Elucidation of 4-Hydroxybenzoic Acid Catabolic Pathways in Pseudarthrobacter phenanthrenivorans Sphe3.

4-hydroxybenzoic acid (4-HBA) is an aromatic compound with high chemical stability, being extensively used in food, pharmaceutical and cosmetic industries and therefore widely distributed in various environments. Bioremediation constitutes the most sustainable approach for the removal of 4-hydroxybenzoate and its derivatives (parabens) from polluted environments. Pseudarthrobacter phenanthrenivorans Sphe3, a strain capable of degrading several aromatic compounds, is able to grow on 4-HBA as the sole carbon and energy source. Here, an attempt is made to clarify the catabolic pathways that are involved in the biodegradation of 4-hydroxybenzoate by Sphe3, applying a metabolomic and transcriptomic analysis of cells grown on 4-HBA. It seems that in Sphe3, 4-hydroxybenzoate is hydroxylated to form protocatechuate, which subsequently is either cleaved in ortho- and/or meta-positions or decarboxylated to form catechol. Protocatechuate and catechol are funneled into the TCA cycle following either the ß-ketoadipate or protocatechuate meta-cleavage branches. Our results also suggest the involvement of the oxidative decarboxylation of the protocatechuate peripheral pathway to form hydroxyquinol. As a conclusion, P. phenanthrenivorans Sphe3 seems to be a rather versatile strain considering the 4-hydroxybenzoate biodegradation, as it has the advantage to carry it out effectively following different catabolic pathways concurrently.

Phenol Degradation by Pseudarthrobacter phenanthrenivorans Sphe3.

Phenol poses a threat as one of the most important industrial environmental pollutants that must be removed before disposal. Biodegradation is a cost-effective and environmentally friendly approach for phenol removal. This work aimed at studying phenol degradation by Pseudarthrobacter phenanthrenivorans Sphe3 cells and also, investigating the pathway used by the bacterium for phenol catabolism. Moreover, alginate-immobilized Sphe3 cells were studied in terms of phenol degradation efficiency compared to free cells. Sphe3 was found to be capable of growing in the presence of phenol as the sole source of carbon and energy, at concentrations up to 1500 mg/L. According to qPCR findings, both pathways of ortho- and meta-cleavage of catechol are active, however, enzymatic assays and intermediate products identification support the predominance of the ortho-metabolic pathway for phenol degradation. Alginate-entrapped Sphe3 cells completely degraded 1000 mg/L phenol after 192 h, even though phenol catabolism proceeds slower in the first 24 h compared to free cells. Immobilized Sphe3 cells retain phenol-degrading capacity even after 30 days of storage and also can be reused for at least five cycles retaining more than 75% of the original phenol-catabolizing capacity.

Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube.

The current study aims at the functional and kinetic characterization of protocatechuate (PCA) 4,5-dioxygenase (PcaA) from Pseudarthrobacter phenanthrenivorans Sphe3. This is the first single subunit Type II dioxygenase characterized in Actinobacteria. RT-PCR analysis demonstrated that pcaA and the adjacent putative genes implicated in the PCA meta-cleavage pathway comprise a single transcriptional unit. The recombinant PcaA is highly specific for PCA and exhibits Michaelis-Menten kinetics with Km and Vmax values of 21 ± 1.6 µM and 44.8 ± 4.0 U × mg-1, respectively, in pH 9.5 and at 20 °C. PcaA also converted gallate from a broad range of substrates tested. The enzymatic reaction products were identified and characterized, for the first time, through in situ biotransformation monitoring inside an NMR tube. The PCA reaction product demonstrated a keto-enol tautomerization, whereas the gallate reaction product was present only in the keto form. Moreover, the transcriptional levels of pcaA and pcaR (gene encoding a LysR-type regulator of the pathway) were also determined, showing an induction when cells were grown on PCA and phenanthrene. Studying key enzymes in biodegradation pathways is significant for bioremediation and for efficient biocatalysts development.

Safety Evaluation, Biogenic Amine Formation, and Enzymatic Activity Profiles of Autochthonous Enterocin-Producing Greek Cheese Isolates of the Enterococcus faecium/durans Group.

Autochthonous single (Ent+) or multiple (m-Ent+) enterocin-producing strains of dairy enterococci show promise for use as bioprotective adjunct cultures in traditional cheese technologies, provided they possess no pathogenic traits. This study evaluated safety, decarboxylase activity, and enzymatic (API ZYM) activity profiles of nine Ent+ or m-Ent+ Greek cheese isolates previously assigned to four distinct E. faecium (represented by the isolates KE64 (entA), GL31 (entA), KE82 (entA-entB-entP) and KE77 (entA-entB-entP-bac31)) and two E. durans (represented by the isolates KE100 (entP) and KE108 (entP-bac31-cyl)) strain genotypes. No strain was ß-hemolytic or harbored vanA and vanB or the virulence genes agg, ace, espA, IS16, hyl, or gelE. All strains were of moderate to high sensitivity to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, penicillin, tetracycline, and vancomycin, except for the E. faecium KE64 and KE82 strains, which were resistant to erythromycin and penicillin. All cheese strains showed moderate to strong esterase-lipase and aminopeptidase activities and formed tyramine, but none formed histamine in vitro. In conclusion, all Ent+ or m-Ent+ strain genotypes of the E. faecium/durans group, except for the cyl-positive E. durans KE108, were safe for use as adjunct cultures in traditional Greek cheeses. Further in situ biotechnological evaluations of the strains in real cheese-making trials are required.

Monitoring Growth Compatibility and Bacteriocin Gene Transcription of Adjunct and Starter Lactic Acid Bacterial Strains in Milk.

ABSTRACT: When developing protective starter cultures for application in cheese technologies, monitoring growth interactions between starter and adjunct lactic acid bacterial (LAB) species and in situ expression of bacteriocin genes in the mixtures is crucial. This study first aimed to monitor the growth of mixed LAB strain populations during milk model fermentations by microbial counts and real-time quantitative PCR. The primary starter strains, Streptococcus thermophilus ST1 and costarter Lactococcus lactis subsp. cremoris M78, served as the basic starter composite coinoculated in all milk treatments. Adjunct bacteriocinogenic Enterococcus faecium strains KE82 and GL31 and the ripening Lactiplantibacillus plantarum H25 strain were added separately to the starter composite, resulting in four LAB combination treatments. The second aim was to quantify gene transcripts of nisin and enterocins B and A synthesized by strains M78, KE82, and GL31, respectively, by reverse transcription-real-time quantitative PCR and to detect the in situ antilisterial effects of the cocultures. Adjunct LAB strains showed growth compatibility with the starter, since all of them exhibited 2- to 3-log-unit increases in their population levels compared to their initial inoculation levels, with ST1 prevailing in all treatments. KE82 grew more competitively than GL31, whereas cocultures with KE82 displayed the strongest in situ antilisterial activity. Nisin gene expression levels were higher at the exponential phase of microbial growth in all treatments. Finally, the expression levels of nisin and enterocin A and B genes were interrelated, indicating an antagonistic activity.