Isolation and identification of a new Bacillus glycinifermentans strain from date palm rhizosphere and its effect on barley seeds under heavy metal stress.

Soil contamination by heavy metals is one of the major problems that adversely decrease plant growth and biomass production. Inoculation with the plant growth-promoting rhizobacteria (PGPR) can attenuate the toxicity of heavy metals and enhancing the plant growth. In this study, we evaluated the potential of a novel extremotolerant strain (IS-2 T) isolated from date palm rhizosphere to improve barley seedling growth under heavy metal stress. The species-level identification was carried out using morphological and biochemical methods combined with whole genome sequencing. The bacterial strain was then used in vitro for inoculating Hordeum vulgare L. exposed to three different Cr, Zn, and Ni concentrations (0.5, 1, and 2 mM) in petri dishes and different morphological parameters were assessed. The strain was identified as Bacillus glycinifermentans species. This strain showed high tolerance to pH (6-11), salt stress (0.2-2 M), and heavy metals. Indeed, the minimum inhibitory concentrations at which bacterium was unable to grow were 4 mM for nickel, 3 mM for zinc, more than 8 mM for copper, and 40 mM for chromium, respectively. It was observed that inoculation of Hordeum vulgare L. under metal stress conditions with Bacillus glycinifermentans IS-2 T stain improved considerably the growth parameters. The capacity of the IS-2 T strain to withstand a range of abiotic stresses and improve barley seedling development under lab conditions makes it a promising candidate for use as a PGPR in zinc, nickel, copper, and chromium bioremediation.

A fast and accurate method for specific detection and quantification of the bloom-forming microalgae Karlodinium veneficum in the marine environment.

Karlodinium veneficum is a toxic benthic globally distributed dinoflagellate which has direct impacts on human health and the environment. Early and accurate detection of this harmful algal bloom-forming species could be useful for potential risks monitoring and management. In the present work, a real-time PCR targeting the internal transcribed spacer ribosomal DNA region for the specific detection and absolute quantification of K. veneficum was designed. Then, the assay conditions were adjusted and validated. The developed qPCR was highly specific for the target species and displayed no cross-reactivity with closely related dinoflagellates and/or other microalgal species commonly distributed along the Tunisian coast. Its lowest detection limit was 5 rDNA copies per reaction, which is often considered satisfying. qPCR assay enumeration accuracy was evaluated using artificially inoculated environmental samples. The comparison of the cell abundance estimates obtained by qPCR assay with the theoretical estimates showed no statistically significant difference across a range of concentrations. We suggest that the qPCR approach developed in the present study may be a valuable tool to investigate the distribution and seasonal dynamics of K. veneficum in marine environments.

In silico evidence of antiviral activity against SARS-CoV-2 main protease of oligosaccharides from Porphyridium sp.

The coronavirus pandemic (COVID-19) has created an urgent need to develop effective strategies for prevention and treatment. In this context, therapies against protease Mpro, a conserved viral target, would be essential to contain the spread of the virus and reduce mortality. Using combined techniques of structure modelling, in silico docking and pharmacokinetics prediction, many compounds from algae were tested for their ability to inhibit the SARS-CoV-2 main protease and compared to the recent recognized drug Paxlovid. The screening of 27 algal molecules including 15 oligosaccharides derived from sulfated and non-sulphated polysaccharides, eight pigments and four poly unsaturated fatty acids showed high affinities to interact with the protein active site. Best candidates showing high docking scores in comparison with the reference molecule were sulfated tri-, tetra- and penta-saccharides from Porphyridium sp. exopolysaccharides (SEP). Structural and energetic analyses over 100 ns MD simulation demonstrated high SEP fragments-Mpro complex stability. Pharmacokinetics predictions revealed the prospects of the identified molecules as potential drug candidates.

Apigenin analogues as SARS-CoV-2 main protease inhibitors: In-silico screening approach.

The COVID-19 new variants spread rapidly all over the world, and until now scientists strive to find virus-specific antivirals for its treatment. The main protease of SARS-CoV-2 (Mpro) exhibits high structural and sequence homology to main protease of SARS-CoV (93.23% sequence identity), and their sequence alignment indicated 12 mutated/variant residues. The sequence alignment of SARS-CoV-2 main protease led to identification of only one mutated/variant residue with no significant role in its enzymatic process. Therefore, Mpro was considered as a high-profile drug target in anti-SARS-CoV-2 drug discovery. Apigenin analogues to COVID-19 main protease binding were evaluated. The detailed interactions between the analogues of Apigenin and SARS-CoV-2 Mpro inhibitors were determined as hydrogen bonds, electronic bonds and hydrophobic interactions. The binding energies obtained from the molecular docking of Mpro with Boceprevir, Apigenin, Apigenin 7-glucoside-4'-p-coumarate, Apigenin 7-glucoside-4'-trans-caffeate and Apigenin 7-O-beta-d-glucoside (Cosmosiin) were found to be -6.6, -7.2, -8.8, -8.7 and -8.0 kcal/mol, respectively. Pharmacokinetic parameters and toxicological characteristics obtained by computational techniques and Virtual ADME studies of the Apigenin analogues confirmed that the Apigenin 7-glucoside-4'-p-coumarate is the best candidate for SARS-CoV-2 Mpro inhibition.

Over-expression of the bacterial phytase US417 in Arabidopsis reduces the concentration of phytic acid and reveals its involvement in the regulation of sulfate and phosphate homeostasis and signaling.

Phytic acid (PA) is the main phosphorus storage form in plant seeds. It is recognized as an anti-nutrient for humans and non-ruminant animals, as well as one of the major sources of phosphorus that contributes to eutrophication. Therefore, engineering plants with low PA content without affecting plant growth capacity has become a major focus in plant breeding. Nevertheless, lack of knowledge on the role of PA seed reserves in regulating plant growth and in maintaining ion homeostasis hinders such an agronomical application. In this context, we report here that the over-expression of the bacterial phytase PHY-US417 in Arabidopsis leads to a significant decrease in seed PA, without any effect on the seed germination potential. Interestingly, this over-expression also induced a higher remobilization of free iron during germination. Moreover, the PHY-over-expressor lines show an increase in inorganic phosphate and sulfate contents, and a higher biomass production after phosphate starvation. Finally, phosphate sensing was altered because of the changes in the expression of genes induced by phosphate starvation or involved in phosphate or sulfate transport. Together, these results show that the over-expression of PHY-US417 reduces PA concentration, and provide the first evidence for the involvement of PA in the regulation of sulfate and phosphate homeostasis and signaling.

Overexpression and biochemical characterization of a thermostable phytase from Bacillus subtilis US417 in Pichia pastoris.

The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50-65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100% of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.

Characterization of the mineral phosphate solubilizing activity of Serratia marcescens CTM 50650 isolated from the phosphate mine of Gafsa.

The mineral phosphate solubilizing (MPS) ability of a Serratia marcescens strain, namely CTM 50650, isolated from the phosphate mine of Gafsa, was characterized on a chemically defined medium (NBRIP broth). Various insoluble inorganic phosphates, including rock phosphate (RP), calcium phosphate (CaHPO(4)), tri-calcium phosphate (Ca(3)(PO(4))(2)) and hydroxyapatite were tested as sole sources of phosphate for bacterial growth. Solubilization of these phosphates by S. marcescens CTM 50650 was very efficient. Indeed, under optimal conditions, the soluble phosphorus (P) concentration it produced reached 967, 500, 595 and 326 mg/l from CaHPO(4), Ca(3)(PO(4))(2), hydroxyapatite and RP, respectively. Study of the mechanisms involved in the MPS activity of CTM 50650, showed that phosphate solubilization was concomitant with significant drop in pH. HPLC-analysis of culture supernatants revealed the secretion of gluconic acid (GA) resulting from direct oxidation pathway of glucose when the CTM 50650 cells were grown on NBRIP containing glucose as unique carbon source. This was correlated with the simultaneous detection by PCR for the first time in a S. marcescens strain producing GA, of a gene encoding glucose dehydrogenase responsible for GA production, as well as the genes pqqA, B, C and E involved in biosynthesis of its PQQ cofactor. This study is expected to lead to the development of an environmental-friendly process for fertilizer production considering the capacity of S. marcescens CTM 50650 to achieve yields of P extraction up to 75% from the Gafsa RP.

Gene cloning and characterization of a thermostable phytase from Bacillus subtilis US417 and assessment of its potential as a feed additive in comparison with a commercial enzyme.

An extracellular phytase from Bacillus subtilis US417 (PHY US417) was purified and characterized. The purified enzyme of 41 kDa was calcium-dependent and optimally active at pH 7.5 and 55 degrees C. The thermal stability of PHY US417 was drastically improved by calcium. Indeed, it recovered 77% of its original activity after denaturation for 10 min at 75 degrees C in the presence of 5 mM CaCl2, while it retained only 22% of activity when incubated for 10 min at 60 degrees C without calcium. In addition, PHY US417 was found to be highly specific for phytate and exhibited pH stability similar to Phyzyme, a commercial phytase with optimal activity at pH 5.5 and 60 degrees C. The phytase gene was cloned by PCR from Bacillus subtilis US417. Sequence analysis of the encoded polypeptide revealed one residue difference from PhyC of Bacillus subtilis VTTE-68013 (substitution of arginine in position 257 by proline in PHY US417) which was reported to exhibit lower thermostability especially in the absence of calcium. With its neutral pH optimum as well as its great pH and thermal stability, the PHY US417 enzyme presumed to be predominantly active in the intestine has a high potential for use as feed additive.