Actinomadura graeca sp. nov.: A novel producer of the macrocyclic antibiotic zelkovamycin.

As part of a screening programme for antibiotic-producing bacteria, a novel Actinomadura species was discovered from a soil sample collected in Santorini, Greece. Preliminary 16S rRNA gene sequence comparisons highlighted Actinomadura macra as the most similar characterised species. However, whole-genome sequencing revealed an average nucleotide identity (ANI) value of 89% with A. macra, the highest among related species. Further phenotypic and chemotaxonomic analyses confirmed that the isolate represents a previously uncharacterised species in the genus Actinomadura, for which the name Actinomadura graeca sp. nov. is proposed (type strain 32-07T). The G+C content of A. graeca 32-07 is 72.36%. The cell wall contains DL-diaminopimelic acid, intracellular sugars are glucose, ribose and galactose, the predominant menaquinone is MK-9(H6), the major cellular lipid is phosphatidylinositol and fatty acids consist mainly of hexadecanoic acid. No mycolic acid was detected. Furthermore, A. graeca 32-07 has been confirmed as a novel producer of the non-ribosomal peptide antibiotic zelkovamycin and we report herein a provisional description of the unique biosynthetic gene cluster.

E40, a novel microbial protease efficiently detoxifying gluten proteins, for the dietary management of gluten intolerance.

Gluten proteins are the causative agent of Celiac Disease (CD), a life-long food intolerance characterized by an autoimmune enteropathy. Inadvertent gluten exposure is frequent even in celiac patients complying with a gluten-free diet, and the supplementation of exogenous gluten-digestive enzymes (glutenases) is indeed a promising approach to reduce the risk of dietary gluten boost. Here we describe Endopeptidase 40, a novel glutenase discovered as secreted protein from the soil actinomycete Actinoallomurus A8, and its recombinant active form produced by Streptomyces lividans TK24. E40 is resistant to pepsin and trypsin, and active in the acidic pH range 3 to 6. E40 efficiently degrades the most immunogenic 33-mer as well as the whole gliadin proteins, as demonstrated by SDS-PAGE, HPLC, LC-MS/MS, and ELISA. T lymphocytes from duodenal biopsies of celiac patients showed a strongly reduced or absent release of IFN-γ when exposed to gluten digested with E40. Data in gastrointestinal simulated conditions suggest that no toxic peptides are freed during gluten digestion by E40 into the stomach to enter the small intestine, thus counteracting the intestinal inflammatory cascade to occur in CD patients. E40 is proposed as a novel candidate in Oral Enzymatic Therapy for the dietary management of gluten toxicity.

Optimized RNA extraction and northern hybridization in streptomycetes.

Northern blot hybridization is a useful tool for analyzing transcript patterns. To get a picture of what really occurs in vivo, it is necessary to use a protocol allowing full protection of the RNA integrity and recovery and unbiased transfer of the entire transcripts population. Many protocols suffer from severe limitations including only partial protection of the RNA integrity and/or loss of small sized molecules. Moreover, some of them do not allow an efficient and even transfer in the entire sizes range. These difficulties become more prominent in streptomycetes, where an initial quick lysis step is difficult to obtain. We present here an optimized northern hybridization protocol to purify, fractionate, blot, and hybridize Streptomyces RNA. It is based on grinding by a high-performance laboratory ball mill, followed by prompt lysis with acid phenol-guanidinium, alkaline transfer, and hybridization to riboprobes. Use of this protocol resulted in sharp and intense hybridization signals relative to long mRNAs previously difficult to detect.