**3. Results**

### **3.1. Molecular identification of the levansucrase producers strains**

#### *3.1.1. 16S rRNA sequences and their analogical electrophoresis*

Six levansucrase producers' bacteria were isolated from different honey sources. The isolates resembled each other in cell morphology where cells were rod-shaped, Gram-positive, motile and spore-forming. Colonies were circular, creamy, and no pigment was formed. They were identified as *Bacillus* spp. based on morphological examination. The identification was confirmed by molecular biological analysis, using 16S rRNA sequencing method. The results showed that the 6 isolates were identified as *Bacillus* spp. (99%), or *B. licheniformis* (99%), or *B. amyloliquefaciens* (99%) ( Fig. 1).

**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 species.

**Figure 2.** The analogical electrophoresis of Bacillus isolates compared to Bacillus strains (from the gene bank) by "AluI",using NEBcutter 2.0. The accession no. of the control Bacillus strains: *B. subtilis* gi|269313996|; *B. subtilis* subsp. subtilis str. AL009126.3; *B. amyloliquefaciens* gi|229484923|; and *B. licheniformis* gi|270356913|.
