Mutagenic effect of acridine orange on the production of EPS in Bacillus sp., its applications as anticoagulant and genetic diversity by ISSR and SCoT technique

Many important substances are produced by some organisms, such as extracellular polysaccharides (EPS). The genus Bacillus is an important one in this field. As a result, in this study, a defined strain of Bacillus sp. BAB3450 (Gen Bank) was used to produce EPS with the goal of increasing productivity by introducing acridine orange mutations (AO). The results were obtained from three mutations that were more productive than the wild type. On the other hand, using primers Inter-Simple Sequence Repeat (ISSR) and Start Codon Targeted Polymorphism (SCoT), a comparison with the three mutants and wild type were made at the molecular level. Eleven ISSR primers were used. The results showed that ISSR-19, with 92.86% polymorphism, was the best primer. Using ten SCoT primers, the results showed that SCoT-2 was the best primer, with 90% polymorphism. For the ISSR primer and SCoT primers, the wild type and three mutants were divided into two major groups based on marker analysis with distance 60 to 75% and 55 to 65%, respectively. Cluster analyses were used to create a dendrogram of Bacillus strains and mutants, revealing high genetic variations between the wild type and mutants. Finally, extracellular polysaccharides (EPS) have a wide range of industrial applications in biomedicine, such as anticoagulant. The EPS from wild type and three mutants were tested for anticoagulant comparing with heparin. Mutant No.1 gave the best anticoagulant (15˃%), while the wild type, mutant No.2, mutant No.3 and mutant No.4 gave 10˃% comparing with heparin (80˃%). Key wards: Genetic Diversity, anticoagulant, Bacillus, acridine orange, ISSR and SCoT. INTRODUCTION Many gram-negative and grampositive bacteria produce extracellular polysaccharides (Wingender et al., 1999; Marvasi et al., 2010). They bind to the cell surface before being released into solution. Microbial EPS may represent a novel source of functional biopolymers for food, industrial and medical applications. Several approaches have been used to improve EPS from Bactria by using mutagenesis (Nassef et al., 2002; Parekh et al., 2000). In this study, a relatively easy method was described in order to induce genetic changes in bacteria using acridine orange (AO). Acridine orange is an aromatic compound that acts as intercalate or DNA base pairs, promoting nucleotide base insertions and deletions during replication. The resulting mutation changes the translated reading frame of the information encoded in the mRNA transcript, resulting in a different amino acid sequence at the point of insertion and/or deletion (Carlton and Brown, 1981; Kapuscinski and Darzynkiewicz, 1984). In molecular biology, molecular markers are primarily used to identify a specific DNA sequence. It is defined as a potentially Received: March 17, 2022; Accepted: March 30, 2022; Available online: April 5, 2022