A new understanding on the prerequisite of antibiotic biodegradation in wastewater treatment: Adhesive behavior between antibiotic-degrading bacteria and ciprofloxacin.

The extensive exploration of antibiotic biodegradation by antibiotic-degrading bacteria in biological wastewater treatment processes has left a notable gap in understanding the behavior of these bacteria when exposed to antibiotics and the initiation of biodegradation processes. This study, therefore, delves into the adhesive behavior of Paraclostridium bifermentans, isolated from a bioreactor treating ciprofloxacin-laden wastewater, towards ciprofloxacin molecules. For the first time, this behavior is observed and characterized through quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy. The investigation further extends to identify key regulatory factors and mechanisms governing this adhesive behavior through a comparative proteomics analysis. The results reveal the dominance of extracellular proteins, particularly those involved in nucleotide binding, hydrolase, and transferase, in the adhesion process. These proteins play pivotal roles through direct chemical binding and the regulation of signaling molecule. Furthermore, QCM-D measurements provide evidence that transferase-related signaling molecules, especially tyrosine, augment the binding between ciprofloxacin and transferases, resulting in enhance ciprofloxacin removal by P. bifermentans (increased by ∼1.2-fold). This suggests a role for transferase-related signaling molecules in manipulating the adhesive behavior of P. bifermentans towards ciprofloxacin. These findings contribute to a new understanding of the prerequisites for antibiotic biodegradation and offer potential strategies for improving the application of antibiotic-degrading bacteria in the treatment of antibiotics-laden wastewater.

The transcription factor CitZAT5 modifies sugar accumulation and hexose proportion in citrus fruit.

Sugars are fundamental to plant developmental processes. For fruits, the accumulation and proportion of sugars play crucial roles in the development of quality and attractiveness. In citrus (Citrus reticulata Blanco.), we found that the difference in sweetness between mature fruits of "Gongchuan" and its bud sport "Youliang" is related to hexose contents. Expression of a SuS (sucrose synthase) gene CitSUS5 and a SWEET (sugars will eventually be exported transporter) gene CitSWEET6, characterized by transcriptome analysis at different developmental stages of these 2 varieties, revealed higher expression levels in "Youliang" fruit. The roles of CitSUS5 and CitSWEET6 were investigated by enzyme activity and transient assays. CitSUS5 promoted the cleavage of sucrose to hexoses, and CitSWEET6 was identified as a fructose transporter. Further investigation identified the transcription factor CitZAT5 (ZINC FINGER OF ARABIDOPSIS THALIANA) that contributes to sucrose metabolism and fructose transportation by positively regulating CitSUS5 and CitSWEET6. The role of CitZAT5 in fruit sugar accumulation and hexose proportion was investigated by homologous transient CitZAT5 overexpression, -VIGS, and -RNAi. CitZAT5 modulates the hexose proportion in citrus by mediating CitSUS5 and CitSWEET6 expression, and the molecular mechanism explained the differences in sugar composition of "Youliang" and "Gongchuan" fruit.

Genome-wide identification and expression analysis of MATE gene family in citrus fruit (Citrus clementina).

Multidrug and toxic compound extrusion (MATE) proteins are a class of secondary active multidrug transporters. In plants, this family has significantly expanded and is involved in numerous plant physiological processes. Although MATE proteins have been identified in an increasing number of species, the understanding about this family in citrus remains unclear. In this study, a total of 69 MATE transporters were identified in the citrus genome (Citrus clementina) and classified into four groups by phylogenetic analysis. Tandem and segmental duplication events were the main causes of the citrus MATE family expansion. RNA-seq and qRT-PCR analyses were performed during citrus fruit development. The results indicated that CitMATE genes showed specific expression profiles in citrus peels and flesh at different developmental stages. Combined with the variations of flavonoids and citrate levels in citrus fruit, we suggested that CitMATE43 and CitMATE66 may be involved in the transport process of flavonoids and citrate in citrus fruit, respectively. In addition, two flavonoids positive regulators, CitERF32 and CitERF33, both directly bind to and activated the CitMATE43 promoter. Our results provide comprehensive information on citrus MATE genes and valuable understanding for the flavonoids and citrate metabolism in citrus fruit.

Ciprofloxacin-degrading Paraclostridium sp. isolated from sulfate-reducing bacteria-enriched sludge: Optimization and mechanism.

Ciprofloxacin (CIP), one of the most widely used fluoroquinolone antibiotics, is frequently detected in the effluents of wastewater treatment plants and aquatic environments. In this study, a CIP-degrading bacterial strain was isolated from the sulfate reducing bacteria (SRB)-enriched sludge, identified as Paraclostridium sp. (i.e., strain S2). The effects of critical operational parameters on CIP removal by the strain S2 were systematically studied and these parameters were optimized via response surface methodology to maximize CIP removal. Furthermore, the pathway and kinetics of CIP removal were investigated by varying the initial CIP concentrations (from 0.1 to 20 mg/L). The CIP removal was characterized by rapid sorption followed by biotransformation with a specific biotransformation rate of 1975.7 ± 109.1 µg/g-cell dry weight/h at an initial CIP concentration of 20 mg/L. Based on the main transformation products, several biotransformation pathways have been proposed including piperazine ring cleavage, OH/F substitution, decarboxylation, and hydroxylation as the major transformation reactions catalyzed by cytochrome P450 and dehydrogenases. Acute toxicity assessment apparently shows that CIP biotransformation by strain S2 resulted in the formation of less toxic intermediates. To the best of our knowledge, this is the very first study in which a key functional microbe, Paraclostridium sp., highly effective in CIP biotransformation, was isolated from SRB-enriched sludge. The findings of this study could facilitate in developing appropriate bioaugmentation strategy, and in designing and operating an SRB-based engineered process for treating CIP-laden wastewater.

Transcription Factor CitERF16 Is Involved in Citrus Fruit Sucrose Accumulation by Activating CitSWEET11d.

Sugars are the primary products of photosynthesis and play an important role in plant growth and development. They contribute to sweetness and flavor of fleshy fruits and are pivotal to fruit quality, and their translocation and allocation are mainly dependent on sugar transporters. Genome-wide characterization of Satsuma mandarin identified eighteen SWEET family members that encode transporters which facilitate diffusion of sugar across cell membranes. Analysis of the expression profiles in tissues of mandarin fruit at different developmental stages showed that CitSWEET11d transcripts were significantly correlated with sucrose accumulation. Further studies indicated that overexpression of CitSWEET11d in citrus callus and tomato fruit showed a higher sucrose level compared to wild-type, suggesting that CitSWEET11d could enhance sucrose accumulation. In addition, we identified an ERF transcription factor CitERF16 by yeast one-hybrid screening assay which could directly bind to the DRE cis-element on the promoter of CitSWEET11d. Overexpression of CitERF16 in citrus callus significantly induced CitSWEET11d expression and elevated sucrose content, suggesting that CitERF16 acts as a positive regulator to promote sucrose accumulation via trans-activation of CitSWEET11d expression.

[Adsorption Mechanisms of Ciprofloxacin by Extracellular Polymeric Substances of Sulfate-reducing Bacteria Sludge].

Extracellular polymeric substances (EPS) in microbial sludge, fulfils a key role in removal of micro-organic pollutants during biological wastewater treatment. In this study, the authors evaluated the removal of ciprofloxacin (CIP) by sulfate-reducing bacteria (SRB) sludge in a sulfate-reducing up-flow sludge bed (SRUSB) reactor, and examined the role of EPS on CIP removal in an SRB sludge system. The results indicated that CIP was removed efficiently through adsorption and biodegradation by SRB sludge, with adsorption the major removal pathway. EPS also played an important role in CIP adsorption by SRB sludge, and the adsorption mechanisms of CIP by EPS were investigated using the three-dimensional excitation-emission matrix fluorescence spectroscopy technologies combined with parallel factor analysis. The functional groups binding CIP onto EPS were identified through Fourier transform infrared (FTIR) spectra analysis. The results suggested that the static quenching of EPS following CIP adsorption led to formation of an EPS-CIP complex, and that the CIP was mainly bound with tryptophan and tyrosine-like protein substances in EPS with the binding constants of 1.43×104 L·mol-1 and 1.02×104 L·mol-1, respectively. The FTIR results suggested that hydroxyl, amino and carboxyl functional groups were mainly responsible for binding of CIP onto EPS. The results revealed the adsorption mechanisms of CIP by EPS in SRB sludge, and enhanced understanding of the role of EPS in sulfur-mediated biological processes for the removal of CIP and other organic micro-pollutants.

Understanding the Role of Extracellular Polymeric Substances on Ciprofloxacin Adsorption in Aerobic Sludge, Anaerobic Sludge, and Sulfate-Reducing Bacteria Sludge Systems.

Extracellular polymeric substances (EPS) of microbial sludge play a crucial role in removal of organic micropollutants during biological wastewater treatment. In this study, we examined ciprofloxacin (CIP) removal in three parallel bench-scale reactors using aerobic sludge (AS), anaerobic sludge (AnS), and sulfate-reducing bacteria (SRB) sludge. The results showed that the SRB sludge had the highest specific CIP removal rate via adsorption and biodegradation. CIP removal by EPS accounted up to 35. 6 ± 1.4%, 23.7 ± 0.6%, and 25.5 ± 0.4% of total removal in AS, AnS, and SRB sludge systems, respectively, at influent CIP concentration of 1000 µg/L, which implied that EPS played a critical role in CIP removal. The binding mechanism of EPS on CIP adsorption in three sludge systems were further investigated using a series of batch tests. The results suggested that EPS of SRB sludge possessed stronger hydrophobicity (proteins/polysaccharides (PN/PS) ratio), higher availability of adsorption sites (binding sites ( n)), and higher binding strength (binding constant ( Kb)) between EPS and CIP compared to those of AS and AnS. The findings of this study provide an insight into the role of EPS in biological process for treating CIP-laden wastewaters.

Cyclic-AMP-dependent protein kinase (PKA) activity assay based on FRET between cationic conjugated polymer and chromophore-labeled peptide.

A sensitive fluorescence turn-on biosensing platform for protein kinase activity assay has been developed based on fluorescence resonance energy transfer (FRET) between a fluorophore labeled peptide and a water soluble cationic conjugated polymer (CCP). The CCP-based assay is based on the electrostatic interaction between the peptide and the CCP. The FRET efficiency will change with the changing charges around the peptide after phosphorylation. The feasibility of this method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.3 mU µL(-1)). Based on its simple mechanism, this assay is also sensitive and robust enough to be applied to the evaluation of PKA inhibitor H-89. The IC50 value, the half maximal inhibitory concentration, was 40 nM. Furthermore, our method has excellent selectivity. CCP-based assay is sensitive, versatile, cost-effective and easy to operate, so, this method is a promising candidate for kinase activity assay and inhibitor screening.

An aptamer-based quartz crystal microbalance biosensor for sensitive and selective detection of leukemia cells using silver-enhanced gold nanoparticle label.

An aptamer-based quartz crystal microbalance (QCM) biosensor was developed for the selective and sensitive detection of leukemia cells. In this strategy, aminophenylboronic acid-modified gold nanoparticles (APBA-AuNPs) which could bind to cell membrane were used for the labeling of cells followed by silver enhancement, through which significant signal amplification was achieved. Both the QCM and fluorescence microscopy results manifested the selectivity of the sensor designed. A good linear relationship between the frequency response and cell concentration over the range of 2×10(3)-1×10(5)cells/mL was obtained, with a detection limit of 1160cells/mL. This approach provides a simple, rapid, and economical method for leukemia cell analysis which might have great potential for further use.

Sensitive and visible detection of apoptotic cells on Annexin-V modified substrate using aminophenylboronic acid modified gold nanoparticles (APBA-GNPs) labeling.

A new strategy for sensitive detection of early stage apoptosis was proposed based on silver-enhanced gold nanoparticle (GNP) label method. Annexin-V modified substrate was constructed via layer-by-layer (LBL) method for specific capture of early stage apoptotic Jurkat cells. A new kind of aminophenylboronic acid modified gold nanoparticle (APBA-GNP) was synthesized and utilized for labeling cells, followed by silver enhancement. Anodic stripping voltammetry (ASV) was applied to sensitive detection of Ag(+) dissolved from the deposited silver particles, which reflected the number of cells. A good linear range from 1 × 10(2) to 3.5 × 10(3) cells was achieved, with a detection limit of 38 apoptotic cells. Moreover, the gray color of silver enhancement could be observed by the naked eye, which could be used to tell apoptotic cells apart from normal cells. Therefore, using the silver-enhanced GNP label method, apoptotic cells could not only be sensitively detected via electrochemical technique, but also can be discriminated from normal cells by the naked eye.

Annexin-V modified QCM sensor for the label-free and sensitive detection of early stage apoptosis.

An easily-made Annexin-V modified quartz crystal microbalance (QCM) sensor was constructed for the quantitative detection of early stage apoptosis for the first time, achieving the goals of specific capture and sensitive detection of target cells in one step without the need for cell labelling.