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Mixed-species genomic microarray analysis of fecal samples reveals differential transcriptional responses of bifidobacteria in breast- And formula-fed infants

Author: Klaassens, E.S. · Boesten, R.J. · Haarman, M. · Knol, J. · Schuren, F.H. · Vaughan, E.E. · Vos, W.M.de
Type:article
Date:2009
Institution: TNO Kwaliteit van Leven
Source:Applied and Environmental Microbiology, 9, 75, 2668-2676
Identifier: 241500
doi: doi:10.1128/AEM.02492-08
Keywords: Biology · Biotechnology · Bifidobacteria · Bifidobacterium · Breast milk · Carbohydrate metabolism · Denaturing gradient gel electrophoresis · Exopolysaccharide · Fecal samples · Fructo-oligosaccharides · Human guts · In-vivo · Metabolic activity · Microarray analysis · Mixed species · Nucleotide sequences · Protein-encoding genes · Quantitative PCR · Quantitative real time PCR · Related functions · RRna genes · Sequence analysis · Species diversity · Transcriptional response · Carbohydrates · Electrophoresis · Encoding (symbols) · Functional groups · Gelation · Metabolism · Nucleic acids · Polysaccharides · RNA · Genes · bacterial RNA · exopolysaccharide · folic acid · fructose oligosaccharide · galactose oligosaccharide · ribosome RNA · abundance · bacterium · feces · gene expression · genomics · hominid · metabolism · polymerase chain reaction · real time · RNA · sugar · article · artificial milk · bacterial metabolism · Bifidobacteriaceae · breast milk · carbohydrate metabolism · denaturing gradient gel electrophoresis · feces analysis · gene expression regulation · gene sequence · human · human experiment · in vivo study · infant · intestine · microarray analysis · microbial community · microbial diversity · normal human · nucleotide sequence · polymerase chain reaction · quantitative analysis · real time polymerase chain reaction · RNA hybridization · RNA isolation · species diversity · transcription regulation · bacterial count · Bifidobacterium · biodiversity · classification · diet · DNA fingerprinting · DNA microarray · feces · gene expression profiling · genetics · microbiology · Bifidobacterium · Bifidobacterium · Biodiversity · Colony Count, Microbial · Diet · DNA Fingerprinting · Feces · Gene Expression Profiling · Humans · Infant · Infant Formula · Milk, Human · Oligonucleotide Array Sequence Analysis

Abstract

Although their exact function remains enigmatic, bifidobacteria are among the first colonizers of the newborn infant gut and further develop into abundant communities, notably in response to diet. Therefore, the transcriptional responses of bifidobacteria in rapidly processed fecal samples from young infants that were fed either breast milk or a formula containing a mixture of galacto- and fructo-oligosaccharides were studied. The presence and diversity of the bifidobacterial fecal communities were determined using PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR for specific species. Changes in the total number of bifidobacteria as well as in species diversity were observed, indicating the metabolic activities of the bifidobacteria within the infant gut. In addition, total RNAs isolated from infant feces were labeled and hybridized to a bifidobacterium-specific microarray comprising approximately 6,000 clones of the major bifidobacterial species of the human gut. Approximately 270 clones that showed the most prominent hybridization with the samples were sequenced. Fewer than 10% of the hybridizing clones contained rRNA genes, whereas the vast majority of the inserts showed matches with protein-encoding genes predicted to originate from bifidobacteria. Although a wide range of functional groups was covered by the obtained sequences, the largest fraction (14%) of the transcribed genes assigned to a functional category were predicted to be involved in carbohydrate metabolism, while some were also implicated in exopolysaccharide production or folate production. A total of three of the above-described protein-encoding genes were selected for quantitative PCR and sequence analyses, which confirmed the expression of the corresponding genes and the expected nucleotide sequences. In conclusion, the results of this study show the feasibility of obtaining insight into the transcriptional responses of intestinal bifidobacteria by analyzing fecal RNA and highlight the in vivo expression of bifidobacterial genes implicated in host-related functions. Copyright © 2009, American Society for Microbiology.