Screening, Identification, and Probiotic Properties of Bacillus Pumilus From Yak.

The yak has a unique physiological structure suited to life in anoxic and cold environments at high altitudes. The aim of this study was to isolate Bacillus species with good probiotic properties from yak feces. A series of tests were performed on the isolated Bacillus: 16S rRNA identification, antibacterial activity, tolerance to gastroenteric fluid, hydrophobicity, auto-aggregation, antibiotic sensitivity, growth performance, antioxidants, and immune indexes. A safe and harmless Bacillus pumilus DX24 strain with good survival rate, hydrophobicity, auto-aggregation, and antibacterial activity was identified in the yak feces. Feeding mice with Bacillus pumilus DX24 increased their daily weight gain, jejunal villus length, villi/Crypt ratio, blood IgG levels, and jejunum sIgA levels. This study confirmed the probiotic effects of Bacillus pumilus isolated from yak feces and provides the theoretical basis for the clinical application and development of new feed additives.

Tuning transcription factor DegU for developing extracellular protease overproducer in Bacillus pumilus.

BACKGROUND: Global transcription machinery engineering (gTME) is an effective approach employed in strain engineering to rewire gene expression and reshape cellular metabolic fluxes at the transcriptional level. RESULTS: In this study, we utilized gTME to engineer the positive transcription factor, DegU, in the regulation network of major alkaline protease, AprE, in Bacillus pumilus. To validate its functionality when incorporated into the chromosome, we performed several experiments. First, three negative transcription factors, SinR, Hpr, and AbrB, were deleted to promote AprE synthesis. Second, several hyper-active DegU mutants, designated as DegU(hy), were selected using the fluorescence colorimetric method with the host of the Bacillus subtilis ΔdegSU mutant. Third, we integrated a screened degU(L113F) sequence into the chromosome of the Δhpr mutant of B. pumilus SCU11 to replace the original degU gene using a CRISPR/Cas9 system. Finally, based on transcriptomic and molecular dynamic analysis, we interpreted the possible mechanism of high-yielding and found that the strain produced alkaline proteases 2.7 times higher than that of the control strain (B. pumilus SCU11) in LB medium. CONCLUSION: Our findings serve as a proof-of-concept that tuning the global regulator is feasible and crucial for improving the production performance of B. pumilus. Additionally, our study established a paradigm for gene function research in strains that are difficult to handle.

Characterization of pumilacidin, a lipopeptide biosurfactant produced from Bacillus pumilus NITDID1 and its prospect in bioremediation of hazardous pollutants.

Highly hydrophobic compounds like petroleum and their byproducts, once released into the environment, can persist indefinitely by virtue of their ability to resist microbial degradation, ultimately paving the path to severe environmental pollution. Likewise, the accumulation of toxic heavy metals like lead, cadmium, chromium, etc., in the surroundings poses an alarming threat to various living organisms. To remediate the matter in question, the applicability of a biosurfactant produced from the mangrove bacterium Bacillus pumilus NITDID1 (Accession No. KY678446.1) is reported here. The structural characterization of the produced biosurfactant revealed it to be a lipopeptide and has been identified as pumilacidin through FTIR, NMR, and MALDI-TOF MS. The critical micelle concentration of pumilacidin was 120 mg/L, and it showed a wide range of stability in surface tension reduction experiments under various environmental conditions and exhibited a high emulsification index of as much as 90%. In a simulated setup of engine oil-contaminated sand, considerable oil recovery (39.78%) by this biosurfactant was observed, and upon being added to a microbial consortium, there was an appreciable enhancement in the degradation of the used engine oil. As far as the heavy metal removal potential of biosurfactant is concerned, as much as 100% and 82% removal was observed for lead and cadmium, respectively. Thus, in a nutshell, the pumilacidin produced from Bacillus pumilus NITDID1 holds promise for multifaceted applications in the field of environmental remediation.

Prediction and validation of enzymatic degradation of aflatoxin M1: Genomics and proteomics analysis of Bacillus pumilus E-1-1-1 enzymes.

Aflatoxins are a class of highly toxic mycotoxins. Aflatoxin M1 (AFM1) is hydroxylated metabolite of aflatoxin B1, having comparable toxicity, which is more commonly found in milk. In this study, the whole genome sequencing of Bacillus pumilus E-1-1-1 isolated from feces of 38 kinds of animals, having aflatoxin M1 degradation ability was conducted. Bacterial genome sequencing indicated that a total of 3445 sequences were finally annotated on 23 different cluster of orthologous groups (COG) categories. Then, the potential AFM1 degradation proteins were verified by proteomics; the properties of these proteins were further explored, including protein molecular weight, hydrophobicity, secondary structure prediction, and three-dimensional structures. Bacterial genome sequencing combined with proteomics showed that eight genes were the most capable of degrading AFM1 including three catalases, one superoxide dismutase, and four peroxidases to clone. These eight genes with AFM1 degrading capacity were successfully expressed. These results indicated that AFM1 can be degraded by Bacillus pumilus E-1-1-1 protein and the most degrading proteins were oxidoreductases.

2-Keto-L-gulonic acid inhibits the growth of Bacillus pumilus and Ketogulonicigenium vulgare.

The typical vitamin C mixed-fermentation process's second stage involves bioconversion of L-sorbose to 2-keto-L-gulonic acid (2-KLG), using a consortium comprising Ketogulonicigenium vulgare and Bacillus spp. (as helper strain). The concentration of the helper strain in the co-fermentation system was closely correlated with K. vulgare cell growth and 2-KLG accumulation. To understand the tolerance and response of the helper strain and K. vulgare to 2-KLG, 2-KLG was added to the single-strain system of Bacillus pumilus and K. vulgare and the basic physiological and biochemical properties were determined. In this study, the addition of 1 mg/mL 2-KLG reduced the number of viable and spore cells, lowered the levels of intracellular reactive oxygen species (ROS), enhanced the intra- and extracellular total antioxidant capacity (T-AOC), and significantly affected the B. pumilus sporulation-related genes expression levels. Furthermore, the addition of 1 mg/mL 2-KLG increased the intracellular ROS levels, decreased the intra- and extracellular T-AOC, and downregulated the antioxidant enzyme-related genes and 2-KLG production enzyme-related genes of K. vulgare. These results suggested that 2-KLG could induce acidic and oxidative stress in B. pumilus and K. vulgare, which could be a guide for a greater understanding of the interaction between the microorganisms.

Transcriptome analysis reveals the regulatory effects of Bacillus amyloliquefaciens and Bacillus pumilus on immune and digestive related genes in the spleen of weanling black goats.

The aim of the present study was to evaluate the effects of Bacillus amyloliquefaciens fsznc-06 and Bacillus pumilus fsznc-09 on the expressions of spleen genes in weanling Jintang black goats. Bacillus amyloliquefaciens fsznc-06 (BA-treated group) and Bacillus pumilus fsznc-09 (BP-treated group) were directly fed to goats, and the spleens were harvested for transcriptome analysis. The KEGG pathway analysis showed that the differentially expressed genes (DEGs) in BA-treated vs CON group were mainly involved in digestive system and immune system, while those in BP-treated vs CON group were mainly involved in immune system, and those in BA-treated vs BP-treated group were mainly involved in digestive system. In conclusion, Bacillus amyloliquefaciens fsznc-06 might promote the expressions of genes related to immune system and digestive system, reduce the expressions of disease genes related to digestive system and might promote mutual accommodation of some immune genes in weanling black goat. Bacillus pumilus fsznc-09 might promote the expressions of genes related to immune system and mutual accommodation of some immune genes in weanling black goat. Bacillus amyloliquefaciens fsznc-06 has advantages over Bacillus pumilus fsznc-09 in promoting the expressions of genes related to digestive system and mutual accommodation of some immune genes.

Engineering non-conserved salt bridges in GH11 xylanase from Bacillus pumilus SSP34 for improved thermal stability: an in-silico evaluation.

GH11 xylanases are commercially important enzymes for degradation of xylan fibers. We have identified the presence of nine non-conserved and five conserved salt bridges in GH11 xylanase from Bacillus pumilus SSP34. We have designed two sets of mutants viz., (1) substitution mutants in which non-conserved charged amino acid residues have been replaced with appropriate hydrophobic residues based on side chain occupancy and hydrophobicity and (2) deletion mutants where non-conserved charged residues have been deleted. The stability of the mutants has been evaluated in-silico by analyzing the contributions of non-covalent interactions like hydrophobic interaction clusters and salt bridges. The stability of the resultant mutants was evaluated using parameters such as radius of gyration, solvent accessible surface area, root mean square deviation, root mean square fluctuations and protein unfolding measurements using molecular dynamic simulations. The deletion of certain charged residues resulted in mutants having lowered radius of gyration and decreased surface areas. However, RMSD and RMSF measurements indicated lowered stability in comparison to substitution mutants. Of the substitution mutants, the SBM 3 was the most stable mutant as indicated by Rg, SASA, RMSF and simulated protein unfolding measurements. The major contributing factors for improved stability could be strengthening of hydrophobic interactions in the GH11 xylanase from B. pumilus. These in-silico stability measurements of salt bridge mutants may lead to better design of GH11 xylanases for commercial applications.Communicated by Ramaswamy H. Sarma.

Regulating Pathways of Bacillus pumilus Adamalysin-like Metalloendopeptidase Expression.

The minor secreted proteinase of B. pumilus 3-19 MprBp classified as the unique bacillary adamalysin-like enzyme of the metzincin clan. The functional role of this metalloproteinase in the bacilli cells is not clear. Analysis of the regulatory region of the mprBp gene showed the presence of potential binding sites to the transcription regulatory factors Spo0A (sporulation) and DegU (biodegradation). The study of mprBp activity in mutant strains of B. subtilis defective in regulatory proteins of the Spo- and Deg-systems showed that the mprBp gene is partially controlled by the Deg-system of signal transduction and independent from the Spo-system.

Effects of Bacillus pumilus on growth performance, serum indexes and muscle development-related gene expression of weanling Jintang black goat.

Antibiotic misuse has been a severe problem in animal husbandry. It is meaningful to replace antibiotics with Bacillus, as feed additives are indeed a research hotspot. Bacillus pumilus plays a certain role in promoting the growth performance and immunological indicators of animals. There are few reports about the function of goat-derived B. pumilus in animals until now. This study aimed to investigate the effects of B. pumilus fsznc-09 on growth performance and immune function of Jintang black goats. B. pumilus-treated group was fed with 1 ml freeze-dried agent of B. pumilus fsznc-09 at a concentration of 109 cfu/ml every 2 days. The growth performance, serum biochemical indexes, the expressions of muscle development and metabolism related genes of Jintang black goats were measured after 30 days. The results showed that the average daily gain and average daily feed intake were significantly increased, and feed conversion ratio was significantly decreased. The activities of total superoxide dismutase, alkaline phosphatase, immunoglobulin G and interferon-γ in serum of goats were significantly increased. However, the activity of malondialdehyde in serum was significantly decreased. The diameters and areas in longissimus dorsi fibre and gluteus fibre of goats were significantly decreased, while the densities in gluteus fibre of goats were significantly increased. The expressions of FAS, LPL, PPAR-γ, CAT, MYOD1, MYOG, MYF5 and MyHCI in longissimus dorsi and gluteus of goats were significantly improved. The expressions of TGFß1, SREBP-1, MyHCIIb and MyHCIIx in longissimus dorsi and gluteus of goats were significantly increased. The expressions of FN1 in longissimus dorsi and MyHCIIa in gluteus of goats were significantly decreased. In conclusion, B. pumilus fsznc-09 can effectively improve the growth performance, immunological indicators and the expressions of muscle development and metabolism related genes of Jintang black goat.

Isolation and Characterization of Two Novel Siphoviruses Novomoskovsk and Bolokhovo, Encoding Polysaccharide Depolymerases Active against Bacillus pumilus.

This study describes two novel bacteriophages infecting members of the Bacillus pumilus group. Even though members of the group are not recognized as pathogenic, several strains belonging to the group have been reported to cause infectious diseases in plants, animals and humans. Bacillus pumilus group species are highly resistant to ultraviolet radiation and capable of forming biofilms, which complicates their eradication. Bacteriophages Novomoskovsk and Bolokhovo were isolated from soil samples. Genome sequencing and phylogenetic analysis revealed that the phages represent two new species of the genus Andromedavirus (class Caudoviricetes). The phages remained stable in a wide range of temperatures and pH values. A host range test showed that the phages specifically infect various strains of B. pumilus. The phages form clear plaques surrounded by halos. Both phages Novomoskovsk and Bolokhovo encode proteins with pectin lyase domains-Putative depolymerases. Obtained in a purified recombinant form, the proteins produced lysis zones on the lawn of a B. pumilus strain. This suggests that Novomoskovsk and Bolokhovo may be effective for the eradication of B. pumilus biofilms.

Bacillus pumilus proteome changes in response to 2,4,6-trinitrotoluene-induced stress.

2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound and is highly resistant to degradation. Most aerobic microorganisms reduce TNT to amino derivatives via formation of nitroso- and hydroxylamine intermediates. Although pathways of TNT degradation are well studied, proteomic analysis of TNT-degrading bacteria was done only for some individual Gram-negative strains. Here, we isolated a Gram-positive strain from TNT-contaminated soil, identified it as Bacillus pumilus using 16S rRNA sequencing, analyzed its growth, the level of TNT transformation, ROS production, and revealed for the first time the bacillary proteome changes at toxic concentration of TNT. The transformation of TNT at all studied concentrations (20-200 mg/L) followed the path of nitro groups reduction with the formation of 4-amino-2,6-dinitrotoluene. Hydrogen peroxide production was detected during TNT transformation. Comparative proteomic analysis of B. pumilus showed that TNT (200 mg/L) inhibited expression of 46 and induced expression of 24 proteins. Among TNT upregulated proteins are those which are responsible for the reductive pathway of xenobiotic transformation, removal of oxidative stress, DNA repair, degradation of RNA and cellular proteins. The production of ribosomal proteins, some important metabolic proteins and proteins involved in cell division are downregulated by this xenobiotic.

Pangenome analyses of Bacillus pumilus, Bacillus safensis, and Priestia megaterium exploring the plant-associated features of bacilli strains isolated from canola.

Previous genome mining of the strains Bacillus pumilus 7PB, Bacillus safensis 1TAz, 8Taz, and 32PB, and Priestia megaterium 16PB isolated from canola revealed differences in the profile of antimicrobial biosynthetic genes when compared to the species type strains. To evaluate not only the similarities among B. pumilus, B. safensis, and P. megaterium genomes but also the specificities found in the canola bacilli, we performed comparative genomic analyses through the pangenome evaluation of each species. Besides that, other genome features were explored, especially focusing on plant-associated and biotechnological characteristics. The combination of the genome metrics Average Nucleotide Identity and digital DNA-DNA hybridization formulas 1 and 3 adopting the universal thresholds of 95 and 70%, respectively, was suitable to verify the identification of strains from these groups. On average, core genes corresponded to 45%, 52%, and 34% of B. pumilus, B. safensis, and P. megaterium open pangenomes, respectively. Many genes related to adaptations to plant-associated lifestyles were predicted, especially in the Bacillus genomes. These included genes for acetoin production, polyamines utilization, root exudate chemoreceptors, biofilm formation, and plant cell-wall degrading enzymes. Overall, we could observe that strains of these species exhibit many features in common, whereas most of their variable genome portions have features yet to be uncovered. The observed antifungal activity of canola bacilli might be a result of the synergistic action of secondary metabolites, siderophores, and chitinases. Genome analysis confirmed that these species and strains have biotechnological potential to be used both as agricultural inoculants or hydrolases producers. Up to our knowledge, this is the first work that evaluates the pangenome features of P. megaterium.

Probiotic Potential of Bacillus licheniformis and Bacillus pumilus Isolated from Tibetan Yaks, China.

Yak (Bos grunniens) inhabit an oxygen-deficient environment at the altitude of 3000 m on the Tibetan Plateau, with a distinctive gut micro-ecosystem. This study evaluated the probiotic potential and physiological property of Bacillus licheniformis and Bacillus pumilus isolated from the gut of yaks. Four strains, two Bacillus licheniformis (named D1 and D2) and two Bacillus pumilus (named X1 and X2), were isolated and identified by 16S rRNA sequencing. All strains had potential antibacterial ability against three indicator pathogens: Escherichia coli C83902, Staphylococcus aureus BNCC186335, and Salmonella enteritidis NTNC13349. The antioxidant activity test showed that D2 sample showed the highest antioxidant activity. Furthermore, all four strains had a higher hydrophobicity, auto-aggregation, acid tolerance, bile tolerance, and antibiotic sensitivity, which all contribute to their survival in the gastrointestinal tract and clinical utility. The animal experimentation (40 KM mice, equally divided into five groups of eight mice each) showed that the strain supplementation not only increased daily weight gain and reduced feed conversion ratio, but also increased the length of the jejunum villi and the value of the V/C (Villi/Crypt). In conclusion, this is the first study demonstrated the probiotic potential of Bacillus licheniformis and Bacillus pumilus isolated from yaks, providing a theoretical basis for the clinical application and development of new feed additives.

Comparative Genome Analysis of Two Bacillus pumilus Strains Producing High Level of Extracellular Hydrolases.

Whole-genome sequencing of a soil isolate Bacillus pumilus, strain 7P, and its streptomycin-resistant derivative, B. pumilus 3-19, showed genome sizes of 3,609,117 bp and 3,609,444 bp, respectively. Annotation of the genome showed 3794 CDS (3204 with predicted function) and 3746 CDS (3173 with predicted function) in the genome of strains 7P and 3-19, respectively. In the genomes of both strains, the prophage regions Bp1 and Bp2 were identified. These include 52 ORF of prophage proteins in the Bp1 region and 38 prophages ORF in the Bp2 region. Interestingly, more than 50% of Bp1 prophage proteins are similar to the proteins of the phi105 in B. subtilis. The DNA region of Bp2 has 15% similarity to the DNA of the Brevibacillus Jimmer phage. Degradome analysis of the genome of both strains revealed 148 proteases of various classes. These include 60 serine proteases, 48 metalloproteases, 26 cysteine proteases, 4 aspartate proteases, 2 asparagine proteases, 3 threonine proteases, and 2 unclassified proteases. Likewise, three inhibitors of proteolytic enzymes were found. Comparative analysis of variants in the genomes of strains 7P and 3-19 showed the presence of 81 nucleotide variants in the genome 3-19. Among them, the missense mutations in the rpsL, comA, spo0F genes and in the upstream region of the srlR gene were revealed. These nucleotide polymorphisms may have affected the streptomycin resistance and overproduction of extracellular hydrolases of the 3-19 strain. Finally, a plasmid DNA was found in strain 7P, which is lost in its derivative, strain 3-19. This plasmid contains five coding DNA sequencing (CDS), two regulatory proteins and three hypothetical proteins.

Improving the decolorization activity of Bacillus pumilus W3 CotA-laccase to Congo Red by rational modification.

Congo Red (CR) is a typical azo dye with highly toxic and carcinogenic properties. This study aimed to improve the decolorization activity of Bacillus pumilus W3 CotA-laccase for azo dye CR. This work analyzed the interaction between CotA-laccase and CR based on homology modeling and molecular docking. The three amino acids (Gly323, Thr377, Thr418) in the substrate-binding pocket were rationally modified through saturation mutation. Finally, the obtained multi-site mutants T377I/T418G and G323S/T377I/T418G decolorized 76.59% and 59.37% of CR within 24 h at pH 8.0 without a mediator, which were 3.15- and 2.44-fold higher than the wild-type CotA. The catalytic efficiency of the multi-site mutants T377I/T418G and G323S/T377I/T418G to CR were increased by 2.21- and 2.01-fold compared with the wild-type CotA, respectively. The mechanism of activity enhancement of mutants was proposed by structural analysis. This evidence suggests that the mutants T377I/T418G and G323S/T377I/T418G could be used as novel bioremediation tools.

An antimicrobial peptide specifically active against Listeria monocytogenes is secreted by Bacillus pumilus SF214.

BACKGROUND: Members of the Bacillus genus produce a large variety of antimicrobial peptides including linear or cyclic lipopeptides and thiopeptides, that often have a broad spectrum of action against Gram-positive and Gram-negative bacteria. We have recently reported that SF214, a marine isolated strain of Bacillus pumilus, produces two different antimicrobials specifically active against either Staphylococcus aureus or Listeria monocytogenes. The anti-Staphylococcus molecule has been previously characterized as a pumilacidin, a nonribosomally synthesized lipopetide composed of a mixture of cyclic heptapeptides linked to fatty acids of variable length. RESULTS: Our analysis on the anti-Listeria molecule of B. pumilus SF214 indicated that it is a peptide slightly smaller than 10 kDa, produced during the exponential phase of growth, stable at a wide range of pH conditions and resistant to various chemical treatments. The peptide showed a lytic activity against growing but not resting cells of Listeria monocytogenes and appeared extremely specific being inactive also against L. innocua, a close relative of L. monocytogenes. CONCLUSIONS: These findings indicate that the B. pumilus peptide is unusual with respect to other antimicrobials both for its time of synthesis and secretion and for its strict specificity against L. monocytogenes. Such specificity, together with its stability, propose this new antimicrobial as a tool for potential biotechnological applications in the fight against the dangerous food-borne pathogen L. monocytogenes.

Genome Mining and Comparative Genome Analysis Revealed Niche-Specific Genome Expansion in Antibacterial Bacillus pumilus Strain SF-4.

The present study reports the isolation of antibacterial exhibiting Bacillus pumilus (B. pumilus) SF-4 from soil field. The genome of this strain SF-4 was sequenced and analyzed to acquire in-depth genomic level insight related to functional diversity, evolutionary history, and biosynthetic potential. The genome of the strain SF-4 harbor 12 Biosynthetic Gene Clusters (BGCs) including four Non-ribosomal peptide synthetases (NRPSs), two terpenes, and one each of Type III polyketide synthases (PKSs), hybrid (NRPS/PKS), lipopeptide, ß-lactone, and bacteriocin clusters. Plant growth-promoting genes associated with de-nitrification, iron acquisition, phosphate solubilization, and nitrogen metabolism were also observed in the genome. Furthermore, all the available complete genomes of B. pumilus strains were used to highlight species boundaries and diverse niche adaptation strategies. Phylogenetic analyses revealed local diversification and indicate that strain SF-4 is a sister group to SAFR-032 and 150a. Pan-genome analyses of 12 targeted strains showed regions of genome plasticity which regulate function of these strains and proposed direct strain adaptations to specific habitats. The unique genome pool carries genes mostly associated with "biosynthesis of secondary metabolites, transport, and catabolism" (Q), "replication, recombination and repair" (L), and "unknown function" (S) clusters of orthologous groups (COG) categories. Moreover, a total of 952 unique genes and 168 exclusively absent genes were prioritized across the 12 genomes. While newly sequenced B. pumilus SF-4 genome consists of 520 accessory, 59 unique, and seven exclusively absent genes. The current study demonstrates genomic differences among 12 B. pumilus strains and offers comprehensive knowledge of the respective genome architecture which may assist in the agronomic application of this strain in future.

A Novel Microbial Zearalenone Transformation through Phosphorylation.

Zearalenone (ZEA) is a mycotoxin widely occurring in many agricultural commodities. In this study, a purified bacterial isolate, Bacillus sp. S62-W, obtained from one of 104 corn silage samples from various silos located in the United States, exhibited activity to transform the mycotoxin ZEA. A novel microbial transformation product, ZEA-14-phosphate, was detected, purified, and identified by HPLC, LC-MS, and NMR analyses. The isolate has been identified as belonging to the genus Bacillus according to phylogenetic analysis of the 16S rRNA gene and whole genome alignments. The isolate showed high efficacy in transforming ZEA to ZEA-14-phosphate (100% transformation within 24 h) and possessed advantages of acid tolerance (work at pH = 4.0), working under a broad range of temperatures (22-42 °C), and a capability of transforming ZEA at high concentrations (up to 200 µg/mL). In addition, 23 Bacillus strains of various species were tested for their ZEA phosphorylation activity. Thirteen of the Bacillus strains showed phosphorylation functionality at an efficacy of between 20.3% and 99.4% after 24 h incubation, suggesting the metabolism pathway is widely conserved in Bacillus spp. This study established a new transformation system for potential application of controlling ZEA although the metabolism and toxicity of ZEA-14-phosphate requires further investigation.

Characterization and engineering of two new GH9 and GH48 cellulases from a Bacillus pumilus isolated from Lake Bogoria.

OBJECTIVES: To search for new alkaliphilic cellulases and to improve their efficiency on crystalline cellulose through molecular engineering RESULTS: Two novel cellulases, BpGH9 and BpGH48, from a Bacillus pumilus strain were identified, cloned and biochemically characterized. BpGH9 is a modular endocellulase belonging to the glycoside hydrolase 9 family (GH9), which contains a catalytic module (GH) and a carbohydrate-binding module belonging to class 3 and subclass c (CBM3c). This enzyme is extremely tolerant to high alkali pH and remains significantly active at pH 10. BpGH48 is an exocellulase, belonging to the glycoside hydrolase 48 family (GH48) and acts on the reducing end of oligo-ß1,4 glucanes. A truncated form of BpGH9 and a chimeric fusion with an additional CBM3a module was constructed. The deletion of the CBM3c module results in a significant decline in the catalytic activity. However, fusion of CBM3a, although in a non native position, enhanced the activity of BpGH9 on crystalline cellulose. CONCLUSIONS: A new alkaliphilic endocellulase BpGH9, was cloned and engineered as a fusion protein (CBM3a-BpGH9), which led to an improved activity on crystalline cellulose.

Salt-tolerant plant growth-promoting Bacillus pumilus strain JPVS11 to enhance plant growth attributes of rice and improve soil health under salinity stress.

Rice (Oryza sativa L.) growth and productivity has been negatively affected due to high soil salinity. However, some salt-tolerant plant growth-promoting bacteria (ST-PGPB) enhance crop growth and reduce the negative impacts of salt stress through regulation of some biochemical, physiological, and molecular features. Total thirty six ST-PGPB were isolated from sodic soil of eastern Uttar Pradesh, India, and screened for salt tolerance at different salt (NaCl) concentrations up to 2000 millimolar (mM). Out of thirty-six, thirteen strains indicated better growth and plant growth properties (PGPs) in NaCl amended medium. Among thirteen, one most effective Bacillus pumilus strain JPVS11 was molecularly characterized, which showed potential PGPs, such as indole-3-acetic acid (IAA),1-aminocyclo propane-1-carboxylicacid (ACC) deaminase activity, P-solubilization, proline accumulation and exopolysaccharides (EPS) production at different concentrations of NaCl (0 -1200 mM). Pot experiment was conducted on rice (Variety CSR46) at different NaCl concentrations (0, 50, 100, 200, and 300 mM) with and without inoculation of Bacillus pumilus strain JPVS11. At elevated concentrations of NaCl, the adverse effects on chlorophyll content, carotenoids, antioxidant activity was recorded in non-inoculated (only NaCl) plants. However, inoculation of Bacillus pumilus strain JPVS11 showed positive adaption and improve growth performance of rice as compared to non-inoculated in similar conditions. A significant (P < 0.05) enhancement plant height (12.90-26.48%), root length (9.55-23.09%), chlorophyll content (10.13-27.24%), carotenoids (8.38-25.44%), plant fresh weight (12.33-25.59%), and dry weight (8.66-30.89%) were recorded from 50 to 300 mM NaCl concentration in inoculated plants as compared to non-inoculated. Moreover, the plants inoculated with Bacillus pumilus strain JPVS11showed improvement in antioxidant enzyme activities of catalase (15.14-32.91%) and superoxide dismutase (8.68-26.61%). Besides, the significant improvement in soil enzyme activities, such as alkaline phosphatase (18.37-53.51%), acid phosphatase (28.42-45.99%), urease (14.77-47.84%), and ß-glucosidase (25.21-56.12%) were recorded in inoculated pots as compared to non-inoculated. These results suggest that Bacillus pumilus strain JPVS11 is a potential ST-PGPB for promoting plant growth attributes, soil enzyme activities, microbial counts, and mitigating the deleterious effects of salinity in rice.