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The global rise of antimicrobial resistance (AMR) presents a critical challenge necessitating the discovery of novel antimicrobial agents. Mangrove microbes are valuable sources of new antimicrobial compounds. This study reports the discovery of a potent antimicrobial peptide (AMP) from Bacillus paralicheniformis NNS4-3, isolated from mangrove sediment, exhibiting significant activity against methicillin-resistant Staphylococcus aureus (MRSA). The AMP demonstrated a minimum inhibitory concentration ranging from 1 to 16 µg/mL in the tested bacteria and exhibited bactericidal effects at higher concentrations. Structural analysis revealed a bacitracin-like configuration and the peptide acted by disrupting bacterial membranes in a time- and concentration-dependent manner. The AMP maintained stability under heat, proteolytic enzymes, surfactants, and varying pH treatments. The ten biosynthetic gene clusters (BGCs) of secondary metabolites were found in the genome. Detailed sequence comparison of the predicted bacitracin BGC indicated distinct DNA sequences compared to previously reported strains. Although the antibiotic resistance genes were found, this strain was susceptible to antibiotics. Our findings demonstrated the potential of Bacillus paralicheniformis NNS4-3 and its AMP as a promising agent in combating AMR. The genetic information could be pivotal for future applications in the healthcare industry, emphasizing the need for continued exploration of marine microbial diversity in drug discovery. [ABSTRACT FROM AUTHOR]

Bacillus paralicheniformis is a new identified species, which was distinguished from Bacillus licheniformis in 2015 through extensive phylogenomic and phylogenetic analyses. In this context, this study aimed to achieve a clear identification of the active plant-growth promoting rhizobacteria (PGPR) B. paralicheniformis isolates among the closely related B. licheniformis through molecular typing, helping for the development of clearly-identified PGPR isolates to be used as biofertilizers. A total of 15 rhizobacteria were isolated from the olive rhizosphere soils. These bacterial isolates exhibited various proprieties in terms of abiotic stress tolerance, biofilm formation under stress conditions, and enzyme activities (i.e., lipases, cellulases, and proteases). In addition, several PGP traits such as phosphate solubilization and the production of siderophores and indol-3 acetic acid were also observed. Molecular identification through 16S rRNA sequencing initially identified all the isolates as Bacillus spp. The multi-locus sequence typing (MLST) scheme, using six housekeeping genes (adk, ccpA, recF, rpoB, sucC, and spo0A) had unveiled distinct allelic profiles resulting in 13 unique sequence types (ST). Notably, a comprehensive analysis indicated no exact allele matches existed between the examined isolates and those documented in the PubMLST database. Among the six housekeeping genes, we noticed that rpoB gene (RNA polymerase, subunit beta) had multiple polymorphic sites that were bordered by conserved sequences. Furthermore, the phylogenetic analysis based on rpoB had identified clusters indicating distinct phylogenetic relationships between B. paralicheniformis and B. licheniformis, and successfully differentiated the two species from a pool of 90 strains. The rpoB partial sequence proved to be effective for accurate species discrimination.

The food enzyme subtilisin (EC 3.4.21.62) is produced with the non‐genetically modified Bacillus paralicheniformis strain AP‐01 by Nagase (Europa) GmbH. It was considered free from viable cells of the production organism. The food enzyme is intended to be used in five food manufacturing processes. Since residual amounts of food enzyme‐total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining four food manufacturing processes. It was estimated to be up to 0.875 mg TOS/kg body weight per day in European populations. The production strain of the food enzyme has the capacity to produce bacitracin and thus failed to meet the requirements of the Qualified Presumption of Safety approach. Bacitracin was detected in the industrial fermentation medium but not in the food enzyme itself. However, the limit of detection of the analytical method used for bacitracin was not sufficient to exclude the possible presence of bacitracin at a level representing a risk for the development of antimicrobial resistant bacteria. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and twenty‐eight matches with respiratory allergens, one match with a contact allergen and two matches with food allergens (melon and pomegranate) were found. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to melon or pomegranate, cannot be excluded, but would not exceed the risk of consuming melon or pomegranate. Based on the data provided, the Panel could not exclude the presence of bacitracin, a medically important antimicrobial, and consequently the safety of this food enzyme could not be established. [ABSTRACT FROM AUTHOR]

In this study, a novel strain for degrading chitin was identified as Bacillus paralicheniformis HL37, and the key chitinase CH1 was firstly mined through recombinant expression in Bacillus amyloliquefaciens HZ12. Subsequently, the sequence composition and catalytic mechanism of CH1 protein were analyzed. The molecular docking indicated that the triplet of Asp526, Asp528, and Glu530 was a catalytic active center. The enzymatic properties analysis revealed that the optimal reaction temperature and pH was 65 °C and 6.0, respectively. Especially, the chitinase activity showed no significant change below 55 °C and it could maintain over 60% activity after exposure to 85 °C for 30 min. Moreover, the optimal host strain and signal peptide were obtained to enhance the expression of chitinase CH1 significantly. As far as we know, it was the first time finding the highly efficient chitin-degrading enzymes in B. paralicheniformis, and detailed explanations were provided on the catalytic mechanism and enzymatic properties on CH1. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme subtilisin (EC 3.4.21.62) is produced with the non‐genetically modified Bacillus paralicheniformis strain AP‐01 by Nagase (Europa) GmbH. It was considered free from viable cells of the production organism. The food enzyme is intended to be used in five food manufacturing processes. Since residual amounts of food enzyme‐total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining four food manufacturing processes. It was estimated to be up to 0.875 mg TOS/kg body weight per day in European populations. The production strain of the food enzyme has the capacity to produce bacitracin and thus failed to meet the requirements of the Qualified Presumption of Safety approach. Bacitracin was detected in the industrial fermentation medium but not in the food enzyme itself. However, the limit of detection of the analytical method used for bacitracin was not sufficient to exclude the possible presence of bacitracin at a level representing a risk for the development of antimicrobial resistant bacteria. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and twenty‐eight matches with respiratory allergens, one match with a contact allergen and two matches with food allergens (melon and pomegranate) were found. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to melon or pomegranate, cannot be excluded, but would not exceed the risk of consuming melon or pomegranate. Based on the data provided, the Panel could not exclude the presence of bacitracin, a medically important antimicrobial, and consequently the safety of this food enzyme could not be established.

Abstract The biosynthetic process of selenium nanoparticles (SeNPs) by specific bacterial strain, whose growth directly affects the synthesis efficiency, has attracted great attentions. We previously reported that Bacillus paralicheniformis SR14, a SeNPs-producing bacteria, could improve intestinal antioxidative function in vitro. To further analyze the biological characteristics of SR14, whole genome sequencing was used to reveal the genetic characteristics in selenite reduction and sugar utilization. The results reviewed that the genome size of SR14 was 4,448,062 bp, with a GC content of 45.95%. A total of 4300 genes into 49 biological pathways was annotated to the KEGG database. EC: 1.1.1.49 (glucose-6-phosphate 1-dehydrogenase) and EC: 5.3.1.9 (glucose-6-phosphate isomerase), were found to play a potential role in glucose degradation and EC:2.7.1.4 (fructokinase) might be involved in the fructose metabolism. Growth profile and selenite-reducing ability of SR14 under different sugar supplements were determined and the results reviewed that glucose had a better promoting effect on the reduction of selenite and growth of bacteria than fructose, sucrose, and maltose. Moreover, RT-qPCR experiment proved that glucose supplement remarkably promoted the expressions of thioredoxin, fumarate reductase, and the glutathione peroxidase in SR14. Analysis of mRNA expression showed levels of glucose-6-phosphate dehydrogenase and fructokinase significantly upregulated under the supplement of glucose. Overall, our data demonstrated the genomic characteristics of SR14 and preliminarily determined that glucose supplement was most beneficial for strain growth and SeNPs synthesis.

The global rise of antimicrobial resistance (AMR) presents a critical challenge necessitating the discovery of novel antimicrobial agents. Mangrove microbes are valuable sources of new antimicrobial compounds. This study reports the discovery of a potent antimicrobial peptide (AMP) from Bacillus paralicheniformis NNS4-3, isolated from mangrove sediment, exhibiting significant activity against methicillin-resistant Staphylococcus aureus (MRSA). The AMP demonstrated a minimum inhibitory concentration ranging from 1 to 16 µg/mL in the tested bacteria and exhibited bactericidal effects at higher concentrations. Structural analysis revealed a bacitracin-like configuration and the peptide acted by disrupting bacterial membranes in a time- and concentration-dependent manner. The AMP maintained stability under heat, proteolytic enzymes, surfactants, and varying pH treatments. The ten biosynthetic gene clusters (BGCs) of secondary metabolites were found in the genome. Detailed sequence comparison of the predicted bacitracin BGC indicated distinct DNA sequences compared to previously reported strains. Although the antibiotic resistance genes were found, this strain was susceptible to antibiotics. Our findings demonstrated the potential of Bacillus paralicheniformis NNS4-3 and its AMP as a promising agent in combating AMR. The genetic information could be pivotal for future applications in the healthcare industry, emphasizing the need for continued exploration of marine microbial diversity in drug discovery.

In this study, a novel strain for degrading chitin was identified as Bacillus paralicheniformis HL37, and the key chitinase CH1 was firstly mined through recombinant expression in Bacillus amyloliquefaciens HZ12. Subsequently, the sequence composition and catalytic mechanism of CH1 protein were analyzed. The molecular docking indicated that the triplet of Asp526, Asp528, and Glu530 was a catalytic active center. The enzymatic properties analysis revealed that the optimal reaction temperature and pH was 65 °C and 6.0, respectively. Especially, the chitinase activity showed no significant change below 55 °C and it could maintain over 60% activity after exposure to 85 °C for 30 min. Moreover, the optimal host strain and signal peptide were obtained to enhance the expression of chitinase CH1 significantly. As far as we know, it was the first time finding the highly efficient chitin-degrading enzymes in B. paralicheniformis, and detailed explanations were provided on the catalytic mechanism and enzymatic properties on CH1.

The biosynthetic process of selenium nanoparticles (SeNPs) by specific bacterial strain, whose growth directly affects the synthesis efficiency, has attracted great attentions. We previously reported that Bacillus paralicheniformis SR14, a SeNPs-producing bacteria, could improve intestinal antioxidative function in vitro. To further analyze the biological characteristics of SR14, whole genome sequencing was used to reveal the genetic characteristics in selenite reduction and sugar utilization. The results reviewed that the genome size of SR14 was 4,448,062 bp, with a GC content of 45.95%. A total of 4300 genes into 49 biological pathways was annotated to the KEGG database. EC: 1.1.1.49 (glucose-6-phosphate 1-dehydrogenase) and EC: 5.3.1.9 (glucose-6-phosphate isomerase), were found to play a potential role in glucose degradation and EC:2.7.1.4 (fructokinase) might be involved in the fructose metabolism. Growth profile and selenite-reducing ability of SR14 under different sugar supplements were determined and the results reviewed that glucose had a better promoting effect on the reduction of selenite and growth of bacteria than fructose, sucrose, and maltose. Moreover, RT-qPCR experiment proved that glucose supplement remarkably promoted the expressions of thioredoxin, fumarate reductase, and the glutathione peroxidase in SR14. Analysis of mRNA expression showed levels of glucose-6-phosphate dehydrogenase and fructokinase significantly upregulated under the supplement of glucose. Overall, our data demonstrated the genomic characteristics of SR14 and preliminarily determined that glucose supplement was most beneficial for strain growth and SeNPs synthesis. Keypoints: Whole genome sequencing was used to reveal the genetic characteristics of SR14 in selenite reduction and sugar utilization. Supplementing glucose had the best promoting effect on growth and selenite reduction of SR14, and the effect was better than supplementing sucrose, fructose, or maltose. EC:1.1.1.49 (glucose-6-phosphate 1-dehydrogenase) and EC:2.7.1.4 (fructokinase) might be associated with the metabolic process of SR14. SR14 might rely on multiple enzymes, such as thioredoxin, fumarate reductase, and glutathione peroxidase, to reduce selenite when supplementing with glucose. [ABSTRACT FROM AUTHOR]

The food enzyme subtilisin (serine endopeptidase, EC 3.4.21.62) is produced with the non‐genetically modified Bacillus paralicheniformis strain DP‐Dzx96 by Genencor International B.V. The food enzyme was considered free from viable cells of the production organism. The food enzyme is intended to be used in five food manufacturing processes: production of protein hydrolysates from plants and fungi, production of protein hydrolysates from meat and fish proteins, production of cooked rice, production of modified meat and fish products, and yeast processing. The production strain of the food enzyme contains known antimicrobial resistance genes. Bacitracin, a medically important antimicrobial, was detected in the food enzyme. The presence of bacitracin represents a risk for the development of antimicrobial resistant bacteria. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and three matches with respiratory and two matches with food allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to muskmelon or pomegranate, cannot be excluded, but would not exceed the risk of consuming these foods. Due to the presence of bacitracin, the Panel concluded that the food enzyme subtilisin produced with the non‐genetically modified Bacillus paralicheniformis strain DP‐Dzx96 cannot be considered safe. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme subtilisin (serine endopeptidase, EC 3.4.21.62) is produced with the non‐genetically modified Bacillus paralicheniformis strain DP‐Dzx96 by Genencor International B.V. The food enzyme was considered free from viable cells of the production organism. The food enzyme is intended to be used in five food manufacturing processes: production of protein hydrolysates from plants and fungi, production of protein hydrolysates from meat and fish proteins, production of cooked rice, production of modified meat and fish products, and yeast processing. The production strain of the food enzyme contains known antimicrobial resistance genes. Bacitracin, a medically important antimicrobial, was detected in the food enzyme. The presence of bacitracin represents a risk for the development of antimicrobial resistant bacteria. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and three matches with respiratory and two matches with food allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to muskmelon or pomegranate, cannot be excluded, but would not exceed the risk of consuming these foods. Due to the presence of bacitracin, the Panel concluded that the food enzyme subtilisin produced with the non‐genetically modified Bacillus paralicheniformis strain DP‐Dzx96 cannot be considered safe.

Cadmium and drought stress are the most destructive of the abiotic stresses with negative effects on both metabolism and photosynthesis. The present experiment aimed to analyze the impact of inoculation with Bacillus paralicheniformis and Trichoderma asperellum on the growth and antioxidant response modulation of maize (Zea mays L.), under drought and Cadmium (Cd) stresses. Regarding plant biomass analysis, fungi inoculation increased leaf dry biomass significantly (11.92%) towards uninoculated ones. The leaf area was affected significantly by bacterial application, 12.15% more than the control. A significant trend (drought+ Cd stress) was observed between fungi-inoculated maize leaves (15.07 µmol/g FW) and bacterial-inoculated leaves (18.71 µmol/g FW) regarding the malondialdehyde quantity. Furthermore, the activities of superoxide dismutase were notably higher (9.63–40.88%) in microorganism-inoculated roots. Similarly, under drought + Cd stress, peroxidase demonstrated a higher activity under bacterial inoculation than fungal ones (92.11% more). The maximum translocation factor was observed in the uninoculated group (under Cd stress), while the bioconcentration factor under drought stress showed a significant increase by microorganisms. The maximum relative water content under bacterial inoculation (82.66%) was achieved. The fungi and bacterial inoculation minimized Cd accumulation in the leaf significantly under drought and drought + Cd stress. Generally, the microorganism inoculation positively and partially maintained the plant's performance, despite the presence of drought and Cd stress. [ABSTRACT FROM AUTHOR]

The food enzyme subtilisin (serine endopeptidase, EC 3.4.21.62) is produced with the non‐genetically modified microorganism Bacillus paralicheniformis strain LMG S‐30155 by ENMEX SA de CV, now part of Kerry Food Ingredients (Cork) Ltd. The food enzyme is intended to be used in oil production, hydrolysis of vegetable/microbial/animal proteins, yeast processing and production of flavouring preparations. The production strain of the food enzyme contains known antimicrobial resistance genes and genes involved in bacitracin biosynthesis. Consequently, it does not fulfil the requirements for the QPS approach to safety assessment. Bacitracin was detected in the food enzyme and the ■■■■■ The presence of bacitracin, a medically important antimicrobial, in the food enzyme represents a risk for the development of resistance in bacteria. Due to the presence of bacitracin, the Panel concluded that the food enzyme subtilisin produced with the non‐genetically modified Bacillus paralicheniformis strain LMG S‐30155 cannot be considered safe. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme subtilisin (serine endopeptidase, EC 3.4.21.62) is produced with the non‐genetically modified microorganism Bacillus paralicheniformis strain LMG S‐30155 by ENMEX SA de CV, now part of Kerry Food Ingredients (Cork) Ltd. The food enzyme is intended to be used in oil production, hydrolysis of vegetable/microbial/animal proteins, yeast processing and production of flavouring preparations. The production strain of the food enzyme contains known antimicrobial resistance genes and genes involved in bacitracin biosynthesis. Consequently, it does not fulfil the requirements for the QPS approach to safety assessment. Bacitracin was detected in the food enzyme and the ■■■■■ The presence of bacitracin, a medically important antimicrobial, in the food enzyme represents a risk for the development of resistance in bacteria. Due to the presence of bacitracin, the Panel concluded that the food enzyme subtilisin produced with the non‐genetically modified Bacillus paralicheniformis strain LMG S‐30155 cannot be considered safe.

Root-knot nematodes (RKNs, Meloidogyne spp.) are obligate plant parasites that constitute a significant pest for agriculture worldwide. They penetrate the plant roots, reducing the uptake of water and nutrients, causing a significant impact on crop yield. One alternative on focus now for nematode management is biological control. Rhizobacteria within the Bacillus genus show multiple modes of action against plant-parasitic nematodes (PPNs) that can act alone or in combination. In this context, we evaluated a dual-strain bacteria combination (B. paralicheniformi FMCH001 and B. subtilis FMCH002) to reduce nematode infection in tomato plants. We evaluated mortality of larvae from Meloidogyne javanica in vitro, as well as eggs hatching after the treatment. Atraction, penetration, establishment, and reproduction assays in vitro or in pots in tomato plants infected with M. javanica and treated/untreated with the dual-strain bacteria combination were also performed. Additionally, morphometric parameters comparing giant cells size from galls of treated and untreated plants by using confocal microscopy were also measured. The results showed that this combination of strains has nematicidal properties in the pre-infection phase by decreasing the egg-hatching, juvenile survival, and attractiveness to the roots. Furthermore, nematode establishment, gall formation, and, remarkably, giant cell development was severely impaired after the bacterial treatment, suggesting interference with morphogenetic mechanisms induced by the nematode during GCs development within the plant. Nematode reproduction in tomato plants was reduced independently of the application mode in soil, before or after bacterial treatment. The dual-strain combination was also effective against other PPNs (i.e. Pratylenchus spp.) and in different crops (soybean). Therefore, combining B. paralicheniformis FMCH001 and B. subtilis FMCH002 is an efficient agent for the biological control of Meloidogyne spp. by interfering with different stages of the nematode cycle as a result of multiple modes of action. [ABSTRACT FROM AUTHOR]

Root-knot nematodes (RKNs, Meloidogyne spp.) are obligate plant parasites that constitute a significant pest for agriculture worldwide. They penetrate the plant roots, reducing the uptake of water and nutrients, causing a significant impact on crop yield. One alternative on focus now for nematode management is biological control. Rhizobacteria within the Bacillus genus show multiple modes of action against plant-parasitic nematodes (PPNs) that can act alone or in combination. In this context, we evaluated a dual-strain bacteria combination (B. paralicheniformi FMCH001 and B. subtilis FMCH002) to reduce nematode infection in tomato plants. We evaluated mortality of larvae from Meloidogyne javanica in vitro, as well as eggs hatching after the treatment. Atraction, penetration, establishment, and reproduction assays in vitro or in pots in tomato plants infected with M. javanica and treated/ untreated with the dual-strain bacteria combination were also performed. Additionally, morphometric parameters comparing giant cells size from galls of treated and untreated plants by using confocal microscopy were also measured. The results showed that this combination of strains has nematicidal properties in the pre-infection phase by decreasing the egg-hatching, juvenile survival, and attractiveness to the roots. Furthermore, nematode establishment, gall formation, and, remarkably, giant cell development was severely impaired after the bacterial treatment, suggesting interference with morphogenetic mechanisms induced by the nematode during GCs development within the plant. Nematode reproduction in tomato plants was reduced independently of the application mode in soil, before or after bacterial treatment. The dual-strain combination was also effective against other PPNs (i.e. Pratylenchus spp.) and in different crops (soybean). Therefore, combining B. paralicheniformis FMCH001 and B. subtilis FMCH002 is an efficient agent for the biological control of Meloidogyne spp. by interfering with different stages of the nematode cycle as a result of multiple modes of action.

The burgeoning global mushroom industry has precipitated challenges related to the efficient and sustainable utilization of spent mushroom substrate (SMS). Composting is regarded as an efficient way for the ecological utilization of SMS. The addition of microbial inoculants can promote the composting process and improve the quality of compost products. This study introduced two bacterial inoculants, Bacillus paralicheniformis HL-05 (BP) and Streptomyces thermoviolaceus LC-10 (ST), into the composting process of SMS. The impact of these inoculants was evaluated through analyses of physicochemical properties, lignocellulose degradation, and high-throughput sequencing to elucidate their ecological roles and optimize the composting process. The results suggest that inoculation with BP and ST significantly prolonged the thermophilic stage by 2-3 days, representing an increase of 22.22-33.33%. Moreover, it boosted the degradation rates of cellulose, hemicellulose, and lignin by 18.37-29.77%, 35.74-50.43%, and 40.32-40.83%, respectively, compared to the control. Furthermore, inoculation rapidly altered the microbial community structure during the rapid temperature-rising stage and strengthened interconnections among composting microorganisms. The microbial inoculation substantially enhanced the proliferation of thermophilic lignocellulose-degrading microorganisms during the thermophilic stage, thereby facilitating the utilization of lignocellulose. This study proposes a novel and effective strategy for SMS composting using microbial inoculants., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Guo-Qing Zhang reports financial support was provided by Beijing University of Agricultur. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We present the complete genome of the halotolerant strain Bacillus paralicheniformis HAS-1., Competing Interests: The authors declare no conflict of interest.

Cadmium and drought stress are the most destructive of the abiotic stresses with negative effects on both metabolism and photosynthesis. The present experiment aimed to analyze the impact of inoculation with Bacillus paralicheniformis and Trichoderma asperellum on the growth and antioxidant response modulation of maize (Zea mays L.), under drought and Cadmium (Cd) stresses. Regarding plant biomass analysis, fungi inoculation increased leaf dry biomass significantly (11.92%) towards uninoculated ones. The leaf area was affected significantly by bacterial application, 12.15% more than the control. A significant trend (drought+ Cd stress) was observed between fungi-inoculated maize leaves (15.07 µmol/g FW) and bacterial-inoculated leaves (18.71 µmol/g FW) regarding the malondialdehyde quantity. Furthermore, the activities of superoxide dismutase were notably higher (9.63–40.88%) in microorganism-inoculated roots. Similarly, under drought + Cd stress, peroxidase demonstrated a higher activity under bacterial inoculation than fungal ones (92.11% more). The maximum translocation factor was observed in the uninoculated group (under Cd stress), while the bioconcentration factor under drought stress showed a significant increase by microorganisms. The maximum relative water content under bacterial inoculation (82.66%) was achieved. The fungi and bacterial inoculation minimized Cd accumulation in the leaf significantly under drought and drought + Cd stress. Generally, the microorganism inoculation positively and partially maintained the plant’s performance, despite the presence of drought and Cd stress.

The inorganic selenium is absorbed and utilized inefficiently, and the range between toxicity and demand is narrow, so the application is strictly limited. Selenium nanoparticles have higher bioactivity and biosafety properties, including increased antioxidant and anticancer properties. Thus, producing and applying eco-friendly, non-toxic selenium nanoparticles in feed additives is crucial. Bacillus paralicheniformis Y4 was investigated for its potential ability to produce selenium nanoparticles and the activity of carboxymethyl cellulases. The selenium nanoparticles were characterized using zeta potential analyses, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). Additionally, evaluations of the anti-α-glucosidase activity and the antioxidant activity of the selenium nanoparticles and the ethyl acetate extracts of Y4 were conducted. B. paralicheniformis Y4 exhibited high selenite tolerance of 400 mM and the selenium nanoparticles had an average particle size of 80 nm with a zeta potential value of −35.8 mV at a pH of 7.0, suggesting that the particles are relatively stable against aggregation. After 72 h of incubation with 5 mM selenite, B. paralicheniformis Y4 was able to reduce it by 76.4%, yielding red spherical bio-derived selenium nanoparticles and increasing the carboxymethyl cellulase activity by 1.49 times to 8.96 U/mL. For the first time, this study reports that the carboxymethyl cellulase activity of Bacillus paralicheniforis was greatly enhanced by selenite. The results also indicated that B. paralicheniformis Y4 could be capable of ecologically removing selenite from contaminated sites and has great potential for producing selenium nanoparticles as feed additives to enhance the added value of agricultural products. [ABSTRACT FROM AUTHOR]

Single cultures and co-cultures of Bacillus licheniformis and Bacillus paralicheniformis isolated from compost were evaluated for their carboxymethyl cellulase (CMCase) and filter paperase (FPase) production potential. Using a medium supplemented with microcrystalline cellulose (MCC), in the co-culture, CMCase and FPase activities increased 8.87- and 2.28-fold and 10.15- and 3.20-fold over B. licheniformis and B. Paralicheniformis monocultures, respectively. The synergistic behavior of the two isolates might be due to the consumption of hydrolysis product (glucose, cellobiose) by one or both of the isolates, which improved their metabolic performance for cellulase secretion. Optimal conditions for cellulase production by this co-culture were a temperature of 45 °C, and pH 7 at 180 rpm in a medium containing rice bran at 1% (w/v) and chicken manure as nitrogen supplement at 2% (w/v). The maximum CMCase and FPase produced under the above conditions were 79.8 U/mL and 12.5 U/mL, respectively. This corresponds to 257.4- and 59.5-fold enhancement in CMCase and FPase activity, respectively, over B. licheniformis monoculture, and 306.9- and 83.3-fold increase with respect to the B. paralicheniformis monoculture. These results indicate that improved cellulase production can be achieved through co-culture and chicken manure nitrogen-supplement. [ABSTRACT FROM AUTHOR]

Featured Application: The Bacillus strains FC37 and Hvs2 could be used as potential biocontrol agents as a sustainable and environmentally friendly control strategy for asparagus fields affected by the decline syndrome. To avoid the use of agrochemicals in agriculture, alternative methods are emerging to control plant pathogens. Some plant growth-promoting rhizobacteria (PGPR) can be used as biocontrol agents since they can induce protection against pathogens. The aim of this study was to evaluate the protective effect of several PGPR strains against the main Fusarium species involved in asparagus decline syndrome (ADS): F. proliferatum, F. oxysporum f. sp. asparagi and F. redolens. In vitro antagonism assays showed that all the bacteria inhibited the mycelium growth of the three Fusarium species. The most effective strains (Streptomyces fradiae Hvs6, Bacillus paralicheniformis Hvs2 and Bacillus velezensis FC37) were tested to evaluate their protective effect on asparagus plants inoculated with pathogenic Fusarium isolates. Strains FC37 and Hvs2 were the most effective in controlling pathogenic F. proliferatum and F. oxysporum f. sp. asparagi, but neither could protect against F. redolens isolates. The production of hydrolytic enzymes such as β-glucosidase, amylase and protease by these bacterial strains could be involved in the structural degradation of the fungal cell wall. In addition, the production of toxic volatile compounds, such as hydrogen cyanide, may inhibit the fungal growth, and the production of phosphate solubilizers could be related to the plant growth promotion. These results suggest that strains FC37 and Hvs2 could be used as potential biocontrol agents as a sustainable and environmentally friendly control strategy for ADS-affected fields. [ABSTRACT FROM AUTHOR]

Thermophilic bacteria, especially from the genus Bacillus, constitute a huge potential source of novel enzymes that could be relevant for biotechnological applications. In this work, we described the cellulose and hemicellulose-related enzymatic activities of the hot spring Bacillus aerius CCMM B940 from the Moroccan Coordinated Collections of Microorganisms (CCMM), and revealed its potential for hemicellulosic biomass utilization. Indeed, B940 was able to degrade complex polysaccharides such as xylan and lichenan and exhibited activity towards carboxymethylcellulose. The strain was also able to grow on agriculture waste such as orange and apple peels as the sole carbon source. Whole-genome sequencing allowed the reclassification of CCMM B940 previously known as B. aerius into Bacillus paralicheniformis since the former species name has been rejected. The draft genome reported here is composed of 38 contigs resulting in a genome of 4,315,004 bp and an average G + C content of 45.87%, and is an important resource for illuminating the molecular mechanisms of carbohydrate metabolism. The annotated genomic sequences evidenced more than 52 genes encoding glycoside hydrolases and pectate lyases belonging to 27 different families of CAZymes that are involved in the degradation of plant cell wall carbohydrates. Genomic predictions in addition to in vitro experiments have revealed broad hydrolytic capabilities of the strain, thus reinforcing its relevance for biotechnology applications. [ABSTRACT FROM AUTHOR]

Abstract The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre‐evaluation of the safety of biological agents, intended for addition to food or feed, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge, safety concerns and occurrence of antimicrobial resistance. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by ‘qualifications’. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS TUs. Schizochytrium limacinum, which is a synonym for Aurantiochytrium limacinum, was added to the QPS list. Of the 78 microorganisms notified to EFSA between October 2020 and March 2021, 71 were excluded; 16 filamentous fungi, 1 Dyella spp., 1 Enterococcus faecium, 7 Escherichia coli, 1 Streptomyces spp., 1 Schizochytrium spp. and 44 TUs that had been previously evaluated. Seven TUs were evaluated: Corynebacterium stationis and Kodamaea ohmeri were re‐assessed because an update was requested for the current mandate. Anoxybacillus caldiproteolyticus, Bacillus paralicheniformis, Enterobacter hormaechei, Eremothecium ashbyi and Lactococcus garvieae were assessed for the first time. The following TUs were not recommended for QPS status: A. caldiproteolyticus due to the lack of a body of knowledge in relation to its use in the food or feed chain, E. hormaechei, L. garvieae and K. ohmeri due to their pathogenic potential, E. ashbyi and C. stationis due to a lack of body of knowledge on their occurrence in the food and feed chain and to their pathogenic potential. B. paralicheniformis was recommended for the QPS status with the qualification ‘absence of toxigenic activity’ and ‘absence of genetic information to synthesize bacitracin’.

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of biological agents, intended for addition to food or feed, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge, safety concerns and occurrence of antimicrobial resistance. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS TUs. Schizochytrium limacinum, which is a synonym for Aurantiochytrium limacinum, was added to the QPS list. Of the 78 microorganisms notified to EFSA between October 2020 and March 2021, 71 were excluded; 16 filamentous fungi, 1 Dyella spp., 1 Enterococcus faecium, 7 Escherichia coli, 1 Streptomyces spp., 1 Schizochytrium spp. and 44 TUs that had been previously evaluated. Seven TUs were evaluated: Corynebacterium stationis and Kodamaea ohmeri were re-assessed because an update was requested for the current mandate. Anoxybacillus caldiproteolyticus, Bacillus paralicheniformis, Enterobacter hormaechei, Eremothecium ashbyi and Lactococcus garvieae were assessed for the first time. The following TUs were not recommended for QPS status: A. caldiproteolyticus due to the lack of a body of knowledge in relation to its use in the food or feed chain, E. hormaechei, L. garvieae and K. ohmeri due to their pathogenic potential, E. ashbyi and C. stationis due to a lack of body of knowledge on their occurrence in the food and feed chain and to their pathogenic potential. B. paralicheniformis was recommended for the QPS status with the qualification 'absence of toxigenic activity' and 'absence of genetic information to synthesize bacitracin'. [ABSTRACT FROM AUTHOR]

Abstract Background Members of the genus Bacillus are important plant growth-promoting rhizobacteria that serve as biocontrol agents. Bacillus paralicheniformis MDJK30 is a PGPR isolated from the peony rhizosphere and can suppress plant-pathogenic bacteria and fungi. To further uncover the genetic mechanism of the plant growth-promoting traits of MDJK30 and its closely related strains, we used comparative genomics to provide insights into the genetic diversity and evolutionary relationship between B. paralicheniformis and B. licheniformis. Results A comparative genomics analysis based on B. paralicheniformis MDJK30 and 55 other previously reported Bacillus strains was performed. The evolutionary position of MDJK30 and the evolutionary relationship between B. paralicheniformis and B. licheniformis were evaluated by studying the phylogeny of the core genomes, a population structure analysis and ANI results. Comparative genomic analysis revealed various features of B. paralicheniformis that contribute to its commensal lifestyle in the rhizosphere, including an opening pan genome, a diversity of transport and the metabolism of the carbohydrates and amino acids. There are notable differences in the numbers and locations of the insertion sequences, prophages, genomic islands and secondary metabolic synthase operons between B. paralicheniformis and B. licheniformis. In particular, we found most gene clusters of Fengycin, Bacitracin and Lantipeptide were only present in B. paralicheniformis and were obtained by horizontal gene transfer (HGT), and these clusters may be used as genetic markers for distinguishing B. paralicheniformis and B. licheniformis. Conclusions This study reveals that MDJK30 and the other strains of lineage paralicheniformis present plant growth-promoting traits at the genetic level and can be developed and commercially formulated in agriculture as PGPR. Core genome phylogenies and population structure analysis has proven to be a powerful tool for differentiating B. paralicheniformis and B. licheniformis. Comparative genomic analyses illustrate the genetic differences between the paralicheniformis-licheniformis group with respect to rhizosphere adaptation.

A novel amylolytic enzyme producing thermoalkaliphilic bacterium, the source of industrially used enzymes was isolated. Isolated strain was identified by morphological, physio-biochemical tests and the 16S rRNA gene sequence analysis. The optimal conditions of enzyme activity were determined. For higher α-amylase production, the variables such as yeast extract, starch, CaCl2, (NH4)2SO4, NaCl and MgSO4 in the α-amylase production medium, the temperature and pH were screened by Plackett–Burman design and optimised using response surface methodology (RSM). The optimal conditions were found to be 0.15 g/L for starch, 0.15 mg/L for CaCl2 and 60°C for temperature. By using RSM model, amylase production increase was achieved sevenfold. It is showed that this method can be utilised to optimize α-amylase production in athermophilic bacteria such as Bacillus paralicheniformis.

The inorganic selenium is absorbed and utilized inefficiently, and the range between toxicity and demand is narrow, so the application is strictly limited. Selenium nanoparticles have higher bioactivity and biosafety properties, including increased antioxidant and anticancer properties. Thus, producing and applying eco-friendly, non-toxic selenium nanoparticles in feed additives is crucial. Bacillus paralicheniformis Y4 was investigated for its potential ability to produce selenium nanoparticles and the activity of carboxymethyl cellulases. The selenium nanoparticles were characterized using zeta potential analyses, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). Additionally, evaluations of the anti-α-glucosidase activity and the antioxidant activity of the selenium nanoparticles and the ethyl acetate extracts of Y4 were conducted. B. paralicheniformis Y4 exhibited high selenite tolerance of 400 mM and the selenium nanoparticles had an average particle size of 80 nm with a zeta potential value of −35.8 mV at a pH of 7.0, suggesting that the particles are relatively stable against aggregation. After 72 h of incubation with 5 mM selenite, B. paralicheniformis Y4 was able to reduce it by 76.4%, yielding red spherical bio-derived selenium nanoparticles and increasing the carboxymethyl cellulase activity by 1.49 times to 8.96 U/mL. For the first time, this study reports that the carboxymethyl cellulase activity of Bacillus paralicheniforis was greatly enhanced by selenite. The results also indicated that B. paralicheniformis Y4 could be capable of ecologically removing selenite from contaminated sites and has great potential for producing selenium nanoparticles as feed additives to enhance the added value of agricultural products.

As close relatives, Bacillus paralicheniformis is often wrongly identified as Bacillus licheniformis. In this study, two genetic markers are presented based on fenC and fenD from the fengycin operon of B. paralicheniformis to rapidly distinguish it from B. licheniformis. The fengycin operon is one of the few present in B. paralicheniformis but absent in B. lichenformis up to date. Using these markers, two presumptive B. paralicheniformis isolates each were recovered from a set of isolates previously identified as B. licheniformis by Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) or identified only to genus level as Bacillus by 16S ribosomal RNA (rRNA) gene sequencing, respectively. Whole genome sequencing of the four isolates confirmed their identity as B. paralicheniformis having the closest similarity with B. paralicheniformis ATCC 9945a (GenBank: CP005965.1) with a 7,682 k-mer score and 97.22% Average Nucleotide Identity (ANI). ANI of 100% suggests that the four isolates are highly similar. Further analysis will be necessary to determine if finer differences exist among these isolates at the level of single nucleotide polymorphisms.

As close relatives, Bacillus paralicheniformis is often wrongly identified as Bacillus licheniformis. In this study, two genetic markers are presented based on fenC and fenD from the fengycin operon of B. paralicheniformis to rapidly distinguish it from B. licheniformis. The fengycin operon is one of the few present in B. paralicheniformis but absent in B. lichenformis up to date. Using these markers, two presumptive B. paralicheniformis isolates each were recovered from a set of isolates previously identified as B. licheniformis by Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) or identified only to genus level as Bacillus by 16S ribosomal RNA (rRNA) gene sequencing, respectively. Whole genome sequencing of the four isolates confirmed their identity as B. paralicheniformis having the closest similarity with B. paralicheniformis ATCC 9945a (GenBank: CP005965.1) with a 7,682 k-mer score and 97.22% Average Nucleotide Identity (ANI). ANI of 100% suggests that the four isolates are highly similar. Further analysis will be necessary to determine if finer differences exist among these isolates at the level of single nucleotide polymorphisms. [ABSTRACT FROM AUTHOR]

Bacillus paralicheniformis MHN12 possesses a 4,245,453-base pair genome with 45.9% G + C content, including 1 CRISPR, 80 tRNA, 8 rRNA genes, and 4,418 predicted coding sequences . MHN12 exhibits high salinity tolerance and plant growth-promoting abilities, making it a promising bioinoculant for enhancing plant growth in saline soils., Competing Interests: The authors declare no conflict of interest.

Abstract Background The increasing spectrum of multidrug-resistant bacteria is a major global public health concern, necessitating discovery of novel antimicrobial agents. Here, members of the genus Bacillus are investigated as a potentially attractive source of novel antibiotics due to their broad spectrum of antimicrobial activities. We specifically focus on a computational analysis of the distinctive biosynthetic potential of Bacillus paralicheniformis strains isolated from the Red Sea, an ecosystem exposed to adverse, highly saline and hot conditions. Results We report the complete circular and annotated genomes of two Red Sea strains, B. paralicheniformis Bac48 isolated from mangrove mud and B. paralicheniformis Bac84 isolated from microbial mat collected from Rabigh Harbor Lagoon in Saudi Arabia. Comparing the genomes of B. paralicheniformis Bac48 and B. paralicheniformis Bac84 with nine publicly available complete genomes of B. licheniformis and three genomes of B. paralicheniformis, revealed that all of the B. paralicheniformis strains in this study are more enriched in nonribosomal peptides (NRPs). We further report the first computationally identified trans-acyltransferase (trans-AT) nonribosomal peptide synthetase/polyketide synthase (PKS/ NRPS) cluster in strains of this species. Conclusions B. paralicheniformis species have more genes associated with biosynthesis of antimicrobial bioactive compounds than other previously characterized species of B. licheniformis, which suggests that these species are better potential sources for novel antibiotics. Moreover, the genome of the Red Sea strain B. paralicheniformis Bac48 is more enriched in modular PKS genes compared to B. licheniformis strains and other B. paralicheniformis strains. This may be linked to adaptations that strains surviving in the Red Sea underwent to survive in the relatively hot and saline ecosystems.

To avoid the use of agrochemicals in agriculture, alternative methods are emerging to control plant pathogens. Some plant growth-promoting rhizobacteria (PGPR) can be used as biocontrol agents since they can induce protection against pathogens. The aim of this study was to evaluate the protective effect of several PGPR strains against the main Fusarium species involved in asparagus decline syndrome (ADS): F. proliferatum, F. oxysporum f. sp. asparagi and F. redolens. In vitro antagonism assays showed that all the bacteria inhibited the mycelium growth of the three Fusarium species. The most effective strains (Streptomyces fradiae Hvs6, Bacillus paralicheniformis Hvs2 and Bacillus velezensis FC37) were tested to evaluate their protective effect on asparagus plants inoculated with pathogenic Fusarium isolates. Strains FC37 and Hvs2 were the most effective in controlling pathogenic F. proliferatum and F. oxysporum f. sp. asparagi, but neither could protect against F. redolens isolates. The production of hydrolytic enzymes such as β-glucosidase, amylase and protease by these bacterial strains could be involved in the structural degradation of the fungal cell wall. In addition, the production of toxic volatile compounds, such as hydrogen cyanide, may inhibit the fungal growth, and the production of phosphate solubilizers could be related to the plant growth promotion. These results suggest that strains FC37 and Hvs2 could be used as potential biocontrol agents as a sustainable and environmentally friendly control strategy for ADS-affected fields.

Background: Members of the genus Bacillus are important plant growth-promoting rhizobacteria that serve as biocontrol agents. Bacillus paralicheniformis MDJK30 is a PGPR isolated from the peony rhizosphere and can suppress plant-pathogenic bacteria and fungi. To further uncover the genetic mechanism of the plant growth-promoting traits of MDJK30 and its closely related strains, we used comparative genomics to provide insights into the genetic diversity and evolutionary relationship between B. paralicheniformis and B. licheniformis. Results: A comparative genomics analysis based on B. paralicheniformis MDJK30 and 55 other previously reported Bacillus strains was performed. The evolutionary position of MDJK30 and the evolutionary relationship between B. paralicheniformis and B. licheniformis were evaluated by studying the phylogeny of the core genomes, a population structure analysis and ANI results. Comparative genomic analysis revealed various features of B. paralicheniformis that contribute to its commensal lifestyle in the rhizosphere, including an opening pan genome, a diversity of transport and the metabolism of the carbohydrates and amino acids. There are notable differences in the numbers and locations of the insertion sequences, prophages, genomic islands and secondary metabolic synthase operons between B. paralicheniformis and B. licheniformis. In particular, we found most gene clusters of Fengycin, Bacitracin and Lantipeptide were only present in B. paralicheniformis and were obtained by horizontal gene transfer (HGT), and these clusters may be used as genetic markers for distinguishing B. paralicheniformis and B. licheniformis. Conclusions: This study reveals that MDJK30 and the other strains of lineage paralicheniformis present plant growth-promoting traits at the genetic level and can be developed and commercially formulated in agriculture as PGPR. Core genome phylogenies and population structure analysis has proven to be a powerful tool for differentiating B. paralicheniformis and B. licheniformis. Comparative genomic analyses illustrate the genetic differences between the paralicheniformis-licheniformis group with respect to rhizosphere adaptation. [ABSTRACT FROM AUTHOR]

Background: The increasing spectrum of multidrug-resistant bacteria is a major global public health concern, necessitating discovery of novel antimicrobial agents. Here, members of the genus Bacillus are investigated as a potentially attractive source of novel antibiotics due to their broad spectrum of antimicrobial activities. We specifically focus on a computational analysis of the distinctive biosynthetic potential of Bacillus paralicheniformis strains isolated from the Red Sea, an ecosystem exposed to adverse, highly saline and hot conditions. Results: We report the complete circular and annotated genomes of two Red Sea strains, B. paralicheniformis Bac48 isolated from mangrove mud and B. paralicheniformis Bac84 isolated from microbial mat collected from Rabigh Harbor Lagoon in Saudi Arabia. Comparing the genomes of B. paralicheniformis Bac48 and B. paralicheniformis Bac84 with nine publicly available complete genomes of B. licheniformis and three genomes of B. paralicheniformis, revealed that all of the B. paralicheniformis strains in this study are more enriched in nonribosomal peptides (NRPs). We further report the first computationally identified trans-acyltransferase (trans-AT) nonribosomal peptide synthetase/polyketide synthase (PKS/ NRPS) cluster in strains of this species. Conclusions: B. paralicheniformis species have more genes associated with biosynthesis of antimicrobial bioactive compounds than other previously characterized species of B. licheniformis, which suggests that these species are better potential sources for novel antibiotics. Moreover, the genome of the Red Sea strain B. paralicheniformis Bac48 is more enriched in modular PKS genes compared to B. licheniformis strains and other B. paralicheniformis strains. This may be linked to adaptations that strains surviving in the Red Sea underwent to survive in the relatively hot and saline ecosystems. [ABSTRACT FROM AUTHOR]

Excessive continuous cropping and soil degradation, such as acidification, hardening, fertility decline, and the degradation of microbial community, lead to the epidemic of soilborne diseases and cause great loss in agriculture production. Application of fulvic acid can improve the growth and yield of various crops and effectively suppress soilborne plant diseases. Bacillus paralicheniformis strain 285-3 producing poly-gamma-glutamic acid is used to remove the organic acid that can cause soil acidification and increase the fertilizer effect of fulvic acid and the effect of improving soil quality and inhibiting soilborne disease. In field experiments, the application of fulvic acid and Bacillus paralicheniformis ferment effectively reduced the incidence of bacterial wilt disease and improved soil fertility. Both fulvic acid powder and B. paralicheniformis ferment improved soil microbial diversity and increased the complexity and stability of the microbial network. For B. paralicheniformis ferment, the molecular weight of poly-gamma-glutamic acid became smaller after heating, which could better improve the soil microbial community and network structure. In fulvic acid and B. paralicheniformis ferment-treated soils, the synergistic interaction between microorganisms increased and the number of keystone microorganisms increased, which included antagonistic bacteria and plant growth-promoting bacteria. Changes in the microbial community and network structure were the main reason for the reduced incidence of bacterial wilt disease. Application of fulvic acid and Bacillus paralicheniformis ferment improved soil physicochemical properties and effectively controlled bacterial wilt disease by changing microbial community and network structure and enriching antagonistic and beneficial bacteria. IMPORTANCE Continuous cropping tobacco has led to soil degradation and caused soilborne bacterial wilt disease. Fulvic acid as a biostimulator was applied to restore soil and control bacterial wilt disease. For improving its effect, fulvic acid was fermented with Bacillus paralicheniformis strain 285-3 producing poly-gamma-glutamic acid. Fulvic acid and B. paralicheniformis ferment inhibited bacterial wilt disease, improved soil quality, enriched beneficial bacteria, and increased microbial diversity and microbial network complexity. Some keystone microorganisms in fulvic acid and B. paralicheniformis ferment-treated soils had potential antimicrobial activity and plant growth-promoting attributes. Fulvic acid and B. paralicheniformis 285-3 ferment could be used to restore soil quality and microbiota and control bacterial wilt disease. This study found new biomaterial to control soilborne bacterial disease by combining fulvic acid and poly-gamma-glutamic acid application.