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Listeria monocytogenes represents one of the main risks for food safety worldwide. Two enzyme-based antimicrobials (enzybiotics) have been combined in a novel treatment against this pathogenic bacterium, resulting in a powerful synergistic effect. One of the enzymes is an endolysin from Listeria phage vB_LmoS_188 with amidase activity (henceforth A10), and the other is an engineered version of glucose oxidase from Aspergillus niger (GOX). Both enzymes, assayed separately against Listeria innocua, showed antibacterial activity at the appropriate doses. The combination of the two enzybiotics resulted in a synergistic effect with a log reduction in viable cells (log N0/N) of 4, whereas, taken separately, the same dose of A10 and GOX caused only 1.2 and 0.2 log reductions, respectively. Flow cytometry and microscopy analyses revealed that A10 treatment alone induced the aggregation of dead cells. L. monocytogenes showed higher resistance to single treatment with GOX or A10 than L. innocua. However, the synergic combination of A10 and GOX resulted in a high lethality of L. monocytogenes with a log N0/N higher than 5 (below the detection limit in our analysis). Altogether, these results represent a novel efficient and eco-friendly antimicrobial treatment against the most lethal food-borne pathogen. [ABSTRACT FROM AUTHOR]

The formation and analysis of amyloid fibers by two β-glucosidases, BglA and BglB, belonging to the GH1 enzyme family, are reported. Both proteins have the (β/α)8 TIM-barrel fold, which is characteristic of this family and is also the most common protein structure. BglA is an octamer, whereas BglB is a monomer. Amyloid fibrillation using pH and temperature as perturbing agents was investigated using fluorescence spectroscopy as a preliminary approach and corroborated using wide-field optical microscopy, confocal microscopy, and field-emission scanning electron microscopy. These analyses showed that both enzymes fibrillate at a wide range of acidic and alkaline conditions and at several temperature conditions, particularly at acidic pH (3–4) and at temperatures between 45 and 65 °C. Circular dichroism spectroscopy corroborated the transition from an α-helix to a β-sheet secondary structure of both proteins in conditions where fibrillation was observed. Overall, our results suggest that fibrillation is a rather common phenomenon caused by protein misfolding, driven by a transition from an α-helix to a β-sheet secondary structure, that many proteins can undergo if subjected to conditions that disturb their native conformation. [ABSTRACT FROM AUTHOR]

A number of single-nucleotide polymorphisms (SNPs) have been identified in the genome of Mycobacterium bovis BCG Pasteur compared with the sequenced strain M. boris 2122/97. The functional consequences of many of these mutations remain to be described; however, mutations in genes encoding regulators may be particularly relevant to global phenotypic changes such as loss of virulence, since alteration of a regulator's function will affect the expression of a wide range of genes. One such SNP falls in bcg3145, encoding a member of the AfsR/Dnrl/SARP class of global transcriptional regulators, that replaces a highly conserved glutamic acid residue at position 159 (E159G) with glycine in a tetratricopeptide repeat (TPR) located in the bacterial transcriptional activation (BTA) domain of BCG3145. TPR domains are associated with protein--protein interactions, and a conserved core (helices T1-T7) of the BTA domain seems to be required for proper function of SARP-family proteins. Structural modelling predicted that the E159G mutation perturbs the third [alpha]-helix of the BTA domain and could therefore have functional consequences. The E159G SNP was found to be present in all BCG strains, but absent from virulent M. bovis and Mycobacterium tuberculosis strains. By overexpressing BCG3145 and Rv3124 in BCG and H37Rv and monitoring transcriptome changes using microarrays, we determined that BCG3145/Rv3124 acts as a positive transcriptional regulator of the molybdopterin biosynthesis moa1 locus, and we suggest that rv3124 be renamed moaR1. The SNP in bcg3145 was found to have a subtle effect on the activity of MoaR1, suggesting that this mutation is not a key event in the attenuation of BCG. DOI: 10.1099/mic.0.037200-0

We describe here an extraordinary purple-colored DNA ligase, LigFa, from the acidophilic ferrous iron-oxidizing archaeon Ferroplasma acidiphilum, a di-ferric enzyme with an extremely low pH activity optimum. Unlike any other DNA ligase studied to date, LigFa contains two [Fe.sup.3+]-tyrosinate centers and lacks any requirement for either [Mg.sup.2+] or [K.sup.+] for activity. DNA ligases from closest phylogenetic and ecophysiological relatives have normal pH optima (6.0-7.5), lack iron, and require [Mg.sup.2+]/[K.sup.+] for activity. Ferric iron retention is pH-dependent, with release resulting in partial protein unfolding and loss of activity. Reduction of the [Fe.sup.3+] to [Fe.sup.2+] results in an 80% decrease in DNA substrate binding and an increase in the pH activity optimum to 5.0. DNA binding induces significant conformational change around the iron site(s), suggesting that the ferric irons of LigFa act both as structure organizing and stabilizing elements and as Lewis acids facilitating DNA binding at low pH. iron metalloenzyme | Picrophilus torridus | Sulfolobus acidocaldarius | Thermoplasma acidophilum | Ferroplasma acidiphilum