Molecular Characterization of Streptomyces albogriseolus Excellent Mutants for Neomycin Production

N-methyl-N-nitro-N-nitrosoguanidine (NTG) mutagen was successfully used to induce hyper producing neomycin mutants. The neomycin-resistant mutants of Streptomyces albogriseolus, which harvested after treatment with NTG mutagen (100μg/ml) and growing with stress of neomycin (1.75 mg/ml), can produce neomycin with higher efficiency than the wild type strain. The T-40-22 mutant strain was the highest in neomycin production which gave 25 mm inhibition zone by using B. subtilis as the tester organism with 166.67 % relation to the wild type strain after tested of 85 mutants were examined after NTG-mutagenesis. When the above result on inhibition zone was compared with the standard data of neomycin concentrations in relation to inhibition area, the mutant T-40-22 which gave a 25 mm inhibition zone was produced 4.0 mg /mL of neomycin compared with 1.5 mg/mL neomycin of type wild strains and therefore the amount of neomycin production was reached to 2.66 folds of the mother strain production. In addition, the polymerase chain reaction (PCR) technique was applied to examine DNA fingerprint by three random primers on a number of excellent strains for neomycin production to link the genetic background of the tested mutants with the data obtained from the neomycin estimation compared to the wild type strain. Several different DNA fingerprints have been detected after random amplified polymorphic DNA (RAPD) analysis by PCR. Finally, phylogenetic analysis was used to divide the studied mutants into clusters which could reflect the genetic diversity of the new superior neomycin mutants.

The DNA-fingerprinting of the selected strains by random amplified polymorphic DNA (RAPD) technique has been shown to be extremely powerful in determination of genetic relationship between different microbial isolates. The RAPD as ideal to study of genomic polymorphism as pre cited literatures and has been applied to compare intraspecific and interspecific variabilities within the bacterial strains (Williams et al., 1990;Caetono-Anolles 1993;Power 1996;Ikeh 2003). In the present literature use of NTG as mutagen or any mutagenic agents to enhance neomycin production by Streptomyces albogriseolus NRRL B-1305 is not reported. Thus, this research was initiated to take advantage of NTG-mutagenesis in S. albogriseolus to obtain neomycin overproducing strains. So, the objective of the present study was to induce mutation by NTG and detect this mutation through RAPD techniques in order to improve the production of neomycin using S. albogriseolus NRRL B-1305 strain.

Estimation of neomycin productivity
The neomycin production of each strain was quantified by using B. subtilis as the tester on Nutrient agar (NA). After inoculation in the broth production medium for 6 days, the antibiotic productivity was examined for the individual strains. The incubated production medium flasks were examined for neomycin yields by a paper disk diffusion method. Standard data for neomycin concentrations was prepared with authentic neomycin (Table 1).

NTG-mutagenesis and isolation of neomycinresistant mutants
In the potent neomycin-producing mother strain which grown on GYM medium for 5 days, mutation was induced by centrifugation of 5 ml S. albogriseolus spores at 8000 rpm for 5 min and suspended in phosphate buffer 0.1 M, pH 7. The bacterial spores were treated with a NTG (100µg / mL) mutagen for 20, 40 and 60 minutes The suspension was then diluted and spread over the surface of the GYM medium with and without neomycin (1.75 mg / mL) to select highly resistant colonies of neomycin. The plates are then incubated for 2 days at 30°C. Developing colonies with high growth rate were transferred on slants for further studies. The resistant mutants with high growth rate were re-cultured on the same medium plates contained neomycin for 2 days at 30°C to confirm their resistance. Total DNA isolation from mutant strains BYF DNA extraction i-genomic Mini Kit, iNtRON Biotechnology Inc., South Korea was used to isolate total DNA from the selected mutants and the wild type strain. The extracted DNA was estimated according to Sambrook et al., (1989) through reading the UV-absorbance at 260 and 280 nm using spectrophotometer (Shimadzu model UV-240) to estimate the DNA quantity and purity.

Molecular characterization of some excellent mutants by PCR-RAPD assay
PCR analysis was done by 2xPCR Master Mix Solution (i-StarTaq), Hot-Start (iNtRON Biotechnology Inc.). Each vial contains all of the necessary reagents, except primer and DNA template, to carry out PCR in a 20µl volume. Three RAPD primers were used in the present study. The first primer (R1) sequence was 5' -CATACC CCCGCCGTT -3'. The second primer (R2) sequence was 5' -GTGTTG TGGTCCACT -3'. The third primer (R3) sequence was 5' -AACCTCCCCCTGACC -3'. All primers were supplied by Operon Technologies Company, Netherlands. To each PCR vial, 15 ng of the used RAPD primer and 80 ng of the purified DNA sample were added. The total volume of the amplification reaction was made to 20µl using sterile distilled water. The amplification protocol was carried out according El-Sherbini and Khattab, (2018). The amplified DNA products from RAPD analysis were electrophoresed on 1.5% agarose gel at consistent 100 volt for about 2 hrs. The different band sizes were determined against Sizer™ -1000 plus DNA Marker (iNtRON Biotechnology Inc.) and the separated bands were stained with 10000X ViSafe Red Gel Stain (Vivantis # SD0103-Malaysia) and the gels were photographed using Gel Documentation System with UV Trans-eliminator.

The genetic distance and phylogenetic relationships of PCR-RAPD assay
The PCR-RAPD amplification patterns for the three mutants as a result of RAPD application were compared to each other as well as to compare them with the wild type strain of S. albogriseolus. DNA fragments were recorded as a binary data, where (1) means presence and (0) means absence of the band. The genetic distance and the phylogenetic relationships were calculated according to (Lynch, 1990;Hammer et al., 2001).

Effect of NTG mutagenesis on Streptomyces albogriseolus spores
The Streptomyces albogriseolus spores suspension was exposed to 100 µg/ml of NTGmutagen for different periods (20, 40 and 60 minutes) and then diluted and overlaid on the GYM medium. Each single colony which appeared on the GYM plates after mutagen treatment was tested on both the GYM and the GYM supplemented with 1.75 mg/ml of neomycin by incubating them at 30°C. Then, the isolates which were able to grow on GYM plates supplemented with 1.75 mg/ml of neomycin were considered as resistant mutants. It was found that the number of resistant mutants increased with the increase of the exposure time up to 40 min and decreased sharply with the 60 min exposure time. i.e., it was 39 mutants for 20 min. exposure, 31 mutants for 40 min. and 15 mutants for 60 min ( Table 2). It was also found that the survival percentages were decreased with the increase of the NTG-exposure time i.e., it was 16.87% for 20 min. and decreased drastically to reach 5.11 and 3.86% after 40 and 60 min of NTG-mutagenesis, respectively. From about 556 colonies obtained after NTG-mutagenesis, 85 mutants showed a desired resistance towards mutagenic agents. In order to improve the genome of bacterial strains, common mutagenic agents such as ultraviolet rays and alkyl mutagenic agents (EMS and NTG), which usually produce different mutations in bacterial DNA, are used. The microbial mutant strains of are used at the industrial level to extensively promote the production of antibiotics through Streptomyces strains.
To test the effect of the NTG mutagenesis on neomycin production, the 85-mutants were evaluated by cultivating these into the fermentation medium for 6 days. Table (3) showed that the mother strain (wild type) gave 15 mm inhibition zone by using B. subtilis as the tester organism as shown also in Fig. 1, only two mutants of them showed neomycin at the same produced by the wild type strain, while three mutants produced lesser neomycin than it, 34 mutants of them showed neomycin produced more than the wild type strain, five of them showed neomycin produced up to 1.47 folds or more than that of the wild type strain. The mutant strain N-20-12 gave 23 mm inhibition zone or 1.53 folds of wild strain and considered as the highest neomycin producing strain after 20 min of NTG-induced mutagenesis followed by the mutant strain N-20-32 which gave 22 mm inhibition zone or 1.46 folds higher production in comparison to wild type as shown in Fig. 1.
Furthermore, when tested the thirty one of neomycin resistant mutants obtained after 40 min. of NTG-treatment for neomycin production (Table 4), only one mutant (T-40-5) were produced neomycin lower than that of the wild type strain, 30 mutants of them showed neomycin produced more than the wild type strain, six of them showed  neomycin. The results obtained were indicated that the survival rate had decreased by increasing the NTG exposure time. This decrease in survival rate might be attributed to damage in nucleic acid and/or defects in other cell components caused by NTG-mutagen. The above data were agreement with those obtained by Yu et al., (2011). They observed that the survival rate in Streptomyces roseosporus after exposure to NTG-mutagen was decreased when increased the exposure time and increased NTG concentration from 0.1 to 0.7 mg/L.

Neomycin productivity after NTG mutagenesis
The mutation produces a change in DNA nucleotide sequence when mutations are induced using mutagenic agents that interact with DNA. Natural mutations are known to be rare compared to induced mutations. Large types of changes in the DNA sequence (base pair substitution, insertion, deletion) are introduced randomly in microbial DNA by applying chemical and physical neomycin produced up to 1.53 folds or more than that of the wild type strain. The mutant strain T-40-22 which gave 25 mm inhibition zone or 1.66 folds of the wild type production and considered as the highest neomycin producer after 40 min of NTG-mutagenesis. The following highest neomycin producer after 40 min. of NTG-mutagenesis was the mutant strain T-40-29 which gave 24 mm inhibition zone or 1.6 folds of the wild type production as shown also in Fig. 1. No mutants were produced neomycin lower than that of the mother strain after 60 min. of NTG-mutagenesis Table 5. The highest neomycin producer after 60 min. of NTG-mutagenesis was the mutant strain G-60-12 which gave 24 mm inhibition zone or 1.6 folds of the mother strain production as shown also in Figure 1. Meanwhile, the following highest neomycin producer after 40 min. of NTGmutagenesis was the mutant G-60-7 strain which gave 23 mm inhibition zone or 1.53 folds of the wild type production.
The obtained results were agreement with those obtained with Yu et al., (2011). They improved the yield of daptomycin by Streptomyces roseosporus after exposure to different concentrations of NTG-mutagen and obtained a mutant strain LC-54-16, which was producing over 5 times higher antibiotic than the wild type strain. Khattab (2011) improved the production of kanamycin by Streptomyces kanamyceticus using UV-light and obtained the resistant mutants to 250µg/ml of Kanamycin which produced kanamycin higher than their wild type strain. Furthermore, Lyutskanova et al., (2005) obtained strains with an increased tylosin production by Streptomyces fradiae using conventional mutagenesis by UV and/ or NTG and recombinant protoplast fusion technology. Also, Jin et al., (2002 a&b) improved the production of rifamycin B producing strain, Amycolatopsis mediterranei, by rational screening after mutagenesis.

Molecular characterization of the highest neomycin producing mutants
Three random primers with three NTG-mutants in comparison of S. albogriseolus NRRL B-1305 (W.T) were applied to detect the molecular variations after NTG-mutagenesis. Fig.  (2) exhibited the amplified banding profiles of the selected three NTG-mutants and wild type strain    In general, the number of the obtained amplified DNA bands as shown in (Table 6) when used the primer R1 and primer R3 were 12 bands while 10 bands were obtained when used the primer R2. On the other hands, the highest polymorphic bands were exhibited with the primer R1 and the lowest polymorphic bands were exhibited with the primer R3. No polymorphic bands were obtained with the primer R2. Furthermore, when used the primer R2 and primer R3, the produced monomorphic bands are the same number (8 bands) and no monomorphic bands produced when used the primer R1. Moreover, when used the primer R1 and primer R2, the produced unique bands are the same number (2 bands) and one unique bands produced when used the primer R3.
It makes sense that the change in DNA nucleotides must have the effect on RAPD fingerprints. In theory therefore, one change base can detect mutations in genomic DNA by this method. The modifications at the annealing sites of the primer may inhibit annealing and can result in loss of corresponding amplified product. Alternatively, new annealing locations can arise from mutations in the template, thus producing new fragments. Many studies using PCR-RAPD fingerprinting protocols to differentiate of .00 G-60- 9 20 133.33 G-60- 10 16 106.67 G-60- 11 18 120.00 G-60- 12 24 160.00 G-60- 13 21 140.00 G-60- 14 20 133.33 G-60- 15 18 120.00    2011) confirmed that using PCR-RAPD, not only the difference between mutant strains and the wild type strain is identified but variable mutants also could be identified.

Phylogenetic tree in relation to PCR-RAPD assay
From the PCR-RAPD data, the genetic distance were performed to generate correct relationships based on different genome sites ( Table 7). The highest percentage of genetic distance was detected between T-40-29 and W.T (90%) followed by (70%) between T-40-22 and T-40-29. Moreover, the lowest genetic distance matrix (57.10%) was obtained between G-60-12 and W.T, T-40-22 and T-40-29.
As shown in (Fig. 5), the dendrogram based on PCR-RAPD distance indicates separated the mutant T-40-29 and the wild type strain of S. albogriseolus into one first cluster and this cluster was linked with the mutant T-40-22 in the second cluster. Finally, the first and second clusters linked with the highly different mutant G-60-12 in the latest cluster.
The above cluster analysis was applied to divide the studied genotypes into groups which could be reflecting the genetic diversity of the excellent neomycin producer mutants. Moreover, the dendogram illustrated that the new genotype (mutant T-40-29) which assumed to be highly neomycin producer was grouped together in one cluster, but the other new genotypes were highly different and located in other clusters. The obtained results are in agreement with those reported by (Yongheng et al., 2011;Shoukry et al., 2013;Khattab et al., 2018). The five different mutants which superior for ochratoxin biosorption and the wild type strain of Lb. delbrueckii NRRL B-1024 were separated into two main clusters based on RAPD analysis .