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Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.



Karen B. Alviar, Krizzia Mae R. Lumangaya, Joan Christine O. Adajar and Mannix S. Pedro
 
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ABSTRACT

Background and Objective: Several species of Bacillus are plant growth-promoting rhizobacteria that can produce the auxin phytohormone Indole-3 acetic acid (IAA) which regulates plant growth and development. This study aimed to amplify key genes, patB and dhaS, two of the three component enzymes found in the IPyA biosynthetic pathway for IAA synthesis and conduct 16S rRNA sequence analysis of two Bacillus isolates from Fermented Plant Juice (FPJ) extracted from Madre de Cacao, Gliricidia sepium. Materials and Methods: Genomic DNA extraction was performed using the CTAB method followed by the amplification of IPyA pathway genes through a polymerase chain reaction. Sequence similarity search was performed using NCBI BLASTn and the protein structure was predicted using Phyre2. Results: The dhaS gene from isolate 1 (B. amyloliquefaciens) had a top nucleotide BLAST hit with B. velezensis strain FJAT-45028 and a translated BLAST hit with an aldehyde dehydrogenase protein. The resulting rank three models in protein structure prediction revealed dhaS which encodes for an enzyme responsible for the conversion of indole-3-acetaldehyde (IAAld) to IAA. Furthermore, phylogenetic trees generated using the Maximum likelihood method revealed the relationship of each isolate with its top five BLAST hits. Conclusion: These amplified IAA-related genes may be further utilized in optimizing phytohormone production that has the potential to improve the capability of Bacillus isolates as biofertilizers.

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Karen B. Alviar, Krizzia Mae R. Lumangaya, Joan Christine O. Adajar and Mannix S. Pedro, 2021. Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.. Biotechnology, 20: 22-30.

DOI: 10.3923/biotech.2021.22.30

URL: https://scialert.net/abstract/?doi=biotech.2021.22.30
 
Copyright: © 2021. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

The genus Bacillus refers to ubiquitous, endospore-forming, rod-shaped bacteria which may be further classified as either gram-positive or gram-variable and aerobic or facultatively anaerobic1. Their ability to produce endospores allow these species to thrive in various environmental conditions2. Colonization of Bacillus spp. in plant roots improves plant functions under various stress conditions such as soil salinity, drought, presence of heavy metals, pests and other pathogenic microorganisms3. Furthermore, metabolites secreted by these species facilitate the nutrient acquisition and contribute to plant hormone production3.

Recent studies on Bacillus spp. were conducted proving its beneficial effects on plant growth and development. In a study by Yuan et al.4, root colonization of B. amyloliquefaciens strain NJN-6 and detection of phytohormone compounds such as Indole-3 acetic acid (IAA) and gibberellin A3 were observed along with the improved growth of banana plants against fusarium wilt. In another study by Reetha et al.5 application of Bacillus subtilis and Pseudomonas fluorescens improved the growth of the onion plant (Allium cepa) and both microorganisms were found out to be capable of producing IAA. With its potential for improvement in crop production, commercialization of Bacillus species, specifically Bacillus ellenbachensis was evident since the late 19th century in a fertilizer named Alinit® as a “bacteriological fertilizer for inoculation of cereals” by Borriss6. Indole-3 acetic acid (IAA) is a naturally occurring auxin, a collection of plant hormones that controls plant growth and development7 through induction of cell division, differentiation and expression. Pathways for IAA biosynthesis has been documented in Arabidopsis thaliana8, ectomycorrhizal fungi9 and plant-beneficial bacteria such as Arthrobacter pascens ZZ2110 and Bacillus amyloliquefaciens SQR911. Multiple IAA biosynthesis pathways particularly indole-3-acetonitrile (IAN), tryptamine (TAM) and indole-3-pyruvic acid (IPyA) pathways were suggested to be present in Bacillus amyloliquefaciens strain SQR911. The IPyA pathway is thought to function in both plants and bacteria, including Bradyrhizobium, Enterobacter, Azospirillum and Rhizobium12. The first step of this pathway involves tryptophan which is converted to IPyA by an aminotransferase113,14. The rate-limiting step in this process is the conversion of IPyA to indole-3-acetaldehyde (IAAld) by indole-3-pyruvate decarboxylase (IPDC) (encoded by ipdC)15,16. The IAAld is then oxidized to IAA by aldehyde dehydrogenase, mutase or oxidase enzymes17. In Bacillus amyloliquefaciens, dhaS is thought to encode indole-3-acetaldehyde dehydrogenase18. The IPyA pathway in this bacterium comprises the genes patB, which codes for the enzyme tryptophan transaminase, yclC, which codes for indole-3 pyruvate decarboxylase and dhaS, which codes for indole-3 acetaldehyde11. In this study, two Bacillus spp. selected from a biofertilizer derived from fermented plant juice of Madre de Cacao, Gliricidia sepium to identify one possible biosynthetic pathway for IAA synthesis. Two genes from the IPyA pathway, dhaS and patB, were amplified and deduced to be highly similar with other organisms’ genes encoding for enzyme tryptophan transaminase and indole-3-acetaldehyde respectively (dhaS and patB).

MATERIALS AND METHODS

Species collection: Two Bacillus isolates from the University of the Philippines Los Baños National Institute of Molecular Biology and Biotechnology (BIOTECH) were grown in LB broth (10 g Peptone, 10 g Yeast Extract and 5 g NaCl per litre of distilled water) using stab culture technique. These Bacillus isolates were incubated overnight with shaking (250 rpm, 37°C) before subjecting to genomic DNA extraction.

Study area: The experiments were carried out at the Insect Physiology laboratory from January, 2019 to February, 2020 at the Institute of Weed Science, Entomology and Plant Pathology, College of Agriculture and Food Science, University of the Philippines Los Banos.

Genomic DNA extraction: The protocol for cetyltrimethylammonium bromide (CTAB) DNA extraction was based on Raymundo and Opulencia19 with some modifications. Ten stab cultures of Bacillus isolates, 5 each from putative B. amyloliquefaciens and B. subtilis were transferred to 1.5 mL Eppendorf tubes and centrifuged at 12000 rpm for 45 sec. The resulting pellet in each tube was resuspended and dispersed in 20 μL Tris-EDTA buffer. The lipid component of the bacterial cell wall was degraded by adding 25 μL of 2% SDS which was followed by incubation at 37°C with shaking (250 rpm) for 1 hr. The addition of 45 μL of 5 M NaCl and 45 μL of 10% CTAB were done for the dissolution and precipitation of proteins and polysaccharides, respectively. After this, the tubes were immersed in a hot water bath (65°C) for 20 min. An equal volume of chloroform: isoamyl alcohol was added for the removal of lipids and separation of aqueous and organic phases. After 30 min, centrifugation was done at 15000 rpm for 10 min. The resulting top layer was pipetted out and an equal volume of isopropanol and 1 mL of 70% ethanol were mixed in the solution.

Table 1:Primer sequences for the amplification of IAA-related genes in two Bacillus isolates
Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.
*bp: Base pairs

Table 2:
PCR reaction components for the amplification of patB and dhaS genes from two Bacillus isolates
Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.
*Micro molar, **Microliter

Another centrifugation was done at 13000 rpm for 5 min followed by the removal of supernatant and drying of the pellet. The extracted genomic DNA was resuspended in 100 μL TE buffer and stored at 4°C.

To determine the concentration and purity of the extracted genomic DNA, spectrophotometric analysis was performed using EpochTM Microplate Spectrophotometer. The extracts were also subjected to agarose gel electrophoresis for visual examination of their quality.

Amplification of IAA-related genes and sequence analysis: The IAA-related genes, patB and dhaS were amplified from specific primers which were designed using the primer designing tool from the National Center for Biotechnology Information (NCBI) based on the sequence of Bacillus amyloliquefaciens (Accession: CP000560) for isolate 1 and Bacillus subtilis (Accession: NC_000964.3) for isolate 2. The identity of the isolates was determined using 16s rRNA as described below. The generated primer sequences given in Table 1 were sent for DIAMED Enterprise for synthesis.

As presented in Table 2, Vivantis 2X Taq Master mix/Promega GoTaq® Colorless Master Mix DNA Amplification product, extracted genomic DNA, generated primers and nuclease-free water was the components utilized for amplification. Combining these components, PCR was then performed with the reaction conditions indicated in Table 3 as suggested in Vivantis DNA Amplification Product 2X Taq Master Mix. The annealing temperature was also varied based on the indicated melting temperature of the primers from Tm calculator tool of Thermo Fisher. The resulting amplicons were subjected to agarose gel electrophoresis to visualize and confirm the presence of the gene in the expected molecular weight.

Table 3:
PCR reaction conditions for the amplification of IAA-related genes from two Bacillus isolates as recommended in Vivantis DNA Amplification Product 2x Taq master mix
Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.
*Seconds

Table 4:Primers used for the amplification of 16S rRNA genes from two Bacillus isolates
Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.

The obtained forward and reverse sequences of amplified IAA-related genes were aligned using Bio-Edit sequence alignment editor and the graphic format of the alignment were exported from CLC Sequence Viewer 8. Similar sequences from databases were also acquired using the Basic Local Alignment Search Tool (BLAST) from NCBI. To further utilize the sequences, the protein structure was predicted using Protein Homology/Analogy Recognition Engine Version 2.0 Phyre 2.

16S rRNA sequence analysis: Amplification of 16S rRNA Sequences from two Bacillus isolates were done using 8F and 1512R primers given in Table 4. The obtained sequences were compared with an online sequence database utilizing BLAST from NCBI. The 16S rRNA sequences from each Bacillus isolate, along with its top BLAST hits were used to generate a phylogenetic tree applying Maximum Likelihood in MEGAX.

RESULTS AND DISCUSSION

Genomic DNA extraction: Assessment of the extracted genomic DNA using spectrophotometry shows that all ten samples having an absorbance ratio of 1.7-2 are purely given in Table 5. This classification was based on the study of Sainz et al.20 in which the resulting absorbance ratio between 1.7-2.0 may be characterized as pure DNA. An increase in RNA absorbance ratio can also be observed in a solution with adjusted pH between 7.5-8.521.

The extracted samples subjected to agarose gel electrophoresis showed intact bands above 12000 bp as observed in Fig. 1. The observed band size above the 1 kb ladder coincides with the previous analysis by Zhang et al.22 that the genome size of B. subtilis is 4.05 Mb while B. amyloliquefaciens is 4.01 Mb. Some of the lanes had a faint or almost unobserved band which may be due to a few or absence of the extracted genomic DNA. Furthermore, the extracts having an intact band and characterized as pure were then used as a template for amplification.

Table 5:Assessment of purity and concentration (ng μL1) of the extracted genomic DNA of two Bacillus isolates using spectrophotometry
Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.

Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.
Fig. 1: Electrophoretogram of the genomic DNA extracts from Bacillus isolates
  Lanes 1-5 are genomic DNA from five different stab cultures of isolate 1 (B. amyloliquefaciens), Lanes 6-10 are genomic DNA from five different stabs cultured of isolate 2 (B. subtilis)

Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.
Fig. 2:
Electrophoretogram of the amplified dhaS gene using the extracted genomic DNA from isolate 1 (B. amyloliquefaciens) as a template at 60°C annealing temperature
 
Lanes 1-4 are PCR products from the genomic DNA template isolated from 4 different stab cultures

Table 6:
Standard nucleotide BLAST (BLASTN) result for the consensus sequence of the amplified dhaS gene from isolate 1 (Bacillus amyloliquefaciens)
Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.

Table 7:Translated BLAST (BLASTX) result for the consensus sequence of the amplified dhaS gene from isolate 1 (B. amyloliquefaciens)
Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.

Amplification of IAA-related genes and bioinformatic analysis of sequences: Amplification of dhaS from isolate 1 at an annealing temperature of 60°C resulted in a distinct band as observed in Fig. 2. The molecular weight of the amplicon lies at approximately 1500 bp mark in the molecular ladder. In contrast with the dhaS gene from Bacillus isolate 2, application of varying annealing temperature ranging from 56-58°C resulted in an amplicon observed at approximately 300 bp in Fig. 3.

On the other hand, degenerate primers were used for the amplification of patB gene for the two isolates. Varying the annealing temperature between 43-45°C resulted in an amplicon having a molecular weight of approximately above 1500 bp in Fig. 4.

As shown in Table 6, the amplified dhaS gene from isolate 1 (B. amyloliquefaciens) had a top BLAST hit with B. amyloliquefaciens strain LM2303 (Accession no. CP018152.1) with percent identity of 98.35% and an E-value of 0.0 for a 99% coverage with a max score of 2591. An E-value of 0.0 was also gathered for B. velezensis strain FJAT-45028 and B. velezensis strain OSY-GA1 suggesting a very similar sequence identity with the isolate being studied. A study by Fan et al.23 suggests that both B. velezensis and B. amyloliquefaciens are included under Clade II of Bacillus subtilis complex having B. velezensis as the taxonomic synonym of B. amyloliquefaciens. Aside from this, the identity of the gene in all of the top BLAST hits had a reference sequence that pertains to 3-hydroxypropionaldehyde dehydrogenase/aldehyde dehydrogenase DhaS which further implies that the aligned sequence shares similarity with the expected gene product of dhaS gene.

Further analysis of the sequence of the amplified dhaS gene in isolate 1 revealed that the top hit in the translated BLAST corresponds to an aldehyde dehydrogenase family protein having a percent identity of 96% given in Table 7. This result also supports the expected enzyme that is coded by dhaS gene which is an aldehyde dehydrogenase.

Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.
Fig. 3:
Electrophoretogram of the amplified dhaS gene using the extracted genomic DNA from isolate 2 (B. subtilis) with varying annealing temperature
 
Lane 1: 56°C, Lane 2: 57°C, Lane 3: 58°C, Lane 4: 58°C, Lane 5: Negative control, genomic DNA templates used were from the five different stab cultures

Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.
Fig. 4:
Electrophoretogram of the amplified patB gene using the extracted genomic DNA from two Bacillus isolates
 
Lane 1: Isolate 2, 43°C; Lane 2: Isolate 2, 44°C; Lane 3: Isolate 2, 45°C; Lane 4: Negative control, 45°C; Lane 5: Isolate 1, 44°C; Lane 6: Isolate 2, 45°C

Table 8:
Overview of the BLAST result using the 16S rRNA sequence from Bacillus isolate 1 with organisms limited to Bacillus (taxid: 1386)
Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.

Table 9:Overview of the BLAST result using the 16S rRNA sequence from Bacillus isolates 2 with organisms limited to Bacillus (taxid: 1386)
Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.

Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.
Fig. 5:Rank three model of the predicted secondary structure of the amino acid translated from the amplified dhaS gene from Bacillus isolate 1 (B. amyloliquefaciens) generated using Phyre2
  Red portion indicates the N-terminus while the Indigo depicts the C-terminus

Prediction of secondary structure using Phyre2 was also done resulting in a top model of a proline dehydrogenase/ delta-1-pyrroline-5-carboxylate24. Aside from this, the rank three models in the structures generated was an aldehyde dehydrogenase with 100% confidence and 25% identity in Fig. 5. The resulting PDB title was described as the structure of a bacterial aldh16 complexed with Nadh24. According to Vasilou et al.25 ALDH16 is a member of the aldehyde dehydrogenase superfamily. This protein has a distinguishing feature of the presence of two protein domains25,26.

A previous study on dhaS gene was conducted by Shao et al.11 in which the gene from B. amyloliquefaciens strain SQR9 was found out to be upregulated in the presence of tryptophan and possibly be involved in the IPyA pathway of IAA biosynthesis. The IPyA pathway is one of the various tryptophan dependent pathways for IAA biosynthesis15. This pathway has been widely observed in bacteria27, such as in Azospirillium brasilense sp.728, Pseudomonas putida GR12-215 and Enterobacter cloacae UW529. Other than the IPyA pathway, dhaS gene has also been suggested to play a role in catalyzing the conversion of Indole-3 acetaldehyde to Indole-3 acetic acid in the terminal step of Tryptamine pathway11. Contrastingly, a study by Idris et al.18 showed that mutation in dhaS gene of B. amyloliquefaciens FZB42 did not affect the IAA production implying that this gene is not involved in IAA biosynthesis. The involvement of dhaS gene in the abovementioned two pathways gives its importance towards IAA biosynthesis. However, it also needs to be taken into consideration that there are differences in the IAA biosynthesis pathway across different strains of the same species which further implies that isolation of the dhaS gene may not be enough to deduce its involvement in the IAA biosynthesis pathway and thus needs further confirmation through in vitro assays and gene knock-out mutation.

16S rRNA sequence analysis: Standard nucleotide BLAST (BLASTN) of 16S rRNA sequence in isolate* 1 showed that the top nucleotide BLAST hit is Bacillus sp. strain 1CY1 with a percent identity of 99.67% in Table 8. Whereas isolate 2 had a top nucleotide BLAST hit with Bacillus subtilis strain GX S-11 having the highest percent identity of 95.65% given in Table 9.

To infer the relationships between 16S rRNA sequences from the two isolates and its top five nucleotide BLAST hits, phylogenetic trees were generated using Maximum Likelihood and Tamura-Nei model with 1000 replicates from MEGA X. In isolate 1, the phylogenetic tree with a maximum log-likelihood of -1812.42 implies that Bacillus isolate 1, Bacillus sp. W4 and Bacillus sp. strain 1CY1 are more closely related to each other than Bacillus badius, Bacillus badius strain KSI 1261 and Bacillus subtilis 30.

Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.
Fig. 6:
Phylogenetic tree of Bacillus isolate 1 and its resulting top five BLAST hits based on 16S rRNA gene sequence generated
  Using maximum likelihood and Tamura-Nei model from MEGA X with 1000 replicates

Image for - Amplification and Sequence Analysis of Indole-3-Pyruvic Acid (IPyA) Pathway Related Genes from Bacillus spp.
Fig. 7:
Phylogenetic tree of Bacillus isolate 2 and its resulting top five BLAST hits based on 16S rRNA gene sequence generated
  Using maximum likelihood and Tamura-Nei model from MEGA X with 1000 replicates

As presented in Fig. 6, the observed highest value (100) in the node indicates that the three species (Bacillus isolate 1, Bacillus sp. W4 and Bacillus sp. strain 1CY1) appeared in the same clade in all of the trees generated whereas a lower value of 42 in the internal node pertains to a less occurrence of the node in the trees replicated.

In contrast with isolate 2, the phylogenetic tree having the highest log-likelihood of -1751.33 suggests that Bacillus isolate 2 lies outside the clade comprising its top five BLAST hits in Fig. 7. The result is also evident in the multiple sequence alignment, in which the sequence from the isolate had several mismatched bases with its top five BLAST hits. Aside from this, the probability of base substitutions and conditional likelihoods affects the tree topology generated using maximum likelihood31.

CONCLUSION

In this study, the indole-3-pyruvic acid pathway is one plausible pathway that may be used by the selected Bacillus species for IAA synthesis. Successful amplification of patB and dhaS from the isolates and the bioinformatics data presented for these two IPyA component genes provides a baseline understanding of the Bacillus spp. isolates found in FPJ biofertilizer. Altogether, the data suggest that IPyA may be the route taken by these species for IAA synthesis which may be further utilized in optimizing phytohormone production that has the potential to improve the capability of Bacillus isolates as biofertilizers.

SIGNIFICANCE STATEMENT

There are reports on IAA biosynthesis pathways in various organisms but only a few studies are available regarding the analysis of each gene involved in these pathways. The bioinformatics data presented here for two-component genes of the IPyA biosynthesis pathway for IAA production provide us with a baseline understanding of the Bacillus spp. isolates found in FPJ biofertilizer. Further identification of the presence of IAA-related genes in several species of Bacillus may pave the way for further understanding of bacterial IAA biosynthesis which has the potential to improve crop production.

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