Evaluation of using a short region of the recA gene for rapid and sensitive speciation of dominant bifidobacteria in the human large intestine.

The feasibility of intragenerically characterizing bifidobacteria by a comparison of a short region within the recA gene was tested. An approximately 300 bp fragment of the recA gene was PCR-amplified from six species from the genus Bifidobacterium using primers directed to two universally conserved regions of the recA gene. A phylogenetic analysis of the sequenced recA products compared favorably to classification based on the 16S rRNA sequences of the species tested. To apply this rapid methodology to unknown human intestinal bifidobacteria, 46 isolates were randomly chosen from the feces of four subjects and initially characterized by RFLP analysis of a PCR-amplified region of their 16S RNA genes. From a representative of the dominant RFLP family in each of the subjects, the recA segment was PCR-amplified, sequenced and phylogenetically analyzed. All four isolates were found to be related to one another and to B. longum and B. infantis. These results illustrate that the recA gene may be useful for intrageneric phylogenetic analysis as well as for the identification of unknown fecal bifidobacteria.

[1]  W. Wenzhi,et al.  Molecular analysis of the composition of the bifidobacterial and lactobacillus microflora of humans , 1996, Applied and environmental microbiology.

[2]  S Karlin,et al.  Bacterial classifications derived from recA protein sequence comparisons , 1995, Journal of bacteriology.

[3]  M. Wilkinson,et al.  Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples , 1995, Applied and environmental microbiology.

[4]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[5]  Y. Bouhnik,et al.  Identification of Bifidobacterium strains by rRNA gene restriction patterns , 1994, Applied and environmental microbiology.

[6]  K. Wilson,et al.  Ribosomal DNA Sequences of Bifidobacteria: Implications for Sequence-based Identification of the Human Colonic Flora , 1993 .

[7]  R. Tanaka,et al.  Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora , 1992, Applied and environmental microbiology.

[8]  K. Dybvig,et al.  Degenerate oligonucleotide primers for enzymatic amplification of recA sequences from gram-positive bacteria and mycoplasmas , 1992, Journal of bacteriology.

[9]  R. Parés,et al.  Selective medium for isolation and enumeration of Bifidobacterium spp , 1988, Applied and environmental microbiology.

[10]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[11]  N. Pace,et al.  Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Felsenstein.  CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP , 1985, Evolution; international journal of organic evolution.

[13]  D. Matteuzzi,et al.  Deoxyribonucleic Acid Homology Relationships Among Species of the Genus Bifidobacterium , 1971 .

[14]  L. Brady,et al.  Differentiation of ingested and endogenous bifidobacteria by DNA fingerprinting demonstrates the survival of an unmodified strain in the gastrointestinal tract of humans. , 1997, The Journal of nutrition.

[15]  H. Philippe,et al.  16S rRNA and 16S to 23S internal transcribed spacer sequence analyses reveal inter- and intraspecific Bifidobacterium phylogeny. , 1996, International journal of systematic bacteriology.

[16]  R. V. Miller,et al.  General microbiology of recA: environmental and evolutionary significance. , 1990, Annual review of microbiology.