Whole-Genome Sequencing and Potassium-Solubilizing Mechanism of Bacillus aryabhattai SK1-7

To analyze the whole genome of Bacillus aryabhattai strain SK1-7 and explore its potassium solubilization characteristics and mechanism, thus providing a theoretical basis for analyzing the utilization and improvement of insoluble potassium resources in soil. Genome information for Bacillus aryabhattai SK1-7 was obtained by using Illumina NovaSeq second-generation sequencing and GridION Nanopore ONT third-generation sequencing technology. The contents of organic acids and polysaccharides in fermentation broth of Bacillus aryabhattai SK1-7 were determined by high-performance liquid chromatography and the anthrone sulfuric acid method, and the expression levels of the potassium solubilization-related genes ackA, epsB, gltA, mdh and ppc were compared by real-time fluorescence quantitative PCR under different potassium source culture conditions. The whole genome of the strain consisted of a complete chromosome sequence and four plasmid sequences. The sequence sizes of the chromosomes and plasmids P1, P2, P3 and P4 were 5,188,391 bp, 136,204 bp, 124,862 bp, 67,200 bp and 12,374 bp, respectively. The GC contents were 38.2, 34.4, 33.6, 32.8, and 33.7%. Strain SK1-7 mainly secreted malic, formic, acetic and citric acids under culture with an insoluble potassium source. The polysaccharide content produced with an insoluble potassium source was higher than that with a soluble potassium source. The expression levels of five potassium solubilization-related genes with the insoluble potassium source were higher than those with the soluble potassium source.

Volatile Organic Compounds from Bacillus aryabhattai MCCC 1K02966 with Multiple Modes against Meloidogyne incognita

Plant-parasitic nematodes cause severe losses to crop production and economies all over the world. Bacillus aryabhattai MCCC 1K02966, a deep-sea bacterium, was obtained from the Southwest Indian Ocean and showed nematicidal and fumigant activities against Meloidogyne incognita in vitro. The nematicidal volatile organic compounds (VOCs) from the fermentation broth of B. aryabhattai MCCC 1K02966 were investigated further using solid-phase microextraction gas chromatography-mass spectrometry. Four VOCs, namely, pentane, 1-butanol, methyl thioacetate, and dimethyl disulfide, were identified in the fermentation broth. Among these VOCs, methyl thioacetate exhibited multiple nematicidal activities, including contact nematicidal, fumigant, and repellent activities against M. incognita. Methyl thioacetate showed a significant contact nematicidal activity with 87.90% mortality at 0.01 mg/mL by 72 h, fumigant activity in mortality 91.10% at 1 mg/mL by 48 h, and repellent activity at 0.01–10 mg/mL. In addition, methyl thioacetate exhibited 80–100% egg-hatching inhibition on the 7th day over the range of 0.5 mg/mL to 5 mg/mL. These results showed that methyl thioacetate from MCCC 1K02966 control M. incognita with multiple nematicidal modes and can be used as a potential biological control agent.

Plant Growth-Promoting Endophytic Bacillus Aryabhattai Triggers Novel Genes to Induce Plant Growth

Background: In nature, plants interact with a wide range of microorganisms. Most of these microorganisms have the ability to promote plant growth through the induction of important molecular pathways. The current work evaluated whether the endophytic bacterium Bacillus aryabhattai encourages plant growth and how transcriptional changes might be implicated in this effect.Results: The endophytic bacterium showed a significant effect on plant growth. Our results revealed that B. aryabhattai promotes the growth of Arabidopsis and tobacco plants. Notably, transcriptional changes in Arabidopsis plants treated with the bacterium were identified. Genes such as cinnamyl alcohol dehydrogenase, apyrase, thioredoxin H8, benzaldehyde dehydrogenase, indoleacetaldoxime dehydratase, berberine bridge enzyme-like and gibberellin-regulated protein were highly expressed. Additionally, endophytic bacterial genes such as arginine decarboxylase, D-hydantoinase, ATP synthase gamma chain and 2-hydroxyhexa-2,4-dienoate hydratase were activated during the interaction with Arabidopsis.Conclusions: The results show that new plant growth-related genes are induced during the interaction endophytic bacterium B. aryabhattai, and these changes may promote plant growth in sustainable agriculture.

Cloning, expression, purification and characterization of chitin deacetylase extremozyme from halophilic Bacillus aryabhattai B8W22

Chitin deacetylase (CDA) (EC 3.5.1.41) is a hydrolytic enzyme that belongs to carbohydrate esterase family 4 as per the CAZY database. The CDA enzyme deacetylates chitin into chitosan. As the marine ecosystem is a rich source of chitin, it would also hold the unexplored extremophiles. In this study, an organism was isolated from 40 m sea sediment under halophilic condition and identified as Bacillus aryabhattai B8W22 by 16S rRNA sequencing. The CDA gene from the isolate was cloned and overexpressed in E. coli Rosetta pLysS and purified using a Ni–NTA affinity chromatography. The enzyme was found active on both ethylene glycol chitin (EGC) and chitooligosaccharides (COS). The enzyme characterization study revealed, maximum enzyme velocity at one hour, optimum pH at 7 with 50 mM Tris–HCl buffer, optimum reaction temperature of 30 ºC in standard assay conditions. The co-factor screening affirmed enhancement in the enzyme activity by 142.43 ± 7.13% and 146.88 ± 4.09% with substrate EGC and COS, respectively, in the presence of 2 mM Mg2+. This activity was decreased with the inclusion of EDTA and acetate in the assay solutions. The enzyme was found to be halotolerant; the relative activity increased to 116.98 ± 3.87% and 118.70 ± 0.98% with EGC and COS as substrates in the presence of 1 M NaCl. The enzyme also demonstrated thermo-stability, retaining 87.27 ± 2.85% and 94.08 ± 0.92% activity with substrate EGC and COS, respectively, upon treatment at 50 ºC for 24 h. The kinetic parameters Km, Vmax, and Kcat were 3.06E−05 µg mL−1, 3.06E + 01 µM mg−1 min−1 and 3.27E + 04 s−1, respectively, with EGC as the substrate and 7.14E−07 µg mL−1, 7.14E + 01 µM mg−1 min−1 and 1.40E + 06 s−1, respectively, with COS as the substrate. The enzyme was found to be following Michaelis–Menten kinetics with both the polymeric and oligomeric substrates. In recent years, enzymatic conversion of chitosan is gaining importance due to its known pattern of deacetylation and reproducibility. Thus, this BaCDA extremozyme could be used for industrial production of chitosan polymer as well as chitosan oligosaccharides for biomedical application.

Expression, biochemical and structural characterization of high-specific-activity β-amylase from Bacillus aryabhattai GEL-09 for application in starch hydrolysis

Background β-amylase (EC 3.2.1.2) is an exo-enzyme that shows high specificity for cleaving the α-1,4-glucosidic linkage of starch from the non-reducing end, thereby liberating maltose. In this study, we heterologously expressed and characterized a novel β-amylase from Bacillus aryabhattai. Results The amino acid-sequence alignment showed that the enzyme shared the highest sequence identity with β-amylase from Bacillus flexus (80.73%) followed by Bacillus cereus (71.38%). Structural comparison revealed the existence of an additional starch-binding domain (SBD) at the C-terminus of B. aryabhattai β-amylase, which is notably different from plant β-amylases. The recombinant enzyme purified 4.7-fold to homogeneity, with a molecular weight of ~ 57.6 kDa and maximal activity at pH 6.5 and 50 °C. Notably, the enzyme exhibited the highest specific activity (3798.9 U/mg) among reported mesothermal microbial β-amylases and the highest specificity for soluble starch, followed by corn starch. Kinetic analysis showed that the Km and kcat values were 9.9 mg/mL and 116961.1 s− 1, respectively. The optimal reaction conditions to produce maltose from starch resulted in a maximal yield of 87.0%. Moreover, molecular docking suggested that B. aryabhattai β-amylase could efficiently recognize and hydrolyze maltotetraose substrate. Conclusions These results suggested that B. aryabhattai β-amylase could be a potential candidate for use in the industrial production of maltose from starch.

Alleviation of Salt Stress in Wheat Seedlings via Multifunctional Bacillus aryabhattai PM34: An In-Vitro Study

Plant growth-promoting rhizobacteria play a substantial role in plant growth and development under biotic and abiotic stress conditions. However, understanding about the functional role of rhizobacterial strains for wheat growth under salt stress remains largely unknown. Here we investigated the antagonistic bacterial strain Bacillus aryabhattai PM34 inhabiting ACC deaminase and exopolysaccharide producing ability to ameliorate salinity stress in wheat seedlings under in vitro conditions. The strain PM34 was isolated from the potato rhizosphere and screened for different PGP traits comprising nitrogen fixation, potassium, zinc solubilization, indole acetic acid, siderophore, and ammonia production, along with various extracellular enzyme activities. The strain PM34 showed significant tolerance towards both abiotic stresses including salt stress (NaCl 2 M), heavy metal (nickel, 100 ppm, and cadmium, 300 ppm), heat stress (60 °C), and biotic stress through mycelial inhibition of Rhizoctonia solani (43%) and Fusarium solani (41%). The PCR detection of ituC, nifH, and acds genes coding for iturin, nitrogenase, and ACC deaminase enzyme indicated the potential of strain PM34 for plant growth promotion and stress tolerance. In the in vitro experiment, NaCl (2 M) decreased the wheat growth while the inoculation of strain PM34 enhanced the germination% (48%), root length (76%), shoot length (75%), fresh biomass (79%), and dry biomass (87%) over to un-inoculated control under 2M NaCl level. The results of experiments depicted the ability of antagonistic bacterial strain Bacillus aryabhattai PM34 to augment salt stress tolerance when inoculated to wheat plants under saline environment.

Cloning of Acetoacetyl-CoA reductase and Polyhydroxybutyrate synthase genes from the local isolate Bacillus aryabhattai 6N-NRC into Escherichia coli

Polyhydroxybutyrate (PHB) is the most known degradable biopolymer, produced by some genera of bacteria under unfavorable growth conditions. Isolation and cloning of acetoacetyl-CoA reductase (phbB) and polyhydroxybutyrate synthase (phbC) genes from local isolate previously identified as Bacillus aryabhattai 6N-NRC (GenBank accession no. MH997667.1) was achieved. Suitable primers designed for the phbB and phbC PCR approach were used to clone the phbB and phbC genes. The phbB and phbC genes were successfully isolated, cloned and the PCR amplicon 744 bp and 1089 bp corresponding to phbB and phbC genes were identified, cloned with the pET-29a (+) carrying the phbB and phbC genes, transformed and expressed in Escherichia coli BL21. The amplification of the phbB and phbC genes using specific primers of pET-29a (+) plasmid was performed. The open reading frame of phbB sequence was found to be 99.06% identical to the sequence of acetoacetyl-CoA reductase of B. aryabhattai (GenBank accession no. CP024035.1), while the open reading frame of phbC sequence was found to be 87.18% identical to the sequence of polyhydroxybutyrate synthase of B. aryabhattai (Gen Bank accession no. CP024035.1) after DNA sequencing. The analysis of the recombinant proteins from E. coli BL21 recombinant colony by tricine-polyacrylamide gel electrophoresis clarified that the expressed phbB and phbC genes in E. coli BL21 strain showed distinct bands of intensity 26.3 KD and 37.5 KD, respectively.