Functional analysis of MFS protein CefT involved in the transport of beta-lactam antibiotics in Acremonium chrysogenum and Saccharomyces cerevisiae

Думина, М. В.; Zhgun, A. A.; Kerpichnikov, I. V.; Domracheva, A. G.; Novak, Metka; Valiachmetov, A. Ya.; Knorre, Dmitry A.; Severin, Fedor F.; Eldarov, M. A.; Bartoshevich, Yu. E.

doi:10.1134/s0003683813040042

Cited by 17 publications

(38 citation statements)

References 23 publications

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“…For microbial infection, fragmented pieces were placed into sterile Petri dishes and saturated with 0.3 ml H 2 O per 1 cm 3 of piece at 25˚C for 48 h. To avoid any contact with water of either side of the material, pieces with mock layers were placed on sterile hydrophobic spacers so that the lid of the Petri dish did not touch the surface of the mock layer. The isolates cultured on CD medium were pre-synchronized for the growth rate on agar using the drop and PLOS ONE dilution method (spot analysis) described earlier [24]. The selected isolates were removed from oblique agar medium, resuspended in 0.9% NaCl to OD 600 = 0.35, subjected to three consecutive 10-fold dilutions in 0.9% NaCl, and 1 μl of each dilution was inoculated into watersaturated mock layers, which were incubated for 4-12 days at 25˚C and analyzed.…”

Section: Preparation and Inoculation Of Mock Layersmentioning

confidence: 99%

Detection of potential biodeterioration risks for tempera painting in 16th century exhibits from State Tretyakov Gallery

Zhgun¹,

Avdanina²,

Shumikhin

et al. 2020

PLoS ONE

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In this study, we investigated biodeterioration of materials used in tempera painting by analyzing the structure of the microbiome in ancient tempera paintings exhibited in State Tretyakov Gallery, Moscow, Russia. Samples were obtained from 16 th-century paintings, including a grand Russian Orthodox icon "The Church Militant" (all exhibits were without visible signs of biodeterioration), and from surrounding walls and ceilings (with vast zones of visible microbial growth). A number of microorganisms isolated from visible signs of environmental bio-damage were also detected in tempera paintings kept in temperature-and humidity-controlled conditions unfavorable for the growth of microflora. To determine the biodegrading potential of the microbiome for tempera paintings, we developed a set of mock layers from paintwork materials used in tempera painting of 16 th century and their modern analogues and inoculated them with cultures containing filamentous fungi and bacteria. The susceptibility to microbial degradation of individual tempera painting materials was examined by micro-Fourier Transform Infrared (FTIR) spectroscopy, which enabled detection of even invisible signs of biodeterioration. The results indicate that the microorganisms isolated from paintings and surrounding areas in the museum are capable of causing significant damage of various tempera materials, among which varnishes were the most resistant; however, the addition of antiseptic (sodium pentachlorophenolate) can inhibit microbial growth on sturgeon glue.

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Section: Preparation and Inoculation Of Mock Layersmentioning

confidence: 99%

Detection of potential biodeterioration risks for tempera painting in 16th century exhibits from State Tretyakov Gallery

Zhgun¹,

Avdanina²,

Shumikhin

et al. 2020

PLoS ONE

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“…chrysogenum the binary pZEN36c vector was constructed. For this purpose the fragment containing the PMA1-TagYFP and SV40 polyA sequences was obtained from pZEN36b as a Spe I-“blunt”/ Hind III 4036 bp DNA fragment and inserted into the Pme I/ Hind III site of pZEN33 [ 26 ]. The description of used plasmids is given in Table 3 .…”

Section: Methodsmentioning

confidence: 99%

“…chrysogenum cef genes for beta-lactam transporters are localized in the “early” biosynthetic cluster [ 23 , 24 ]. It was assumed that CPC export to the culture medium is directed by CefT belonging to the major facilitator superfamily (MFS) antiporters [ 25 ], however, this observation was questioned in further studies [ 26 ]. The MFS transporters use the electrochemical gradient generated by the plasma membrane H + -ATPase (PMA) [ 27 ].…”

Section: Introductionmentioning

confidence: 99%

“…This strain typically produces 9–12 grams of CPC during laboratory fermentation in shake flasks, 200–300 times higher than WT strains [ 29 ]. We have previously shown that the overproduction phenotype correlates with upregulation of cef genes [ 31 ], chromosomal rearrangements [ 5 ], as well as alterations in polyamine metabolism [ 32 ], in cell wall structure [ 33 ], in size of filamentous hyphae and conidia formation [ 34 ], in colony size and coloration [ 26 ], and has other physiological changes. The PMA1 defect may be one of the reasons for reduced strain growth rate and overall fitness, diminished resistance to abiotic stress and proficiency in nutrients [ 30 ].…”

Section: Introductionmentioning

confidence: 99%

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The critical role of plasma membrane H+-ATPase activity in cephalosporin C biosynthesis of Acremonium chrysogenum

Zhgun¹,

Думина²,

Valiakhmetov

et al. 2020

PLoS ONE

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The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H + -ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A . chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A . chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A . chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae , under the control of the constitutive gpdA promoter from Aspergillus nidulans , results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A . chrysogenum HY OE :: PMA1 strains has been increased by 50–100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A . chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production.

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“…The filamentous fungi were cultivated on agarized Czapek-Dox (CD) medium (30 g/L sucrose, 2 g/L NaNO 3 , 1 g/L K 2 HPO 4 , 0.5 g/L MgSO 4 ×7 H 2 O, 0.5 g/L KCl, 0.01 g/L FeSO 4 ×7 H 2 O, 20 g/L agar, pH 7.0-7.4). To determine the toxic effect of AO-Agm, APA, DFMA and DFMO on the growth of fungal cells the drop-dilution method was used with some modifications as described earlier [40,41]. Cells were collected from agar slants and diluted with 0.9% NaCl solution up to OD 600 = 0.5 (basic concentration), followed by serial tenfold dilutions with the same solution.…”

Section: Fungal Growth Inhibitionmentioning

confidence: 99%

Hydroxylamine Analogue of Agmatine: Magic Bullet for Arginine Decarboxylase

et al. 2020

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The biogenic polyamines, spermine, spermidine (Spd) and putrescine (Put) are present at micro-millimolar concentrations in eukaryotic and prokaryotic cells (many prokaryotes have no spermine), participating in the regulation of cellular proliferation and differentiation. In mammalian cells Put is formed exclusively from L-ornithine by ornithine decarboxylase (ODC) and many potent ODC inhibitors are known. In bacteria, plants, and fungi Put is synthesized also from agmatine, which is formed from L-arginine by arginine decarboxylase (ADC). Here we demonstrate that the isosteric hydroxylamine analogue of agmatine (AO-Agm) is a new and very potent (IC50 3•10−8 M) inhibitor of E. coli ADC. It was almost two orders of magnitude less potent towards E. coli ODC. AO-Agm decreased polyamine pools and inhibited the growth of DU145 prostate cancer cells only at high concentration (1 mM). Growth inhibitory analysis of the Acremonium chrysogenum demonstrated that the wild type (WT) strain synthesized Put only from L-ornithine, while the cephalosporin C high-yielding strain, in which the polyamine pool is increased, could use both ODC and ADC to produce Put. Thus, AO-Agm is an important addition to the set of existing inhibitors of the enzymes of polyamine biosynthesis, and an important instrument for investigating polyamine biochemistry.

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Functional analysis of MFS protein CefT involved in the transport of beta-lactam antibiotics in Acremonium chrysogenum and Saccharomyces cerevisiae

Cited by 17 publications

References 23 publications

Detection of potential biodeterioration risks for tempera painting in 16th century exhibits from State Tretyakov Gallery

Detection of potential biodeterioration risks for tempera painting in 16th century exhibits from State Tretyakov Gallery

The critical role of plasma membrane H+-ATPase activity in cephalosporin C biosynthesis of Acremonium chrysogenum

Hydroxylamine Analogue of Agmatine: Magic Bullet for Arginine Decarboxylase

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