Genome mining conformance to metabolite profile of Bacillus strains to control potato pathogens.

Biocontrol agents are safe and effective methods for controlling plant disease pathogens, such as Fusarium solani, which causes dry wilt, and Pectobacterium spp., responsible for potato soft rot disease. Discovering agents that can effectively control both fungal and bacterial pathogens in potatoes has always presented a challenge. Biological controls were investigated using 500 bacterial strains isolated from rhizospheric microbial communities, along with two promising biocontrol strains: Pseudomonas (T17-4 and VUPf5). Bacillus velezensis (Q12 and US1) and Pseudomonas chlororaphis VUPf5 exhibited the highest inhibition of fungal growth and pathogenicity in both laboratory (48%, 48%, 38%) and greenhouse (100%, 85%, 90%) settings. Q12 demonstrated better control against bacterial pathogens in vivo (approximately 50%). Whole-genome sequencing of Q12 and US1 revealed a genome size of approximately 4.1 Mb. Q12 had 4413 gene IDs and 4300 coding sequences, while US1 had 4369 gene IDs and 4255 coding sequences. Q12 exhibited a higher number of genes classified under functional subcategories related to stress response, cell wall, capsule, levansucrase synthesis, and polysaccharide metabolism. Both Q12 and US1 contained eleven secondary metabolite gene clusters as identified by the antiSMASH and RAST servers. Notably, Q12 possessed the antibacterial locillomycin and iturin A gene clusters, which were absent in US1. This genetic information suggests that Q12 may have a more pronounced control over bacterial pathogens compared to US1. Metabolic profiling of the superior strains, as determined by LC/MS/MS, validated our genetic findings. The investigated strains produced compounds such as iturin A, bacillomycin D, surfactin, fengycin, phenazine derivatives, etc. These compounds reduced spore production and caused deformation of the hyphae in F. solani. In contrast, B. velezensis UR1, which lacked the production of surfactin, fengycin, and iturin, did not affect these structures and failed to inhibit the growth of any pathogens. Our findings suggest that locillomycin and iturin A may contribute to the enhanced control of bacterial pectolytic rot by Q12.

Comprehensive genome analysis of Pseudomonas sp. SWRIQ11, a new plant growth-promoting bacterium that alleviates salinity stress in olive.

The study presents the genome analysis of a new Pseudomonas sp. (SWRIQ11), which can alleviate salinity stress effects on growth of olive seedlings in greenhouse study. The strain SWRIQ11 can tolerate salinity up to 6%, produce siderophores, indole acetic acid (IAA), aminocyclopropane-1-carboxylate (ACC) deaminase, and has the phosphate-solubilizing capability. The SWRIQ11 genome contained an assembly size of 6,196,390 bp with a GC content of 60.1%. According to derived indices based on whole-genome sequences for species delineation, including tetra nucleotide usage patterns (TETRA), genome-to-genome distance (GGDC), and average nucleotide identity (ANI), Pseudomonas sp. SWRIQ11 can be considered a novel species candidate. The phylogenetic analysis revealed SWRIQ11 clusters with Pseudomonas tehranensis SWRI196 in the same clade. The SWRIQ11 genome was rich in genes related to stress sensing, signaling, and response, chaperones, motility, attachments, colonization, and enzymes for degrading plant-derived carbohydrates. Furthermore, the genes for production of exopolysaccharides, osmoprotectants, phytohormones, and ACC deaminase, ion homeostasis, nutrient acquisition, and antioxidant defenses were identified in the SWRIQ11 genome. The results of genome analysis (identification of more than 825 CDSs related to plant growth-promoting and stress-alleviating traits in the SWRIQ11 genome which is more than 15% of its total CDSs) are in accordance with laboratory and greenhouse experiments assigning the Pseudomonas sp. SWRIQ11 as a halotolerant plant growth-promoting bacterium (PGPB). This research highlights the potential safe application of this new PGPB species in agriculture as a potent biofertilizer.

Gene cloning and characterization of a novel recombinant 40-kDa heat shock protein from Mesobacillus persicus B48.

The present study reports the recognition and characterization of the gene encoding the co-chaperone DnaJ in the halophilic strain Mesobacillus persicus B48. The new extracted gene was sequenced and cloned in E. coli, followed by protein purification using a C-terminal His-tag. The stability and function of the recombinant DnaJ protein under salt and pH stress conditions were evaluated. SDS-PAGE revealed a band on nearly 40-kDa region. The homology model structure of new DnaJ demonstrated 56% similarity to the same protein from Streptococcus pneumonia. Fluorescence spectra indicated several hydrophobic residues located on the protein surface, which is consistent with the misfolded polypeptide recognition function of DnaJ. Spectroscopic results showed 56% higher carbonic anhydrase activity in the presence of the recombinant DnaJ homolog compared to its absence. In addition, salt resistance experiments showed that the survival of recombinant E. coli+DnaJ was 2.1 times more than control cells in 0.5 M NaCl. Furthermore, the number of recombinant E. coli BL21+DnaJ colonies was 7.7 times that of the control colonies in pH 8.5. Based on the results, DnaJ from the M. persicus can potentially be employed for improving the functional features of enzymes and other proteins in various applications.

Salinity stress endurance of the plants with the aid of bacterial genes.

The application of plant growth-promoting bacteria (PGPB) is vital for sustainable agriculture with continuous world population growth and an increase in soil salinity. Salinity is one of the severe abiotic stresses which lessens the productivity of agricultural lands. Plant growth-promoting bacteria are key players in solving this problem and can mitigate salinity stress. The highest of reported halotolerant Plant growth-promoting bacteria belonged to Firmicutes (approximately 50%), Proteobacteria (40%), and Actinobacteria (10%), respectively. The most dominant genera of halotolerant plant growth-promoting bacteria are Bacillus and Pseudomonas. Currently, the identification of new plant growth-promoting bacteria with special beneficial properties is increasingly needed. Moreover, for the effective use of plant growth-promoting bacteria in agriculture, the unknown molecular aspects of their function and interaction with plants must be defined. Omics and meta-omics studies can unreveal these unknown genes and pathways. However, more accurate omics studies need a detailed understanding of so far known molecular mechanisms of plant stress protection by plant growth-promoting bacteria. In this review, the molecular basis of salinity stress mitigation by plant growth-promoting bacteria is presented, the identified genes in the genomes of 20 halotolerant plant growth-promoting bacteria are assessed, and the prevalence of their involved genes is highlighted. The genes related to the synthesis of indole acetic acid (IAA) (70%), siderophores (60%), osmoprotectants (80%), chaperons (40%), 1-aminocyclopropane-1-carboxylate (ACC) deaminase (50%), and antioxidants (50%), phosphate solubilization (60%), and ion homeostasis (80%) were the most common detected genes in the genomes of evaluated halotolerant plant growth-promoting and salinity stress-alleviating bacteria. The most prevalent genes can be applied as candidates for designing molecular markers for screening of new halotolerant plant growth-promoting bacteria.

Functional and phylogenetic analyses of camel rumen microbiota associated with different lignocellulosic substrates.

Rumen microbiota facilitates nutrition through digestion of recalcitrant lignocellulosic substrates into energy-accessible nutrients and essential metabolites. Despite the high similarity in rumen microbiome structure, there might be distinct functional capabilities that enable different ruminant species to thrive on various lignocellulosic substrates as feed. Here, we applied genome-centric metagenomics to explore phylogenetic diversity, lignocellulose-degrading potential and fermentation metabolism of biofilm-forming microbiota colonizing 11 different plant substrates in the camel rumen. Diversity analysis revealed significant variations in the community of rumen microbiota colonizing different substrates in accordance with their varied physicochemical properties. Metagenome reconstruction recovered genome sequences of 590 bacterial isolates and one archaeal lineage belonging to 20 microbial phyla. A comparison to publicly available reference genomes and rumen metagenome-assembled genomes revealed that most isolates belonged to new species with no well-characterized representatives. We found that certain low abundant taxa, including members of Verrucomicrobiota, Planctomycetota and Fibrobacterota, possessed a disproportionately large number of carbohydrate active enzymes per Mb of genome, implying their high metabolic potential to contribute to the rumen function. In conclusion, we provided a detailed picture of the diversity and functional significance of rumen microbiota colonizing feeds of varying lignocellulose composition in the camel rumen. A detailed analysis of 591 metagenome-assembled genomes revealed a network of interconnected microbiota and highlighted the key roles of certain taxonomic clades in rumen function, including those with minimal genomes (e.g., Patescibacteria). The existence of a diverse array of gene clusters encoding for secondary metabolites unveiled the specific functions of these biomolecules in shaping community structure of rumen microbiota.

Cloning, purification and biochemical characterization of two glutathione S-transferase isoforms from Rutilus frisii kutum.

Glutathione S-transferases are an important multifunctional family of intracellular enzymes that their detoxification function has been reported in fishes since 1970, but no studies have been conducted on Rutilus frisii kutum GSTs yet. In the present study, RkGSTA and RkGSTM encoding genes were cloned and sequenced and their nucleotide sequences were submitted to NCBI GenBank. In order to reduce the expression challenges of recombinant proteins including low solubility, low yield and insufficient purity issues in E. coli, the pKJE7 chaperone plasmid was used to increase the recovery of expressed proteins in the soluble fractions. Best expression clone was selected for purification by Ni-NTA affinity chromatography. The three-dimensional structural models were constructed by I-TASSER. The optimum temperature of purified RkGSTA and RkGSTM was 35 and 30 °C, with optimum activity at pH 9.0 and 8.5, respectively. The thermostability and pH stability results indicated that RkGSTA is more heat-tolerant than RkGSTM though both of them retained more than 80% of their activities at pH 6.5 to 9.0. Overall, this study represents a comprehensive perspective on the structural and biochemical aspects of this enzyme that would be even used in further researches such as drug design studies in order to eliminate toxicant compounds from the body and environment of fishes to protect them against undesired harmful damages.

Molecular cloning, expression, and functional characterization of 70-kDa heat shock protein, DnaK, from Bacillus halodurans.

In the present study, we report cloning, sequencing, and functional characterization dnaK gene of B. halodurans that is the central component in cellular network of molecular chaperones. The 3D structures of DnaK obtained by I-TASSER server showed that the overall structures of DnaK from B. halodurans and human HSP70 chaperone BiP are very similar with a homology of 88.8%. The purified recombinant DnaK consists of a His-tag at C-terminus and show a band on approximately 70-kDa region in SDS-PAGE. The resultant refolding assay revealed that the refolding rate was considerably improved by the addition of the novel DnaK chaperone for the refolding of heat-denatured carbonic anhydrase. Also, salt resistance experiments indicated that E. coli + DnaK survival had enhanced by 4.4-fold as compared with control cells in 0.4 M NaCl. The number of E. coli + DnaK colonies was 2.5-fold higher than control colonies in pH 9.5. We showed that DnaK refolding functions were decreased by increasing Cd2+ in nanomolar concentrations. Hg2+ had a biphasic effect on recombinant DnaK refolding function: inhibition at low and stimulation at high concentrations. It was concluded that the DnaK from B. halodurans can potentially be employed for improving functional properties of proteins in various applications.

Molecular cloning, prokaryotic expression, purification, structural studies and functional implications of Heat Shock Protein 70 (Hsp70) from Rutilus frisii kutum.

A novel Hsp70 chaperone from Rutilus frisii kutum was identified, cloned, expressed, purified and its functional characteristics revealed. The 3D structure of Hsp70 from Rutilus kutum was constructed using the crystal structure of E. coli Hsp70 as the template, with 47% sequence identity. The in vitro ATPase activity assay after 60min, ATP hydrolysis of purified recombinant Hsp70 (8µM) was improved by binding to denatured thermally luciferase (3µM) about 2.5-fold compared with that of Hsp70 alone. Based on the results, it was found that the purified Hsp70 chaperone was able to considerably suppress heat-induced aggregation of luciferase by binding to DnaJ co-chaperone (5µM) more than 70% after 10min at 42°C. In addition, Hsp70 DnaJ complex improved the refolding of heat-shocked luciferase nearly 40% after 60min at 25°C. It was concluded that Hsp70 protein from Rutilus frisii kutum has the critical role in preventing heat-induced aggregation of luciferase and refolding of heat-denatured luciferase was strictly dependent on the activity of Hsp70, thus, this protein can potentially be used for improving the functional properties of luciferase in various applications.

The dynamics of the bacterial communities developed in maize silage.

Ensilage provides an effective means of conserving summer-grown green forage to supply as winter feed to ruminants. The fermentation process involved in the ensilage process relies on lactic acid bacteria (LAB). Here, 16S ribosomal DNA amplicon pyrosequencing was used to follow the dynamic behaviour of the LAB community during the ensilage of maize biomass, with a view to identify the key species involved in the process. The biomass used for ensilage was a single-cross maize hybrid, harvested at the milk-line stage. The crop was grown at three contrasting locations. Aspects of the physico-chemical composition of the material and the LAB species present were sampled at 0, 3, 6, 14, 21 and 32 days after ensilage was initiated. In all three cases, members of the Leuconostocaceae family dominated the epiphytic bacterial community, notably Leuconostoc and Weissella, but some variation was noted in the abundance of certain Leuconostocaceae and Lactobacillaceae species, as well as that of some Acetobacteraceae, Enterobacteriaceae and Moraxellaceae species. The constellation of the microbiome which developed during the ensilage process differed markedly from that of the epiphytic one, with Lactobacillaceae, particularly Lactobacillus and Pediococcus spp. dominating. The abundance of heterofermentative Leuconostocaceae spp. in the epiphytic community and the extent of the transition from hetero- to homo-fermentation during the initial ensilage period are important factors in determining silage quality.

Purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis (X5B).

This study reports the purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis, strain X5B. The enzyme was purified to homogeneity by acetone precipitation, ultrafiltration and carboxymethyl (CM) cation exchange chromatography, respectively. The purified protease was a monomeric enzyme with a relative molecular mass of 48-50 kDa and it was inhibited by PMSF indicating that it is a serine-protease. The optimum pH, temperature and NaCl concentration were 9.5, 35 °C and 0.98 M, respectively. The enzyme showed a significant tolerance to salt and alkaline pH. It retained approximately 50% of activity at 2.5 M NaCl and about 70% of activity at highly alkaline pH of 11.0; therefore, it was a moderately halophilic and also can be activated by metals, especially by Ca(2+). The specific activity of the purified protease was measured to be 425.23 µmol of tyrosine/min per mg of protein using casein as a substrate. The apparent K m and V max values were 0.126 mM and 0.523 mM/min, respectively and the accurate value of k cat was obtained as 3.284 × 10(-2) s(-1). These special and important characteristics make this serine protease as valuable tool for industrial applications.

Complete genome sequence of Oceanimonas sp. GK1, a halotolerant bacterium from Gavkhouni Wetland in Iran.

Oceanimonas sp. GK1 (IBRC-M 10197) is a marine halotolerant gammaproteobacterium which was characterized as producing large amounts of poly-ß-hydroxybutyrate. Here we present the whole-genome sequence of Oceanimonas sp. GK1, which consists of a single circular chromosome of 3,514,537 bp and two plasmids 8,462 and 4,245 bp in length.

Draft genome sequence of Nesterenkonia sp. strain F, isolated from Aran-Bidgol Salt Lake in Iran.

The draft genome of the aerobic, Gram-positive, halophilic chemoorganotroph Nesterenkonia sp. strain F consists of a 2,812,133-bp chromosome. This study is the first to report the shotgun-sequenced draft genome of a member of the genus Nesterenkonia.