The Streptomyces coelicolor small ORF trpM stimulates growth and morphological development and exerts opposite effects on actinorhodin and calcium-dependent antibiotic production

In actinomycetes, antibiotic production is often associated with a morpho-physiological differentiation program that is regulated by complex molecular and metabolic networks. Many aspects of these regulatory circuits have been already elucidated and many others still deserve further investigations. In this regard, the possible role of many small open reading frames (smORFs) in actinomycete morpho-physiological differentiation is still elusive. In Streptomyces coelicolor, inactivation of the smORF trpM (SCO2038) – whose product modulates L-tryptophan biosynthesis – impairs production of antibiotics and morphological differentiation. Indeed, it was demonstrated that TrpM is able to interact with PepA (SCO2179), a putative cytosol aminopeptidase playing a key role in antibiotic production and sporulation. In this work, a S. coelicolor trpM knock-in (Sco-trpMKI) mutant strain was generated by cloning trpM into overexpressing vector to further investigate the role of trpM in actinomycete growth and morpho-physiological differentiation. Results highlighted that trpM: (i) stimulates growth and actinorhodin (ACT) production; (ii) decreases calcium-dependent antibiotic (CDA) production; (iii) has no effect on undecylprodigiosin production. Metabolic pathways influenced by trpM knock- in were investigated by combining two-difference in gel electrophoresis/nanoliquid chromatography coupled to electrospray linear ion trap tandem mass spectrometry (2D- DIGE/nanoLC-ESI-LIT-MS/MS) and by LC-ESI-MS/MS procedures, respectively. These analyses demonstrated that over-expression of trpM causes an over-representation of factors involved in protein synthesis and nucleotide metabolism as well as a down-representation of proteins involved in central carbon and amino acid metabolism. At the metabolic level, this corresponded to a differential accumulation pattern of different amino acids – including aromatic ones but tryptophan – and central carbon intermediates. PepA was also down-represented in Sco-trpMKI. The latter was produced as recombinant His-tagged protein and was originally proven having the predicted aminopeptidase activity. Altogether, these results highlight the stimulatory effect of trpM in S. coelicolor growth and ACT biosynthesis, which are elicited through the modulation of various metabolic pathways and PepA representation, further confirming the complexity of regulatory networks that control antibiotic production in actinomycetes

Kinetic features of carbonyl reductase 1 acting on glutathionylated aldehydes

The attempt to evaluate the human carbonyl reductase 1 (CBR1) activity on 3-glutathionylated-4-hydroxyalkanals through the classical spectrophotometric assay, in which NADPH oxidation is monitored at 340 nm, failed. This was due to the ability of the enzyme to catalyze the reduction of the free aldehyde form and at the same time the oxidation of the hemiacetal structure of this class of substrates, thus leading to the occurrence of a disproportion reaction sustained by a redox recycle of the pyridine cofactor. Making use of glutathionylated alkanals devoid of the 4 hydroxyl group, and thus unable to structurally arrange into a cyclic hemiacetal form, the susceptibility to inhibition of CBR1 to polyphenols was tested. Flavones, that were much more effective than isoflavones, resulted able to modulate the reductase activity of the enzyme on this new peculiar class of substrates

Differential proteomic analysis highlights metabolic strategies associated with balhimycin production in Amycolatopsis balhimycina chemostat cultivations

Background Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina batch cultivations. Combining chemostat fermentation technology, where cells proliferate with constant parameters in a highly reproducible steady-state, and differential proteomics, the relationships between physiological status and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted. Results Two minimal defined media, one with low Pi (0.6 mM; LP) and proficient glucose (12 g/l) concentrations and the other one with high Pi (1.8 mM) and limiting (6 g/l; LG) glucose concentrations, were developed to promote and repress antibiotic production, respectively, in A. balhimycina chemostat cultivations. Applying the same dilution rate (0.03 h-1), both LG and LP chemostat cultivations showed a stable steady-state where biomass production yield coefficients, calculated on glucose consumption, were 0.38+/-0.02 and 0.33+/-0.02 g/g (biomass dry weight/glucose), respectively. Notably, balhimycin was detected only in LP, where quantitative RT-PCR revealed upregulation of selected bal genes, devoted to balhimycin biosynthesis, and of phoP, phoR, pstS and phoD, known to be associated to Pi limitation stress response. 2D-Differential Gel Electrophoresis (DIGE) and protein identification, performed by mass spectrometry and computer-assisted 2D reference-map (http://www.unipa.it/ampuglia/Abal-proteome-maps) matching, demonstrated a differential expression for proteins involved in many metabolic pathways or cellular processes, including central carbon and phosphate metabolism. Interestingly, proteins playing a key role in generation of primary metabolism intermediates and cofactors required for balhimycin biosynthesis were upregulated in LP. Finally, a bioinformatic approach showed PHO box-like regulatory elements in the upstream regions of nine differentially expressed genes, among which two were tested by electrophoresis mobility shift assays (EMSA). Conclusion In the two chemostat conditions, used to generate biomass for proteomic analysis, mycelia grew with the same rate and with similar glucose-biomass conversion efficiencies. Global gene expression analysis revealed a differential metabolic adaptation, highlighting strategies for energetic supply and biosynthesis of metabolic intermediates required for biomass production and, in LP, for balhimycin biosynthesis. These data, confirming a relationship between primary metabolism and antibiotic production, could be used to increase antibiotic yield both by rational genetic engineering and fermentation processes improvement

Poplar woody root proteome during the transition dormancy-active growth

Woody plants living in temperate climates finely regulate their growth and development in relation to seasonal changes; their transition from vegetative to dormancy phase represents an adaptation to their environment. Events occurring in the shoot during onset/release from dormancy have been largely investigated, whereas in woody roots they remain completely unknown. In recent years, we have been interested in understanding the molecular and physiological events occurring in poplar woody root during release from dormancy. Here, we propose the results of a comparative analysis of the proteome of poplar woody root sampled at different time points: T0 (dormancy condition), T1 (release from dormancy), and T2 (full vegetative condition). This study identified proteins that may be involved in the long-term survival of a dormant root or landmarking a specific time point

Human carbonyl reductase 1 as efficient catalyst for the reduction of glutathionylated aldehydes derived from lipid peroxidation

Human recombinant carbonyl reductase 1 (E.C. 1.1.1.184, hCBR1) is shown to efficiently act as aldehyde reductase on glutathionylated alkanals, namely 3-glutathionyl-4-hydroxynonanal (GSHNE), 3-glutathionyl-nonanal, 3-glutathionyl-hexanal and 3-glutathionyl-propanal. The presence of the glutathionyl moiety appears as a necessary requirement for the susceptibility of these compounds to the NADPH-dependent reduction by hCBR1. In fact the corresponding alkanals and alkenals, and the cysteinyl and γ-glutamyl-cysteinyl alkanals adducts were either ineffective or very poorly active as CBR1 substrates. Mass spectrometry analysis reveals the ability of hCBR1 to reduce GSHNE to the corresponding GS-dihydroxynonane (GSDHN) and at the same time to catalyze the oxidation of the hemiacetal form of GSHNE, generating the 3-glutathionylnonanoic–δ-lactone. These data are indicative of the ability of the enzyme to catalyze a disproportion reaction of the substrate through the redox recycle of the pyridine cofactor. A rationale for the observed preferential activity of hCBR1 on different GSHNE diastereoisomers is given by molecular modelling. These results evidence the potential of hCBR1 acting on GSHNE to accomplish a dual role, both in terms of HNE detoxification and, through the production of GSDHN, in terms of involvement into the signalling cascade of the cellular inflammatory response

Purification and characterization of a Cys-Gly hydrolase from the gastropod mollusk, Patella caerulea

A magnesium-dependent cysteinyl-glycine hydrolyzing enzyme from the gastropod mollusk Patella caerulea was purified to electrophoretic homogeneity through a simple and rapid purification protocol. The molecular masses of the native protein and the subunit suggest that the enzyme has a homohexameric structure. Structural data in combination with kinetic parameters determined with Cys-Gly and compared with Leu-Gly as a substrate, indicate that the purified enzyme is a member of the peptidase family M17. The finding that an enzyme of the peptidase family M17 is responsible also in mollusks for the breakdown of Cys-Gly confirms the important role of this peptidase family in the glutathione metabolism