APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2002, p. 6421–6424 0099-2240/02/$04.00⫹0 DOI: 10.1128/AEM.68.12.6421–6424.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. Vol. 68, No. 12 Competitive PCR for Quantitation of a Cytophaga-Flexibacter-Bacteroides Phylum Bacterium Associated with the Tuber borchii Vittad. Mycelium Elena Barbieri,1 Giulia Riccioni,1 Anna Pisano,1 Davide Sisti,2 Sabrina Zeppa,1 Deborah Agostini,1 and Vilberto Stocchi1* “Giorgio Fornaini” Institute of Biochemistry1 and Institute of Botany,2 University of Urbino, 61029 Urbino, Pesaro-Urbino, Italy Received 12 April 2002/Accepted 29 August 2002 A recent straightforward molecular study aimed at surveying potential endo- and epiphytic bacteria within the mycelium of the ectomycorrhizal fungus Tuber borchii Vittad. revealed the presence of a novel uncultured bacterium associated with the hyphae of this fungus (1). The phylogenetic analysis unequivocally placed the bacterial sequences in a single new rRNA branch in the Sphingobacterium subgroup of the CytophagaFlexibacter-Bacteroides (CFB) phylum (6, 18). A few CFB cells per septum of mycelium were detected by the fluorescent in situ hybridization technique, with both general and specific oligonucleotide probes (1). The occurrence of this novel bacterium associated with the T. borchii fungus will shed light on the relative importance of this bacterial species as a new, significant partner in fungusplant symbiosis. Thus, in order to estimate the abundance of this strain within the fungal host tissue, we used a competitive PCR (cPCR) technique (3, 7, 10, 11, 15, 22). This represents the first molecular attempt to enumerate an uncultured bacterium associated with a mycorrhizal fungus. Mycelia and bacterial strain. Four mycelium strains of T. borchii Vittad. (1BO [ATCC 96540], 10RA, 17BO, and Z43), in which the presence of the CFB bacterium (1) had been revealed, were used in this study. Mycelial cultures were grown in MNN liquid medium (21) and analyzed after 30 days. The 16S rRNA gene (rDNA) from the associated CFB bacterium was amplified by PCR following the procedures described by Barbieri et al. (1). In addition, Sphingobacterium heparinum ATCC 13125 was chosen from among the most closely related culturable strains to determine its rRNA operon copy number. Ergosterol assay. Ergosterol is a component of the fungal membrane and provides a reliable indication of metabolically active fungal biomass (4). The assay for ergosterol was carried out on 30-day-old cultures of the four T. borchii mycelium strains utilized in this study, as described by Zeppa et al. (21). Construction of the QPCR competitor. The 16S rDNA amplification product from the mycelium strain 17BO was cloned into the TA cloning vector pGEM (Promega) and sequenced. The sequence obtained was identical (1,450 of 1,450 nucleotides) to the b-17BO 16S rDNA sequence (GenBank no. AF070444). The recombinant DNA plasmid pb-17BO was cleaved with AgeI (cleavage sites, positions 1243 and 1422, 5⬘-3⬘), generating a 179-bp DNA fragment and the competitor pb-17BO-c, a new plasmid which has a deletion but has the same priming sequences as the DNA target. After ligation, the 179-bp deletion was confirmed by sequencing. An internal primer, SH-878f (5⬘ CGA TGA TAC GCG AGG 3⬘; Escherichia coli positions 878 to 893) (2) was designed based on the 16S rDNA sequences of clones b-17BO, b-Z43, b-10RA, and b-1BO of S. heparinum, available in GenBank/EMBL/DDBJ. The primer SH-878f was used in combination with a universal primer reverse primer (1). The quantitative PCR (QPCR) procedure consisted of 22 cycles (unless specified otherwise): denaturation at 94°C for 45 s, annealing at 58°C for 45 s, and elongation at 72°C for 2 min. The specificity of the primer was checked by cloning and sequencing the amplification product obtained from the T. borchii mycelium strains (1BO, 10RA, 17BO, and Z43). The sequences obtained confirmed that only the CFB bacterium from the samples was amplified. We determined the amplification kinetics of the target sequences and the competitor over a range of 35 cycles. PCR products were quantified as a function of the cycle number by measuring the ethidium bromide-stained DNA with the Gel Doc 2000 Quantity One software program (Bio-Rad), in which the pixel densities of the bands were transformed into pixel intensity ratios (14). Secondly, a calibration curve was constructed by amplifying a range of masses of the 16S rDNA target from a CFB bacterium which is present in the four T. borchii mycelium strains used in this study, in the presence of a constant mass (or number of molecules) of competitor pb-17BO-c. The two fragments obtained in cPCR were resolved by electrophoresis using a 1.5% agarose gel containing ethidium bromide (1 ␮g/ml). The intensities of the ethidium bromide staining of the 540and 361-bp PCR bands were then plotted as a function of the * Corresponding author. Mailing address: Istituto di Chimica Biologica “Giorgio Fornaini,” University of Urbino, Via A. Saffi, 2, 61029 Urbino (PU), Italy. Phone: 39 0722 305262. Fax: 39 0722 320188. E-mail: v.stocchi@uniurb.it. 6421 Downloaded from http://aem.asm.org/ on June 1, 2016 by guest An uncultured bacterium associated with the ectomycorrhizal fungus Tuber borchii Vittad. was identified as a novel member of the Cytophaga-Flexibacter-Bacteroides group. Utilizing a quantitative PCR targeting the 16S rRNA gene, we relatively quantified this bacterium in the host. The estimated number of bacteria was found to be approximately 106 cells per 30-day-old T. borchii mycelium culture. This represents the first molecular attempt to enumerate an uncultured bacterium associated with a mycorrhizal fungus. 6422 BARBIERI ET AL. FIG. 1. QPCR of pb-17BO and competitor. Increasing numbers of molecules (from 100 to 107) of pb-17BO target (F) were coamplified with a fixed amount (104 molecules) of competitor, pb-17BO-c (E). The intensity value incorporated the band area for each PCR band visualized in the agarose gel was plotted as a function of the number of pb-17BO molecules. The intersection of the two curves shows the point of equivalence. The determined value of (1.2 ⫾ 0.122) ⫻ 104 molecules for the input pb-17BO sequence corresponds with the actual input of 1.2 ⫻ 104 molecules of the competitor pb-17BO-c. Three independent QPCRs were carried out, and some points in the figure are hidden. itory compounds (3) such as fungal polysaccharides, which could interfere with the PCR, or to the low density of the bacterial cells within the mycelial host tissue. Moreover, the differences in rrn copy number between a lab culture and environmental bacterial cells may affect the PCR amplification of target 16S rDNA (5). The amplification rates of the other samples, 1BO, Z43, and 10RA, were nearly identical, and their amplification efficiencies were comparable to those of 17BO (data not shown). FIG. 2. Amplification efficiency of the 16S rDNA from the 17BO T. borchii mycelium (■) and the competitor pb-17BOc (E). Band intensities were calculated as described for Fig. 1. Efficiency was calculated from the slope of each regression. Downloaded from http://aem.asm.org/ on June 1, 2016 by guest log10 of the known competitor copy number. The point of equivalence was that at which the intensities from the competitor and the target were equal and represented the relative number of copies of the 16S rDNA in the T. borchii mycelium strains. rRNA operon copy number determination for S. heparinum. Since the CFB bacterium is not yet culturable, S. heparinum (strain ATCC 13125), the most closely related culturable bacterium, was chosen to relatively quantify the rrn copies of the CFB bacterial cell in the fungal tissue. Genomic DNA from S. heparinum was digested with three different restriction enzymes (SacI, PstI, and PvuII; Promega Inc.) noncutting for 16S rDNA. rRNA operon copy numbers were determined by Southern blotting analysis of gel-separated restriction digests using a labeled DNA probe complementary to nearly the full length of the 16S rRNA gene of S. heparinum (positions 8 to 1456). Hybridization was carried out at 65°C and washing was done under stringent conditions, as described by Sambrook et al. (16). Genomic DNA from E. coli was used as a positive control. QPCR competitor. The target sequence is not present in pure cultures of the microorganism. For this reason, the target used to construct the calibration curve had to be a cloned version of the CFB bacterium 16S rDNA within the T. borchii mycelium. Fungal strain 17BO was the first to be analyzed, and pb-17BO was the clone designed. The competitor pb-17BO-c, obtained by deletion of the AgeI restriction site, was 179 bp smaller than the original clone. Both pb-17BO and pb-17BO-c were amplified by using the QPCR condition previously described and quantified as a function of the cycle number. The competitive control (pb-17BO-c) amplifies with equal efficiency and achieves a plateau simultaneously with the target DNA (pb-17BO) in a linear range of DNA masses (or molecules). From these results, it was possible to apply pb-17BO-c as an internal standard for the relative quantification of the bacterial DNA within T. borchii mycelium strains. Figure 1 shows a progressive competition between variable quantities of pb-17BO (from 100 to 107 molecules) and a fixed amount of pb-17BO-c (104 molecules). Three experiments were carried out to confirm test reproducibility; the point of equivalence at the intersection of the two curves corresponded to a mean of 1.2 [1.078; 1.332] ⫻ 104 molecules of pb-17BO (95% confidence interval). Copies of the CFB bacterium 16S rDNA in T. borchii mycelium. To determine the quantitative frame of QPCR amplification of the 16S rDNA targets from the mycelium strains 1BO, 17BO, 10RA, and Z43 and the competitor (pb-17BO-c), their kinetics were compared by a PCR cycle test. The cPCR amplification efficiencies of target and competitor were calculated from the slope of the curves of amplification, between cycles 18 and 28, following the linear regression shown in Fig. 2, (efficiency [eff] ⫽ 10␣ ⫺ 1, where ␣ is the slope of the regression line). The efficiency of pb-17BO-c was slightly higher than that of the 17BO target (efftarget ⫽ 0.441; effcompetitor ⫽ 0.464). However, they reached saturation at the same number of cycles (35 cycles), and the efficiency ratio of target and competitor, between cycles 18 and 28, showed a constant value of 1.052. This slight difference in efficiency between target and competitor may be due to the presence of minimal traces of inhib- APPL. ENVIRON. MICROBIOL. VOL. 68, 2002 QPCR FOR BACTERIAL DNA WITHIN T. BORCHII MYCELIUM 6423 rRNA operon copy number determination for S. heparinum. To determine the rRNA operon copy number in S. heparinum strain ATCC 13125, its 16S rRNA sequence (GenBank accession no. M11657.1) (19) was examined. These analyses revealed that the 16S rRNA gene did not contain any SacI, PstI, or PvuII sites. Digestion of S. heparinum chromosomal DNA with each of the noncutting restriction enzymes and subsequent Southern hybridization of the 16S rRNA gene results in three bands in each lane (data not shown). E. coli genomic DNA containing seven rRNA operon copies (http://rrndb .cme.msu.edu) (8) was used as a control. These results confirmed that S. heparinum strain ATCC 13125 has three copies of the rRNA operon. QPCR of the 16S rDNA in T. borchii mycelium strains. After construction of the competitor and determination of the internal control concentration, a QPCR was carried out by coamplifying a range of masses of the target DNA from the T. borchii mycelia, strain 17BO, ranging from 1.5 to 50 ng in the presence of a constant mass of competitor (0.025 pg, corresponding to 1.20 ⫻ 104 molecules of pb-17BO). As shown in Fig. 3, the logarithm of the intensity ratios of the 16S rDNA amplification products from the mycelium strain 17BO to that of the competitor was plotted as a function of the initial number of target DNA molecules. The Zachar equation (20) was applied: log(Nn1/Nn2) ⫽ log(No1/No2) ⫹ n log(eff1/eff2), where Nn1 and Nn2 are the PCR products (intensity of ethidium bromide staining), No1 and No2 are the initial number of molecules or mass (nanograms) of target and competitor templates, respectively, n is the PCR cycle number, and eff1 and eff2 are the efficiencies of template amplifications (10␣ ⫺ 1, as reported above). This equation is valid assuming that the efficiency of amplification of both target and competitor remains constant for each cycle in the amplification reaction (14, 20, 22). QPCR assays were carried out by amplifying the range of masses of the other T. borchii mycelium strains (1BO, Z43, and 10RA) using the same procedure and concentration as for the 17BO strain (Table 1). The molecular identification of the CFB bacterium within T. borchii mycelium clearly placed this strain among the Sphingobacter subgroup of the CFB phylum; however, attempts to isolate and grow this bacterium from the mycelium have been unsuccessful. Thus, a molecular approach has been applied for the enumeration of this strain. Through a cPCR, we could relatively estimate its rrn copy number within T. borchii mycelium tissue. Since the rrn copy number of S. heparinum, the most closely related strain, was found to be three (three operons), it is likely that the CFB is present at a level of 106 cells per 30-day-old T. borchii mycelial culture. Moreover, in order to better relate the number of CFB cells to the fungal biomass, the content of free ergosterol in 30-day-old mycelium strains is reported in Table 1. The number of CFB bacteria estimated by QPCR includes TABLE 1. Relationship between CFB cells and T. borchii mycelium strainsa T. borchii strain Dry wt (mg) Amt of total DNA (ng) Ergosterol concn (␮g/mg) No1 in QPCR (ng)b 17BO 10RA 1BO Z43 9 ⫾ 3.2 13 ⫾ 4.1 14 ⫾ 4.9 8 ⫾ 2.6 7,450.8 ⫾ 1,490 9,233.6 ⫾ 2,585.2 9,747.0 ⫾ 2,241.5 7,069.2 ⫾ 1,767.3 3.214 ⫾ 0.013 3.569 ⫾ 0.018 3.862 ⫾ 0.013 2.890 ⫾ 0.017 8.13 [7.98; 8.28] 12.50 [12.29; 15.17] 10.26 [10.15; 10.37] 8.08 [7.68; 8.48] a b No. of CFB cells 1 rrn copy (107) 3 rrn copies (106) 1.56 ⫾ 0.37 1.98 ⫾ 0.39 2.01 ⫾ 0.30 1.15 ⫾ 0.67 5.19 ⫾ 1.25 6.60 ⫾ 1.31 6.71 ⫾ 1.01 3.81 ⫾ 2.28 Data are means ⫾ standard deviations. Quantity of DNA from T. borchii mycelium extrapolated by the Zachar equation at the equivalence point obtained by QPCR (mean [95% interval of confidence]). Downloaded from http://aem.asm.org/ on June 1, 2016 by guest FIG. 3. cPCR for 16S rRNA gene from the uncultured CFB bacterium in the host T. borchii mycelium. DNAs extracted from T. borchii mycelium (strain 17BO) with masses ranging from 50 to 1.5 ng were coamplified with a fixed amount of competitor, 0.025 pg (1.20 ⫻ 104 molecules). The relative intensities of the PCR bands, corresponding to the target and the competitor of the agarose gel (A), were used for the determination of the competition equivalence point (B). Three determinations are plotted. 6424 BARBIERI ET AL. This work was supported by Progetto Strategico CNR “Biotecnologia dei funghi eduli ectomicorrizici: dalle applicazioni agro-forestali a quelle agro-alimentari.” We thank A. Zambonelli, University of Bologna, Bologna, Italy, for providing mycelium strains. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. REFERENCES 1. Barbieri, E., L. Potenza, I. Rossi, D. Sisti, G. Giomaro, S. Rossetti, C. Beimfohr, and V. Stocchi. 2000. 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Downloaded from http://aem.asm.org/ on June 1, 2016 by guest live as well as dead bacterial cells. However, the detection of these bacteria within the hyphae of T. borchii by in situ hybridization experiments using eubacterial and specific 16S rRNAbased probes (1) was possible due to the of presence of mature rRNA in live cells, a condition sine qua non for the hybridization assays. Thus, we consider that 106 cells per 30-day-old mycelium culture of T. borchii is representative of a probable live population. Since the fungus T. borchii is utilized as a competent fungus for in vitro ectomycorrhizal synthesis and expression studies (9, 13, 17), the presence of prokaryotic sequences or genes within DNA extracted from T. borchii mycelium strains might be considered for further biological investigations or biotechnological applications. The biological function of this CFB bacterium in the fungalbacterial interaction is still unknown, but since similar bacterial sequences have also been found in Tuber aestivum and Tuber uncinatum mycelium species (12), we cannot exclude coevolutionary events. Molecular studies are in progress for a better understanding of this fungal-bacterial interaction. APPL. ENVIRON. MICROBIOL.