Differentiation of Bifidobacterium species using partial RNA polymerase β-subunit (rpoB) gene sequences

Abstract

Partial RNA polymerase b-subunit gene (rpoB) sequences (315 bp) were determined and used to differentiate the type strains of 23 species of the genus Bifidobacterium. The sequences were compared with those of the partial hsp60 (604 bp) and 16S rRNA genes (1475 or 1495 bp). The rpoB gene sequences showed nucleotide sequence similarities ranging from 84.1 % to 99.0 %, while the similarities of the hsp60 sequences ranged from 78.5 % to 99.7 % and the 16S rRNA gene sequence similarities ranged from 89.4 % to 99.2 %. The phylogenetic trees constructed from the sequences of these three genes showed similar clustering patterns, with the exception of several species. The Bifidobacterium catenulatum-Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum subsp. pseudolongum-Bifidobacterium pseudolongum subsp. globosum and Bifidobacterium gallinarum-Bifidobacterium pullorum-Bifidobacterium saeculare groups were more clearly differentiated in the partial rpoB and hsp60 gene sequence trees than they were in the 16S rRNA gene tree. Based on sequence similarities and tree topologies, the newly determined rpoB gene sequences are suitable molecular markers for the differentiation of species of the genus Bifidobacterium and support various other molecular tools used to determine the relationships among species of this genus.Bifidobacteria are Gram-positive, non-spore-forming, nonmotile, non-gas-producing, anaerobic, bifid-shaped rods that colonize the gastrointestinal tracts of humans, as well as other mammals and some insects. There has been increased interest in these organisms recently due to their beneficial effects on gastrointestinal health. Indeed, they have been used as probiotics and evaluated for their ability to reduce several gastrointestinal tract diseases and protect against pathogens (Guarner & Malagelada, 2003;Mitsuoka & Kaneuchi, 1977;Ventura et al., 2008).Bacillus bifidus was first isolated from a healthy breast-fed infant by Tissier in 1899. The genus Bifidobacterium was later designated as an independent taxon by Orla-Jensen (1924) (Ventura et al., 2008), after which many species belonging to this genus were identified and classified. Traditionally, phenotypic analyses based on morphology and the patterns of various carbohydrate fermentation tests have been used to differentiate species of the genus Bifidobacterium. However, it has been suggested that these classification criteria may be unclear and may provide questionable results (Mitsuoka & Kaneuchi, 1977). Therefore, improved methods of identification and differentiation are necessary. Accordingly, various molecular tools that are currently available may be useful for the reliable identification of such organisms.The recent rapid developments in molecular biology have resulted in the use of various new methods for the identification and differentiation of species of the genus Bifidobacterium. These include PCR-linked methods such as nucleotide-dependent phylogenetic analysis targeting housekeeping genes (Jian et al., 2001;Ventura et al., 2004aVent...

High-Throughput Sequencing Reveals the Incomplete, Short-Term Recovery of Infant Gut Microbiota following Parenteral Antibiotic Treatment with Ampicillin and Gentamicin

“…A set of PCR primers, which were used previously to facilitate the identification of bifidobacteria (43) and which amplify a 351-bp region from the Bifidobacterium RNA polymerase ␤-subunit gene, rpoB, were also utilized in this study. Twelve samples from week 4 and 12 from week 8 were selected and amplified using these primers, which had MID tags and 454 adapters attached, allowing pooling of the samples for sequencing while also enabling downstream separation of individual results for analysis (see Table S1 in the supplemental material).…”

Bifidobacterium aerophilum sp. nov., Bifidobacterium avesanii sp. nov. and Bifidobacterium ramosum sp. nov.: Three novel taxa from the faeces of cotton-top tamarin ( Saguinus oedipus L.)

“…Thus, the phylogenetic location of the novel strains was verified by the analysis of A c c e p t e d M a n u s c r i p t 7 six genetic markers (hsp60, rpoB, dnaG, dnaJ and clpC) [CJR4], which have been shown to be discriminative 169 for the classification of the genus Bifidobacterium [8,13,40]. 170…”

“…A c c e p t e d M a n u s c r i p t 6 TCGATCGGGCACATACGG-3') and BifR2 (5'-CGACCACTTCGGCAACCG-3') [13], DnaJ1-uni (5'-137 GAGAAGTTCAAGGACATCTC-3') and DnaJ1-rev (5'GCTTGCCCTTGCCGG-3'), ClpC-uni (5'-138…”

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