 2004). Sequence analyses of 16S rRNA and 16S–23S spacers (Leblond-Bourget et al., 1996, Kwon et al., 2005), but also of highly conserved genes such as tuf, recA (Ventura and Zink, 2003) and groEL (HSP60 family) gene sequences (Jian et al., 2001), have been used for elucidation of the Bifidobacterium taxonomy. However, controversy exists with respect to the phylogenetic position of some industrial strains such as B. animalis that is also described as B. lactis (Ventura and Zink, 2002). Furthermore, the taxonomic classification of B. longum-related strains is still not completely resolved (Mattarelli et al., 2008).

While the number of sequenced genomes is rapidly accumulating, so far only the B. longum NCC2705 genome has been sequenced and annotated completely (Schell et al., 2002). Complete genomes of a variety of other Bifidobacterium species have reported to be completed (Liu et al., 2005, Ventura et al., 2007a); including those of B. animalis, B. breve and B. adolescentis, but only that of the latter one has been deposited in a public database. This may be due to their application potential, as is illustrated by the discovery of a serpin-encoding gene in the B. longum genome that has the capacity to inhibit eukaryotic elastase-like serine proteases and may play an important role in the interaction between these commensal bacteria and their host (Ivanov et al., 2006).

One of the outstanding characteristics of the bifidobacterial genome is the presence of numerous genes that allow acquisition, transport and metabolism of a broad range of complex dietary polysaccharides that cannot be processed by the human host. This arsenal of genes encoding proteins involved in carbohydrate depolymerization, uptake and metabolism is known as glycobiome. Over 8% of the annotated genes of B. breve UCC2003 and B. longum biotype longum NCC2705 encode enzymes involved in the carbohydrate metabolism (Ventura et al., 2007b). Some sugar-degrading abilities are restricted to certain species or strains of a particular species (Ward et al., 2007, LoCascio et al., 2007). Comparative genome hybridization suggested that these sugar-degrading genes and those that encode restriction–modification systems belong to the variable clusters within the Bifidobacterium genome that have been acquired via horizontal gene transfer (Schell et al., 2002, Ventura et al., 2007b). In addition, mobile elements such as prophage-like elements and plasmids, although not ubiquitous in bifidobacteria, can also cause variation between strains and species (Ventura et al., 2007a).

In addition, a B. longum and an unpublished B. breve microarray have been used as genotyping tools in comparative genome hybridizations (Parche et al., 2007, Ventura et al., 2007a). To provide an alternative in the absence of published genome sequences, we developed an approach based on random clone-based microarrays (Vlaminckx et al., 2007, Leavis et al., 2007). This approach is ideally suited for determination of genomic differences of not or not yet completely sequenced genomes. A genomic DNA-based microarray was constructed, containing random-clones of six Bifidobacterium species relevant for the intestinal tract of humans. In this study we show the applicability of this microarray as a diagnostic tool in the analysis of diversity and function of bifidobacteria.

Section snippets

Bacterial strains, culturing, DNA isolation and 16S rRNA sequencing

The origin and other information of the used bacterial strains is summarized in Table 1. All Bifidobacterium strains were grown anaerobically in MRS broth (Difco, Detroit, USA) supplemented with 0.05% l-cysteine hydrochloride monohydrate (Sigma-Aldrich, Steinheim, D) and incubated at 37 °C. Genomic DNA (gDNA) was extracted using a protocol based on enzymatic lysis (Baess, 1974). Additionally, after precipitation the supernatant was discarded and the pellet was washed using 250 μl of 70% ethanol

Design, construction and application of the BMS microarray

A Bifidobacterium mixed-species microarray was constructed, carrying genomic DNA (gDNA) of six Bifidobacterium species, derived from the adult colon, except for B. pseudolongum that was included to address its comparison with the intestinal B. longum (Table 1). This so called BMS-array was used to study genomic differences both within a single Bifidobacterium species and between bifidobacterial phylogenetic clusters (Fig. 1). First, gDNA of the six used Bifidobacterium spp. was hybridized to a

Acknowledgements

This work was supported by the Dutch Ministry of Economic Affairs through the Innovation Oriented Research Program on Genomics (IOP Genomics: IGE01016). We would like to thank Martien Caspers from TNO Quality of Life for his excellent input with respect to the data analysis, Robert van den Berg for correlation mapping assistance, and Eline Klaassens for providing Bifidobacterium isolates and database searches.

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