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Microorganisms May 2024Lactoperoxidase (LP) is an important enzyme of the salivary and mammary glands. It has been proven to increase the shelf life of raw milk by inhibiting the growth of...
Lactoperoxidase (LP) is an important enzyme of the salivary and mammary glands. It has been proven to increase the shelf life of raw milk by inhibiting the growth of bacteria, especially , , , and spp. The aim of this work was to verify the use of LP to extend the shelf life of meat products. In vitro experiments showed inhibitory effects on the selected bacteria ( (ATCC 33090), (CP054440.1), and (ATCC 13525) due to a prolongation of the lag phase of growth curves. A lower increase in viable counts ( < 0.05) was also found by testing pork cubes' surface treated with LP solution (5%) + and stored for 7 days at 15 °C. LP has also been studied at concentrations of 0.25 and 0.50% in meat products (pork ham and pâté) during refrigerated storage (4 °C for 28 days). Lower viable counts were observed throughout the storage experiment, especially for 0.50% LP ( < 0.05). Meat products containing LP also showed lower levels of oxidation (MAD) ( < 0.05). According to these results, LP could extend the shelf life of a wider range of products.
PubMed: 38792839
DOI: 10.3390/microorganisms12051010 -
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 -
Journal of Microbiological Methods Jul 2024Flow cytometry (FCM) provides unique information on bacterial viability and physiology, allowing a real-time early warning antimicrobial and antibiofilm monitoring...
Flow cytometry (FCM) provides unique information on bacterial viability and physiology, allowing a real-time early warning antimicrobial and antibiofilm monitoring system for preventing the spread risk of foodborne disease. The present work used a combined culture-based and FCM approach to assess the in vitro efficacy of essential oils (EOs) from condiment plants commonly used in Mediterranean Europe (i.e., thyme EO, oregano EO, basil EO, and lemon EO) against planktonic and sessile cells of food-pathogenic Listeria monocytogenes 56 LY, and contaminant and alterative species Escherichia coli ATCC 25922 and Pseudomonas fluorescens ATCC 13525. Evaluation of the bacterial response to the increasing concentrations of natural compounds posed FCM as a crucial technique for the quantification of the live/dead, and viable but non-culturable (VBNC) cells when antimicrobial agents exert no real bactericidal action. Furthermore, the FCM results displayed higher numbers of viable bacteria expressed as Active Fluorescent Units (AFUs) with a greater level of repeatability compared with outcomes of the plate-count method. Overall, accurate counting of viable microbial cells is a critically important parameter in food microbiology, and flow cytometry provides an innovative approach with high-throughput potential for applications in the food industry as "flow microbiology".
Topics: Flow Cytometry; Biofilms; Pseudomonas fluorescens; Listeria monocytogenes; Oils, Volatile; Escherichia coli; Microbial Viability; Food Microbiology; Anti-Bacterial Agents; Thymus Plant; Origanum; Microbial Sensitivity Tests; Citrus; Ocimum basilicum
PubMed: 38759758
DOI: 10.1016/j.mimet.2024.106956 -
Probiotics and Antimicrobial Proteins May 2024Bacteriocins produced by lactic acid bacteria (LAB) have good potential for use as food biopreservatives. Lacticaseibacillus paracasei Zhang (L. paracasei Zhang) is both...
Bacteriocins produced by lactic acid bacteria (LAB) have good potential for use as food biopreservatives. Lacticaseibacillus paracasei Zhang (L. paracasei Zhang) is both a food use and a probiotic bacterium. This study aimed to purify and preliminary characterize the active antibacterial metabolite of L. paracasei Zhang. The cell-free supernatant of L. paracasei Zhang was collected and purified by ultrafiltration and gel filtration chromatography. The 1-3 kDa active fraction could inhibit the growth of Staphylococcus aureus but not Escherichia coli. Further antibacterial activity assays revealed its capacity to suppress various foodborne and human opportunistic pathogens (including Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Listeria monocytogenes, and Bacillus cereus), but not fungi. The antibacterial activity showed good tolerance to heat (40 to 100 °C), acid-base (pH 2-3 and pH 6-10), and digestions by a number of industrial and animal/human enzymes (such as trypsin, pepsin, α-amylase, and protease K, except papain); these desired properties make it a suitable biopreservative to be used in harsh and complex industrial production processes. The high papain sensitivity suggested a proteinaceous/peptide nature of the bioactivity. Moreover, our genomic data mining for bacteriocin through BAGEL4 revealed an area of interest encoding a complete set of putative genes required for bacteriocin production. In conclusion, our study showed that L. paracasei Zhang can produce extracellular functional antibacterial metabolite, likely a class II bacteriocin. Our preliminary extraction and characterization of the active metabolite demonstrated that it has good potential to be used as a biopreservative or an agent for suppressing gastrointestinal infections.
PubMed: 38748307
DOI: 10.1007/s12602-024-10249-9 -
Folia Microbiologica May 2024There is an increasing demand for bioinoculants based on plant growth-promoting rhizobacteria (PGPR) for use in agricultural ecosystems. However, there are still...
There is an increasing demand for bioinoculants based on plant growth-promoting rhizobacteria (PGPR) for use in agricultural ecosystems. However, there are still concerns and limited data on their reproducibility in different soil types and their effects on endemic rhizosphere communities. Therefore, this study explored the effects of inoculating the PGPR, Pseudomonas fluorescens strain UM270, on maize growth (Zea mays L.) and its associated rhizosphere bacteriome by sequencing the 16S ribosomal genes under greenhouse conditions. The results showed that inoculation with PGPR P. fluorescens UM270 improved shoot and root dry weights, chlorophyll concentration, and total biomass in the three soil types evaluated (clay, sandy-loam, and loam) compared to those of the controls. Bacterial community analysis of the three soil types revealed that maize plants inoculated with the UM270 strain showed a significant increase in Proteobacteria and Acidobacteria populations, whereas Actinobacteria and Bacteroidetes decreased. Shannon, Pielou, and Faith alpha-biodiversity indices did not reveal significant differences between treatments. Beta diversity revealed a bacterial community differential structure in each soil type, with some variation among treatments. Finally, some bacterial groups were found to co-occur and co-exclude with respect to UM270 inoculation. Considered together, these results show that PGPR P. fluorescens UM270 increases maize plant growth and has an important effect on the resident rhizobacterial communities of each soil type, making it a potential agricultural biofertilizer.
PubMed: 38748205
DOI: 10.1007/s12223-024-01171-2 -
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 -
International Journal of Biological... Jun 2024Optically pure 1,2,3,4-tetrahydroquinolines (THQs) represent a class of important motifs in many natural products and pharmaceutical agents. While recent advances on...
Optically pure 1,2,3,4-tetrahydroquinolines (THQs) represent a class of important motifs in many natural products and pharmaceutical agents. While recent advances on redox biocatalysis have demonstrated the great potential of amine oxidases, all the transformations focused on 2-substituted THQs. The corresponding biocatalytic method for the preparation of chiral 4-substituted THQs is still challenging due to the poor activity and stereoselectivity of the available enzyme. Herein, we developed a biocatalytic kinetic resolution approach for enantiodivergent synthesis of 4-phenyl- or alkyl-substituted THQs. Through structure-guided protein engineering of cyclohexylamine oxidase derived from Brevibacterium oxidans IH-35 A (CHAO), the variant of CHAO (Y215H/Y214S) displayed improved specific activity toward model substrate 4-phenyl substituted THQ (0.14 U/mg, 13-fold higher than wild-type CHAO) with superior (R)-stereoselectivity (E > 200). Molecular dynamics simulations show that CHAO Y215H/Y214S allows a suitable substrate positioning in the expanded binding pocket to be facilely accessed, enabling enhanced activity and stereoselectivity. Furthermore, a series of 4-alkyl-substituted THQs can be transformed by CHAO Y215H/Y214S, affording R-isomers with good yields (up to 50 %) and excellent enantioselectivity (up to ee > 99 %). Interestingly, the monoamine oxidase from Pseudomonas fluorescens Pf0-1 (PfMAO1) with opposite enantioselectivity was also mined. Together, this system enriches the kinetic resolution methods for the synthesis of chiral THQs.
Topics: Kinetics; Stereoisomerism; Quinolines; Biocatalysis; Brevibacterium; Substrate Specificity; Molecular Dynamics Simulation; Monoamine Oxidase
PubMed: 38729465
DOI: 10.1016/j.ijbiomac.2024.132102 -
The Science of the Total Environment Jul 2024Plant growth regulators (PGR) and plant growth-promoting bacteria (PGPB) have the potential in phytoremediation of heavy metals (HMs) contaminated soils. However, their...
Enhancing the phytoextraction efficiency of heavy metals in acidic and alkaline soils by Sedum alfredii Hance: A study on the synergistic effect of plant growth regulator and plant growth-promoting bacteria.
Plant growth regulators (PGR) and plant growth-promoting bacteria (PGPB) have the potential in phytoremediation of heavy metals (HMs) contaminated soils. However, their sole application may not yield the optimal results, thus necessitating the combined application. The present study aimed to enhance the phytoremediation efficiency of Sedum alfredii Hance (S. alfredii) in acidic and alkaline soils through the combination of PGR (Brassinolide, BR) and PGPB (Pseudomonas fluorescens, P. fluorescens). The combination of BR and P. fluorescens (BRB treatment) effectively increased the removal efficiency of S. alfredii for Cd, Pb, and Zn by 355.2 and 155.3 %, 470.1 and 128.9 %, and 408.4 and 209.6 %, in acidic and alkaline soils, respectively. Moreover, BRB treatment led to a substantial increase in photosynthetic pigments contents and antioxidant enzymes activities, resulting in a remarkable increase in biomass (86.71 and 47.22 %) and dry mass (101.49 and 42.29 %) of plants grown in acidic and alkaline soils, respectively. Similarly, BRB treatment significantly elevated the Cd (109.4 and 71.36 %), Pb (174.9 and 48.03 %), and Zn levels (142.8 and 104.3 %) in S. alfredii shoots, along with cumulative accumulation of Cd (122.7 and 79.47 %), Pb (183.8 and 60.49 %), and Zn (150.7 and 117.9 %), respectively. In addition, the BRB treatment lowered the soil pH and DTPA-HMs contents, while augmenting soil enzymatic activities, thereby contributing soil microecology and facilitating the HMs absorption and translocation by S. alfredii to over-ground tissues. Furthermore, the evaluation of microbial community structure in phyllosphere and rhizosphere after remediation revealed the shift in microbial abundance. The combined treatment altered the principal effects on S. alfredii HMs accumulation from bacterial diversity to the soil HMs availability. In summary, our findings demonstrated that synergistic application of BR and P. fluorescens represents a viable approach to strengthen the phytoextraction efficacy of S. alfredii in varying soils.
Topics: Sedum; Biodegradation, Environmental; Soil Pollutants; Metals, Heavy; Plant Growth Regulators; Soil; Pseudomonas fluorescens; Soil Microbiology
PubMed: 38719039
DOI: 10.1016/j.scitotenv.2024.173029