Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual determinations of ergot contamination.

We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes.

Biochemical, transcriptional and translational evidences of the phenol-meta-degradation pathway by the hyperthermophilic Sulfolobus solfataricus 98/2.

Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D) and meta involving a catechol 2,3 dioxygenase (C23D). Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively) and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg x L(-1)) suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.

Hydroxytyrosol from tyrosol using hydroxyphenylacetic acid-induced bacterial cultures and evidence of the role of 4-HPA 3-hydroxylase.

Hydroxytyrosol (HTyr) is a potent natural antioxidant found in olive mill wastewaters. Bacterial conversion of 4-tyrosol (2-(4-hydroxyphenyl)-ethanol) to HTyr was reported in a limited number of bacterial species including Pseudomonas aeruginosa. In this work, we studied this conversion, taking as a model the newly isolated Halomonas sp. strain HTB24. It was first hypothesized that the enzyme responsible for 4-tyrosol hydroxylation in HTyr was a 4-hydroxyphenylacetic acid 3-hydroxylase (HPAH, EC 1.14.13.3), previously known to convert 4-hydroxyphenylacetic acid (4-HPA) into 3,4-dihydroxyphenylacetic acid (3,4-DHPA) in P. aeruginosa. Cloning and expression of hpaB (oxygenase component) and hpaC (reductase component) genes from P. aeruginosa confirmed this hypothesis. Furthermore, using cultures of HTB24 containing 4-tyrosol, it was shown that 4-HPA accumulation preceded 4-tyrosol hydroxylation. We further demonstrated that the synthesis of HPAH activity was induced by 4-HPA, with the latter compound being formed from 4-tyrosol oxidation by aryl-dehydrogenases. Interestingly, similar results were obtained with other 4-HPA-induced bacteria, including P. aeruginosa, Serratia marcescens, Escherichia coli, Micrococcus luteus and other Halomonas, thus demonstrating general hydroxylating activity of 4-tyrosol by the HPAH enzyme. E. coli W did not have aryl-dehydrogenase activity and hence were unable to oxidize 4-tyrosol to 4-HPA and HTyr to 3,4-DHPA, making this bacterium a good candidate for achieving better HTyr production.