Actinomycetol. (2002) 16:1–5 VOL. 16, NO. 1 Application of PCR for Selection of Gram-Positive Bacteria with High DNA G+C Content among New Isolates Liyan Yu1, Yoko¯ Takahashi1, 2, Atsuko Matsumoto2, Akio Seino1, Yuzuru Iwai1 and Satoshi O¯ mura1, 2* 1Research Center for Biological Function, The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan 2Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan (Received Dec. 10, 2001 / Accepted May 1, 2002) A PCR condition was established to distinguish strains of Actinobacteria from both Gram-negative bacte- ria and Gram-positive bacteria with low G+C content of DNA by taking advantage of a stable, specific and common insertion of a 100 bp sequence in the domain III of the 23S ribosomal RNA gene of class Actinobacteria. The PCR was designed to amplify a 380 bp fragment from Actinobacteria and a 270 bp fragment from the above bacteria. PCR-based survey of known strains as well as newly isolated soil bac- teria confirmed that this PCR worked well to select Actinobacteria. In the survey it was unexpectedly found that several strains provided both 270 bp and 380 bp amplified fragments (the sequences of these two fragments obtained from Agrococcus jenensis were determined), indicating the presence of at least two copies of the domain III of the 23S rDNA gene. INTRODUCTION characteristic, we establish a simple PCR method to dis- criminate Actinobacteria strains from new isolates in this Since the discovery of streptomycin1), a large number of study. antibiotics have been isolated from cultures of a wide vari- MATERIALS AND METHODS ety of streptomycetes and rare actinomycetes. To improve the possibility of discovering new and useful bioactive Organisms and culture conditions: compounds from microorganisms, the scope of microbial Forty-one strains were isolated from four soil samples resources should be expanded to bacterial strains other using water agar medium containing 1% L-proline with 25 than actinomycetes, which have been recognized as the µg/mL nalidixic acid and 25 µg/mL aztreonam. These most promising resource of microbial products. In this antibiotics were added to inhibit some Gram-negative and context, we focused on Actinobacteria strains other than Gram-positive bacteria with a low DNA G+C content. filamentous actinomycetes as a new potential microbial Thirty-two Gram-positive strains selected by Gram stain- resource, because these microorganisms have not been ing from the 41 isolates were cultured in TSB medium at searched so much so far. 27 °C to exponential phase for PCR analysis and quantita- Significant rRNA homology has been reported between tive analysis of G+C content of DNA. Strains used for a actinomycetes and a group of Gram-positive bacteria with comparative study are listed in Table 1. high G+C content of DNA2). Stackebrandt et al. (1997) proposed designating these organisms as class PCR conditions: Actinobacteria3). The class Actinobacteria contains five Aliquots (0.5 mL) of exponential phase culture broth subclasses, Actinobacteridae, Acidimicrobidae, were placed in 1.5 mL microtubes, washed once with dis- Coriobacteridae, Sphaerobacteridae and Rubrobacteridae, tilled water followed by Tris-EDTA buffer (TE) contain- and more than 120 genera. Subclass Actinobacteridae is ing 10 mM Tris base and 1 mM EDTA, pH 8.0), and the biggest group containing actinomycetes and many resuspended in 0.5 mL TE buffer. The suspension was other genera forming bacteria-like colony. While actino- subjected to sonication for 5 min in a room temperature mycete strains form generally hard colonies on agar media, water bath (BRANSON 3200, BRANSON Ultrasonic Co. other Actinobacteria strains form the soft colonies. Thus, USA), followed by centrifugation at 10 000×g for 5 min. the latter group of bacteria contains many strains morpho- The resulting supernatant was stored at −20 °C until use logically indistinguishable from Gram-negative bacteria for PCR . and Gram-positive bacteria with a low G+C content. The primer pair (23InsV, 5’-MADGCGTAGNC- A stable 100 bp insertion within domain III (helix 54a) GAWGG-3’ and 23InsR, 5’-GTGWCGGTTTNBGGTA- of the 23S ribosomal RNA gene has been identified as a 3’)6) was used for amplification of 270 bp and/or 380 bp phylogenetic marker unique to Actinobacteria4–7), particu- fragments from the domain III of 23S rRNA/DNA. PCR larly subclass Actinobacteridae. Based on this genetic reactions of 25 µL total volume contained 2.5 µL 10 xTaq * Corresponding author. Present address: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Tiantan, Beijing, China 100050. 1 ACTINOMYCETOLOGICA VOL. 16, NO. 1 Table 1. Strains used for comparative study described by Hopwood et al.8). The G+C content of DNA Name Strain was determined by high performance liquid chromatogra- Gram-negative bacteria phy following the treatment with P1 nuclease and alkaline 1. Pseudomonas aeruginosa IFO-3080 phosphatase according to the method of Tamaoka and 9) 2. Escherichia coli IFO-12734 Komagata . 3. Salmonella typhi KB-17 (H901 W) Gram-positive bacteria with low DNA G+C content RESULTS AND DISCUSSION 4. Staphylococcus aureus ATCC 6538P 5. Cytophaga lytica JCM-8516T Optimization of PCR conditions: 6. Bacillus subtilis KB-27 (PCI 219) PCR is an in vitro method that allows the specific Actinobacteria amplification of any particular segment of DNA of which 7. Aeromicrobium fastidiosum JCM-8088 T nucleotide sequence is known. However, to get good PCR 8. Terrabacter tumescens JCM-1365 T result, supplier’s PCR protocol may be modified. For the 9. Micrococcus luteus KB-189 amplification of the domain III of 23S rDNA, concentra- 10. Luteococcus japonicus JCM-9415 T tions of enzyme, deoxynucleotide triphoshate, magnesium, 11. Agrococcus jenensis JCM-9950 T and primer were examined (data not shown) and the 12. Rhodococcus erythropolis JCM-3201 T opitimized concentrations were described in MATERIALS 13. Rhodococcus rhodochrous JCM-3202 T AND METHODS. The sequences of primers 23InsV and 14. Brachybacterium faecium IFO-14762 T 23InsR were confirmed by the alignment of full rDNA 15. Cellulomonas turbata JCM-3160 T sequences (from DDBJ) of three Frankia species, 16. Nocardia asteroides JCM-3384 T Streptomyces ambofaciens, S. lividans S. griseus, S. rimo- 17. Streptomyces hygroscopicus JCM-4772 T sus, Micrococcus luteus, Mycobacterium kansas, M. lep- 18. Streptomyces violaceoruber IFO-15146 rae, M. smegmatis and Bacillus subtilis. For discriminative amplification of target PCR products (270 bp and 380 bp fragments), effect of annealing temper- DNA polymerase buffer (TaKaRa, Tokyo), 1 µL dNTP ature was examined using template DNAs from three mix (2.5 mM dATP, dCTP, dGTP and dTTP, TaKaRa), 1 strains each of Gram-negative and -positive bacteria and µL each 100 µM primers, 1 µL genomic DNA (stored Actinobacteria as shown in Fig. 1. The 270 bp band was supernatant), and 0.2 µL 5 U/µL Taq DNA polymerase observed in all of the Gram-negative and -positive bacteria (TaKaRa). The reaction mixture was overlaid with mineral tested (lanes 1–6), while the 380 bp band was observed oil (Sigma). PCR was carried out in a TaKaRa 480 only in Actinobacteria (lanes 7–8) at all of three annealing Thermal Cycler under the following conditions; 95 °C for temperatures (59, 61 and 63 °C). However, the use of 2 min → 33 x (94 °C for 30 sec → 63 °C for 45 sec. → annealing temperatures of 59 °C and 61 °C yielded many 72 °C for 1 min) → 72 °C for 5 min. PCR products were non-specific bands (Fig. 1a and b) in both Gram-negative monitored by agarose [1% (W/V)] gel electrophoresis and Gram-positive bacteria tested. These non-specific (AGE). bands were not yielded when annealing temperature was elevated to 63 °C (Fig. 1c). It was further demonstrated Cloning and sequencing of PCR products: that PCR experiment using the 63 °C annealing tempera- The target bands of interest were cut out of the AGE ture is obviously useful to distinguish Actinobacteria from agarose to purify PCR products with QIAquick Gel Gram-negative and Gram-positive bacteria as shown in Extraction Kit (QIAGEN Co., Germany). The purified Fig. 2. Only 270 bp band was specifically and commonly PCR product was ligated with pT7 Blue T-Vector detected on all the lanes (1–6) of Gram-negative and -posi- (Novagen Inc., USA) to transform E. coli DH10B. tive bacteria, whereas 380 bp band was commonly Ampicillin-resistant transformed cells were inoculated into observed only on the lanes (7–18) of Actinobacteria 3 mL LB medium containing 50 µg/mL ampicillin and although 270 bp band was also detected on several grown at 37 °C. Recombinant DNA was extracted from Actinobacteria lanes. cultures using High PureTM Plasmid Isolation Kits (Roche Diagnostic, Tokyo) and sequenced using BigDye TM Sequencing and characterization of PCR products of Primer Cycle Sequencing Ready Reaction Kits (PE Agrococcus jenensis: Applied Biosystems), on a model 377 Prism Automatic Among the 270 bp and 380 bp PCR products derived DNA Sequencer (PE Applied Biosystems). The resulting from Actinobacteria species, sequences of those from A. rDNA sequences from three clones each were aligned jenensis were determined and compared with 23S rDNA against sequences obtained from the DDBJ databases. sequences from the DDBJ databases of B. subtilis and M. luteus. The sequence of the 270 bp fragment from A. Analysis of Guanine-plus-cytosine contents of DNA: jenensis showed high homology to that of the correspond- Genomic DNA was extracted according to the method ing region of B. subtilis 23S rDNA (Fig. 3a). The 380 bp 2 ACTINOMYCETOLOGICA VOL. 16, NO. 1 Fig. 1. Effect of PCR annealing temperature on the discriminative amplification of rDNA fragments. Annealing temperatures tested: 59 °C (a), 61 °C (b), and 63°C (c).