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Use of the Gyrb Gene for the Identification of Pandoraea Species

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Use of the Gyrb Gene for the Identification of Pandoraea Species FEMS Microbiology Letters 208 (2002) 15^19 www.fems-microbiology.org Use of the gyrB gene for the identi¢cation of Pandoraea species Tom Coenye *, John J. LiPuma Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, 8301 MSRB III, Box 0646, 1150 W. Med. Ctr. Dr., Ann Arbor, MI 48109-0646, USA Received 9 November 2001; accepted 27 November 2001 First published online 29 January 2002 Abstract The recently described genus Pandoraea consists of five named species and four unnamed genomospecies, several of which have been identified in clinical specimens including respiratory secretions from persons with cystic fibrosis. We investigated whether it is possible to distinguish species of the genus Pandoraea by means of restriction fragment length polymorphism (RFLP) analysis and direct sequencing of the gyrB gene. Sixty-seven Pandoraea isolates were included. Species-specific RFLP patterns were obtained following digestion of the PCR- amplified gyrB gene with MspI. Specificity of RFLP groupings was confirmed by direct sequencing of several representative isolates. Our results indicate that RFLP analysis and sequencing of the gyrB gene are useful for the identification of Pandoraea species. We also found that further taxonomic studies within the L-Proteobacteria using the gyrB gene would benefit from the development of additional primers allowing more efficient amplification of the gyrB gene. Our data also indicate that the taxonomic status of Pandoraea genomospecies 2 should be reinvestigated. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: gyrB; Identi¢cation; Cystic ¢brosis; Pandoraea; Burkholderia cepacia complex 1. Introduction rial molecular systematics studies include recA [7,8], rpoD [9] and gyrB. The gyrB gene, encoding a bacterial DNA It is generally accepted that the phylogenetic relation- gyrase (topoisomerase type II), has been used in phyloge- ships between microorganisms can be deduced from se- netic studies of the genera Pseudomonas [9^11], Acineto- quence comparisons of conserved macromolecules. Ribo- bacter [12,13], Vibrio [14], Micromonospora [15] and My- somal RNA genes are one of the best targets for cobacterium [16] and for members of the Cytophaga^ phylogenetic studies because they are universally present Flavobacterium^Bacteroides complex [17]; results from and functionally constant and have a mosaic structure of these studies have indicated that groupings based on highly conserved and more variable domains [1]. The di- gyrB sequences are generally in agreement with DNA^ rect sequencing of 16S and or 23S rDNA molecules by DNA hybridization data. PCR technology provides a phylogenetic framework which The genus Pandoraea contains ¢ve species (Pandoraea serves as the backbone for modern microbial taxonomy apista, Pandoraea sputorum, Pandoraea norimbergensis, [2]. However there is no threshold value for 16S rDNA Pandoraea pnomenusa and Pandoraea pulmonicola) and homology for species recognition and due to the slow evo- four unnamed genomospecies (designated Pandoraea ge- lution of the 16S rDNA genes, recently diverged species nomospecies 1, 2, 3 and 4) [18,19]. Isolates belonging to may not be recognizable [3^6]. To circumvent these limi- the genus Pandoraea have mainly been recovered from the tations it has been suggested that phylogenetic informa- respiratory secretions of cystic ¢brosis patients but can tion derived from other genes could be used to comple- also be isolated from other clinical samples (including ment the information obtained from 16S rDNA sequence blood) and the environment [18,19]. Sequence analysis of analysis [5,6]. Other genes that have been used for bacte- the 16S rDNA gene of the di¡erent named and unnamed Pandoraea species has revealed high similarity values [18,19] and although identi¢cation of most Pandoraea spe- * Corresponding author. cies using recently developed 16S rDNA-based PCR as- Tel.: +1 (734) 9369767; Fax: +1 (734) 6154770. E-mail addresses: [email protected] (T. Coenye), says is possible, not all species can be separated using [email protected] (T. Coenye). these assays [20]. Phenotypic identi¢cation of Pandoraea 0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0378-1097(01)00589-4 FEMSLE 10329 25-3-02 16 T. Coenye, J.J. LiPuma / FEMS Microbiology Letters 208 (2002) 15^19 species is also not straightforward and reliable identi¢ca- tion often requires a polyphasic approach [18,19]. The aim of this study was to determine whether restriction frag- ment length polymorphism (RFLP) analysis and direct sequencing of the gyrB gene could be used for the identi- ¢cation of Pandoraea species. 2. Materials and methods 2.1. Bacterial strains and growth conditions All 67 Pandoraea isolates included in this study have previously been described [18^20]. We included ¢ve P. norimbergensis isolates, three P. pulmonicola isolates, 26 P. apista isolates, 13 P. sputorum isolates and 15 P. pno- Fig. 1. MspI RFLP analysis of the PCR-ampli¢ed gyrB gene of selected menusa isolates. In addition we also included strains be- Pandoraea isolates. Lanes: M, 100-bp DNA ladder (Gibco BRL); 1, P. longing to the unnamed Pandoraea genomospecies 1, 2, 3 apista HI2804; 2, P. apista AU2303; 3, P. apista AU0170; 4, P. apista (one isolate each) and 4 (two isolates). Strains were grown LMG 16407T ;5,P. pulmonicola LMG 18106T ;6,P. norimbergensis aerobically on Mueller Hinton broth (Becton Dickinson) LMG 13019; 7, P. norimbergensis LMG 16603; 8, P. norimbergensis T supplemented with 1.8% (w/v) agar and incubated over- LMG 18379 ;9,P. sputorum AU0125; 10, P. sputorum AU0103; 11, Pandoraea genomospecies 2 LMG 20601; 12, P. pnomenusa LMG night at 32³C. All strains were previously identi¢ed using a 18087T ; 13, P. pnomenusa LMG 18820; 14, P. pnomenusa HI2800; 15, polyphasic^taxonomic approach and/or genus- and spe- Pandoraea genomospecies 3 LMG 20602; 16, Pandoraea genomospecies cies-speci¢c 16S rDNA-based PCR assays [18^20]. 4 LMG 20603; 17, Pandoraea genomospecies 4 AU1775. 2.2. DNA preparation DNA was prepared by heating one or two colonies enzymes used included MspI (Promega), RsaI, TaqI, (picked from an overnight grown culture) at 95³C for 15 HaeIII and DdeI (Gibco). Restriction fragments were sep- min in 20 Wl lysis bu¡er, containing 0.25% (v/v) SDS and arated in 10U15-cm 2% agarose gels (Genepure) in 0.05 M NaOH. Following lysis, 180 Wl sterile distilled 0.5UTBE bu¡er at 100 V for 75 min. A 100-bp DNA water was added to the lysis bu¡er and the DNA solutions ladder (Gibco) was included on all gels to allow standard- were stored at 320³C. ization and sizing. Gels were stained with ethidium bro- mide and visualized using the GelDoc System (Bio-Rad). 2.3. RFLP analysis of the gyrB gene Densitometric analysis, normalization and interpolation of the patterns and numerical analysis using the Dice coe¤- PCR reactions were performed in 25-Wl reaction mix- cient were performed using the Quantity One 4.1 (Bio- tures, containing 2 Wl DNA solution, 1 U Taq polymerase Rad) and Molecular Analyst (Bio-Rad) software pack- (Qiagen), 250 mM (each) deoxynucleotide triphosphate ages. (Gibco), 1UPCR bu¡er (Qiagen) and 20 pmol of each oligonucleotide primer. The primers used were the 2.4. Partial sequence determination of the gyrB gene universal primers described by Yamamoto and Harayama [10]: UP-1 (5P-GAAGTCATCATGACCGTTCTGCAY- The gyrB gene was ampli¢ed using PCR, as described GCNGGNGGNAARTTYGA-3P) and UP-2r (5P-AGCA- above for the RFLP analysis. The PCR product was pu- GGGTACGGATGTGCGAGCCRTCNACRTCNGCRT- ri¢ed using the Promega Wizard PCR Preps kit (Promega) CNGTCAT-3P). Ampli¢cation was carried out using a according to the manufacturer's instructions. Sequence PTC-100 programmable thermal cycler (MJ Research). analysis was performed with an Applied Biosystems 3700 After initial denaturation for 2 min at 94³C, 30 ampli¢ca- DNA sequencer and the protocols of the manufacturer tion cycles were completed, each consisting of 1 min at (PE Applied Biosystems) using the BigDye Terminator 94³C, 1 min at 65³C and 1 min at 72³C. A ¢nal extension Cycle Sequencing Ready Reaction kit. The sequencing of 10 min at 72³C was applied. Restriction digests were primer used was the universal sequencing primer UP-1S performed in 20-Wl reaction mixtures, containing 5 U (5P-GAAGTCATCATGACCGTTCTGCA-3P). All the se- restriction enzyme, 1Uappropriate bu¡er, 0.2 Wl bovine quence reads were trimmed to approximately 550 bp. Phy- serum albumin (10 mg ml31) and 8 Wl PCR product. logenetic trees based on the neighbor-joining method [21] Restriction digests were carried out at the temperature were constructed using the MegAlign (DNAStar Inc.) soft- recommended by the manufacturer for 3 h. Restriction ware package. FEMSLE 10329 25-3-02 T. Coenye, J.J. LiPuma / FEMS Microbiology Letters 208 (2002) 15^19 17 2.5. Nucleotide accession numbers 3. Results All partial gyrB sequences have been deposited in Gen- 3.1. RFLP analysis of the gyrB gene Bank with the following accession numbers: AF439524 (LMG 18819T), AF439525 (LMG 18106T), AF439526 Using universal primer pair UP-1 and UP-2r, the com- (LMG 13019), AF439527 (LMG 16407T), AF439528 plete gyrB gene (approximately 1200 bp) was successfully (LMG 18087T), AF439529 (LMG 18379T), AF439530 ampli¢ed from 66 of the 67 Pandoraea strains included in (HI2800), AF439531 (HI2801), AF439532 (HI2802), this study. Despite repeated attempts we were not able to AF439533 (LMG 20601), AF439534 (HI2804), amplify the gyrB gene from Pandoraea genomospecies 1 AF439535 (LMG 20602) and AF439536 (LMG 20603). isolate R-5199. The e¤ciency of ampli¢cation of the gyrB Fig. 2. Dendrogram derived from the UPGMA linkage of Dice coe¤cients between the gyrB RFLP patterns generated by digestion with MspI. FEMSLE 10329 25-3-02 18 T.
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