Evaluation of the hypervariable region in the 16S rDNA sequence as an index for rapid species identification in the genus Paenibacillus.

The genus Paenibacillus (Ash et al., 1993) comprising aerobic or facultatively anaerobic endospore-forming bacilli is represented by rRNA group 3 and contains 28 validated species and 2 subspecies.The genus is phenotypically similar to other members of the family Bacillaceae but is difficult to distinguish from the other genera by morphology alone.Recently a highly specific primer set directed to the 16S rDNA of Paenibacillus was described (Shida et al., 1997), which enabled the rapid identification of Paenibacillus to the genus level.However the identification of Paenibacillus below the genus level remains difficult and time-consuming due to the absence of well-defined phenotypes that differentiate species (Reva et al., 2001).In our previous studies, we have demonstrated that a hypervariable (HV) region, corresponding to the 5Ј end region of 16S rDNA (nucleotide positions 70 to 344; Bacillus subtilis numbering), is a useful marker for the rapid identification and grouping of Alicyclobacillus species (Goto et al., 2002b, c) and Bacillus species (Goto et al., 2000).In this study, 28 type strains and 31 additional strains of Paenibacillus species were subjected to a sequence comparison of the HV regions and the use of this HV region for molecular typing of the various species of the genus Paenibacillus was evaluated.The 59 strains used in this study were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA), DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany), IAM (Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan), IFO (Institute for Fermentation, Osaka, Japan) and NCIMB (National Collection of Industrial and Marine Bacteria, Aberdeen, UK).The Paenibacillus strains were cultivated by the methods recommended in the ATCC, DSM, IAM, IFO and NCIMB strain catalogues, respectively.The two newest species, P. azoreducens (Meehan et al., 2001) and P. jamilae (Aguilera et al., 2001), were not included because these type strains were not available for distribution at the time of this study.Extraction and purification of genomic DNA, sequencing of the 16S rDNA and HV region, and phylogenetic analysis were performed as described in our previous papers (Goto et al., 2000(Goto et al., , 2002a, b), b).Sequences determined during the present work have been deposited in the DNA data bank of Japan (DDBJ) under the accession numbers AB073186 to AB073206 and AB073362 Evaluation of the hypervariable region in the 16S rDNA sequence as an index for rapid species identification in the genus Paenibacillus