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1.
Sequential Primary and Secondary Shunt Metabolism in Penicillium chrysogenum 总被引:2,自引:1,他引:1
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Penicillium chrysogenum was grown on a rich medium and on a more sparse medium which favored penicillin production. Mycelia grown on both media were examined for changes in lipid, mannitol, erythritol, glycerol, pentitol, trehalose, and residual mycelium, and the filtrates were examined for penicillin. Penicillin production took place after the bulk of trehalose, polyol, and lipid had accumulated, and hence the sequential pattern of primary and secondary shunt metabolism, as observed in the case of ergot alkaloid production by Claviceps purpurea, was demonstrated in this example of penicillin production. 相似文献
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Production of Penicillin by Fungi Growing on Food Products: Identification of a Complete Penicillin Gene Cluster in Penicillium griseofulvum and a Truncated Cluster in Penicillium verrucosum
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Mycobiota growing on food is often beneficial for the ripening and development of the specific flavor characteristics of the product, but it can also be harmful due to the production of undesirable compounds such as mycotoxins or antibiotics. Some of the fungi most frequently isolated from fermented and cured meat products such as Penicillium chrysogenum and Penicillium nalgiovense are known penicillin producers; the latter has been shown to be able to produce penicillin when growing on the surface of meat products and secrete it to the medium. The presence of penicillin in food must be avoided, since it can lead to allergic reactions and the arising of penicillin resistance in human-pathogenic bacteria. In this article we describe a study of the penicillin production ability among fungi of the genus Penicillium that are used as starters for cheese and meat products or that are frequently isolated from food products. Penicillium griseofulvum was found to be a new penicillin producer and to have a penicillin gene cluster similar to that of Penicillium chrysogenum. No other species among the studied fungi were found to produce penicillin or to possess the penicillin biosynthetic genes, except P. verrucosum, which contains the pcbAB gene (as shown by hybridization and PCR cloning of fragments of the gene) but lacks pcbC and penDE. Antibacterial activities due to the production of secondary metabolites other than penicillin were observed in some fungi. 相似文献
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A specific and sensitive radioimmunoassay of trace amounts of penicillin G in biological fluids is proposed. It is based on the quantitative transformation of penicillin into penicilloyl by rupture of the β-lactam ring of the penicillin molecule, and then the determination of the penicilloyl groups thus formed. Using ?-aminocaproic acid as an acceptor which covalently binds with the penicilloyl groups formed, at alkaline pH, allows a complete and reproducible aminolysis of penicillin. The inhibition of the binding to antipenicilloyl antibodies and the association constant appear to be identical whether penicilloyl groups derive from penicillin or are present under standard penicilloyl ?-aminocaproate form. Besides this application to the radioimmunoassay of penicillin, the study of the in vitro aminolysis of penicillin should permit better understanding of the complex mechanism of the covalent binding of penicilloyl groups to proteins observed in vivo after penicillin therapy, binding which leads to the allergenic metabolite of penicillin. 相似文献
5.
D. N. Wright 《Journal of bacteriology》1967,93(1):185-190
When penicillin was added to cultures of Mycoplasma neurolyticum in amounts up to 1,000 units per ml, lag time and generation time were increased and the total population was reduced in proportion to the antibiotic concentration. Although growth suppression by penicillin was complete, the death rate was slow and linear over periods up to 12 days. Growth after induced lag was due to a decay in penicillin activity and was not the result of mutant selection. However, repeated transfer in media which contained increasing concentrations of penicillin resulted in normal growth of M. neurolyticum at penicillin levels as high as 1,000 units per ml. Penicillinase did not play a role in recovery from penicillin-induced lag, and the inactive penicillin molecule did not prevent normal growth of M. neurolyticum. Removal of penicillin from the medium by washing or penicillinase during the induced lag was immediately followed by normal growth of the organism. These results suggest a reversible antibiotic function for penicillin which prevents multiplication of the organism by means unrelated to cell wall formation. 相似文献
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Background
Penicillin resistance in Streptococcus pneumoniae is mediated by a mosaic of genes encoding altered penicillin-binding proteins (PBPs). Nonetheless, S. pneumoniae has also developed non-PBP mechanisms implicated in penicillin resistance. In this study, whole genome sequencing of resistant organisms was used to discover mutations implicated in resistance to penicillin.Results
We sequenced two S. pneumoniae isolates selected for resistance to penicillin in vitro. The analysis of the genome assemblies revealed that six genes were mutated in both mutants. These included three pbp genes, and three non-pbp genes, including a putative iron permease, spr1178. The nonsense mutation in spr1178 always occurred in the first step of the selection process. Although the mutants had increased resistance to penicillin, the introduction of altered versions of PBPs into a penicillin-susceptible strain by sequential transformation led to strains with a minimal increase in resistance, thus implicating other genes in resistance. The introduction by transformation of the non-PBP recurrent mutations did not increase penicillin resistance, but the introduction of the nonsense mutation in the putative iron permease spr1178 led to a reduced accumulation of reactive oxygen species following exposure to penicillin and to other bactericidal antibiotics as well.Conclusions
This study indicates that the selection of resistance to penicillin in S. pneumoniae involves the acquisition of mutations conferring tolerance to the antibiotic-induced accumulation of oxidants, which translates into an increased survival that putatively enables the selection of major resistance determinants such as mutations in PBPs. 相似文献7.
Aspergillus nidulans is able to synthesize penicillin and serves as a model to study the regulation of its biosynthesis. Only three enzymes are required to form the beta lactam ring tripeptide, which is comprised of l-cysteine, l-valine and l-aminoadipic acid. Whereas two enzymes, AcvA and IpnA localize to the cytoplasm, AatA resides in peroxisomes. Here, we tested a novel strategy to improve penicillin production, namely the change of the residence of the enzymes involved in the biosynthesis. We tested if targeting of AcvA or IpnA (or both) to peroxisomes would increase the penicillin yield. Indeed, AcvA peroxisomal targeting led to a 3.2-fold increase. In contrast, targeting IpnA to peroxisomes caused a complete loss of penicillin production. Overexpression of acvA, ipnA or aatA resulted in 1.4, 2.8 and 3.1-fold more penicillin, respectively in comparison to wildtype. Simultaneous overexpression of all three enzymes resulted even in 6-fold more penicillin. Combination of acvA peroxisomal targeting and overexpression of the gene led to 5-fold increase of the penicillin titer. At last, the number of peroxisomes was increased through overexpression of pexK. A strain with the double number of peroxisomes produced 2.3 times more penicillin. These results show that penicillin production can be triggered at several levels of regulation, one of which is the subcellular localization of the enzymes. 相似文献
8.
Hybrids have been obtained by protoplast fusion of penicillin-producing strains of Aspergillus nidulans with strains of A. rugulosus which do not produce the antibiotic at detectable levels. Induced haploidization of the hybrids allowed the recovery of stable segregants, which were screened for penicillin production. Approximately half of the segregants from each cross produced penicillin, indicating segregation of one or several linked genes which are essential for penicillin biosynthesis. These essential genes were found to be located on linkage-group VI of A. nidulans by observing segregation in multi-marked strains. The penicillin-producing segregants showed a wide range of titres. Some segregants, which had recombinant phenotypes, produced significantly more penicillin than the A. nidulans parent. 相似文献
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Sulfur Regulation of the Sulfate Transporter Genes sutA and sutB in Penicillium chrysogenum
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Mart van de Kamp Theo A. Schuurs Arnold Vos Ted R. van der Lende Wil N. Konings Arnold J. M. Driessen 《Applied microbiology》2000,66(10):4536-4538
Penicillium chrysogenum uses sulfate as a source of sulfur for the biosynthesis of penicillin. Sulfate uptake and the mRNA levels of the sulfate transporter-encoding sutB and sutA genes are all reduced by high sulfate concentrations and are elevated by sulfate starvation. In a high-penicillin-yielding strain, sutB is effectively transcribed even in the presence of excess sulfate. This deregulation may facilitate the efficient incorporation of sulfur into cysteine and penicillin. 相似文献
11.
Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus
Leticia L. Torres ángel Cantero Mercedes del Valle Anabel Marina Fernando López-Gallego José M. Guisán José Berenguer Aurelio Hidalgo 《Applied and environmental microbiology》2013,79(5):1555-1562
A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or β24 improved the Km for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site. 相似文献
12.
Rathinaswamy P Gaikwad SM Suresh CG Prabhune AA Brannigan JA Dodson GG Pundle AV 《International journal of biological macromolecules》2012,50(1):25-30
The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148 kDa. The apparent Km of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40 mM and 0.63 mM, respectively, and the association constants were 8.93 × 102 M−1 and 2.51 × 105 M−1, respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride. 相似文献
13.
Rachel J. Skilton Lesley T. Cutcliffe David Barlow Yibing Wang Omar Salim Paul R. Lambden Ian N. Clarke 《PloS one》2009,4(11)
Background
Chlamydia trachomatis is a major human pathogen with a unique obligate intracellular developmental cycle that takes place inside a modified cytoplasmic structure known as an inclusion. Following entry into a cell, the infectious elementary body (EB) differentiates into a non - infectious replicative form known as a reticulate body (RB). RBs divide by binary fission and at the end of the cycle they redifferentiate into EBs. Treatment of C.trachomatis with penicillin prevents maturation of RBs which survive and enlarge to become aberrant RBs within the inclusion in a non - infective persistent state. Persistently infected individuals may be a reservoir for chlamydial infection. The C.trachomatis genome encodes the enzymes for peptidoglycan (PG) biosynthesis but a PG sacculus has never been detected. This coupled to the action of penicillin is known as the chlamydial anomaly. We have applied video microscopy and quantitative DNA assays to the chlamydial developmental cycle to assess the effects of penicillin treatment and establish a framework for investigating penicillin induced chlamydial persistence.Principal Findings
Addition of penicillin at the time of cell infection does not prevent uptake and the establishment of an inclusion. EB to RB transition occurs but bacterial cytokinesis is arrested by the second binary fission. RBs continue to enlarge but not divide in the presence of penicillin. The normal developmental cycle can be recovered by the removal of penicillin although the large, aberrant RBs do not revert to the normal smaller size but remain present to the completion of the developmental cycle. Chromosomal and plasmid DNA replication is unaffected by the addition of penicillin but the arrest of bacterial cytokinesis under these conditions results in RBs accumulating multiple copies of the genome.Conclusions
We have applied video time lapse microscopy to the study of the chlamydial developmental cycle. Linked with accurate measures of genome replication this provides a defined framework to analyse the developmental cycle and to investigate and provide new insights into the effects of antibiotic treatments. Removal of penicillin allows recovery of the normal developmental cycle by 10–20 hrs and the process occurs by budding from aberrant RBs. 相似文献14.
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Based on experimental results, two different kinetic models and reaction mechanisms of penicillin amidase (penicillin amidohydrolase EC 3.5.1.11) have been studied and analysed. The enzyme from Escherichia coli shows an ordered uni-bi reaction mechanism while the enzyme from Bacillus megaterium shows kinetics of double inhibition by the reaction products. The difference in the reaction mechanism is elucidated by two possible mechanistic models on a theoretical basis. Also suggested is the analytical method by which two different reaction mechanisms can be tested and confirmed. 相似文献
16.
《Anaerobe》2000,6(2):81-85
The aim of this study was to evaluate the prevalence of anaerobes in patients with thoracic empyema over a period of 30 months and to assess the susceptibility of the isolates to penicillin, clindamycin and metronidazole. Seventy-nine pleural fluid specimens were obtained from 75 adult patients with empyema. Anaerobic isolates were identified by Crystal anaerobes identification system and routine methods. Susceptibility testing was conducted using broth microdilution method and limited agar dilution test. Anaerobic bacteria were found in 50 (66.7%) of the patients and included 96 isolates representing 16 genera. The predominant Gram-positive anaerobes were Peptostreptococcus species (19 isolates) and Streptococcus intermedius (10), and the commonest Gram-negative species were Fusobacterium nuleatum (13),Fusobacterium necrophorum (6) and Prevotella inermedia (3). From two to four anaerobes per specimen were present in 57.4% of the specimens yielding anaerobic bacteria. The susceptibility of the Gram-negative anaerobic isolates to penicillin and that of the Gram-positive anaerobes to clindamyin and metronidazole were unpredictable. The variable resistance patterns among anaerobes and the predominance of mixed anaerobic infections highlight the role of the anaerobic dignostics in case of serious pleuropulmonary diseases. 相似文献
17.
Marta Fernández-Aguado Ricardo V. Ullán Fernando Teijeira Raquel Rodríguez-Castro Juan F. Martín 《Applied microbiology and biotechnology》2013,97(7):3073-3084
Penicillium chrysogenum, an industrial microorganism used worldwide for penicillin production, is an excellent model to study the biochemistry and the cell biology of enzymes involved in the synthesis of secondary metabolites. The well-known peroxisomal location of the last two steps of penicillin biosynthesis (phenylacetyl–CoA ligase and isopenicillin N acyltransferase) requires the import into the peroxisomes of the intermediate isopenicillin N and the precursors phenylacetic acid and coenzyme A. The mechanisms for the molecular transport of these precursors are still poorly understood. In this work, a search was made, in the genome of P. chrysogenum, in order to find a Major Facilitator Superfamily (MFS) membrane protein homologous to CefT of Acremonium chrysogenum, which is known to confer resistance to phenylacetic acid. The paaT gene was found to encode a MFS membrane protein containing 12 transmembrane spanners and one Pex19p-binding domain for Pex19-mediated targeting to peroxisomal membranes. RNA interference-mediated silencing of the paaT gene caused a clear reduction of benzylpenicillin secretion and increased the sensitivity of P. chrysogenum to the penicillin precursor phenylacetic acid. The opposite behavior was found when paaT was overexpressed from the glutamate dehydrogenase promoter that increases phenylacetic acid resistance and penicillin production. Localization studies by fluorescent laser scanning microscopy using PaaT–DsRed and EGFP–SKL fluorescent fusion proteins clearly showed that the protein was located in the peroxisomal membrane. The results suggested that PaaT is involved in penicillin production, most likely through the translocation of side-chain precursors (phenylacetic acid and phenoxyacetic acid) from the cytosol to the peroxisomal lumen across the peroxisomal membrane of P. chrysogenum. 相似文献
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Gonorrhea has increased in incidence over the past five years in Manitoba as elsewhere. Cases which did not respond to routine penicillin treatment were noted to be occuring more often. In 1948, strains of N. gonorrhoeae isolated in Canada were all sensitive to 0.06 unit of penicillin per c.c. in vitro. Commencing in May 1962, strains of N. gonorrhoeae were isolated from 100 patients of each sex attending the clinic at the St. Boniface General Hospital. Eighteen per cent required concentrations of more than 0.05 unit of penicillin per ml. to inhibit growth; 31% were not inhibited by the 2 μg. disc of dihydrostreptomycin; but only one strain was found resistant to sulfisoxazole and none to oxytetracycline. Results of penicillin treatment were markedly less successful in the patients with strains that demonstrated reduced penicillin sensitivity. Use of streptomycin and a sulfonamide proved to be a satisfactory substitute. Intramuscular oxytetracycline was less successful despite the laboratory findings. 相似文献
20.
The ring expansion enzyme (desacetoxycephalosporin C synthetase) of cephalosporin C biosynthesis in Cephalosporium acremonium has been purified 40-fold, using gel filtration on LKB Ultrogel AcA54. Purity was about twice that previously attained. The purified enzyme showed an almost absolute requirement for α-ketoglutarate, and most other acids were inactive or only weakly active. The order of addition of the cofactors and reactants was found to exert a major effect on enzyme activity. The major negative effect was caused by α-ketoglutarate or penicillin N being added to the enzyme first. The most effective order of addition to the enzyme was found to be the simultaneous addition of Fe2+, ascorbate and ATP, followed by α-ketoglutarate and then penicillin N. The negative effect of the required reactant, α-ketoglutarate, when added too early, coincides with observations made about other α-ketoglutarate-dependent dioxygenases even though, in the case of the synthetase, one atom of oxygen does not end up in the product, desacetoxycephalosporin C. 相似文献