New Insights into Pseudomonas spp.-Produced Antibiotics: Genetic Regulation of Biosynthesis and Implementation in Biotechnology.

Pseudomonas bacteria are renowned for their remarkable capacity to synthesize antibiotics, namely mupirocin, gluconic acid, pyrrolnitrin, and 2,4-diacetylphloroglucinol (DAPG). While these substances are extensively employed in agricultural biotechnology to safeguard plants against harmful bacteria and fungi, their potential for human medicine and healthcare remains highly promising for common science. However, the challenge of obtaining stable producers that yield higher quantities of these antibiotics continues to be a pertinent concern in modern biotechnology. Although the interest in antibiotics of Pseudomonas bacteria has persisted over the past century, many uncertainties still surround the regulation of the biosynthetic pathways of these compounds. Thus, the present review comprehensively studies the genetic organization and regulation of the biosynthesis of these antibiotics and provides a comprehensive summary of the genetic organization of antibiotic biosynthesis pathways in pseudomonas strains, appealing to both molecular biologists and biotechnologists. In addition, attention is also paid to the application of antibiotics in plant protection.

Resistance towards and biotransformation of a Pseudomonas-produced secondary metabolite during community invasion.

The role of antagonistic secondary metabolites produced by Pseudomonas protegens in suppression of soil-borne phytopathogens has been clearly documented. However, their contribution to the ability of P. protegens to establish in soil and rhizosphere microbiomes remains less clear. Here, we use a four-species synthetic community (SynCom) in which individual members are sensitive towards key P. protegens antimicrobial metabolites (DAPG, pyoluteorin, and orfamide A) to determine how antibiotic production contributes to P. protegens community invasion and to identify community traits that counteract the antimicrobial effects. We show that P. protegens readily invades and alters the SynCom composition over time, and that P. protegens establishment requires production of DAPG and pyoluteorin. An orfamide A-deficient mutant of P. protegens invades the community as efficiently as wildtype, and both cause similar perturbations to community composition. Here, we identify the microbial interactions underlying the absence of an orfamide A mediated impact on the otherwise antibiotic-sensitive SynCom member, and show that the cyclic lipopeptide is inactivated and degraded by the combined action of Rhodococcus globerulus D757 and Stenotrophomonas indicatrix D763. Altogether, the demonstration that the synthetic community constrains P. protegens invasion by detoxifying its antibiotics may provide a mechanistic explanation to inconsistencies in biocontrol effectiveness in situ.

Pyoluteorin regulates the biosynthesis of 2,4-DAPG through the TetR family transcription factor PhlH in Pseudomonas protegens Pf-5.

Soil and rhizosphere bacteria act as a rich source of secondary metabolites, effectively fighting against a diverse array of pathogens. Certain Pseudomonas species harbor biosynthetic gene clusters for producing both pyoluteorin and 2,4-diacetylphloroglucinol (2,4-DAPG), which are polyketides that exhibit highly similar antimicrobial spectrum against bacteria and fungi or oomycete. A complex cross talk exists between pyoluteorin and 2,4-DAPG biosynthesis, and production of 2,4-DAPG was strongly repressed by pyoluteorin, yet the underlying mechanism is still elusive. In this study, we find that the TetR family transcription factor PhlH is involved in the cross talk between pyoluteorin and 2,4-DAPG biosynthesis. PhlH binds to a palindromic sequence within the promoter of phlG (PphlG), which encodes a C-C bond hydrolase responsible for degrading 2,4-DAPG. As a signaling molecule, pyoluteorin disrupts the PhlH-PphlG complex by binding to PhlH, leading to decreased levels of 2,4-DAPG. Proteomics data suggest that pyoluteorin regulates multiple physiological processes including fatty acid biosynthesis and transportation of taurine, siderophore, and amino acids. Our work not only reveals a novel mechanism of cross talk between pyoluteorin and 2,4-DAPG biosynthesis, but also highlights pyoluteorin's role as a messenger in the complex communication network of Pseudomonas.IMPORTANCEAntibiosis serves as a crucial defense mechanism for microbes against invasive bacteria and resource competition. These bacteria typically orchestrate the production of multiple antibiotics in a coordinated fashion, wherein the synthesis of one antibiotic inhibits the generation of another. This strategic coordination allows the bacterium to focus its resources on producing the most advantageous antibiotic under specific circumstances. However, the underlying mechanisms of distinct antibiotic production in bacterial cells remain largely elusive. In this study, we reveal that the TetR family transcription factor PhlH detects the secondary metabolite pyoluteorin and mediates the cross talk between pyoluteorin and 2,4-DAPG biosynthesis in the biocontrol strain Pseudomonas protegens Pf-5. These findings hold promise for future research, potentially informing the manipulation of these systems to enhance the effectiveness of biocontrol agents.

Investigating the Impact of Tillage and Crop Rotation on the Prevalence of phlD-Carrying Pseudomonas Potentially Involved in Disease Suppression.

Winter oilseed rape (OSR) is becoming an increasingly popular crop in rotations as it provides a cash crop and reduces the incidence of take-all fungal disease (caused by Gaeumannomyces graminis) in subsequent wheat production. The exact mechanism of this inhibition of fungal pathogens is not fully understood; however, the selective recruitment of bacterial groups with the ability to suppress pathogen growth and reproduction is thought to play a role. Here we examine the effect of tillage practice on the proliferation of microbes that possess the phlD gene involved in the production of the antifungal compound 2,4-diacetylphloroglucinol (2,4-DAPG), in the rhizospheres of both winter oilseed rape and winter wheat grown in rotation over a two-year period. The results showed that conservation strip tillage led to a significantly greater phlD gene copy number, both in the soil and in the roots, of oilseed rape and wheat crops, whereas crop rotation of oilseed rape and wheat did not increase the phlD gene copy number in winter wheat.

Regulation of 2,4-diacetylphloroglucinol biosynthesis and biocontrol capacity by the BolA family protein IbaG in Pseudomonas fluorescens 2P24.

The monothiol glutaredoxin GrxD plays an essential role in the biosynthesis of the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and the biocontrol capacity of the soil bacterium Pseudomonas fluorescens 2P24. However, the detailed mechanism underlying GrxD-mediated activation of the production of 2,4-DAPG remains unclear. Here, we found that GrxD directly interacted with IbaG, a BolA protein family member. The mutation of ibaG significantly decreased 2,4-DAPG production. Furthermore, expressing ibaG restored the production of 2,4-DAPG in the grxD ibaG double mutant to wild-type levels in the presence of dithiothreitol, suggesting that IbaG was required for GrxD-mediated regulation of 2,4-DAPG production. Transcriptome sequencing analyses revealed that IbaG plays a global role in gene regulation by affecting the expression of numerous genes throughout the genome. We also demonstrated that IbaG is an important regulator of several cellular processes, including swarming motility, biofilm formation, siderophore production, and acid resistance. Altogether, our data suggest that IbaG has an essential role in 2,4-DAPG production, motility, and biofilm formation. We also propose a regulatory mechanism linking GrxD to 2,4-DAPG production via IbaG. IMPORTANCE The production of 2,4-diacetylphloroglucinol (2,4-DAPG) is positively influenced by the monothiol glutaredoxin GrxD in Pseudomonas fluorescens 2P24. However, the regulatory mechanism underlying GrxD-mediated regulation of 2,4-DAPG biosynthesis is mostly uncharacterized. Here, we show the function of the BolA-like protein IbaG in 2,4-DAPG biosynthesis. We also demonstrate that GrxD directly interacts with IbaG and influences the redox state of IbaG. Altogether, this work provides new insights into the role of the highly conserved IbaG protein in regulating 2,4-DAPG synthesis, biofilm formation, and other biocontrol traits of P. fluorescens.

Citrate Synthase GltA Modulates the 2,4-Diacetylphloroglucinol Biosynthesis of Pseudomonas fluorescens 2P24 and is Essential for the Biocontrol Capacity.

Carbon metabolism is critical for microbial physiology and remarkably affects the outcome of secondary metabolite production. The production of 2,4-diacetylphloroglucinol (2,4-DAPG), a bacterial secondary metabolite with a broad spectrum of antibiotic activity, is a major mechanism used by the soil bacterium Pseudomonas fluorescens 2P24 to inhibit the growth of plant pathogens and control disease occurrence. Strain 2P24 has evolved a complex signaling cascade to regulate the production of 2,4-DAPG. However, the role of the central carbon metabolism in modulating 2,4-DAPG production has not been fully determined. In this study, we report that the gltA gene, which encodes citrate synthase, affects the expression of the 2,4-DAPG biosynthesis gene and is essential for the biocontrol capacity of strain 2P24. Our data showed that the mutation of gltA remarkably decreased the biosynthesis of 2,4-DAPG. Consistent with this result, the addition of citrate in strain 2P24 resulted in increased 2,4-DAPG production and decreased levels of RsmA and RsmE. In comparison with the wild-type strain, the gltA mutant was severely impaired in terms of biocontrol activity against the bacterial wilt disease of tomato plants caused by Ralstonia solanacearum. Moreover, the gltA mutant exhibited increased antioxidant activity, and the expression of oxidative, stress-associated genes, including ahpB, katB, and oxyR, was significantly upregulated in the gltA mutant compared to the wild-type strain. Overall, our data indicate that the citrate synthase GltA plays an important role in the production of 2,4-DAPG and oxidative stress and is required for biocontrol capacity.

RpoZ regulates 2,4-DAPG production and quorum sensing system in Pseudomonas fluorescens 2P24.

Introduction: Pseudomonas fluorescens 2P24 was isolated from soil of natural decay associated with wheat take-all and it can effectively control soil-borne diseases caused by a variety of plant pathogens. 2,4-diacetylphloroglucinol (2,4-DAPG), is produced by P. fluorescens 2P24 and plays an important role in the prevention and control of plant diseases. To understand the resistant mechanism, in this study, we conducted experiments to explore the regulation role of rpoZ in the synthesis of the antibiotic 2,4-DAPG and regulation of QS system. Methods: A random mini-Tn5 mutagenesis procedure was used to screen regulators for phlA transcription in stain PM901, which containing a phlA∷lacZ transcriptional fusion reporter plasmid. We identified 12 insertion mutants could significantly change phlA gene expression. By analyzing the amino acid sequences of the interrupted gene, we obtained a mutant strain Aa4-29 destroyed the rpoZ gene, which encodes the omiga subunit. We constructed the plasmid of rpoZ mutant (pBBR-△rpoZ) transformed into competent cells of P. fluorescens 2P24 by electro-transformation assay. The strains of P. fluorescens 2P24/pBBR, 2P24-△rpoZ/pBBR, 2P24-△rpoZ/pBBR-rpoZ were used to evaluate the regulation role of rpoZ in 2,4-DAPG production and quorum sensing system. Results: According to ß-galactosidase activity, we found that rpoZ positively regulated the expression of phlA (a synthesis gene of 2,4-DAPG) and PcoI (a synthesis gene of PcoI/PcoR QS signal system) at the transcriptional level. The production of 2,4-DAPG antibiotic and signal molecule AHL was influenced by rpoZ. Further, rpoZ was involved in regulating rsmA expression. RpoZ also has a certain regulatory effect on rpoS transcription, but no effect on the transcription of phlF, emhABC and emhR. According to the biocontrol assay, P. fluorescens 2P24 strains with rpoZ showed obvious antagonism ability against the Rhizoctonia solani in cotton, while the mutant strain of rpoZ lost the biocontrol effect. RpoZ had a significant effect on the swimming and biofilm formation in P. fluorescens 2P24. Conclusion: Our data showed that rpoZ was an important regulator of QS system, 2,4-DAPG in P. fluorescens 2P24. This may imply that P. fluorescens 2P24 has evolved different regulatory features to adapt to different environmental threats.

Investigation of the dual role of acyl phloroglucinols as a new hope for antibacterial and anti-SARS-CoV-2 agents employing integrated in vitro and multi-phase in silico approaches.

With the rapid spread of multi-drug-resistant bacteria and more infectious or aggressive variants of SARS-CoV-2, it is critical to develop drugs that can quickly adapt to evolving bacterial and viral mutations. In this study, encouraged by nature, we synthesized a series of phloroglucinol (PG) derivatives, acyl phloroglucinols (ACPLs) 2a-4c by mimicking the structure of the natural antifungal 2,4-diacetylphloroglucinol 3a (2,4-DAPG). According to the quantum chemical calculation, these compounds were expected to be exceptionally favourable for intermolecular interaction with protein receptors. Intriguingly, the in vitro study of Staphylococcus aureus (S. aureus) ATCC 25923 and Escherichia coli (E. coli) ATCC 25922, showed that the four ACPLs 3a-4a had good antibacterial activity and selectivity against gram-positive bacteria, S. aureus. These results were then supported by in silico molecular docking and molecular dynamics simulations (MDs) between these potent compounds and the S. aureus FtsA protein (PDB ID: 3WQU). Furthermore, with the aid of the knowledge base Virus-CKB along with the molecular docking study, it was found that the three ACPLs 4a-4c showed potential inhibitors against SARS-CoV-2 PLpro (PDB ID: 7CMD). Additionally, 100 ns of MDs was carried out in order to assess the stability behaviour of ACPLs at the docked site. Moreover, in silico ADME/T and drug likeness of all studied ACPLs were also predicted. Finally, shape-based screening of FDA-approved drugs was performed using the most prominent synthesized ACPLs as a template, enabling us to include several medications that could be utilized as antibacterial and antiviral drug candidates.Communicated by Ramaswamy H. Sarma.

2,4-Dipropylphloroglucinol inhibits the growth of human lung and colorectal cancer cells through induction of apoptosis.

Scientists are finding the most effective chemotherapeutic agents for the treatment of cancer. In the present study, we evaluated the anticancer mechanism of DPPG, a derivative of DAPG (2,4-diacetylphloroglucinol), for the first time. DPPG and DAPG inhibited 83 and 59% of human colorectal cancer HCT116 cell growth at 40.0 µg/ml, and 74 and 57% of human lung cancer A549 cell growth at 10.0 µg/ml concentrations respectively. Furthermore, DPPG and DAPG inhibited 97 and 73% colony formation of the HCT116 cells at 20.0 µg/ml concentration. DPPG and DAPG induced apoptosis in the HCT116 and A549 cells that was confirmed by Hoechst 33342 and FITC-annexin V staining. This result also revealed that ROS generated in both the HCT116 and A549 cells after treatment with DPPG. However, no ROS production was observed in HCT116 and A549 cells after treatment with DAPG. Both DAPG and DPPG significantly increased the CASP3 protein expression that was detected by staining the cells with the super-view 488-CASP3 substrate. Expression of WNT1 gene was eliminated in DPPG and DAPG treated HCT116. Expression of MAPK1 gene was entirely abolished in DPPG treated cells, whereas a significant decrease was observed for DAPG. An intense band of CASP8 gene product was observed agarose gel for DPPG treated HCT116 cells than DAPG. Molecular docking simulation showed the high binding affinities (≥ 6.5 kcal/mol) of DPPG and DAPG with target proteins WNT1, MAPK1, CASP8, and CASP3 in HCT116 cells. This manuscript demonstrated that DAPG and DPPG inhibited lung and colorectal cancer cells by inducing apoptosis. DAPG and DPPG inhibited A549 and HCT116 cells growth by inducing apoptosis.

Cloning and Molecular Characterization of the phlD Gene Involved in the Biosynthesis of "Phloroglucinol", a Compound with Antibiotic Properties from Plant Growth Promoting Bacteria Pseudomonas spp.

phlD is a novel kind of polyketide synthase involved in the biosynthesis of non-volatile metabolite phloroglucinol by iteratively condensing and cyclizing three molecules of malonyl-CoA as substrate. Phloroglucinol or 2,4-diacetylphloroglucinol (DAPG) is an ecologically important rhizospheric antibiotic produced by pseudomonads; it exhibits broad spectrum anti-bacterial and anti-fungal properties, leading to disease suppression in the rhizosphere. Additionally, DAPG triggers systemic resistance in plants, stimulates root exudation, as well as induces phyto-enhancing activities in other rhizobacteria. Here, we report the cloning and analysis of the phlD gene from soil-borne gram-negative bacteria-Pseudomonas. The full-length phlD gene (from 1078 nucleotides) was successfully cloned and the structural details of the PHLD protein were analyzed in-depth via a three-dimensional topology and a refined three-dimensional model for the PHLD protein was predicted. Additionally, the stereochemical properties of the PHLD protein were analyzed by the Ramachandran plot, based on which, 94.3% of residues fell in the favored region and 5.7% in the allowed region. The generated model was validated by secondary structure prediction using PDBsum. The present study aimed to clone and characterize the DAPG-producing phlD gene to be deployed in the development of broad-spectrum biopesticides for the biocontrol of rhizospheric pathogens.

Identification and Characterization of Bacteria-Derived Antibiotics for the Biological Control of Pea Aphanomyces Root Rot.

Antibiosis has been proposed to contribute to the beneficial bacteria-mediated biocontrol against pea Aphanomyces root rot caused by the oomycete pathogen Aphanomyces euteiches. However, the antibiotics required for disease suppression remain unknown. In this study, we found that the wild type strains of Pseudomonas protegens Pf-5 and Pseudomonas fluorescens 2P24, but not their mutants that lack 2,4-diacetylphloroglucinol, strongly inhibited A. euteiches on culture plates. Purified 2,4-diacetylphloroglucinol compound caused extensive hyphal branching and stunted hyphal growth of A. euteiches. Using a GFP-based transcriptional reporter assay, we found that expression of the 2,4-diacetylphloroglucinol biosynthesis gene phlAPf-5 is activated by germinating pea seeds. The 2,4-diacetylphloroglucinol producing Pf-5 derivative, but not its 2,4-diacetylphloroglucinol non-producing mutant, reduced disease severity caused by A. euteiches on pea plants in greenhouse conditions. This is the first report that 2,4-diacetylphloroglucinol produced by strains of Pseudomonas species plays an important role in the biocontrol of pea Aphanomyces root rot.

A Mycorrhizal Bacteria Strain Isolated From Polyporus umbellatus Exhibits Broad-Spectrum Antifungal Activity.

The microbes in the rhizosphere (or mycorrhizosphere) could promote plant growth, however, it is unclear whether mycorrhizosphere microbes could fight multiple fungal pathogens. In this study, twenty-one bacterial strains distributed in 6 genera, including 5 Pseudomonas strains, were isolated from mycorrhizal samples of Polyporus umbellatus that rely on other fungi during their life cycles. Further screening and pot experiments showed that the Pseudomonas strain ZL8 not only inhibited the growth of phytopathogenic fungi, but also promoted the growth of Salvia miltiorrhiza through inhibiting its wilting. In addition, strain ZL8 was found to have the ability to dissolve phosphate, produce IAA and siderophore. Nineteen compounds were identified from the fermentation broth of strain ZL8, of which 2,4-diacetylphloroglucinol (DAPG) had a significant inhibitory effect on phytopathogenic fungi with a minimum inhibitory concentration of 3.12-25 µg/mL. Molecular docking predicted that DAPG could bind to myosin I at two unique sites, which may be responsible to the inhibition of fungal growth. The evaluation results showed that strain ZL8 can be used to develop a dual-purpose biocontrol agents and biofertilizer. These results also provide new insights into the discovery and utilization of new resources for biocontrol agents and biolfertilizers.

Effect of the Monothiol Glutaredoxin GrxD on 2,4-Diacetylphloroglucinol Biosynthesis and Biocontrol Activity of Pseudomonas fluorescens 2P24.

Pseudomonas fluorescens 2P24 is a plant root-associated bacterium that suppresses several soilborne plant diseases due to its production of the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG). The biosynthesis of 2,4-DAPG is controlled by many regulatory elements, including the global regulator of the Gac/Rsm regulon and the pathway-specific repressor PhlF. In this work, a novel genetic element grxD, which encodes the monothiol glutaredoxin GrxD, was identified and characterized in the production of 2,4-DAPG in P. fluorescens 2P24. Our data showed that the mutation of grxD remarkably decreased 2,4-DAPG production. GrxD lost its ability to alter the production of 2,4-DAPG when the active-site CGFS motif of GrxD was mutated by site-directed mutagenesis. Further studies showed that the RsmA and RsmE proteins were essential for the GrxD-mediated regulation of 2,4-DAPG and exoprotease production. In addition, our data revealed that the deletion of grxD increased the expression of phlF, which negatively regulated the production of 2,4-DAPG. In addition, the grxD mutant was severely impaired in the biocontrol effect against the bacterial wilt of tomato. Overall, our results indicated that the monothiol glutaredoxin GrxD is involved in the production of 2,4-DAPG of P. fluorescens by influencing the Gac/Rsm global signaling pathway and transcriptional regulator PhlF and is essential for the biocontrol properties.

Characterization and Assessment of 2, 4-Diacetylphloroglucinol (DAPG)-Producing Pseudomonas fluorescens VSMKU3054 for the Management of Tomato Bacterial Wilt Caused by Ralstonia solanacearum.

Microbial bio-products are becoming an appealing and viable alternative to chemical pesticides for effective management of crop diseases. These bio-products are known to have potential to minimize agrochemical applications without losing crop yield and also restore soil fertility and productivity. In this study, the inhibitory efficacy of 2,4-diacetylphloroglucinol (DAPG) produced by Pseudomonas fluorescens VSMKU3054 against Ralstonia solanacearum was assessed. Biochemical and functional characterization study revealed that P. fluorescens produced hydrogen cyanide (HCN), siderophore, indole acetic acid (IAA) and hydrolytic enzymes such as amylase, protease, cellulase and chitinase, and had the ability to solubilize phosphate. The presence of the key antimicrobial encoding gene in the biosynthesis of 2,4-diacetylphloroglucinol (DAPG) was identified by PCR. The maximum growth and antimicrobial activity of P. fluorescens was observed in king's B medium at pH 7, 37 °C and 36 h of growth. Glucose and tryptone were found to be the most suitable carbon and nitrogen sources, respectively. DAPG was separated by silica column chromatography and identified by various methods such as UV-Vis, FT-IR, GC-MS and NMR spectroscopy. When R. solanacearum cells were exposed to DAPG at 90 µg/mL, the cell viability was decreased, reactive oxygen species (ROS) were increased and chromosomal DNA was damaged. Application of P. fluorescens and DAPG significantly reduced the bacterial wilt incidence. In addition, P. fluorescens was also found effective in promoting the growth of tomato seedlings. It is concluded that the indigenous isolate P. fluorescens VSMKU3054 could be used as a suitable biocontrol agent against bacterial wilt disease of tomato.

2,4-Diacetylphloroglucinol producing Pseudomonas fluorescens JM-1 for management of ear rot disease caused by Fusarium moniliforme in Zea mays L.

Maize (Zea mays L.) is a major cereal crop grown in a large number of countries. Loss in maize yield due to biotic stresses including fungal phytopathogens is a matter of immense concern. Control measures applied for eradication of fungal phytopathogens in maize are not up to the mark and more often involve harsh chemical(s)/pesticide(s) that cause deleterious effects both in humans and soil biota. Greener alternatives, such as the use of rhizosphere microbes in the form of bioinoculants, have proven to be very successful in terms of enhancing crop yield and suppressing fungal phytopathogens. In the present study, fluorescent pseudomonads were isolated from the maize rhizosphere and monitored for their plant growth-promoting (PGP) and biocontrol activities against Fusarium moniliforme. Based on various PGP traits and biocontrol potential, isolate JM-1 was found to be most effective and as per 16S rRNA gene sequencing analysis was identified as Pseudomonas fluorescens. Further experiments showed that the biocontrol potential of JM-1 against ear rot fungus involved the production of antifungal compound 2,4-diacetylphloroglucinol (DAPG). When examined for antagonistic interaction under scanning electron microscopy (SEM), structural abnormality, hyphal lysis, and deformity in fungal mycelium were observed. In the pot experiment, application of talc-based JM-1 containing bioformulation (in pot trials) showed significant enhancement in maize growth parameters (including the seed number and weight) in comparison to control even in presence of the phytopathogen. Ear fresh weight, dry weight, number of seeds per plant, and 100-grain weight were found to increase significantly by 34, 34, 52, and 18% respectively, in comparison to control. P. fluorescens JM-1 can therefore be used as a bioinoculant for ear rot disease control and sustainably enhancing maize yield. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03201-7.

Pseudomonas protegens FJKB0103 Isolated from Rhizosphere Exhibits Anti-Methicillin-Resistant Staphylococcus aureus Activity.

Staphylococcus aureus is amongst the most virulent pathogens, causing chronic and life-threatening human infections. Methicillin-resistant S. aureus (MRSA) are multidrug-resistant strains, and the ability of forming a biofilm reduces their sensitivity to antibiotics. Thus, the alternative compounds inhibiting both resistant strains and biofilm formation are in high demand. In our study, the strain FJKB0103 was isolated from the rhizosphere of Garcinia mangostana, showing strong anti-MRSA activity. We performed molecular phylogenic analysis, analyzed average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH), and biochemical characteristics to identify strain FJKB0103 as Pseudomonas protegens. Herein, the genome of strain FJKB0103 was sequenced and subjected to antiSMASH platform, mutational, and functional analyses. The FJKB0103 draft genome was 6,776,967 bp with a 63.4% G + C content, and 16 potential secondary compound biosynthetic clusters in P. protegens FJKB0103 were predicted. The deletion mutant and complementary analysis suggested that DAPG was the anti-MRSA compound. Further tests showed that MRSA strains were sensitive to DAPG, and the lysis of bacterial cells was observed at a high concentration of DAPG. Additionally, DAPG inhibited the biofilm formation of MRSA at subinhibitory concentration. These results suggested that DAPG might be a good alternative treatment to control infections caused by MRSA.

Pseudomonas bijieensis Strain XL17 within the P. corrugata Subgroup Producing 2,4-Diacetylphloroglucinol and Lipopeptides Controls Bacterial Canker and Gray Mold Pathogens of Kiwifruit.

Kiwifruit worldwide suffers from the devastating diseases of bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) and gray mold caused by Botrytis cinerea. Here, an endophytic bacterium XL17 isolated from a rape crown gall was screened out for its potent antagonistic activities against Psa and B. cinerea. Strain XL17 and its cell-free culture filtrate (CF) inhibited the growth of Psa and B. cinerea, Psa-associated leaf necrosis, and B. cinerea-associated kiwifruit necrosis. Electron microscopy showed that XL17 CF could damage the cell structures of Psa and B. cinerea. Genome-based taxonomy revealed that strain XL17 belongs to Pseudomonas bijieensis within the P. corrugata subgroup of the P. fluorescens species complex. Among the P. corrugata subgroup containing 31 genomospecies, the presence of the phl operon responsible for the biosynthesis of the phenolic polyketide 2,4-diacetylphloroglucinol (DAPG) and the absence of the lipopeptide/quorum sensing island can serve as the genetic marker for the determination of a plant-protection life style. HPLC detected DAPG in extracts from XL17 CF. MALDI-TOF-MS analysis revealed that strain XL17 produced cyclic lipopeptides of the viscosin family and orfamide family. Together, phenotypic, genomic, and metabolic analyses identified that P. bijieensis XL17 producing DAPG and cyclic lipopeptides can be used to control bacterial canker and gray mold pathogens of kiwifruit.

Inhibitory efficacy of 2, 4-diacetylphloroglucinol against SARS-COV-2 proteins: in silico study.

The pandemic of Severe Acute Respiratory Syndrome Coronavirus-2 has affected millions of people worldwide with common symptoms of fever, cough, and respiratory complications. The pandemic has posed a huge challenge to emergency health services due to unavailability of potent therapeutic drugs. The proteins associated with the viral pathogenesis has been identified as suitable targets for drug design and warrants effective drug discovery to abate COVID-19. The papain-like protease (PLpro), nucleocapsid (N), main protease (Mpro) and non-structural protein (nsp12) of SARS-CoV-2, key component of processing of viral polyproteins, transcription, assembly and replication. On this streak, present study evaluated the interaction of ligand 2,4-diacetylphloroglucinol (DAPG) with viral proteins using molecular docking with (i) AutoDock 4.2.6 and (ii) AutoDock Vina followed by molecular dynamic simulation studies of protein-ligand complex configuration. The analysis revealed that PLpro (3E9S) and N (4J3K) protein corresponds to the highest docking score and therefore, selected for molecular dynamics simulation study (100 ns). The study comprised analysis of parameters: (i) RMSD and RMSF, (ii) radius of gyration- which indicated interaction of protein entities with ligand supported steadiness of the complex, (iii) Coulombic and Lennard-Jones interactions, which played a significant role in complex stability. DAPG showed a good number of H-bonds with PLpro and MM-PBSA binding energy when compared to the N protein. This study showed DAPG as a potential bioactive molecule to act as an inhibitor for the PLpro thereby, DAPG can be used as potential inhibitor against SARS-CoV-2 and is potential drug candidate against COVID-19. Supplementary Information: The online version contains supplementary material available at 10.1007/s11756-021-00979-4.

Biofertilizers: An ecofriendly technology for nutrient recycling and environmental sustainability.

Modern intensive agricultural practices face numerous challenges that pose major threats to global food security. In order to address the nutritional requirements of the ever-increasing world population, chemical fertilizers and pesticides are applied on large scale to increase crop production. However, the injudicious use of agrochemicals has resulted in environmental pollution leading to public health hazards. Moreover, agriculture soils are continuously losing their quality and physical properties as well as their chemical (imbalance of nutrients) and biological health. Plant-associated microbes with their plant growth- promoting traits have enormous potential to solve these challenges and play a crucial role in enhancing plant biomass and crop yield. The beneficial mechanisms of plant growth improvement include enhanced nutrient availability, phytohormone modulation, biocontrol of phytopathogens and amelioration of biotic and abiotic stresses. Solid-based or liquid bioinoculant formulation comprises inoculum preparation, addition of cell protectants such as glycerol, lactose, starch, a good carrier material, proper packaging and best delivery methods. Recent developments of formulation include entrapment/microencapsulation, nano-immobilization of microbial bioinoculants and biofilm-based biofertilizers. This review critically examines the current state-of-art on use of microbial strains as biofertilizers and the important roles performed by these beneficial microbes in maintaining soil fertility and enhancing crop productivity.

ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6.

Many Pseudomonas protegens strains produce the antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), both of which have antimicrobial properties. The biosynthesis of these metabolites is typically controlled by multiple regulatory factors. Virulence factor regulator (Vfr) is a multifunctional DNA-binding regulator that modulates 2,4-DAPG biosynthesis in P. protegens FD6. However, the mechanism by which Vfr regulates this process remains unclear. In the present study, chromatin immunoprecipitation of FLAG-tagged Vfr and nucleotide sequencing analysis were used to identify 847 putative Vfr binding sites in P. protegens FD6. The consensus P. protegens Vfr binding site predicted from nucleotide sequence alignment is TCACA. The qPCR data showed that Vfr positively regulates the expression of phlF and phlG, and the expression of these genes was characterized in detail. The purified recombinant Vfr bound to an approximately 240-bp fragment within the phlF and phlG upstream regions that harbor putative Vfr consensus sequences. Using electrophoretic mobility shift assays, we localized Vfr binding to a 25-bp fragment that contains part of the Vfr binding region. Vfr binding was eliminated by mutating the TACG and CACA sequences in phlF and phlG, respectively. Taken together, our results show that Vfr directly regulates the expression of the 2,4-DAPG operon by binding to the upstream regions of both the phlF and phlG genes. However, unlike other Vfr-targeted genes, Vfr binding to P. protegens FD6 does not require an intact binding consensus motif. Furthermore, we demonstrated that vfr expression is autoregulated in this bacterium. These results provide novel insights into the regulatory role of Vfr in the biocontrol agent P. protegens.