**3.2. Identification by 16–23S intergenic region**

202 The Complex World of Polysaccharides

species.

**Figure 1.** Phylogenetic neighbor-joining tree obtained by 16S rRNA sequence analysis of the tested isolates and other *Bacillus* spp. present in the gene bank database (accession numbers in parentheses).

The DNA of the isolates was extracted as described in Section 2 and the 1.5 kb 16S rRNA gene was amplified for each DNA by PCR using primers bac-F and bac-R. The PCR amplification, purification and sequencing were performed as described previously. The 1.5 kb obtained sequences were aligned and clustered with sequences from the NCBI database. 16S rRNA gene sequence analysis indicated that the six isolates (K, M, A, C, E, and G) were *Bacillus* spp. with 99% identity any of these three species *B. subtilis, or B. licheniformis, or B. amyloliquefaciens* and they clustered into a monophyletic line in a phylogenetic tree. To distinguish and clear identification of these strains on the species level the analogical electrophoresis, using NEB cutter was applied to identify the 16S rRNA results, which have been sequenced, as the strains of the same species expected to have almost the same sites when digested with AluI. Fig. 2 showed that the isolates M and G have the same size fragments as *B. subtilis* gi 269313996 while the other isolates A, C, E; K showed different AluIfragments which differ to the AluI fragments generated from 16S rRNA sequence of *B. subtilis, or B. licheniformis or B. amyloliquefaciens*. It was clear that 16S rRNA gene alone could not distinguish these three closely related Bacillus To distinguish between these three closely related strains the 16–23S intergenic region was amplified by primers L516SF × L523SR, and then the sequence was determined for the six isolates. The homology results for the 16–23S intergenic region showed that: K and M strains showed 100% similarity to *B. subtilis* but strains A, C, E, G could be either *B. subtilis (99%) or B. amyloliquefaciens* (85%) **(Table. 1.).** 


