¹Biological Science Dept., Faculty of Sciences, King Abdul-Aziz Univ., KSA

²Princess Doctor Najla Bint Saud Al Saud Distinguished Research Centre for Biotechnology Jeddah, Saudi Arabia

³Biology Department, Faculty of Science, Taif University, Taif, Zip Code 21974, P.B 888, KSA

⁴Cell Biology Department National Research Center, Dokki, Giza Egypt.

⁵Microbial Genetics Department Genetic Engineering & Biotechnology Division  National Research Center, Dokki, 12622 Giza Egypt

Corresponding author email: saboaba@kau.edu.sa

Article Publishing History

INTRODUCTION

The microbes in the rhizosphere are a diverse mixture of microorganisms that can actively interact with the host plant in different ways. The studies on the microbial rhizosphere communities have shown the significant influence of plant species and cultivars in shaping microbial communities in the rhizosphere (Lei et al., 2018).Soil is an important environment and contain all number of additives such as mineral with organic elements for plant growth, also contain huge number of microorganisms safe and non-safe for plant (Wołejko et al., 2020). The soil conditions are important for plant and microorganisms such as physical respectabilities and chemical constituents (Tajik et al., 2020).

Many microorganisms isolated and identified from soil all over the world by scientist’s, such as bacteria, algae, fungi, etc. all these microorganisms are benefits for plant production and, also fight plant pathogens. So, it plays a main role for biological control of plant pathogens. Significant characters from soil bacteria such as gibberellic acid (GA3), indole-3-acetic acid (IAA) which are phytohormones, nitrogen fixation, and nutrient solubilization show main factors in plants production (Tabatabaei,et al., 2016, Wołejko et al., 2020).

Bacillus strains have been extensively studied specially Bacillus thuringiensis because of its huge benefits and safe for human being. There are too many applications of these bacillus under study in biological control because it used for fight many types of insects (Sanahuja,et al., 2011). B. amyloliquefaciens, B. subtilis, B. megaterium and B. thuringiensis and other similar bacterial strains isolated from soil types when microbiome in soil studied by (Boottanun et al., 2017; Kui et al., 2019; Panneerselvam et al., 2019). Isolation of bacillus strains and species from soil is easy way, it isolated from various depth (Labo et al., 2018: Kui et al., 2019).

The 16S rRNA gene sequence is used for phylogenetic studies based on the produced sequences (Woese et al., 1977). Bacterial 16S rRNA genes are located within the rRNA operons, which also contain genes for 23S rRNA, 5S rRNA, tRNA, and associated intergenic spacer regions (Woese et al., 1990). Identification of bacterial strains to the genus and species level is preferred according to sequencing of rRNA genes because this gene contain highly conserved and hypervariable regions (Anda et al., 2015). 16S rRNA sequence analysis used for identification of bacterial isolates this method is reliable and fast than classical conventional methods. (Woo et al., 2008). Phenotypic characteristics analysis including Gram staining, cell morphology, and biochemical properties are still the first steps in species identification, and 16S rRNA sequence analysis may be performed only when the phenotypic results are not definitive (Clarridge. 2004).

In the genome of some single strains there are variation in 16S rRNA gene sequences such as the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions among the multiple 16S rRNA copies (Sacchi et al., 2002;Acinaset al., 2004). By universal 16S rRNA designed primers to target conserved sites and targeted the 16S rRNA gene in isolated DNA from any bacterial strain and sequencing of such amplified products, now useful method for bacterial identification (Petti,et al., 2015). In this study isolation and molecular identification of some bacillus strains from Saudi Arabia soil were done. Bioinformatics analysis and comparison of multiple 16S rRNA genes sequencing from isolated bacterial strains were done.

MATERIAL AND METHODS

Four different soil samples were collected from different regions of Jeddah City. Isolation of Bacillus strains were done. 0.5g of soil samples was suspended in 10 ml of sterile distilled water, soil samples was homogenized vigorously with vortex. After mixing 1 ml of the supernatant was stored at80^oC for some minutes to kill most non-spore-forming bacteria.100 µL of suspension spread over nutrient agar plates. The apparent colonies were spread on the entire surface of nutrient agar m plates with triplicate. After incubation at 30^oC for two days all colonies like bacillus colonies were chosen. The obtained single colony re-purified by re-streaking over Nutrient agar plates several times, stored in Nutrient agar slants at 4^oC, and long term storage in Nutrient agar broth with 30% glycerol at -80^oC (Masoud et al., 2015; Al-Yahyawy et al., 2019).

Extraction of DNA: Extraction of DNAs was carried out by I mL of each isolated strains was centrifuged to collect the pellet. 200µL of TES buffer (50mM EDTA, 100mM Tris pH8, and 10% SDS) and 20µL Lysozyme added to the bacterial pellet with gentle pipetting at 37oCfor 60 min., 20µL of proteinase K solution added to the mix incubated at 37^oC for 60 min., the pellet suspension transferred to ice path for 5 min. then 250 µL of Sodium acetate 4 Madded. The mix was spin down for 5 min. at 10.000 rpm for 5 min. clear supernatant was transferred to a new tube and 250 µL of chloroform/isoamyle alcohol added to the supernatant in ice path for 5 min. centrifugation was done at 10.000 rpm for 5 min. the collected DNA pellet dried for 10
min in laminar flow cabinet. 50 µL of TE (10mM Tris pH8, 1mM EDTA) added to each tube. The DNA were detected by electrophoresis in 1.5% agarose gel (Sabir, et al., 2013; Al-Yahyawy,et al., 2019.).

16s rDNA isolation and analysis: PCR amplification and sequencing of 16S rDNA16S gene sequencing and analysiswere done. The amplified PCR products of the 16S rRNA gene bacterial gene fragments were detected by electrophoresis in 1.5% agarose gel. The amplified fragments were purified and sequenced at MACROGEN sequencing company, Seoul, Korea using the automated sequencer ABI 3100 (Applied Biosystems) with Big Dye Terminator Kit v. 3.1 (Applied Biosystems). Primers 518 F (5’ C CA GCAG CC GC GG TAATACG – 3′) and 800 R (5′- TA CC AG GG TA TCTA AT CC -3′) were used for sequencing. The sequences obtained were edited with the software Vector NTI Suite 9, and compared with the NCBI database through BLAST searches. In this comparison, sequences of type strains most closely related to the sequences of the isolates were searched. For the definition of operational taxonomic units (OTUs), a similarity limit of 97% was adopted (Sabir, et al., 2013; Al-Yahyawy,et al., 2019).

Bioinformatics and data analysis: All the 39 sequences were subjected to ncbi BLAST search tool http://blast.ncbi.nlm.nih.gov to detect non-chance sequence similarity. BLAST search was restricted to 16S ribosomal RNA (Bacteria and Archaea), where models (XM/XP) as well as unclultured/environmental samples were also filtered out, such that more reliable results would be attained. Each individual sequence was solely blastd, where blast hit with the lowest expect-value (which indicate number of non-chance alignments) was picked. In order to ensure that Blast out puts were governed by expected-value (aka e-value), Blast algorithm parameter was decreased such the expected threshold was set to more stringent value of 1e−6. Alignment of the 39 sequence was carried out using version 2 of Clustalx (Larkin et al., 2007)

Exploratory data and phylogenetic analyses were carried out under R Project for Statistical Computing (R Core Team, 2017). Where Exploratory data analysis was done using Seqinr (Charif&Lobry, 2007) R package. Phylogenetic analysis was carried out by ape package (Paradis, et al., 2004). Reconstruction of the phylogenetic tree was done using Neighbor joint method (Nei, 1987).DnaSP (LibradoandRozas, 2009) software was used to analyze the haplotype diversity (Hd), the average number of nucleotide differences, the average number of nucleotide differences (Tajima, 1983), the nucleotide diversity (π). The polymorphic site (S), the singleton variable sites (SP), and the parsimony informative sites (PIP) for each gene, and the average number of nucleotide substitutions per site between species (Dxy) (Lynch and Crease, 1990).

RESULTS AND DISCUSSION

Bacterial samples: Out of 36 Bacillus bacteria from soil samples near Jeddah were isolated on nutrient agar plates as pure culture in this study(Table 1) and fig. (1).The isolated strains were re-purified and streaked over the same plates (Fig. 1). 16s rDNA gene of the isolates were done as shown in fig (2). The16S rRNA were identified by 16S rRNA sequence analysis at Macrogen Company Korea, with Macrogen universal primer as identified in materials and methods. The results of 16 s gene isolation as shown pure 16S rRNA gene isolation in fig (2).

Figure 1: An example for purification of Bacillus isolates appeared over nutrient agar plates.

Table 1. Soil Samples Locations

Sample number	city	location
1	Tabuk	28.378089,36.565889
2	Baljurashi	19.865853,41.582135
3	Khobar	26.213500,50.188347
4	Jeddah	21.490086, 39.202683

Figure 2: 1 16S rRNA isolation on agarose gel electrophoresis

NCBI BLAST query: BLAST results are shown in Table (2) for all the 39 isolates. The criteria used for query sequence aimed to narrow down the search space (database), as the smaller the database the more likely to contain sequence of interest. For all the 39 queries zero E-values were attained indicated that all alignments were non-chance alignments. The query cover percentage ranged from 93 to 100 % with the only one exception for isolate 33. The vast majority of values of identical percent (% of how similar the query sequence is to the target sequence) ranged from 97% to 100% with only 2 isolates have identical percent of 80% and 82%  table( 2).

The distribution of isolates/species/distribution is presented graphically in Figure (3)the NCBI BLAST query resulted in 17 species,  mostly  belong  to Bacillus sp,namely Species number 11 B.paramycoides, 4 B.oceanisediminis, 3 B.wiedmannii, 2 for each of B.aryabhattai, B.drentensis, B.megaterium, B.niacini, B.simplex, Lysinibacillusfusiformis and Lysinibacillusmacroides, where only one isolate was found to be belong to each of B.firmus, B.onubensis,B.persicus,B.thioparans,B.toyonensis,Lysinibacilluspakistanensis and Oceanobacilluslongus.

Figure 3: The distribution of isolated bacterial strains at species level

Table 2. blast results of isolated Bacillus strains

Isolate	BLAST Results Species	Query cover%	E-value	% identical
11-27F	B.paramycoides	100	0.00	99
12-27F	B.paramycoides	100	0.00	100
13-27F	B.paramycoides	99	0.00	100
14-27F	B.paramycoides	100	0.00	99
15-27F	B.paramycoides	100	0.00	99
27-27F	B.paramycoides	99	0.00	100
32-27F	B.paramycoides	100	0.00	100
34-27F	B.paramycoides	99	0.00	100
36-27F	B.paramycoides	98	0.00	99
46-27F	B.paramycoides	99	0.00	100
6-27F	B.paramycoides	99	0.00	100
30-27F	B.oceanisediminis	95	0.00	99
37-27F	B.oceanisediminis	93	0.00	99
49-27F	B.oceanisediminis	95	0.00	99
29-27F	B.oceanisediminis	96	0.00	99
C2-27F	B.wiedmannii	99	0.00	100
18-27F	B.wiedmannii	99	0.00	100
10-27F	B.wiedmannii	100	0.00	99
19-27F	B.aryabhattai	99	0.00	100
20-27F	B.aryabhattai	99	0.00	100
2-27F	B.megaterium	99	0.00	100
8-27F	B.megaterium	99	0.00	100
22-27F	B.simplex	100	0.00	99
28-27F	B.simplex	97	<0.00	82
43-27F	B.niacini	99	0.00	99
5-27F	B.niacini	99	0.00	98
31-27F	B.drentensis	100	0.00	99
4-27F	B.drentensis	100	0.00	97
40-27F	Lysinibacillusfusiformis	100	0.00	98
41-27F	Lysinibacillusfusiformis	100	0.00	98
50-27F	Lysinibacillusmacroides	99	0.00	99
52-27F	Lysinibacillusmacroides	99	0.00	99
1-27F	B.onubensis	95	0.00	99
44-27F	B.firmus	100	0.00	97
45-27F	B.thioparans	99	0.00	98
48-27F	Lysinibacilluspakistanensis	99	0.00	96
9-27F	B. persicus	99	0.00	99
C1-27F	B. toyonensis	99	0.00	99
33-27F	Oceanobacilluslongus	72	<0.00	80

Exploratory data analysis: Table (3) shows a description of the isolated species as well as their sequence length and the percentages of GC content of each isolate.  Sequence length varied greatly among the 39 isolates ranged from 463 bp for B. oceanisediminis to 500 bp for B. onubensis. The percentage of GC content were found to be constant within species with no similar trend was observed for the Saudi isolates. Generally speaking, % GC ranged from 52 for B.megaterium to 56 for B.persicus. Base frequencies for all the 39 isolates is shown graphically in figure (4)No noticeable variation were observed in base frequencies for all the 39 isolate exceptfor B.simplex where this species has a noticeable access in cytosine base.

Table 3. The sequence length and percentage of GC ratio of isolated bacillus strains

Species	sequence Length	GC%
12-B. paramycoides	474	0.53
15-B. paramycoides	471	0.53
32-B. paramycoides	476	0.53
46-B. paramycoides	476	0.53
27-B. paramycoides	467	0.54
13-B. paramycoides	477	0.53
36-B. paramycoides	476	0.53
6-B. paramycoides	476	0.53
11-B. paramycoides	479	0.53
14-B. paramycoides	478	0.53
34-B. paramycoides	475	0.53
29-B. oceanisediminis	463	0.55
30-B. oceanisediminis	472	0.55
49-B. oceanisediminis	467	0.55
37-B. oceanisediminis	480	0.55
10-B. wiedmannii	483	0.53
18-B. wiedmannii	477	0.53
C2-B. wiedmannii	476	0.53
2-B. megaterium	475	0.52
8-B. megaterium	477	0.52
40-L. fusiformis	479	0.53
41-L. fusiformis	480	0.53
50-L. macroides	475	0.53
52-L. macroides	468	0.53
48-L. pakistanensis	481	0.52
19-B. aryabhattai	474	0.53
20-B. aryabhattai	474	0.53
43-B. niacini	473	0.55
5-B. niacini	476	0.55
4-B. drentensis	481	0.55
31-B. drentensis	466	0.56
22-B. simplex	474	0.54
28-B. simplex	470	0.52
44-B. firmus	478	0.55
45-B. thioparans	476	0.54
1-B. onubensis	500	0.52
33-O. longus	499	0.52
C1-B. toyonensis	471	0.54
9-B. persicus	474	0.56

Cluster & phylogenetic analysis: Cluster analysis and heatmap were carried out as pre-processing step to glean an insight into the data distribution. Results of cluster analysis are shown graphically in figure (5), where 2 large clusters are shown. The first cluster comprises merely 2 species B.simples and Oceanobacilluslongus, which indeicate that these two speices are distantly densely related to the rest of species.  All other 37 species were clurested together in the second cluster occupying different clades.

Figure 4: Base frequencies for all the 39 isolates bacillus isolated strains

Table 4. Length of conserved regions, conservation, homzigosity and P-valuesof 39 isolates

Region Start-End	Conservation	Homozigosity	P-value
61-138	0.61	0.1	0.04
63-140	0.61	0.9	0.04
65-157	0.60	0.9	0.02
82-160	0.61	0.9	0.03
209-290	0.60	0.95	0.04
244-344	0.60	0.97	0.02
271-393	0.60	0.97	0.01

Figure 5: Cluster analysis of 36 isolated bacillus strains

Evolutionary relationships of taxa was inferred by reconstructing phylogenetic tree using the Neighbor-Joining method (Saitou & Nei, 1987). The optimal tree with the sum of branch length = 1.2 is shown (figure 6). The results of phylogenetic analysis deeply sports the findings of cluster analysis. B.simples and Oceanobacillus longus were found to have the longest branches of the phylogenetic tree, which again indicate that these two species are distantly densely related to the rest of species. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method are in the units of the number of base substitutions per site. The analysis involved 39 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 437 positions in the final dataset.

Figure 6: Phylogenetic tree using the Neighbor-Joining method Maximum Likelihood method indicated and reconstructing the evolutionary relationships of isolated bacillus strains.

Polymorphism and Genetic Diversity among isolated bacillus strains: All 39 isolates were aligned using both Clustalx and Maft build in ape package (Paradis et al., 2004). As the alignment results of the two software were found to be identical, the robustness of the alignment method is ensured. General information about the polymorphisms on the isolates is found   in a table (4). The number of sites was 442, of which 233 sites were fond monomorphic, 209 sites were polymorphic. These polymorphic sites divided into 104 parsimony informative sites (i.e. sites that have a minimum of two nucleotides that are present at least twice) and 105 singleton variable sites (non- informative).In the present data set number of haplotypes were 22 where haplotype diversity was 0.94±0.03 Nucleotide diversity (π) is defined as the average number of nucleotide differences per site between two sequences Nei (1987). Estimate of nucleotide diversity was found to be 0.10, where the average number of nucleotide differences was 44 (table 5).

Table 4. Estimated parameters of the polymorphic sites for 39 bacillus isolated strains

Isolate      No.	No. Mono.	No. Poly	Parimony	Singleton
Sites	-morphic sites	-morphic sites	informative sites	variable sites
442	233	209	104	105

Table 5. Estimated parameters of DNA polymorphism for 39 bacillus isolated strain

Table (6) shows the conserved regions along the 39 isolates and measurements of conservation (C), homosigoisty and P- value. Conservation (C) is calculated as the proportion of conserved sites in the alignment region, where homosigosity is measured as 1- heterzygosity. A total of seven conserved regions were observed, 6 of which are overlapping. The P-value for 6 of these seven con- served region were 0.05, where the P-value for the seventh conserved region was0.01

Table 6. Length of conserved regions, conservation, homzigosity and P-values of all 39 bacillus isolated strains

Region Start-End	Conservation	Homozigosity	P-value
61-138	0.61	0.1	0.04
63-140	0.61	0.9	0.04
65-157	0.60	0.9	0.02
82-160	0.61	0.9	0.03
209-290	0.60	0.95	0.04
244-344	0.60	0.97	0.02
271-393	0.60	0.97	0.01

16S rRNA gene sequencing has been a widely used method for the identification of bacterial isolates from different environmental samples. The traditional culture methods for identification depending on phenotypic characteristics and biochemical testing. These methods require minimum equipment but differentiate only between common species (Springer, et al., 2001; Tortoli, et al, 2001). The 16S rRNA gene sequencing has been described and available technique for bacterial identification (Mauti, et al., 2019). The timing needed for 16S rRNA gene sequencing confirmd as speed and accuracy for identification of bacterial isolates comparing to normal traditional identification methods (Cloud, et al., 2002; Kirschner,et al., 2000; Turenne, et al., 2001; Sabir, et al., 2013; Al-Yahyawyet al., 2019).

Comparing of microbial communities with the sequenceswith closely related species in the similar 16s r RNA sequences of databases help all scientists all over the world to identify the bacterial species in their samples (Burns, et al., 2015; Claesson, et al., 2010). The 16S rRNA gene become more commonly used for rapid identification of unknown isolates environmental samples and for confirmation of variant sequence data that might have been generated by the higher-throughput methods (Clarridge 2004). The number of 16S rRNA gene sequences in different databases is increased daily (Boudewijns, et al., 2006). Our results confirmed that a description of the isolated species as well as their sequence length and the percentages of GC content of each isolate these data are inagreement with literatures  (Turenne, et al., 2001; Sabir, et al., 2013; Al-Yahyawyet al., 2019).

No noticeable variation in the base frequencies for all the 39 isolates except for B.simplex where this species has a noticeable access in cytosine base. Also obtained results shown all 37 species were clurested together in the second cluster occupying different clades (Dréan,et al., 2015).I n the present data set we show number of nucleotide diversity according to Nei (1987). A total of seven conserved regions were observed along the 39 isolates and measurements of conservation (C), homosigoisty and P- value. Conservation (C) is calculated as the proportion of conserved sites in the alignment region, where homosigosity is measured as 1- heterzygosity (Paradis,et al., 2004).

CONCLUSION

In conclusion, these preliminary results confirm the fact that species identification by bioinformatics analysis approach may be sufficient for analyzing cultured and uncultured bacterial communities from environmental samplesto species level in some cases. By the public databases it resolve wasting time by traditional bacterial identification methods, but should be confirmed by complementary DNA sequencing. High-quality DNA sequences with SNP analysis could increase the power of databases to facilitate bacterial identification and discrimination between closely related species (Uelze, et al., 2020).

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