Design of a degenerate primer pair to target a bacterial functional community: The hppd bacterial gene coding for the enzyme targeted by herbicides, a study case.

The present work aimed to design a degenerate primer pair to target a large part of the hppd soil bacterial community, possibly affected by herbicides. We validated these primers by qPCR and high-throughput sequencing analysis of soil samples.

Development of a PCR-free DNA-based assay for the specific detection of Vibrio species in environmental samples by targeting the 16S rRNA.

A novel PCR-free DNA-based assay was developed for the detection of Vibrio spp. A sandwich hybridization format using an immobilized capture probe and a labeled signal probe was selected and combined with chemiluminescent method for the detection of the RNA target. In a first step, probes were validated using positive controls (PCs). A linearity was observed between 0.1 and 2.5 nM of PC, and detection limit was determined as 0.1 nM. In a second step, specificity was checked by using RNA extracted from a panel of 31 environmental bacterial strains. Detection limit of 5 ng µL-1 of total fragmented RNA was obtained, and the assay allowed a good discrimination between the 21 Vibrio and the 10 non-Vibrio strains tested. Finally, the DNA-based assay was successfully applied to analysis of spiked and natural environmental samples. Stability and analysis time of the DNA-based assay were also investigated to optimize working conditions. We demonstrated that microplates can be coated beforehand with capture probe and stored at 4 °C without any buffer in wells for at least 30 days. The use of the pre-made plates enables the assay to be completed in 2 h. The developed assay appeared as an interesting tool to determine the presence of bacteria in environmental samples.

Oxovanadium-salen and -salan complexes as effective labels for electrochemical immunosensing: a case study for estradiol detection.

Oxovanadium complexes are presented as new labels for the development of electrochemical immunosensors. The concept was successfully applied to the accurate detection of estradiol, an emerging environmental pollutant, at concentrations ranging from 4 ng L(-1) to 5 µg L(-1).

Aptasensor and genosensor methods for detection of microbes in real world samples.

The increasing concerns about food and environmental safety have prompted the desire to develop rapid, specific, robust and highly sensitive methods for the detection of microorganisms to ensure public health. Although traditional microbiological methods are available, they are labor intensive, unsuitable for on-site and high throughput analysis, and need well-trained personnel. To circumvent these drawbacks, many efforts have been devoted towards the development of biosensors, using nucleic acid as bio-recognition element. In this review, we will focus on recent significant advances made in two types of DNA-based biosensors, namely genosensors, and aptasensors. In genosensor approach, DNA or RNA target is detected through the hybridization reaction between DNA or RNA and ssDNA sensing element, while in aptasensor method, DNA or RNA aptamer, capable of binding to a target molecule with high affinity and specificity, plays the role of receptor. The goal of this article is to review the innovative methods that have been emerged in genosensor and aptasensor during recent years. Particular attention is given to recent advances and trends in selection of biorecognition element, DNA immobilization strategies and sensing formats.

Expression in Escherichia coli of native and chimeric phenolic acid decarboxylases with modified enzymatic activities and method for screening recombinant E. coli strains expressing these enzymes.

Four bacterial phenolic acid decarboxylases (PAD) from Lactobacillus plantarum, Pediococcus pentosaceus, Bacillus subtilis, and Bacillus pumilus were expressed in Escherichia coli, and their activities on p-coumaric, ferulic, and caffeic acids were compared. Although these four enzymes displayed 61% amino acid sequence identity, they exhibit different activities for ferulic and caffeic acid metabolism. To elucidate the domain(s) that determines these differences, chimeric PAD proteins were constructed and expressed in E. coli by exchanging their individual carboxy-terminal portions. Analysis of the chimeric enzyme activities suggests that the C-terminal region may be involved in determining PAD substrate specificity and catalytic capacity. In order to test phenolic acid toxicity, the levels of growth of recombinant E. coli displaying and not displaying PAD activity were compared on medium supplemented with different concentrations of phenolic acids and with differing pHs. Though these acids already have a slight inhibitory effect on E. coli, vinyl phenol derivatives, created during decarboxylation of phenolic acids, were much more inhibitory to the E. coli control strain. To take advantage of this property, a solid medium with the appropriate pH and phenolic acid concentration was developed; in this medium the recombinant E. coli strains expressing PAD activity form colonies approximately five times smaller than those formed by strains devoid of PAD activity.

Inducible metabolism of phenolic acids in Pediococcus pentosaceus is encoded by an autoregulated operon which involves a new class of negative transcriptional regulator.

Pediococcus pentosaceus displays a substrate-inducible phenolic acid decarboxylase (PAD) activity on p-coumaric acid. Based on DNA sequence homologies between the three PADs previously cloned, a DNA probe of the Lactobacillus plantarum pdc gene was used to screen a P. pentosaceus genomic library in order to clone the corresponding gene of this bacteria. One clone detected with this probe displayed a low PAD activity. Subcloning of this plasmid insertion allowed us to determine the part of the insert which contains a 534-bp open reading frame (ORF) coding for a 178-amino-acid protein presenting 81.5% of identity with L. plantarum PDC enzyme. This ORF was identified as the padA gene. A second ORF was located just downstream of the padA gene and displayed 37% identity with the product of the Bacillus subtilis yfiO gene. Subcloning, transcriptional analysis, and expression studies with Escherichia coli of these two genes under the padA gene promoter, demonstrated that the genes are organized in an autoregulated bicistronic operonic structure and that the gene located upstream of the padA gene encodes the transcriptional repressor of the padA gene. Transcription of this pad operon in P. pentosaceus is acid phenol dependent.

Knockout of the p-coumarate decarboxylase gene from Lactobacillus plantarum reveals the existence of two other inducible enzymatic activities involved in phenolic acid metabolism.

Lactobacillus plantarum NC8 contains a pdc gene coding for p-coumaric acid decarboxylase activity (PDC). A food grade mutant, designated LPD1, in which the chromosomal pdc gene was replaced with the deleted pdc gene copy, was obtained by a two-step homologous recombination process using an unstable replicative vector. The LPD1 mutant strain remained able to weakly metabolize p-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids. We have shown that L. plantarum has a second acid phenol decarboxylase enzyme, better induced with ferulic acid than with p-coumaric acid, which also displays inducible acid phenol reductase activity that is mostly active when glucose is added. Those two enzymatic activities are in competition for p-coumaric and ferulic acid degradation, and the ratio of the corresponding derivatives depends on induction conditions. Moreover, PDC appeared to decarboxylate ferulic acid in vitro with a specific activity of about 10 nmol. min(-1). mg(-1) in the presence of ammonium sulfate. Finally, PDC activity was shown to confer a selective advantage on LPNC8 grown in acidic media supplemented with p-coumaric acid, compared to the LPD1 mutant devoid of PDC activity.

Molecular characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum: gene cloning, transcriptional analysis, overexpression in Escherichia coli, purification, and characterization.

By using degenerate primers designed from the first 19 N-terminal amino acids of Lactobacillus plantarum p-coumaric acid decarboxylase (PDC), a 56-bp fragment was amplified from L. plantarum in PCRs and used as a probe for screening an L. plantarum genomic bank. Of the 2,880 clones in the genomic bank, one was isolated by colony hybridization and contained a 519-bp open reading frame (pdc gene) followed by a putative terminator structure. The pdc gene is expressed on a monocistronic transcriptional unit, which is transcribed from promoter sequences homologous to Lactococcus promoter sequences. No mRNA from pdc and no PDC activity were detected in uninduced cell extracts, indicating that the expression is transcriptionally regulated by p-coumaric acid, which corresponds to an activation factor up to 6,000. The pdc gene was overexpressed constitutively in Escherichia coli, and the recombinant enzyme was purified and characterized.