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Microorganisms May 2024To optimize the application of plant growth-promoting rhizobacteria (PGPR) in field trials, tracking methods are needed to assess their shelf life and to determine the...
To optimize the application of plant growth-promoting rhizobacteria (PGPR) in field trials, tracking methods are needed to assess their shelf life and to determine the elements affecting their effectiveness and their interactions with plants and native soil microbiota. This work developed a real-time PCR (qtPCR) method which traces and quantifies bacteria when added as microbial consortia, including five PGPR species: , , , , and Through a literature search and in silico sequence analyses, a set of primer pairs which selectively tag three bacterial species (, and ) was retrieved. The primers were used to trace these microbial species in a field trial in which the consortium was tested as a biostimulant on two wheat varieties, in combination with biochar and the mycorrhizal fungus . The qtPCR assay demonstrated that the targeted bacteria had colonized and grown into the soil, reaching a maximum of growth between 15 and 20 days after inoculum. The results also showed biochar had a positive effect on PGPR growth. In conclusion, qtPCR was once more an effective method to trace the fate of supplied bacterial species in the consortium when used as a cargo system for their delivery.
PubMed: 38792831
DOI: 10.3390/microorganisms12051002 -
International Journal of Molecular... May 2024Next-generation sequencing has transformed the acquisition of vast amounts of genomic information, including the rapid identification of target gene sequences in...
Next-generation sequencing has transformed the acquisition of vast amounts of genomic information, including the rapid identification of target gene sequences in metagenomic databases. However, dominant species can sometimes hinder the detection of rare bacterial species. Therefore, a highly sensitive amplification technique that can selectively amplify bacterial genomes containing target genes of interest was developed in this study. The rolling circle amplification (RCA) method can initiate amplification from a single locus using a specific single primer to amplify a specific whole genome. A mixed cell suspension was prepared using ATCC17400 (targeting nonribosomal peptide synthetase [NRPS]) and (non-target), and a specific primer designed for the was used for the RCA reaction. The resulting RCA product (RCP) amplified only the genome. The was successfully amplified using RCP as a template from even five cells, indicating that the single-priming RCA technique can specifically enrich the target genome using gene-specific primers. Ultimately, this specific genome RCA technique was applied to metagenomes extracted from sponge-associated bacteria, and sequences were successfully obtained from an unknown sponge-associated bacterium. Therefore, this method could be effective for accessing species-specific sequences of in unknown bacteria, including viable but non-culturable bacteria.
Topics: Peptide Synthases; Nucleic Acid Amplification Techniques; Genome, Bacterial; High-Throughput Nucleotide Sequencing; Escherichia coli; Pseudomonas fluorescens; Sequence Analysis, DNA; Metagenome
PubMed: 38791129
DOI: 10.3390/ijms25105089 -
Microbial Cell Factories May 2024Aminopyrrolnitrin (APRN), a natural halogenated phenylpyrrole derivative (HPD), has strong antifungal and antiparasitic activities. Additionally, it showed 2.8-fold...
Aminopyrrolnitrin (APRN), a natural halogenated phenylpyrrole derivative (HPD), has strong antifungal and antiparasitic activities. Additionally, it showed 2.8-fold increased photostability compared to pyrrolnitrin, a commercially available HPD with antimicrobial activity. For microbial production of APRN, we first engineered anthranilate phosphoribosyltransferase encoded by trpD from Corynebacterium glutamicum, resulting in a TrpD mutation that exhibits feedback-resistant against L-tryptophan and higher substrate affinity compared to wild-type TrpD. Plasmid-borne expression of trpD in C. glutamicum TP851 strain with two copies of trpD in the genome led to the production of 3.1 g/L L-tryptophan in flask culture. Subsequent step for L-tryptophan chlorination into 7-chloro-L-tryptophan was achieved by introducing diverse sources of genes encoding tryptophan 7-halogenase (PrnA or RebH) and flavin reductase (Fre, PrnF, or RebF). The combined expression of prnA from Serratia grimesii or Serratia plymuthica with flavin reductase gene from Escherichia coli, Pseudomonas fluorescens, or Lechevalieria aerocolonigenes yielded higher production of 7-chloro-L-tryptophan in comparison to other sets of two-component systems. In the next step, production of putative monodechloroaminopyrrolnitrin (MDAP) from 7-chloro-L-tryptophan was achieved through the expression of prnB encoding MDAP synthase from S. plymuthica or P. fluorescens. Finally, an artificial APRN biosynthetic pathway was constructed by simultaneously expressing genes coding for tryptophan 7-halogenase, flavin reductase, MDAP synthase, and MDAP halogenase (PrnC) from different microbial sources within the L-tryptophan-producing TP851 strain. As prnC from S. grimesii or S. plymuthica was introduced into the host strain, which carried plasmids expressing prnA from S. plymuthica, fre from E. coli, and prnB from S. plymuthica, APN3639 and APN3638 accumulated 29.5 mg/L and 28.1 mg/L of APRN in the culture broth. This study represents the first report on the fermentative APRN production by metabolically engineered C. glutamicum.
Topics: Corynebacterium glutamicum; Metabolic Engineering; Pyrrolnitrin; Fermentation; Bacterial Proteins; Tryptophan; Escherichia coli; Oxidoreductases
PubMed: 38783320
DOI: 10.1186/s12934-024-02424-y -
International Journal of Molecular... Apr 2024Aphids are insect pests that suck phloem sap and introduce salivary proteins into plant tissues through saliva secretion. The effector of salivary proteins plays a key...
Aphids are insect pests that suck phloem sap and introduce salivary proteins into plant tissues through saliva secretion. The effector of salivary proteins plays a key role in the modulation of host plant defense responses and enhancing aphid host adaptation. Based on previous transcriptome sequencing results, a candidate effector cyclin-dependent kinase-like (CDK) was identified from the grain aphid . In this study, the function of SaCDK in wheat defense response and the adaptation of was investigated. Our results showed that the transient overexpression of SaCDK in tobacco suppressed cell death triggered by mouse pro-apoptotic protein-BAX or PAMP-INF1. SaCDK, delivered into wheat cells through a -mediated bacterial type III secretion system, suppressed callose deposition in wheat seedlings, and the overexpression of SaCDK in wheat significantly decreased the expression levels of salicylic acid and jasmonic acid signaling pathway-related genes phenylalanine ammonia lyase (), pathogenesis-related 1 protein (), lipoxygenase () and Ω-3 fatty acid desaturase (). In addition, aphid bioassay results showed that the survival and fecundity of were significantly increased while feeding on the wheat plants carrying SaCDK. Taken together, our findings demonstrate that the salivary protein SaCDK is involved in inhibiting host defense response and improving its host adaptation, which lays the foundation to uncover the mechanism of the interaction of cereal aphids and host plants.
Topics: Animals; Aphids; Triticum; Salivary Proteins and Peptides; Insect Proteins; Adaptation, Physiological; Plant Diseases; Gene Expression Regulation, Plant; Nicotiana; Cyclopentanes; Oxylipins
PubMed: 38731798
DOI: 10.3390/ijms25094579 -
Microbiology Spectrum Jun 2024Swarming motility in pseudomonads typically requires both a functional flagellum and the production/secretion of a biosurfactant. Published work has shown that the...
Swarming motility in pseudomonads typically requires both a functional flagellum and the production/secretion of a biosurfactant. Published work has shown that the wild-type Pf0-1 is swarming deficient due to a point mutation in the gene, which until recently was thought to inactivate rather than attenuate the Gac/Rsm pathway. As a result, little is known about the underlying mechanisms that regulate swarming motility by Pf0-1. Here, we demonstrate that a Δ Δ Δ mutant, which phenotypically mimics Gac/Rsm pathway overstimulation, is proficient at swarming motility. RsmA and RsmE appear to play a key role in this regulation. Transposon mutagenesis of the Δ Δ Δ mutant identified multiple factors that impact swarming motility, including pathways involved in flagellar synthesis and biosurfactant production/secretion. We find that loss of genes linked to biosurfactant Gacamide A biosynthesis or secretion impacts swarming motility, as does loss of the alternative sigma factor FliA, which results in a defect in flagellar function. Collectively, these findings provide evidence that Pf0-1 can swarm if the Gac/Rsm pathway is activated, highlight the regulatory complexity of swarming motility in this strain, and demonstrate that the cyclic lipopeptide Gacamide A is utilized as a biosurfactant for swarming motility.IMPORTANCESwarming motility is a coordinated process that allows communities of bacteria to collectively move across a surface. For Pf0-1, this phenotype is notably absent in the parental strain, and to date, little is known about the regulation of swarming in this strain. Here, we identify RsmA and RsmE as key repressors of swarming motility modulating the levels of biosurfactant production/secretion. Using transposon mutagenesis and subsequent genetic analyses, we further identify potential regulatory mechanisms of swarming motility and link Gacamide A biosynthesis and transport machinery to swarming motility.
Topics: Pseudomonas fluorescens; Movement; Bacterial Proteins; Methyltransferases; Surface-Active Agents; Mutagenesis; Sigma Factor
PubMed: 38687073
DOI: 10.1128/spectrum.00166-24 -
World Journal of Clinical Cases Apr 2024The clinical incidence of spinal infection is gradually increasing, and its onset is insidious, easily leading to missed diagnosis and misdiagnosis, which may lead to...
BACKGROUND
The clinical incidence of spinal infection is gradually increasing, and its onset is insidious, easily leading to missed diagnosis and misdiagnosis, which may lead to serious complications such as nervous system dysfunction, spinal instability and/or deformity, and cause a huge burden on society and families. Early identification of the causative agent and precision medicine will greatly reduce the suffering of patients. At present, the main pathogenic bacteria that cause spinal infection are , Streptococcus, Pneumococcus, , and Klebsiella. There are no reports of spinal infection caused by .
CASE SUMMARY
We report a 32-year-old female patient with spinal infection. She presented with flank pain, initially thought to be bone metastases or bone tuberculosis, and had a family background of tumors. Her clinical features and changes in imaging and laboratory tests led to the suspicion of thoracic spine infection. Histopathology of the lesion showed inflammation, tissue culture of the lesion was negative several times, and the possible pathogen - was found after gene sequencing of the lesion. The patient recovered completely after a full course of antibiotic treatment.
CONCLUSION
This report increases the range of pathogens involved in spinal infections, highlights the unique advantages of gene sequencing technology in difficult-to-diagnose diseases, and validates conservative treatment with a full course of antibiotics for spinal infections without complications.
PubMed: 38680271
DOI: 10.12998/wjcc.v12.i12.2099 -
International Journal of Molecular... Apr 2024Molecular studies about cyanide biodegradation have been mainly focused on the hydrolytic pathways catalyzed by the cyanide dihydratase CynD or the nitrilase NitC. In...
Molecular studies about cyanide biodegradation have been mainly focused on the hydrolytic pathways catalyzed by the cyanide dihydratase CynD or the nitrilase NitC. In some strains, the assimilation of cyanide has been linked to NitC, such as the cyanotrophic model strain CECT 5344, which has been recently reclassified as CECT 5344. In this work, a phylogenomic approach established a more precise taxonomic position of the strain CECT 5344 within the species . Furthermore, a pan-genomic analysis of and other species with cyanotrophic strains, such as and , allowed for the comparison and identification of the and genes involved in cyanide resistance, and the and genes required for the assimilation of cyanide or cyanate, respectively. While cyanide resistance genes presented a high frequency among the analyzed genomes, genes responsible for cyanide or cyanate assimilation were identified in a considerably lower proportion. According to the results obtained in this work, an in silico approach based on a comparative genomic approach can be considered as an agile strategy for the bioprospection of putative cyanotrophic bacteria and for the identification of new genes putatively involved in cyanide biodegradation.
Topics: Cyanides; Biodegradation, Environmental; Pseudomonas; Phylogeny; Genome, Bacterial; Genomics; Bacterial Proteins; Aminohydrolases; Pseudomonas pseudoalcaligenes
PubMed: 38674043
DOI: 10.3390/ijms25084456 -
Foods (Basel, Switzerland) Apr 2024is an edible mushroom that suffers significant losses due to fungal contamination and bacteriosis. The Pseudomonadaceae family represents one of the most frequent...
is an edible mushroom that suffers significant losses due to fungal contamination and bacteriosis. The Pseudomonadaceae family represents one of the most frequent etiologic agents. Grapefruit seed extract (GSE) is a plant extract that contains different bioactive components, such as naringin, and exhibits a strong antibacterial and antioxidant activity. Over the last decade, GSE use as an alternative to chemical treatments in the food sector has been tested. However, to our knowledge, its application on mushroom crops has never been investigated. This study focuses on evaluating GSE efficacy in preventing yellowing. GSE antibiotic activity, inhibitory and bactericidal concentrations, and antibiofilm activity against several microorganisms were tested with the Kirby-Bauer disk diffusion assay, the broth microdilution susceptibility test, and the Crystal violet assay, respectively. In vitro, the extract exhibited antimicrobial and antibiofilm activity against 6538 and MRSA (wild type), ATCC 8739, and spp. 9027, (wild type)). GSE application in vivo, in pre- and post-sprouting stages, effectively prevented bacterial infections and subsequent degradation in the mushroom crops: none of the treated manifested bacteriosis. Our findings support the use of GSE as an eco-friendly and sustainable alternative to chemical treatments for protecting crops from bacterial contamination, consequently ensuring food safety and preventing financial losses due to spoilage. Furthermore, GSE's potential health benefits due to its content in naringin and other bioactive components present new possibilities for its use as a nutraceutical in food fortification and supplementation.
PubMed: 38672835
DOI: 10.3390/foods13081161 -
Microbial Ecology Apr 2024Microorganisms produce siderophores, which are low-molecular-weight iron chelators when iron availability is limited. The present analyzed the role of LNPF1 as...
Purification and Characterization of Desferrioxamine B of Pseudomonas fluorescens and Its Application to Improve Oil Content, Nutrient Uptake, and Plant Growth in Peanuts.
Microorganisms produce siderophores, which are low-molecular-weight iron chelators when iron availability is limited. The present analyzed the role of LNPF1 as multifarious PGPR for improving growth parameters and nutrient content in peanut and soil nutrients. Such multifarious PGPR strains can be used as effective bioinoculants for peanut farming. In this work, rhizosphere bacteria from Zea mays and Arachis hypogaea plants in the Salem area of Tamil Nadu, India, were isolated and tested for biochemical attributes and characteristics that stimulate plant growth, such as the production of hydrogen cyanide, ammonia (6 µg/mL), indole acetic acid (76.35 µg/mL), and solubilizing phosphate (520 µg/mL). The 16S rRNA gene sequences identified the isolate LNPF1 as Pseudomonas fluorescens with a similarity percentage of 99% with Pseudomonas sp. Isolate LNPF1 was evaluated for the production of siderophore. Siderophore-rich supernatant using a Sep Pack C18 column and Amberlite-400 Resin Column (λmax 264) produced 298 mg/L and 50 mg/L of siderophore, respectively. The characterization of purified siderophore by TLC, HPLC, FTIR, and 2D-NMR analysis identified the compound as desferrioxamine, a hydroxamate siderophore. A pot culture experiment determined the potential of LNPF1 to improve iron and oil content and photosynthetic pigments in Arachis hypogaea L. and improve soil nutrient content. Inoculation of A. hypogea seeds with LNPF1 improved plant growth parameters such as leaf length (60%), shoot length (22%), root length (54.68%), fresh weight (47.28%), dry weight (37%), and number of nuts (66.66) compared to the control (untreated seeds). This inoculation also improved leaf iron content (43.42), short iron content (38.38%), seed iron (46.72%), seed oil (31.68%), carotenoid (64.40%), and total chlorophyll content (98.%) compared to control (untreated seeds). Bacterized seeds showed a substantial increase in nodulation (61.65%) and weight of individual nodules (95.97) vis-à-vis control. The results of the present study indicated that P. fluorescens might be utilized as a potential bioinoculant to improve growth, iron content, oil content, number of nuts and nodules of Arachishypogaea L., and enrich soil nutrients.
Topics: Arachis; Deferoxamine; Pseudomonas fluorescens; India; RNA, Ribosomal, 16S; Nutrients; Siderophores; Iron; Soil
PubMed: 38630182
DOI: 10.1007/s00248-024-02377-0 -
Plants (Basel, Switzerland) Mar 2024Milpa is an agroecological production system based on the polyculture of plant species, with corn featuring as a central component. Traditionally, the milpa system does...
Milpa is an agroecological production system based on the polyculture of plant species, with corn featuring as a central component. Traditionally, the milpa system does not require the application of chemicals, and so pest attacks and poor growth in poor soils can have adverse effects on its production. Therefore, the application of bioinoculants could be a strategy for improving crop growth and health; however, the effect of external inoculant agents on the endemic microbiota associated with corn has not been extensively studied. Here, the objective of this work was to fertilize a maize crop under a milpa agrosystem with the PGPR UM270, evaluating its impact on the diversity of the rhizosphere (rhizobiome) and root endophytic (root endobiome) microbiomes of maize plants. The endobiome of maize roots was evaluated by 16S rRNA and internal transcribed spacer region (ITS) sequencing, and the rhizobiome was assessed by metagenomic sequencing upon inoculation with the strain UM270. The results showed that UM270 inoculation of the rhizosphere of UM270 did not increase alpha diversity in either the monoculture or milpa, but it did alter the endophytic microbiome of maize plant roots by stimulating the presence of bacterial operational taxonomic units (OTUs) of the genera and (in a monoculture), whereas, in the milpa system, the PGPR stimulated greater endophytic diversity and the presence of genera such as , , and N-fixing rhizobia genera, including , , and . No clear association was found between fungal diversity and the presence of strain UM270, but beneficial fungi, such as and , were detected in the Milpa system. In addition, network analysis revealed unique interactions with species such as sp., , and , which could potentially play beneficial roles in the plant. Finally, the UM270 strain does not seem to have a strong impact on the microbial diversity of the rhizosphere, but it does have a strong impact on some functions, such as trehalose synthesis, ammonium assimilation, and polyamine metabolism. The inoculation of UM270 biofertilizer in maize plants modifies the rhizo- and endophytic microbiomes with a high potential for stimulating plant growth and health in agroecological crop models.
PubMed: 38611483
DOI: 10.3390/plants13070954