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International Journal of Antimicrobial... Oct 2023The goal of this study was to determine the effects of phenolic extracts from grape (GrPE), pomegranate (PoPE), and persimmon (PePE) by-products on bacterial virulence...
OBJECTIVE
The goal of this study was to determine the effects of phenolic extracts from grape (GrPE), pomegranate (PoPE), and persimmon (PePE) by-products on bacterial virulence activities such as biofilms, motility, energy-dependent efflux pumps, and β-lactamase activity, which are modulated primarily by quorum sensing (QS), defining their potential applications.
METHOD
The microdilution method was used to determine the minimum inhibitory concentration (MIC) and sub-inhibitory concentrations (SICs) of the extracts against reference pathogenic bacteria. The antibacterial mode of action was determined by labelling bacterial cells in in vivo cell-tracking experiments.
RESULTS
Antibiograms showed that PoPE inhibited bacteria at lower concentrations, and PePE had a stronger effect against Klebsiella pneumoniae. Both extracts caused significant cell membrane damage (CMD), whereas GrPE did not. At SICs, all extracts showed anti-QS activity, especially PePE, which inhibited violacein and pyocyanin production at 1/128 × MIC. Additionally, QS autoinducers found in Chromobacterium violaceum and Pseudomonas aeruginosa were modulated by the extracts; PePE showed the highest modulation. Antibiofilm assays revealed that GrPE, at MIC and 2 × MIC, acted as a potent antibiofilm agent against biofilms of Pseudomonas putida, Bacillus cereus, and Staphylococcus aureus, which was related to disruption of swarming motility by GrPE. All extracts, especially PoPE, exerted a potent effect against the activation of efflux pumps of P. aeruginosa as well as β-lactamase activity in K. pneumoniae.
CONCLUSION
Results suggest that the anti-virulence potential of the extracts may be related to their effect as extracellular autoinducer modulators. This study allowed to define potential applications of these extracts.
PubMed: 37517626
DOI: 10.1016/j.ijantimicag.2023.106937 -
Plants (Basel, Switzerland) Dec 2023Salinity inhibits plant growth by affecting physiological processes, but soil microorganisms like plant growth-promoting rhizobacteria (PGPR) can alleviate abiotic...
Salinity inhibits plant growth by affecting physiological processes, but soil microorganisms like plant growth-promoting rhizobacteria (PGPR) can alleviate abiotic stress and enhance crop productivity. However, it should be noted that rhizobacteria employ different approaches to deal with salt stress conditions and successfully colonize roots. The objective of this study was to investigate the effect of salt stress on bacterial survival mechanisms such as mobility, biofilm formation, and the autoaggregation capacity of three plant growth-promoting strains: SJ04, WCS417r, and GB03. These strains were grown in diluted LB medium supplemented with 0, 100, 200, or 300 mM NaCl. Swimming and swarming mobility were evaluated in media supplemented with 0.3 and 0.5% agar, respectively. Biofilm formation capacity was quantified using the crystal violet method, and the autoaggregation capacity was measured spectrophotometrically. In addition, we evaluated in vitro the capacity of the strains to ameliorate the effects of saline stress in . The study found that the GB03 strain exhibited enhanced swarming mobility when the salt concentration in the medium increased, resulting in a two-fold increase in the halo diameter at 300 mM. However, high concentrations of NaCl did not affect the swimming mobility. In contrast, swimming motility was reduced in WCS417r and SJ04 under salt stress. On the other hand, exposure to 300 mM NaCl resulted in a 180% increase in biofilm formation and a 30% rise in the percentage of autoaggregation in WCS417r. Conversely, the autoaggregation percentage of the strains SJ04 and GB03 remained unaffected by saline stress. However, for GB03, biofilm formation decreased by 80% at 300 mM. Simultaneously, inoculation with the three evaluated strains alleviated the detrimental effects of salinity on plant growth. Under 150 mM salt stress, all strains showed increased fresh weight, with GB03 and WCS417r improving by 40% and SJ04 exhibiting the most remarkable effect with a 70% rise compared to non-inoculated plants. Despite their different strategies for mitigating salt stress, the application of these strains presents a promising strategy for effectively mitigating the negative consequences of salt stress on plant cultivation.
PubMed: 38068694
DOI: 10.3390/plants12234059 -
Microbiology Spectrum Dec 2023The genus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol, and synthetic biology. Though several...
The genus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol, and synthetic biology. Though several strains have been identified with beneficial properties, chromosomal manipulations to further improve these strains for commercial applications have been limited due to the lack of efficient genetic tools that have been tested across this genus. Here, we test the recombineering efficiencies of five phage-derived recombinases across three biotechnologically relevant strains: KT2440, Pf-5, and CHA0. These results demonstrate a method to generate targeted mutations quickly and efficiently across these strains, ideally introducing a method that can be implemented across the genus and a strategy that may be applied to develop analogous systems in other nonmodel bacteria.
Topics: Pseudomonas; Recombinases; Bacteriophages
PubMed: 37882574
DOI: 10.1128/spectrum.03176-23 -
Frontiers in Microbiology 2023Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of...
Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of the microbiome allows us to create an artificial microbiome without the microorganism of interest and to evaluate its functions and interactions with other constituent bacteria. However, few techniques that can specifically remove only a single species from a large number of microorganisms and can be applied universally to a variety of microorganisms have been developed. Antisense peptide nucleic acid (PNA) is a potent designable antimicrobial agent that can be delivered into microbial cells by conjugating with a cell-penetrating peptide (CPP). Here, we tested the efficacy of the conjugate of CPP and PNA (CPP-PNA) as microbiome modifiers. The addition of CPP-PNA specifically inhibited the growth of and in an artificial bacterial consortium comprising , , , and . Moreover, the growth inhibition of promoted the growth of and inhibited the growth of . These results indicate that CPP-PNA can be used not only for precise microbiome engineering but also for analyzing the growth relationships among constituent microorganisms in the microbiome.
PubMed: 38260881
DOI: 10.3389/fmicb.2023.1321428 -
Bioresource Technology Nov 2023Pseudomonas putida is a promising strain for lignin valorisation. However, there is a dearth of stable and efficient systems for secreting enzymes to enhance the...
Pseudomonas putida is a promising strain for lignin valorisation. However, there is a dearth of stable and efficient systems for secreting enzymes to enhance the process. Therefore, a novel secretion system for recombinant lignin-depolymerising peroxidase was developed. By adopting a flagellar type III secretion system, P. putida KT-M2, a secretory host strain, was constructed and an optimal secretion signal fusion partner was identified. Application of the dye-decolourising peroxidase of P. putida to this system resulted in efficient oxidation activity of the cell-free supernatant against various chemicals, including lignin model compounds. This peroxidase-secreting strain was examined to confirm its lignin utilisation capability, resulting in the efficient assimilation of various lignin substrates with 2.6-fold higher growth than that of the wild-type strain after 72 h of cultivation. Finally, this novel system will lead efficient bacterial lignin breakdown and utilization through enzyme secretion, paving the way for sustainable lignin-consolidated bioprocessing.
Topics: Lignin; Pseudomonas putida; Peroxidase; Peroxidases; Oxidoreductases; Coloring Agents
PubMed: 37739186
DOI: 10.1016/j.biortech.2023.129779 -
Microbiology Resource Announcements Jun 2024We report the complete genome of strain WBB028, which exhibits broad-spectrum antifungal activity. This strain was isolated from leaf litter collected at Walker Branch...
We report the complete genome of strain WBB028, which exhibits broad-spectrum antifungal activity. This strain was isolated from leaf litter collected at Walker Branch Watershed located on the Oak Ridge Reservation in eastern Tennessee (35.9614 N 84.2864 W). The genome is 6.3 Mbp with a 62.5% GC content.
PubMed: 38860815
DOI: 10.1128/mra.00234-24 -
ACS Omega Jul 2023Acetate is an end-product of anaerobic biodegradation and one of the major metabolites of microbial fermentation and lingo-cellulosic hydrolysate. Recently, acetate has...
Acetate is an end-product of anaerobic biodegradation and one of the major metabolites of microbial fermentation and lingo-cellulosic hydrolysate. Recently, acetate has been highlighted as a feedstock to produce value-added chemicals. This study examined acetate conversion to succinate by citrate synthase ()-overexpressed under microaerobic conditions. The acetate metabolism is initiated with the gltA enzyme, which converts acetyl-CoA to citrate. -overexpressing (-KT) showed an ∼50% improvement in succinate production compared to the wild type. Under the optimal pH of 7.5, the accumulation of succinate (4.73 ± 0.6 mM in 36 h) was ∼400% higher than that of the wild type. Overall, overexpression alone resulted in 9.5% of the maximum theoretical yield in a minimal medium with acetate as the sole carbon source. This result shows that citrate synthase is important in acetate conversion to succinate by under microaerobic conditions.
PubMed: 37521642
DOI: 10.1021/acsomega.3c02520 -
Communications Biology Oct 2023In all domains of life, transfer RNAs (tRNAs) contain post-transcriptionally sulfur-modified nucleosides such as 2- and 4-thiouridine. We have previously reported that a...
In all domains of life, transfer RNAs (tRNAs) contain post-transcriptionally sulfur-modified nucleosides such as 2- and 4-thiouridine. We have previously reported that a recombinant [4Fe-4S] cluster-containing bacterial desulfidase (TudS) from an uncultured bacterium catalyzes the desulfuration of 2- and 4-thiouracil via a [4Fe-5S] cluster intermediate. However, the in vivo function of TudS enzymes has remained unclear and direct evidence for substrate binding to the [4Fe-4S] cluster during catalysis was lacking. Here, we provide kinetic evidence that 4-thiouridine-5'-monophosphate rather than sulfurated tRNA, thiouracil, thiouridine or 4-thiouridine-5'-triphosphate is the preferred substrate of TudS. The occurrence of sulfur- and substrate-bound catalytic intermediates was uncovered from the observed switch of the S = 3/2 spin state of the catalytic [4Fe-4S] cluster to a S = 1/2 spin state upon substrate addition. We show that a putative gene product from Pseudomonas putida KT2440 acts as a TudS desulfidase in vivo and conclude that TudS-like enzymes are widespread desulfidases involved in recycling and detoxifying tRNA-derived 4-thiouridine monophosphate nucleosides for RNA synthesis.
Topics: Thiouridine; RNA, Transfer; Bacteria; Catalysis; Sulfur
PubMed: 37891428
DOI: 10.1038/s42003-023-05450-5 -
Metabolic Engineering Mar 2024Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a...
Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane) and has been produced in several engineered microorganisms. Recently, Pseudomonas putida has gained interest as a future host for isoprenol bioproduction as it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the "IPP-bypass" pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational modeling and strain engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.
Topics: Pseudomonas putida; Carbon; Metabolic Engineering
PubMed: 38369052
DOI: 10.1016/j.ymben.2024.02.004 -
ACS Synthetic Biology Nov 2023Type I polyketide synthases (T1PKSs) hold enormous potential as a rational production platform for the biosynthesis of specialty chemicals. However, despite great...
Type I polyketide synthases (T1PKSs) hold enormous potential as a rational production platform for the biosynthesis of specialty chemicals. However, despite great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on the protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in , , and . Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enabled the production of an unnatural polyketide in each of these hosts. Furthermore, we developed a free online tool (https://basebuddy.lbl.gov) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. In this work, we not only highlight the significance of codon optimization but also establish the groundwork for the high-throughput assembly and characterization of PKS pathways in alternative hosts.
Topics: Polyketide Synthases; Codon; Polyketides
PubMed: 37851920
DOI: 10.1021/acssynbio.3c00367