Bacterial infections have become increasingly difficult to treat due to the occurrence of antibiotic-resistant strains. A promising strategy to increase the efficacy of therapy is to combine ...antibacterials with agents that decrease pathogen virulence
via
the modulation of the quorum sensing (QS). Lactonases inhibit acylated homoserine lactone (AHL)-mediated QS in Gram-negative bacteria, including the leading nosocomial pathogen
Pseudomonas aeruginosa
. Here we describe the characteristics of heterologously expressed YtnP lactonase from
Bacillus paralicheniformis
ZP1 (YtnP-ZP1) isolated from agricultural soil using the culture enrichment method. Purified YtnP-ZP1 hydrolyzed different AHLs with preference to substrates with long acyl residues as evaluated in assays with biosensors and HPLC. The enzyme showed good thermostability and activity in a wide temperature range. YtnP-ZP1 in 50 μg mL
–1
concentration reduced the amount of
P. aeruginosa
-produced long-chain AHLs by 85%, while it hydrolyzed 50% of short-chain AHLs. Incubation of
P. aeruginosa
PAO1 with YtnP-ZP1 reduced its swarming motility and elastolytic activity without bactericidal effect. YtnP-ZP1 caused the inhibition of biofilm formation and disintegration of mature biofilms in
P. aeruginosa
PAO1 and multiresistant clinical strain BR5H that was visualized by crystal violet staining. The treatment with YtnP-ZP1 in concentrations higher than 25 μg mL
–1
improved the survival of
P. aeruginosa
PAO1-infected zebrafish (
Danio rerio
), rescuing 80% of embryos, while in combination with tobramycin or gentamicin survival rate increased to 100%. The treatment of
P. aeruginosa
PAO1 biofilms on infected zebrafish tail wounds with 50 μg mL
–1
YtnP-ZP1 and 2 × MIC tobramycin led to infection clearing in 2 days. The extensive toxicity studies proved YtnP-ZP1 was non-toxic to human cells and zebrafish. In conclusion, novel YtnP-ZP1 lactonase with its effective anti-virulence activity could be used to increase the efficacy of clinically approved antibiotics in clearing both systemic and biofilm-associated
P. aeruginosa
infections.
Candidiasis caused by Candida albicans is one of the most common fungal infections in modern society. The limited arsenal of clinical drugs, their side effects and emerging resistance, largely ...contribute to the low efficacy of current antifungal therapies. The morphogenetic yeast-to-hyphae transition is the key virulence feature for the establishment of local and systemic C. albicans infections. We show that a delfinidin-rich fruit extract from Aristotelia chilensis (Molina, Stuntz) (maqui berry) inhibits filamentation of C. albicans, in both laboratory and clinical strains. The extract acts synergistically with nystatin, with filament formation completely prevented by the combination of ¼×MIC of nystatin and 0.125 mg/ml of the extract. The combination treatment results in increased survival of C. albicans-infected zebrafish embryos compared to treatment with nystatin. Neither the extract nor its combination with nystatin was toxic at effective doses. These results warrant further investigation of maqui berry extract as adjuvant antifungal treatment.
Since adaptations are an increasingly common type of activity architectural practice, and buildings out-of-use are a problem faced by a growing number of cities, the paper discusses the structure and ...segments of multi-criteria models created in different contexts, which are developed to contribute to better decision-making in the process of adaptation, to achieve maximum economic, environmental and social sustainability. The choice of the appropriate architectural and structural intervention is influenced by several criteria, among which, mostly, the decisive ones are those related to the economic viability of the entire adaptation process. The paper presents a comparative analysis of criteria, applied valorization, and applications of five multi-criteria models. The criteria considered within all types of adaptations have been determined, and those that are specific to a certain type of intervention or context have been pointed out. Regarding applied valorization systems, the advantages and disadvantages of MCDA methods used to develop the analyzed tools, quantitative and qualitative scales of evaluation of criteria and indicators are recognized, as well as the possibility of changing their weighting factor. Since the models are intended for non-professional users, the simplicity of the application was considered. Although the focus of research is on the adaptation of office buildings by adaptive reuse, models that include other types of adaptations are also analyzed, because certain segments of these models may be important for creating new models with a greater degree of personalization, which application may extend the period of the useful life of adapted buildings.
Laccases are multicopper-oxidases with variety of biotechnological applications. While predominantly used, fungal laccases have limitations such as narrow pH and temperature range and their ...production via heterologous protein expression is more complex due to posttranslational modifications. In comparison, bacterial enzymes, including laccases, usually possess higher thermal and pH stability, and are more suitable for expression and genetic manipulations in bacterial expression hosts. Therefore, the aim of this study was to identify, recombinantly express, and characterize novel laccases from Pseudomonas spp. A combination of approaches including DNA sequence analysis, N-terminal protein sequencing, and genome sequencing data analysis for laccase amplification, cloning, and overexpression have been used. Four active recombinant laccases were obtained, one each from P. putida KT2440 and P. putida CA-3, and two from P. putida F6. The new laccases exhibited broad temperature and pH range and high thermal stability, as well as the potential to degrade selection of synthetic textile dyes. The best performing laccase was CopA from P. putida F6 which degraded five out of seven tested dyes, including Amido Black 10B, Brom Cresol Purple, Evans Blue, Reactive Black 5, and Remazol Brilliant Blue. This work highlighted species of Pseudomonas genus as still being good sources of biocatalytically relevant enzymes.
Petrochemical plastics are generally recalcitrant to microbial degradation and accumulate in the environment. Biodegradable polymers obtained synthetically like poly(ε-caprolactone) (PCL) or ...polyhydroxyalkanoates (PHA), obtained biotechnologically, have shown great potential as a replacement for petroleum-based plastics. Nevertheless, their biodegradation and environmental faith have been less examined. In this study, thin films of PCL (200 μm) and medium chain length PHA (mcl-PHA, 70 M fraction of 3-hydroxyoctanoate and 30 M fraction of 3-hydroxydecanoate, 600 μm) were exposed to total protein preparations (extracellular proteins combined with a crude cell extract) of soil isolates Pseudomonas chlororaphis B-561 and Streptomyces sp. BV315 that had been grown on waste cooking oil as a sole carbon source. Biodegradation potential of two polyesters was evaluated in buffer with total protein preparations and in a laboratory compost model system augmented with selected bacteria. Overall, PCL showed better biodegradation properties in comparison to mcl-PHA. Both materials showed surface erosion after 4-weeks of exposure to total protein preparations of both strains, with a moderate weight loss of 1.3% when P. chlororaphis B-561 was utilized. In laboratory compost model system PCL and mcl-PHA showed significant weight loss ranging from 13 to 17% when Streptomyces sp. BV315 culture was used. Similar weight loss of PCL and mcl-PHA was achieved for 4 and 8 weeks, respectively indicating slower degradation of mcl-PHA. Growth on waste cooking oil as a sole carbon source increased the potential of both tested strains to degrade PCL and mcl-PHA, making them good candidates for augmentation of compost cultures in waste management of both waste cooking oils and biodegradable polymers.
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•Pseudomonas and Streptomyces grown on waste cooking oil for biopolymers degradation.•Enzymes from Pseudomonas chlororaphis B-561 hydrolyzed PCL better than mcl-PHA.•Mcl-PHA degraded more efficiently in model compost with Streptomyces sp. BV315.
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•Bacillus laccase and whole-cells expressing it oxidized selected 1,4-dihydropyridines.•Whole-cell biocatalyst was stable after 1-month storage at 4 °C.•Multicopper oxidase CueO from ...the expression host enhanced the oxidation activity.•Laccase based biocatalysts were immobilized on bacterial nanocellulose.
Biocatalytic oxidations mediated by laccases are gaining importance due to their versatility and beneficial environmental effects. In this study, the oxidation of 1,4-dihydropyridines has been performed using three different types of bacterial laccase-based catalysts: purified laccase from Bacillus licheniformis ATCC 9945a (BliLacc), Escherichia coli whole cells expressing this laccase, and bacterial nanocellulose (BNC) supported BliLacc catalysts. The catalysts based on bacterial laccase were compared to the commercially available Trametes versicolor laccase (TvLacc). The oxidation product of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate was obtained within 7–24 h with good yields (70–99%) with all three biocatalysts. The substrate scope was examined with five additional 1,4-dihydropyridines, one of which was oxidized in high yield. Whole-cell biocatalyst was stable when stored for up to 1-month at 4 °C. In addition, evidence has been provided that multicopper oxidase CueO from the E. coli expression host contributed to the oxidation efficiency of the whole-cell biocatalyst. The immobilized whole-cell biocatalyst showed satisfactory activity and retained 37% of its original activity after three biotransformation cycles.
The purpose of this paper was to initiate broad research aimed to establish the preferred color of light of LEDs from a driver's point of view. Two street lighting installations (one with 3000 K and ...the other with 4000 K LEDs) were evaluated both objectively and subjectively. The objective evaluation, realized using a CCD camera, included detection of small targets and pedestrians. A slight advantage was identified for the 3000 K lighting installation regarding both types of target. As for subjective evaluation (realized through a questionnaire), the task of the participants (drivers) was to choose the more appropriate between the two lighting installations regarding six lighting parameters, as well as the overall visibility. The 3000 K LED installation was evaluated as a better solution for most analyzed parameters, as well as for the overall visibility. However, only the results regarding the color of the light (in favor of the 3000 K LEDs) and the detection of small light-colored obstacles (in favor of 4000 K LEDs) were convincing, which was confirmed by the statistical analysis. Due to the obtained mild preference for the 3000 K LEDs and several limitations/challenges of the conducted surveys, it was concluded that additional research is needed in order to decide on the preferred color of light of LEDs from a driver's perspective.
The adaptability of halophytes to increased soil salinity is related to complex rhizosphere interactions. In this study, an integrative approach, combining culture-independent and culture-dependent ...techniques was used to analyze the bacterial communities in the endorizosphere of indigenous succulent halophytes Salicornia europaea, Suaeda maritima, and Camphorosma annua from the natural salt marshes of Slano Kopovo (Serbia). The 16 S rDNA analyses gave, for the first time, an insight into the composition of the endophytic bacterial communities of S. maritima and C. annua. We have found that the composition of endophyte microbiomes in the same habitat is to some extent influenced by plant species. A cultivable portion of the halophyte microbiota was tested at different NaCl concentrations for the set of plant growth promoting (PGP) traits. Through the mining of indigenous halotolerant endophytes, we obtained a collection representing a core endophyte microbiome conferring desirable PGP traits. The majority (65%) of the selected strains belonged to the common halotolerant/halophilic genera Halomonas, Kushneria, and Halobacillus, with representatives exhibiting multiple PGP traits, and retaining beneficial traits in conditions of the increased salinity. The results suggest that the root endosphere of halophytes is a valuable source of PGP bacteria supporting plant growth and fitness in salt-affected soils.
Three Streptomyces sp. strains with a multitude of target enzymatic activities confirmed by functional screening, namely BV129, BV286 and BV333, were subjected to genome sequencing aiming at the ...annotation of genes of interest, in-depth bioinformatics characterization and functional expression of the biocatalysts. A whole-genome shotgun sequencing followed by de novo genome assembly and annotation was performed revealing genomes of 6.4, 9.4 and 7.3 Mbp, respectively. Functional annotation of the proteins of interest resulted in between 2047 and 2763 putative targets. Among the various enzymatic activities that the three Streptomyces strains demonstrated to produce by functional screening, we focused our attention on transaminases (TAs) and laccases due to their high biocatalytic potential. Bioinformatics search allowed the identification of a putative TA from Streptomyces sp. BV333 as a potentially novel broad substrate scope TA and a putative laccase from Streptomyces sp. BV286 as potentially novel blue multicopper oxidase. The two sequences were cloned and overexpressed in Escherichia coli and the two novel enzymes, transaminase Sbv333-TA and laccase Sbv286-LAC, were characterized. Interestingly, both enzymes resulted to be exceptionally thermostable, Sbv333-TA showing a melting temperature (TM = 85 °C) only slightly lower compared to the TM of the most thermostable transaminases described to date (87–88 °C) and Sbv286-LAC being even thermoactivated at temperature >60 °C. Moreover, Sbv333-TA showed a broad substrate scope and remarkably demonstrated to be active in the transamination of β-ketoesters, which are rarely accepted by currently known TAs. On the other hand, Sbv286-LAC showed an improved activity in the presence of the cosolvent acetonitrile. Overall, it was shown that a combination of approaches from standard microbiological and biochemical screens to genome sequencing and analysis is required to afford novel and functional biocatalysts.
We have studied the effect of compositions and methods of preparation on the physico-chemical and biocompatible behavior of the hydrogel matrices. Hydrogel matrices are synthesized by free radical ...polymerization of 2-hydroxyethyl methacrylate net and with gelatin. Highly porous hydrogel structures were obtained by porogenation, and by cryogenic treatment followed by freeze-drying. All samples were characterized for structural, morphological, absorption, degradation and in vitro (healthy human fibroblast cell line) and in vivo (zebrafish
Danio rerio
) biocompatible properties. The obtained results show that cryo samples, especially with gelatin show better, favorable absorption, morphological and biocompatible properties in comparison with PHEMA samples, which makes these materials highly attractive for biomedical uses.
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