The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of the novel antibiotic dalbavancin. Previous studies have shown that phosphate limitation results in enhanced A40926 production. The A40926 biosynthetic gene (dbv) cluster, which consists of 37 genes, encodes two putative regulators, Dbv3 and Dbv4, as well as the response regulator (Dbv6) and the sensor-kinase (Dbv22) of a putative two-component system. Reverse transcription-PCR (RT-PCR) and real-time RT-PCR analysis revealed that the dbv14-dbv8 and the dbv30-dbv35 operons, as well as dbv4, were negatively influenced by phosphate. Dbv4 shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, the transcriptional activator of streptomycin biosynthesis in Streptomyces griseus. Dbv4 was expressed in Escherichia coli as an N-terminal His 6 -tagged protein. The purified protein bound the dbv14 and dbv30 upstream regions but not the region preceding dbv4. Bbr, a Dbv4 ortholog from the gene cluster for the synthesis of the glycopeptide balhimycin, also bound to the dbv14 and dbv30 upstream regions, while Dbv4 bound appropriate regions from the balhimycin cluster. Our results provide new insights into the regulation of glycopeptide antibiotics, indicating that the phosphate-controlled regulator Dbv4 governs two key steps in A40926 biosynthesis: the biosynthesis of the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine and critical tailoring reactions on the heptapeptide backbone.

Aims: Larvae of the red palm weevil (RPW) Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) feed inside palm stem tissues, making galleries and producing a wet fermenting frass. We characterized the culturable micro-organisms associated with frass produced by tunnelling larvae inside the Canary island date palms and investigated the role of frass and gut bacteria in plant polymers breakdown. \ud Methods and Results: A culture-dependent method was used to isolate bacteria from frass and noninfested palm tissues. Bacterial isolates were grouped into operational taxonomic units based on polymorphisms in the ITS-PCR profiles, and representative isolates were identified by partial sequencing of the 16S rRNA gene. Frass bacteria were dominated by 2,3-butanediol fermenter Enterobacteriaceae. None of the bacterial isolates was able to degrade cellulose; however, cellulolytic and hemicellulolytic bacteria were isolated from the larval gut enrichment cultures.\ud Conclusions: Frass bacteria are specifically associated with the RPW larvae and might play beneficial roles for RPW, other than nutritional, that deserve further investigations. Breakdown of plant polymers probably occurs inside the larvae digestive system.\ud Significance and Impact of the Study: Frass and gut micro-organisms of R. ferrugineus should be included in studies of the interactions between RPW, its plant hosts, and its enemies

A bacterial artificial chromosomal library of Nonomuraea sp. ATCC39727 was constructed using Escherichia coli-Streptomyces artificial chromosome (ESAC) and screened for the presence of dbv genes known to be involved in the biosynthesis of the glycopeptide A40926. dbv genes were cloned as two large, partially overlapping, fragments and transferred into the host Streptomyces lividans, thus generating strains S. lividansColon, two colonsNmESAC50 and S. lividansColon, two colonsNmESAC57. The heterologous expression of Nonomuraea genes in S. lividans was successfully demonstrated by using combined RT-PCR and proteomic approaches. MALDI-TOF analysis revealed that a Nonomuraea ABC transporter is expressed as two isoforms in S. lividans. Moreover, its expression may not require a Nonomuraea positive regulator at all, as it is present at similar levels in both clones even though S. lividansColon, two colonsNmESAC57 lacks regulatory genes. Considered together, these results show that S. lividans expresses Nonomuraea genes from their own promoters and support the idea that S. lividans can be a good host for genetic analysis of Nonomuraea.

Termite gut is host to a complex microbial community consisting of prokaryotes, and in some cases flagellates, responsible for the degradation of lignocellulosic material. Here we report data concerning the analysis of the gut microbiota of Reticulitermes lucifugus (Rossi), a lower termite species that lives in underground environments and is widespread in Italy, where it causes damage to wood structures of historical and artistic monuments. A 16S rRNA gene clone library revealed that the R. lucifugus gut is colonized by members of five phyla in the domain Bacteria: Firmicutes (49 % of clones), Proteobacteria (24 %), Spirochaetes (14 %), the candidatus TG1 phylum (12 %), and Bacteroidetes (1 %). A collection of cellulolytic aerobic bacteria was isolated from the gut of R. lucifugus by enrichment cultures on different cellulose and lignocellulose substrates. Results showed that the largest amount of culturable cellulolytic bacteria of R. lucifugus belongs to Firmicutes in the genera Bacillus and Paenibacillus (67 %). These isolates are also able to grow on xylan and show the largest clear zone diameter in the Congo red test. Reticulitermes lucifugus hosts a diverse community of bacteria and could be considered an acceptable source of hydrolytic enzymes for biotechnological applications.

The glycopeptide A40926, produced by the actinomycete Nonomuraea gerenzanensis, is the precursor of dalbavancin, a second-generation glycopeptide antibiotic approved for clinical use in the USA and Europe in 2014 and 2015, respectively. The final product of the biosynthetic pathway is an O-acetylated form of A40926 (acA40926). Glycopeptide biosynthesis in N. gerenzanensis is dependent upon the dbv gene cluster that encodes, in addition to the two essential positive regulators Dbv3 and Dbv4, the putative members of a two-component signal transduction system, specifically the response regulator Dbv6 and the sensor kinase Dbv22. The aim of this work was to assign a role to these two genes. Our results demonstrate that deletion of dbv22 leads to an increased antibiotic production with a concomitant reduction in glycopeptide resistance. Deletion of dbv6 results in a similar phenotype, although the effects are not as strong as in the Δdbv22 mutant. Consistently, quantitative RT-PCR analysis showed that Dbv6 and Dbv22 negatively regulate the regulatory genes (dbv3 and dbv4), as well as some dbv biosynthetic genes (dbv23 and dbv24), whereas Dbv6 and Dbv22 positively regulate transcription of the single, cluster-associated resistance gene. Finally, we demonstrate that exogenously added acA40926 and its precursor A40926 can modulate transcription of dbv genes but with an opposite extent: A40926 strongly stimulates transcription of the Dbv6/Dbv22 target genes while acA40926 has a neutral or negative effect on transcription of those genes. We propose a model in which glycopeptide biosynthesis in N. gerenzanensis is modulated through a positive feedback by the biosynthetic precursor A40926 and a negative feedback by the final product acA40926. In addition to previously reported control systems, this sophisticated control loop might help the producing strain cope with the toxicity of its own product. This work, besides leading to improved glycopeptide producing strains, enlarges our knowledge on the regulation of glycopeptide biosynthesis in actinomycetes, setting N. gerenzanensis and its two-component system Dbv6-Dbv22 apart from other glycopeptide producers. The glycopeptide antibiotic A40926 (Fig. 1A) is the precursor for dalbavancin (Fig. 1B), a semisynthetic lipoglycopeptide clinically used for treatment of acute skin infections caused by methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Streptococcus pyogenes. A40926 is produced by the actinomycete N. gerenzanensis as a complex of related compounds, differing mostly by the type of N-acyl chain attached to the glucuronic acid moiety. The core structure of A40926 consists of a heptapeptide containing the proteinogenic amino acid tyrosine and the non-proteinogenic amino acids 3,5-dihydroxyphenylglycine (DPG) and 4-hydroxyphenylglycine (HPG). The heptapeptide is assembled by a nonribosomal peptide synthetase (NRPS) and modified by the action of various enzymes: four oxygenases, one halogenase, one hydroxylase, two glycosyltransferases, one hexose ox...

The effect of actinomycin D, cycloheximide and glucocorticoids on the intestinal absorption of phosphate was studied. The effective intestinal absorption of 32P and 47Ca was determined simultaneously in intact rats in vivo using a whole body counter. Both, actinomycin D and cycloheximide caused a significant diminution of the intestinal absorption of phosphate whereas calcium absorption was not altered. Experiments with the in situ ligated loop technique were performed to eliminate the possibility that the action of the protein synthesis inhibitors could be due to an altered intestinal motility effect. Phosphate absorption was also significantly diminished under this condition. On the other hand, the administration of glucocorticoids produced a significant inhibition of phosphate and calcium absorption in the rat in vivo. The reported results indicate that proteins and/or enzymes with a rapid turn-over are involved in the mechanism of phosphate intestinal absorption, and confirm previous observations that phosphate and calcium are transported across the intestine by different mechanisms.

Phospholipid hydroperoxide glutathione peroxidase (GPx4/PHGPx) is a most abundant selenoprotein in the brain and GPx4 null mice die in utero at midgestation or knockdown of GPx4 during mammalian embryogenesis disturbs brain development. The current experiments were designed to investigate, by semiquantitative RT-PCR, the cerebral function of GPx4 of the lizards collected in both polluted (Sarno River, Italy) and unpolluted (Lago Matese, Italy) areas in order to provide useful molecular information on this widely distributed seleno-enzyme in adult as well through the development of the lizard, Podarcis sicula. Our research demonstrates the lizard GPx4 hormonal control. Treatments with HCG increases immunoreactivity while the anti-estrogen ICI 182-780 induces its reduction. The partial region of the cloned cDNA is of 340 bp; conserved protein domains reports its membership to selenoprotein and analysis amino acid residues the value as an antioxidant. The semiquantitative expression of GPx 4 of adult lizard brain in polluted station resulted generally very high and let sustain its importance as lizard neuroendocrine disruptor alarm

No abstract