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Ciudad Real, Spain

Santos-Beneit F.,Institute Biotecnologia Of Leon | Martin J.F.,University of Leon
Journal of Global Antimicrobial Resistance

Vancomycin is an essential antibiotic to treat infections caused by multidrug-resistant bacteria. Several bacteria show resistance to vancomycin, including the model actinomycete Streptomyces coelicolor. In this study, vancomycin disk diffusion tests were performed to determine vancomycin resistance in S. coelicolor M145 under rich (TSA medium) or defined (MMCGT medium) growth conditions. A vancomycin-susceptible phenotype was observed when the TSA rich medium was used, whereas a resistant phenotype was obtained when the low-phosphate MMCGT medium was used. To identify which component was responsible for the vancomycin-resistant phenotype, all the components of the MMCGT medium were added individually to the TSA medium, and vice versa. Addition of phosphate to the MMCGT medium (the phosphate concentration is much higher in TSA than in MMCGT) produced a vancomycin-susceptible phenotype in MMCGT. Phosphate regulation of vancomycin resistance is not PhoP-dependent since the same minimum inhibitory concentrations were obtained in S. coelicolor parental and ΔphoP mutant strains. This phosphate regulation was not observed in the vancomycinproducer Amycolatopsis orientalis NRRL 2452, which was always resistant both in TSA and MMCGT (with or without phosphate addition) media. On the other hand, other Streptomyces spp. were susceptible to vancomycin in all conditions tested, including Streptomyces toyocaensis, the producer of a glycopeptide antibiotic different from vancomycin. In conclusion, the phosphate concentration clearly affects the resistance of S. coelicolor to vancomycin. © 2013 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved. Source

Martin J.,Institute Biotecnologia Of Leon | Garcia-Estrada C.,Institute Biotecnologia Of Leon | Kosalkova K.,Institute Biotecnologia Of Leon | Ullan R.V.,Institute Biotecnologia Of Leon | And 2 more authors.
Fungal Genetics and Biology

We described previously that an autoinducer molecule, identified as 1,3-diaminopropane (1,3-DAP), is secreted by Penicillium chrysogenum and Acremonium chrysogenum. Using pH-controlled fermentor cultures we have observed in this work that 1,3-DAP and spermidine clearly stimulate the biosynthesis of benzylpenicillin in P. chrysogenum, both in defined and in complex penicillin production media. Both 1,3-DAP and spermidine, but not putrescine (1,4-diaminobutane), produce a drastic increase in the transcript levels of the penicillin biosynthetic genes pcbAB, pcbC and penDE. These polyamines do not affect the expression of the global pH-stress regulator pacC gene, thus excluding that the effect of 1,3-DAP and spermidine is due to a modification of the pH control mechanism. Expression of the three penicillin biosynthetic genes is drastically reduced in a laeA-knock-down mutant of P. chrysogenum, which produces very low levels of benzylpenicillin. Interestingly, 1,3-DAP and spermidine revert the effect of the laeA knock-down mutation, completely restoring the levels of penicillin production. Furthermore, 1,3-DAP and spermidine enhanced the expression of laeA in the parental strain and restored the levels of laeA transcripts in the laeA knock-down mutant. Taken together these results indicate that the stimulatory effect of the inducer molecules 1,3-DAP and spermidine is exerted, at least in part, through the stimulation of the expression of laeA, a global regulator that acts epigenetically on the expression of secondary metabolite genes by heterochromatin reorganization. © 2012 Elsevier Inc. Source

Fernandez-Martinez L.T.,Institute Biotecnologia Of Leon | Fernandez-Martinez L.T.,John Innes Center | Santos-Beneit F.,Institute Biotecnologia Of Leon | Martin J.F.,Institute Biotecnologia Of Leon | Martin J.F.,University of Leon
Molecular Genetics and Genomics

Two-component regulatory systems play a key role in the cell metabolism adaptation to changing nutritional and environmental conditions. The fidelity between the two cognate proteins of a two-component system is important since it determines whether a specific response regulator integrates the signals transmitted by different sensor kinases. Phosphate regulation in Streptomyces coelicolor is mostly mediated by the PhoR-PhoP two-component system. Previous studies elucidated the mechanisms that control phosphate regulation as well as the genes directly regulated by the response regulator PhoP (pho regulon) in this organism. However, the role of the histidine kinase PhoR in Streptomyces coelicolor had not been unveiled so far. In this work, we report the characterization of a non-polar ΔphoR deletion mutant in S. coelicolor that keeps its native promoter. Induction of the phoRP operon was dependent upon phosphorylation of PhoP, but the ΔphoR mutant expressed phoP at a basal level. RT-PCR and reporter luciferase assays demonstrated that PhoR plays a key role in the activation of the pho regulon in this organism. Our results point towards a strict cognate partner specificity in terms of the phosphorylation of PhoP by PhoR thus corroborating the tight interaction between the two-components of this system. © Springer-Verlag 2012. Source

Garcia-Estrada C.,Institute Biotecnologia Of Leon | Barreiro C.,Institute Biotecnologia Of Leon | Jami M.-S.,Institute Biotecnologia Of Leon | Martin-Gonzalez J.,Institute Biotecnologia Of Leon | Martin J.-F.,University of Leon
Journal of Proteomics

In this article we studied the differential protein abundance of Penicillium chrysogenum in response to either 1,3-diaminopropane (1,3-DAP) or spermidine, which behave as inducers of the penicillin production process. Proteins were resolved in 2-DE gels and identified by tandem MS spectrometry. Both inducers produced largely identical changes in the proteome, suggesting that they may be interconverted and act by the same mechanism. The addition of either 1,3-DAP or spermidine led to the overrepresentation of the last enzyme of the penicillin pathway, isopenicillin N acyltransferase (IAT). A modified form of the IAT protein was newly detected in the polyamine-supplemented cultures. Both inducers produced a rearrangement of the proteome resulting in an overrepresentation of enzymes involved in the biosynthesis of valine and other precursors (e.g. coenzyme A) of penicillin. Interestingly, two enzymes of the homogentisate pathway involved in the degradation of phenylacetic acid (a well-known precursor of benzylpenicillin) were reduced following the addition of either of these two inducers, allowing an increase of the phenylacetic acid availability. Both inducers produced also an increase in the intracellular content of vesicles that derived to vacuoles in late stages and promoted sporulation of P. chrysogenum in solid medium. Biological significance: The analysis of global protein changes produced in response to polyamines 1,3-DAP and spermidine provides a valuable information for the understanding of the molecular mechanisms underlying the production of penicillin. This represents useful information to improve the production of this antibiotic and many other bioactive secondary metabolites not only in P. chrysogenum, but in other filamentous fungi as well. © 2013 Elsevier B.V. Source

Jami M.-S.,University of Leon | Garcia-Estrada C.,Institute Biotecnologia Of Leon | Barreiro C.,Institute Biotecnologia Of Leon | Cuadrado A.-A.,University of Oviedo | And 3 more authors.
Molecular and Cellular Proteomics

The filamentous fungus Penicillium chrysogenum is well-known by its ability to synthesize β-lactam antibiotics as well as other secondary metabolites. Like other filamentous fungi, this microorganism is an excellent host for secretion of extracellular proteins because of the high capacity of its protein secretion machinery. In this work, we have characterized the extracellular proteome reference map of P. chrysogenum Wisconsin 54-1255 by two-dimensional gel electrophoresis. This method allowed the correct identification of 279 spots by peptide mass finger-printing and tandem MS. These 279 spots included 328 correctly identified proteins, which corresponded to 131 different proteins and their isoforms. One hundred and two proteins out of 131 were predicted to contain either classical or nonclassical secretion signal peptide sequences, providing evidence of the authentic extracellular location of these proteins. Proteins with higher representation in the extracellular proteome were those involved in plant cell wall degradation (polygalacturonase, pectate lyase, and glucan 1,3-β-glucosidase), utilization of nutrients (extracellular acid phosphatases and 6-hydroxy-D-nicotine oxidase), and stress response (catalase R). This filamentous fungus also secretes enzymes specially relevant for food industry, such as sulfydryl oxidase, dihydroxy-acid dehydratase, or glucoamylase. The identification of several antigens in the extracellular proteome also highlights the importance of this microorganism as one of the main indoor allergens. Comparison of the extracellular proteome among three strains of P. chrysogenum, the wild-type NRRL 1951, the Wis 54-1255 (an improved, moderate penicillin producer), and the AS-P-78 (a penicillin high-producer), provided important insights to consider improved strains of this filamentous fungus as versatile cell-factories of interest, beyond antibiotic production, for other aspects of white biotechnology. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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