Specific oligonucleotide primers for detection of endoglucanase positive Bacillus subtilis by PCR

A polymerase chain reaction (PCR) assay was developed for discrimination of Bacillus subtilis from other members of B. subtilis group as well as rapid identification from environmental samples. Primers ENIF and EN1R from endoglucanase gene were used to amplify a1311 bp DNA fragment. The specificity of the primers was tested with seven reference strains and 28 locally isolated strains of endoglucanase positive Bacillus species. The PCR product was only produced from B. subtilis. The results demonstrated high specificity of two oligonucleotides for B. subtilis. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of B. subtilis. To our knowledge this is the first report of a B. subtilis specific primer set.


Introduction
Genus Bacillus is a Gram-positive, spore-forming, fermentative, aerobic and rodshaped bacteria. Several species of this group are non-pathogenic, simple to cultivate and secrete enzymes such as proteases, amylases and cellulases that are useful for a number of industrial applications (Arbige et al. 1993). The Bacillus subtilis group contains the closely related taxa Bacillus subtilis subsp. subtilis (Smith et al. 1964;Nakamura et al. 1999), Bacillus licheniformis (Skerman et al. 1980), Bacillus amyloliquefaciens (Priest et al. 1987), Bacillus atrophaeus (Nakamura 1989), Bacillus mojavensis (Roberts et al. 1994), Bacillus vallismortis (Roberts et al. 1996), Bacillus subtilis subsp. spizizenii (Nakamura et al. 1999). Classical identification methods based on biochemical tests or fatty acid methyl ester profiling were laborious and hence not applicable for the purpose of a rapid screening. These taxa can be differentiated from one another by fatty acid composition analysis, restriction digest analysis and DNA-DNA hybridization analysis, but are quite difficult to differentiate by phenotypic characteristics (Roberts et al. 1994;Nakamura et al. 1999). The use of the 16S rRNA sequence as a target for genetic detection was therefore considered. Numerous Bacillus species described so far have been found to display rather conserved 16S rRNA sequences compared to other genera. Thus the use of this taxonomic marker is sometimes inadequate for species definition according to generally accepted criteria (Stackebrandt and Goebel 1994). Such unusual similarities exist for members of the 'Bacillus 16S rRNA groupI', including B. subtilis, which displays 99.3 % similarity at the 16S rRNA level to B. atrophaeus and 98.3 % to B. licheniformis and B. amyloliquefaciens (Ash et al. 1991). In the present study, it has been shown that specific primers for detection of endoglucanase gene could be used for identification of B. subtilis.

Materials and methods
Bacterial strains and culture conditions A total 35 Bacillus strains were used in this study (Table 1). Of 12 B. subtilis, ten strains were isolates from pond sediments. For Bacillus cereus, one ATCC strain and nine pond S. Ashe Á U. J. Maji Á R. Sen Á S. Mohanty Á N. K. Maiti (&) Division of Fish Health Management, Central Institute of Freshwater Aquaculture, Kaushalyaganga, Bhubaneswar 751002, Orissa, India e-mail: maitink@yahoo.co.in sediment isolates were tested. Five pond isolates that had been classified as Bacillus pumilus and two as B. amyloliquefaciens were also included in the test. For Bacillus megaterium, B. licheniformis and Bacillus thuringiensis, ATCC strains were analysed. All the pond sediment isolates were identified by 16S rDNA sequencing and available in our laboratory.
Soil samples 24 Soil samples collected from agriculture field and fish culture ponds were also included.

DNA isolation
The total genomic DNA was extracted from bacterial suspension (after 12 h incubation in LB) using DNA extraction kit (Merck Bioscience, India) following the manufacture's instruction. Soil genomic DNA was extracted by using ultra clean soil DNA isolation kits (MoBio, USA).

Polymerase chain reaction
The PCR reaction mixtures (50 ll) contained, dNTPs each 100 lmol; 1X PCR buffer (10 mM Tris Cl, 50 mM KCl, 1.5 mM MgCl 2 and 0.01 % gelatin); each primer 10 pmol; Taq DNA polymerase (NEB) 0.75U and bacterial DNA 100 ng. The touch down PCR in a volume of 50 ll was carried out with initial denaturation of 94°C for 5 min followed by ten cycles of touch down program (94°C for 30 s, 70°C for 20 s and 74°C for 45 s, followed by a 1°C decrease of the annealing temperature every cycle). After completion of the touch down program, 25 cycles were subsequently performed (94°C for 30 s, 60°C for 20 s and 74°C for 45 s) and ending with a 10 min extension at 74°C. PCR reactions were run on a 1.5 % agarose gel in 1X TBE.

Cloning and sequencing
Band was excised from the gel and PCR product was purified by using the QIAquick gel purification kit according to the manufacturer's instructions (QIAGEN, Germany). The purified PCR product was cloned in pGEM Ò -T Easy vector following manufacturer's protocol (promega) and transformed into DH5a cells. Sequencing of the positive clones were done by Sanger method using 96 capillary high through put sequencer; ABI 3,730 XL (Xcelris, India) with T7 and SP6 universal primer. Based on multiple alignments of endo-b-1,4-glucanase genes, a specific consensus motif was identified in the As expected, the test turned out to be positive only for B. subtilis among the different species of Genus Bacillus PCR amplification with genomic DNA isolated from in vitro cultured B. subtilis resulted in a reproducible amplification of 1,311 bp product with primer combinations EN1F/EN1R. To determine the sensitivity of PCR, endpoint titration with serial dilutions of genomic DNA isolated from the standard strain of B. subtilis was carried out and positive results obtained as little as 500 ficogram of DNA (Fig. 1).

Results and discussion
To assess the range of specificity of the PCR test, a number of endoglucanase positive Bacillus species were assayed. Given the considerable number of species established to date under B. subtilis group, our choice to assess the range of specificity was restricted to B. subtilis subsp. subtilis that was representative of the B. subtilis group.
To test the specificity of the amplified products, control experiments were performed under the same conditions with DNA from different members of B. subtilis group as well as B. cereus group. The test found to be positive only for B. Subtilis (Fig. 2). It is noteworthy that the species detected as positive with this test are very close from a taxonomic point of view when phylogenetic tree was constructed on the basis of endoglucanase gene sequences of different species of Genus Bacillus (Fig. 3). Attempt to detect B. subtilis directly from soil samples collected from agriculture field and fish culture ponds were successful, out of 24 soil samples collected from fish pond and agricultural field ten samples were positive for amplification. Cloning and sequencing confirmed the amplicon to be endoglucanase gene of B. subtilis. However, after enrichment of negative soil samples on TSB 20 % increased positivity rate was obtained by PCR, demonstrating that the initial  concentration of B. subtilis was at a proportion below the detection limit. The primers not only differentiated B. subtilis from other species but also differentiated at subspecies level as expected product size could not be predicted from B. subtilis subsp. spizizenii by primer blast (NCBI).
In this report, a PCR method has been established for identification of B. subtilis. Endo-b-1,4-glucanase gene has been chosen to design primers that could be useful for identification and direct detection of B. subtilis from environmental samples. Detection of B. subtilis has been shown to be specific, although the primers showed specificity for G. stearothermophilus and P. campinasensis in primer blast, and predicted amplicon to be 1,311 bp. However, B. subtilis could be differentiated from G. stearothermophilus and P. campinasensis by sequencing of the pcr product. 16S rRNA gene sequence analysis is the most commonly used method for identifying bacteria or for constructing bacterial phylogenetic relationships (Woese 1987;Vandamme et al. 1996;Joung and Cote 2002); however, its usefulness is limited because of the high percentage of sequence similarity between closely related species (Ash et al. 1991;Marti'nez-Murcia et al. 1992;Christensen et al. 1998). The use of protein-encoding genes as phylogenetic markers is now a common approach (Yamamoto and Harayama 1998; Ko et al. 2004;Chelo et al. 2007). Detailed investigations have demonstrated that sequences from protein-encoding genes can accurately predict genome relatedness and may replace DNA-DNA hybridization for species identification and delineation in the future (Stackebrandt et al. 2002;Zeigler 2003). Wang et al. (2007) clearly showed that in the B. subtilis group, within which species differentiation is very difficult, core genes such as gyrB allow differentiation on genetic basis. Compared to other genera, Bacillus species are having conserved 16s rRNA sequences and are difficult to identify at species level using this marker (Stackebrandt and Goebel 1994).

Conclusion
In the present study, the demonstrated specificity of the oligonucleotides used as PCR primers and results of experiments with soil samples provide the basis to develop a diagnostic assay for identification and detection B. subtilis form environment samples. As the primers used in this study have been found to be specific to endoglucanase gene of G. stearothermophilus and P. campinasensis, we suggest to use these primers as supplementary PCR assay to 16s rRNA sequencing for identification of B. subtilis.