The Actinomycete Nonomuraea produces the glycopeptide A40926, precursor of dalbavancin, a novel lipoglycopeptide homologous to vancomycin that could be used against emerging resistant Gram-positive bacteria. The A40926 biosynthetic gene cluster (dbv), which consists of 37 genes, encodes two regulators, Dbv3 (LuxR-like) and Dbv4 (StrR-like), as well as the response regulator (Dbv6) and the sensor-kinase (Dbv22) of a putative two-component system. Previous work demonstrated a positive role of Dbv4 in antibiotic production by activating the transcription of 13 dbv genes; up to now no role was assigned to Dbv3, Dbv6 and Dbv22. In addition, an IclR-like protein is thought to be involved in dbv4 expression. The iclR gene is not localized in the dbv cluster and encodes a putative DNA binding protein that controls growth, primary and secondary metabolism in other bacteria. Mutants in dbv3, dbv4, dbv6, dbv22 and iclR genes were independently generated, revealing that Dbv3, Dbv4 and IclR are necessary for antibiotic production, while the response regulator Dbv6 and the sensor-kinase Dbv22 do not seem to influence A40926 production. Phenotypic analysis of the dbv6 and dbv22 mutants revealed that Dbv6 (VanR like) and Dbv22 (VanS like) do not influence bacterial growth, but they could control resistance; indeed, the mutants are more sensitive to vancomycin (a commercially available glycopeptide) in respect to the wild type. Dbv3 belongs to the LAL family (Large ATP-binding regulators of the LuxR family). The LuxR transcriptional regulator is a key player of a large variety of quorum sensing processes, regulating the expression of genes related to antibiotic biosynthesis, motility, nodulation, plasmid transfer, bioluminescence, pathogenicity, biofilm formation, etc. Kinetic growth curves of dbv3 mutant (Ko dbv3) revealed that Dbv3 does not influence bacterial growth in liquid rich medium (R3) but that it is necessary for antibiotic production. Semi-quantitative RT-PCR analysis after 48 hours of growth demonstrated that Dbv3 controls some genes involved in the antibiotic production. To further investigate the role of Dbv3 in differentiation and growth, strains that over-express dbv3 were generated. Phenotypic analyses revealed no differences in growth on R3 medium, while antibiotic production was about two fold more abundant than in the wild type. In experiments of affinity chromatography using as bait a DNA fragment upstream of the gene dbv4 (called P4) a protein IclR-like was isolated, that could have a role in regulating the expression of dbv4 gene. IclR family comprises regulators acting as repressors, activators and proteins with a dual role (activator and repressor). Members of the IclR family control genes whose products are involved in the glyoxylate shunt, multidrug resistance, degradation of aromatics, inactivation of quorum-sensing signals, determinants of plant pathogenicity, antibiotic production and sporulation. Since P4 is localized downstream dbv4 transcriptional start site, mapped by S1-mapping experiments, a negative role to IclR was hypothesized. Indeed, qRT-PCR analysis showed that the levels of transcription of genes iclR and dbv4 have an opposite trend: iclR is more transcribed when a low yield of antibiotic is produced; dbv4, vice versa, is more transcribed when antibiotic production is high. These preliminary data suggest a possible involvement of IclR in A40926 production. Three knock out iclR mutants were generated. Morphological and physiological analysis revealed that they lost the capacity to grow on minimal medium, they were not pigmented in the rich medium R3-agar and produced the antibiotic after 9 days of growth, while wild type produced after 3 days in the same growth condition. Different carbon sources and aminoacids were added to minimal medium to restore growth and pigmentation and to understand which metabolic pathways was under IclR control. At the same time, morphological and physiological analysis of five iclR over-expression mutants revealed that IclR has a negative effect on antibiotic production confirming its involvement in A40926 biosynthesis. To investigate whether IclR directly regulates antibiotic biosynthesis, an His-tagged IclR was overproduced in Escherichia coli and purified. Gel mobility shift assays of different kind of DNA probe were carried out using the purified His6-tagged IclR. No bindings were detected. This could be due to an incorrect protein folding or to the need of some cofactors to help the protein to bind DNA. This study provides new insights into gene expression in gram-positive bacteria, specifically focusing on the complex regulatory network governing both growth and secondary metabolism.
|Titolo:||Regulation of physiological differentiation in Nonomuraea sp. ATCC 39727|
|Settore Scientifico Disciplinare:||Settore BIO/19 - Microbiologia Generale|
|Data di pubblicazione:||25-feb-2014|
|Altre informazioni significative:||dottoranda XXIV ciclo - Biologia Cellulare (e dello Sviluppo)|
|Citazione:||(2014). Regulation of physiological differentiation in Nonomuraea sp. ATCC 39727. (Tesi di dottorato, , 2014).|
|Tipologia:||Tesi di dottorato|
|Appare nelle tipologie:||07 - Tesi di dottorato pre 2013|