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1.
Cell Chem Biol ; 30(3): 235-247.e12, 2023 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-36863346

RESUMO

Malignant tumors can evade destruction by the immune system by attracting immune-suppressive regulatory T cells (Treg) cells. The IKZF2 (Helios) transcription factor plays a crucial role in maintaining function and stability of Treg cells, and IKZF2 deficiency reduces tumor growth in mice. Here we report the discovery of NVP-DKY709, a selective molecular glue degrader of IKZF2 that spares IKZF1/3. We describe the recruitment-guided medicinal chemistry campaign leading to NVP-DKY709 that redirected the degradation selectivity of cereblon (CRBN) binders from IKZF1 toward IKZF2. Selectivity of NVP-DKY709 for IKZF2 was rationalized by analyzing the DDB1:CRBN:NVP-DKY709:IKZF2(ZF2 or ZF2-3) ternary complex X-ray structures. Exposure to NVP-DKY709 reduced the suppressive activity of human Treg cells and rescued cytokine production in exhausted T-effector cells. In vivo, treatment with NVP-DKY709 delayed tumor growth in mice with a humanized immune system and enhanced immunization responses in cynomolgus monkeys. NVP-DKY709 is being investigated in the clinic as an immune-enhancing agent for cancer immunotherapy.


Assuntos
Neoplasias , Fatores de Transcrição , Animais , Humanos , Camundongos , Fator de Transcrição Ikaros , Imunoterapia , Neoplasias/terapia , Neoplasias/metabolismo , Linfócitos T Reguladores/metabolismo , Fatores de Transcrição/metabolismo
2.
Early Interv Psychiatry ; 17(6): 597-607, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36196478

RESUMO

BACKGROUND: Cardiovascular and metabolic diseases are the leading contributors to the early mortality associated with psychotic disorders. To date, it has not been possible to disentangle the effect of medication and non-medication factors on the physical health of people with a first episode of psychosis (FEP). This study aimed to isolate the effects of antipsychotic medication on anthropometric measurements, fasting glucose and lipids. METHODS: This study utilized data from a triple-blind randomized placebo-controlled trial comparing two groups of antipsychotic-naïve young people with a FEP who were randomized to receive a second-generation antipsychotic medication (FEP-medication group) or placebo (FEP-placebo group) for 6 months. Twenty-seven control participants were also recruited. RESULTS: Eighty-one participants commenced the trial; 69.1% completed at least 3 months of the intervention and 33.3% completed the full 6 months. The FEP-placebo group gained a mean of 2.4 kg (±4.9) compared to 1.1 kg (±4.9) in the control participants (t = 0.76, p = .45). After controlling for multiple analyses, there was no difference in blood pressure, waist circumference or heart rate between the FEP-placebo group and controls. After 6 months, the FEP medication group had gained 4.1 kg (±4.5), higher than those receiving placebo but not statistically significant (t = 0.8, p = .44). There were no differences in fasting glucose or lipids between the FEP groups after 3 months. CONCLUSIONS: While limited by small numbers and high attrition, these findings indicate that some of the metabolic complications observed in psychotic disorders could be attributable to factors other than medication. This emphasizes the need to deliver physical health interventions early in the course of FEP.


Assuntos
Antipsicóticos , Transtornos Psicóticos , Humanos , Adolescente , Antipsicóticos/efeitos adversos , Transtornos Psicóticos/complicações , Lipídeos/uso terapêutico , Glucose
3.
Artigo em Inglês | MEDLINE | ID: mdl-28947474

RESUMO

A major challenge in treating patients is the selection of the "right" antibiotic regimen. Given that the optimal ß-lactam/ß-lactamase inhibitor pair is dependent upon the spectrum of ß-lactamase enzymes produced and the frequency of resistance to the ß-lactamase inhibitor, it might be useful if a stand-alone were available for the clinician to pair with the "right" ß-lactam rather than only in a fixed combination. We describe herein a one-compartment in vitro infection model studies conducted to identify the magnitudes of the pharmacokinetic-pharmacodynamic (PK-PD) index for a ß-lactamase inhibitor, CB-618, that would restore the activity of four ß-lactam partner agents (cefepime, ceftazidime, ceftolozane, and meropenem) with various doses (1 or 2 g) and dosing intervals (8 or 12 h). The challenge panel included Klebsiella pneumoniae (n = 5), Escherichia coli (n = 2), and Enterobacter cloacae (n = 1) strains, which produced a wide variety of ß-lactamase enzymes (AmpC, CTXM-15, KPC-2, KPC-3, FOX-5, OXA-1/30, OXA-48, SHV-1, SHV-11, SHV-27, and TEM-1). Free-drug human concentration-time profiles were simulated for each agent, and specimens were collected for drug concentration and bacterial density determinations. CB-618 restored the activity of each ß-lactam partner. The magnitudes of the CB-618 ratio of the area under the concentration-time curve from 0 to 24 h to the MIC (i.e., the AUC/MIC ratio) associated with net bacterial stasis and 1- and 2-log10 CFU/ml reductions from baseline at 24 h were 11.2, 32.9, and 136.3, respectively. These data may provide a PK-PD basis for the development of a stand-alone ß-lactamase inhibitor.


Assuntos
Antibacterianos/farmacologia , Ceftazidima/farmacologia , Cefalosporinas/farmacologia , Tienamicinas/farmacologia , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/farmacocinética , Cefepima , Simulação por Computador , Enterobacter cloacae/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Meropeném , Testes de Sensibilidade Microbiana , beta-Lactamases/metabolismo
4.
Antimicrob Agents Chemother ; 60(7): 3891-6, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27001820

RESUMO

The usefulness of ß-lactam antimicrobial agents is threatened as never before by ß-lactamase-producing bacteria. For this reason, there has been renewed interest in the development of broad-spectrum ß-lactamase inhibitors. Herein we describe the results of dose fractionation and dose-ranging studies carried out using a one-compartment in vitro infection model to determine the exposure measure for CB-618, a novel ß-lactamase inhibitor, most predictive of the efficacy when given in combination with meropenem. The challenge panel included Enterobacteriaceae clinical isolates, which collectively produced a wide range of ß-lactamase enzymes (KPC-2, KPC-3, FOX-5, OXA-48, SHV-11, SHV-27, and TEM-1). Human concentration-time profiles were simulated for each drug, and samples were collected for drug concentration and bacterial density determinations. Using data from dose fractionation studies and a challenge Klebsiella pneumoniae isolate (CB-618-potentiated meropenem MIC = 1 mg/liter), relationships between change from baseline in log10 CFU/ml at 24 h and each of CB-618 area under the concentration-time curve over 24 h (AUC0-24), maximum concentration (Cmax), and percentage of the dosing interval that CB-618 concentrations remained above a given threshold were evaluated in combination with meropenem at 2 g every 8 h (q8h). The exposure measures most closely associated with CB-618 efficacy in combination with meropenem were the CB-618 AUC0-24 (r(2) = 0.835) and Cmax (r(2) = 0.826). Using the CB-618 AUC0-24 indexed to the CB-618-potentiated meropenem MIC value, the relationship between change from baseline in log10 CFU/ml at 24 h and CB-618 AUC0-24/MIC ratio in combination with meropenem was evaluated using the pooled data from five challenge isolates; the CB-618 AUC0-24/MIC ratio associated with net bacterial stasis and the 1- and 2-log10 CFU/ml reductions from baseline at 24 h were 27.3, 86.1, and 444.8, respectively. These data provide a pharmacokinetics-pharmacodynamics (PK-PD) basis for evaluating potential CB-618 dosing regimens in combination with meropenem in future studies.


Assuntos
Antibacterianos/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Tienamicinas/farmacologia , Inibidores de beta-Lactamases/farmacologia , Antibacterianos/farmacocinética , Klebsiella pneumoniae/metabolismo , Meropeném , Testes de Sensibilidade Microbiana , Tienamicinas/farmacocinética , Inibidores de beta-Lactamases/farmacocinética
5.
Antimicrob Agents Chemother ; 59(4): 2102-12, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25624330

RESUMO

Viridans group streptococci (VGS) are part of the normal flora that may cause bacteremia, often leading to endocarditis. We evaluated daptomycin against four clinical strains of VGS (MICs = 1 or 2 µg/ml) using an in vitro-simulated endocardial vegetation model, a simulated bacteremia model, and kill curves. Daptomycin exposure was simulated at 6 mg/kg of body weight and 8 mg/kg every 24 h for endocardial and bacteremia models. Total drug concentrations were used for analyses containing protein (albumin and pooled human serum), and free (unbound) drug concentrations (93% protein bound) were used for analyses not containing protein. Daptomycin MICs in the presence of protein were significantly higher than those in the absence of protein. Despite MICs below or at the susceptible breakpoint, all daptomycin regimens demonstrated limited kill in both pharmacodynamic models. A reduction of approximately 1 to 2 log10 CFU was seen for all isolates and dosages except daptomycin at 6 mg/kg, which achieved a reduction of 2.7 log10 CFU/g against one strain (Streptococcus gordonii 1649) in the endocardial model. Activity was similar in both pharmacodynamic models in the presence or absence of protein. Similar activity was noted in the kill curves over all multiples of the MIC. Regrowth by 24 h was seen even at 8× MIC. Postexposure daptomycin MICs for both pharmacodynamic models increased to >256 µg/ml for all isolates by 24 and 72 h. Despite susceptibility to daptomycin by standard MIC methods, these VGS developed high-level daptomycin resistance (HLDR) after a short duration following drug exposure not attributed to modification or inactivation of daptomycin. Further evaluation is warranted to determine the mechanism of resistance and clinical implications.


Assuntos
Antibacterianos/farmacologia , Daptomicina/farmacologia , Estreptococos Viridans/efeitos dos fármacos , Bacteriemia/microbiologia , Cálcio/metabolismo , Contagem de Colônia Microbiana , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Endocardite Bacteriana/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Infecções Estreptocócicas/microbiologia , Streptococcus gordonii/efeitos dos fármacos
6.
J Antibiot (Tokyo) ; 64(1): 111-6, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21102593

RESUMO

A potent new lipopeptide antibiotic, A54145E(Asn(3)Asp(9)), was isolated from the fermentation broth of Streptomyces fradiae DA1489 engineered to delete genes encoding enzymes involved in hydroxylation of Asn(3) and methoxylation of Asp(9). The chemical structure predicted from the genetic changes in the biosynthetic pathway was determined by analyses of chemical transformations, D, L-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS and 2D NMR techniques. These studies confirmed the primary amino acid sequence of A54145E(Asn(3)Asp(9)) predicted from the genetic engineering strategy, and also confirmed the structure and locations of three D-amino acids predicted from bioinformatic studies.


Assuntos
Antibacterianos/biossíntese , Streptomyces/metabolismo , Antibacterianos/química , Asparagina/análogos & derivados , Asparagina/biossíntese , Asparagina/química , Asparagina/genética , Lipoproteínas/biossíntese , Lipoproteínas/química , Lipoproteínas/genética , Espectroscopia de Ressonância Magnética , Rotação Ocular , Sarcosina/análogos & derivados , Sarcosina/biossíntese , Sarcosina/química , Sarcosina/genética , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , Streptomyces/genética
7.
J Antibiot (Tokyo) ; 64(1): 79-87, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21102596

RESUMO

A54145 is a complex of lipopeptide antibiotics produced by Streptomyces fradiae. A54145 factors are structurally related to daptomycin, with four modified amino acids, only one of which is present in daptomycin. We generated three mutants defective in lptJ, lptK or lptL, whose gene products are involved in the formation of hydroxy-Asn(3) (hAsn(3)) and methoxy-Asp(9) (moAsp(9)). Each of the mutants produced novel lipopeptides related to A54145 and the profiles allowed assignment of functions for those genes. We constructed strains carrying different combinations of these genes coupled with a mutation in the lptI gene involved in the biosynthesis of 3-methyl-Glu(12) (3mGlu(12)), and all recombinants produced novel lipopeptides. One of the compounds displayed very good antibacterial activity in the presence of bovine surfactant, which interacts with daptomycin or A54145E to inhibit their antibacterial activities.


Assuntos
Antibacterianos/metabolismo , Lipoproteínas/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , DNA/química , DNA/genética , Fermentação , Teste de Complementação Genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Testes de Sensibilidade Microbiana , Mutagênese Insercional , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
8.
Appl Environ Microbiol ; 76(20): 6877-87, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20802082

RESUMO

A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the φC31 or φBT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu12), at the expense of factors containing 3-methyl-glutamic acid (3mGlu12). This provided a practical route to produce high levels of pure Glu12-containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.


Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Família Multigênica , Streptomyces/genética , Streptomyces/metabolismo , Produtos Biológicos/biossíntese , Produtos Biológicos/genética , Cromossomos Artificiais Bacterianos , Conjugação Genética , Escherichia coli/genética , Teste de Complementação Genética , Engenharia Genética/métodos , Vetores Genéticos , Genética Microbiana/métodos , Lipoproteínas/biossíntese , Lipoproteínas/genética , Plasmídeos , Streptococcus pneumoniae/efeitos dos fármacos
9.
Antimicrob Agents Chemother ; 54(4): 1404-13, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20086142

RESUMO

Daptomycin is a cyclic lipopeptide antibiotic approved for the treatment of skin and skin structure infections caused by Gram-positive pathogens and for that of bacteremia and right-sided endocarditis caused by Staphylococcus aureus. Daptomycin failed to meet noninferiority criteria for the treatment of community-acquired pneumonia, likely due to sequestration in pulmonary surfactant. Many analogues of daptomycin have been generated by combinatorial biosynthesis, but only two displayed improved activity in the presence of bovine surfactant, and neither was as active as daptomycin in vitro. In the present study, we generated hybrid molecules of the structurally related lipopeptide A54145 in Streptomyces fradiae and tested them for antibacterial activity in the presence of bovine surfactant. Hybrid A54145 nonribosomal peptide synthetase (NRPS) biosynthetic genes were constructed by genetic engineering and were expressed in combination with a deletion of the lptI methyltransferase gene, which is involved in the formation of the 3-methyl-glutamic acid (3mGlu) residue at position 12. Some of the compounds were very active against S. aureus and other Gram-positive pathogens; one compound was also highly active in the presence of bovine surfactant, had low acute toxicity, and showed some efficacy against Streptococcus pneumoniae in a mouse model of pulmonary infection.


Assuntos
Antibacterianos/farmacologia , Daptomicina/análogos & derivados , Animais , Antibacterianos/biossíntese , Antibacterianos/química , Bovinos , Daptomicina/biossíntese , Daptomicina/química , Daptomicina/farmacologia , Feminino , Genes Bacterianos , Bactérias Gram-Positivas/efeitos dos fármacos , Lipoproteínas/biossíntese , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pneumonia Pneumocócica/tratamento farmacológico , Engenharia de Proteínas , Surfactantes Pulmonares/administração & dosagem , Staphylococcus aureus/efeitos dos fármacos , Streptomyces/genética
10.
J Bacteriol ; 189(16): 5867-74, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17573474

RESUMO

Biosynthesis of cephamycin C in Streptomyces clavuligerus involves the initial conversion of lysine to alpha-aminoadipic acid. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase carry out this two-step reaction, and genes encoding each of these enzymes are found within the cephamycin C gene cluster. However, while mutation of the lat gene causes complete loss of cephamycin production, pcd mutants still produce cephamycin at 30% to 70% of wild-type levels. Cephamycin production by pcd mutants could be restored to wild-type levels either by supplementation of the growth medium with alpha-aminoadipic acid or by complementation of the mutation with an intact copy of the pcd gene. Neither heterologous PCR nor Southern analyses showed any evidence for the presence of a second pcd gene. Furthermore, cell extracts from pcd mutants lack detectable PCD activity. Cephamycin production in the absence of detectable PCD activity suggests that S. clavuligerus must have some alternate means of producing the aminoadipyl-cysteinyl-valine needed for cephamycin biosynthesis.


Assuntos
Proteínas de Bactérias/metabolismo , Cefamicinas/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento
11.
Proc Natl Acad Sci U S A ; 103(46): 17462-7, 2006 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-17090667

RESUMO

Daptomycin, a cyclic lipopeptide produced by Streptomyces roseosporus, is the active ingredient of Cubicin (daptomycin-for-injection), a first-in-class antibiotic approved for treatment of skin and skin-structure infections caused by Gram-positive pathogens and bacteremia and endocarditis caused by Staphylococcus aureus, including methicillin-resistant strains. Genetic engineering of the nonribosomal peptide synthetase (NRPS) in the daptomycin biosynthetic pathway was exploited for the biosynthesis of novel active antibiotics. lambda-Red-mediated recombination was used to exchange single or multiple modules in the DptBC subunit of the NRPS to modify the daptomycin cyclic peptide core. We combined module exchanges, NRPS subunit exchanges, inactivation of the tailoring enzyme glutamic acid 3-methyltransferase, and natural variations of the lipid tail to generate a library of novel lipopeptides, some of which were as active as daptomycin against Gram-positive bacteria. One compound was more potent against an Escherichia coli imp mutant that has increased outer membrane permeability. This study established a robust combinatorial biosynthesis platform to produce novel peptide antibiotics in sufficient quantities for antimicrobial screening and drug development.


Assuntos
Antibacterianos/biossíntese , Daptomicina/análogos & derivados , Daptomicina/biossíntese , Sequência de Aminoácidos , Antibacterianos/química , Daptomicina/química , Deleção de Genes , Dados de Sequência Molecular , Estrutura Molecular , Mutação/genética , Alinhamento de Sequência , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Streptomyces/química , Streptomyces/metabolismo
12.
Microbiology (Reading) ; 150(Pt 12): 4137-45, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15583166

RESUMO

CcaR is a positive-acting transcriptional regulator involved in cephamycin C and clavulanic acid biosynthesis in Streptomyces clavuligerus. Previous sequence analyses of the ccaR gene revealed two possible start codons, an ATG, and a GTG located in-frame 18 bp downstream of the ATG. To determine the true start codon, ccaR was expressed, either from the GTG or ATG codon, in Escherichia coli. A protein product was only obtained from the ATG construct. Similarly, ccaR constructs originating from ATG or GTG and designed for expression from a glycerol-regulated promoter in Streptomyces species were prepared and used to complement a S. clavuligerus ccaR mutant. Bioassays showed that only the ATG construct could complement the ccaR mutant to restore cephamycin C production, and Western analysis confirmed the presence of CcaR in the mutant complemented with the ATG construct only. To ensure that expression of ccaR from its native promoter also initiated at the ATG rather than GTG, a conservative point mutation was introduced into ccaR, converting the GTG to GTC. The GTC construct still fully complemented a ccaR mutant, confirming that ATG is the true start codon. Inspection of the region upstream of ccaR by S1 nuclease protection and primer extension analyses indicated the presence of two transcript start points that mapped to residues located 74 and 173 bp upstream of the ATG codon.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cefamicinas/biossíntese , Ácido Clavulânico/biossíntese , Regulação Bacteriana da Expressão Gênica , Streptomyces/metabolismo , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Mutação , Biossíntese de Proteínas , Streptomyces/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica
13.
J Antibiot (Tokyo) ; 57(7): 436-45, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15376556

RESUMO

Gentamicin is a 4,6-disubstituted aminocyclitol antibiotic complex synthesised by some members of the actinomycete genus Micromonospora. In a search for the gentamicin biosynthetic gene cluster we identified, using a cosmid library approach, a region of the M. echinospora ATCC15835 chromosome that encodes homologues of aminoglycoside biosynthesis genes including gntB-a close homologue of the 2-deoxy-scyllo-inosose synthase gene (btrC) from butirosin-producing Bacillus circulans. Insertional inactivation was achieved by homologous recombination with an internal gntB fragment-containing suicide plasmid, delivered by conjugal transfer from Escherichia coli. gntB disruptants were gentamicin nonproducing mutants as assayed by an ELISA antibiotic detection system, proving the association of gntB (or a downstream region) with gentamicin biosynthesis. The function of some open reading frames within the cluster, predicted by nucleotide database homology searching, is discussed with regards to their potential roles in gentamicin biosynthesis. The discovery of this genetic region represents the first report of a gene cluster involved in the biosynthesis of a 4,6-disubstituted aminocyclitol antibiotic.


Assuntos
Gentamicinas/biossíntese , Micromonospora/genética , Família Multigênica , Fases de Leitura Aberta
14.
Microbiology (Reading) ; 149(Pt 9): 2443-2453, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12949170

RESUMO

Micromonospora carbonacea var. africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome. Sequence analysis of a 4.4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP). Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp. by conjugation from Escherichia coli. Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNAHis gene in the chromosome. The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3' end of the tRNA. Integration of pMLP1-based plasmids in M. carbonacea var. africana caused a loss of the pMLP1 phage. Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site. Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.


Assuntos
Bacteriófagos/genética , Integrases/genética , Micromonospora/genética , Micromonospora/virologia , RNA de Transferência de Histidina/química , Sítios de Ligação Microbiológicos/genética , Sequência de Bases , Cromossomos Bacterianos , DNA Bacteriano/genética , DNA Viral/genética , Escherichia coli , Genes Bacterianos , Vetores Genéticos , Biblioteca Genômica , Micromonospora/classificação , Dados de Sequência Molecular , Plasmídeos , RNA de Transferência de Histidina/genética , Recombinação Genética , Integração Viral/genética
15.
Microbiology (Reading) ; 148(Pt 3): 643-656, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11882698

RESUMO

In Streptomyces coelicolor bldA encodes the principal leucyl tRNA for translation of UUA codons and controls pigmented antibiotic production by the presence of TTA codons in the genes encoding the pathway-specific activators of actinorhodin and undecylprodigiosin biosynthesis. In Streptomyces clavuligerus the gene encoding the pathway-specific activator of both cephamycin C and clavulanic acid production, ccaR, also contains a TTA codon and was expected to exhibit bldA-dependence. A cloned S. clavuligerus DNA fragment containing a sequence showing 91% identity to the S. coelicolor bldA-encoded tRNA was able to restore antibiotic production and sporulation to bldA mutants of S. coelicolor and the closely related Streptomyces lividans. A null mutation of the bldA gene in S. clavuligerus resulted in the expected sporulation defective phenotype, but unexpectedly had no effect on antibiotic production. Transcript analysis showed no difference in the levels of ccaR transcripts in the wild-type and bldA mutant strains, ruling out any effect of elevated levels of the ccaR mRNA. Furthermore, when compared to the wild-type strain, the bldA mutant showed no differences in the levels of CcaR, suggesting that the single TTA codon in ccaR is mistranslated efficiently. The role of codon context in bldA dependence is discussed.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mutação , Biossíntese de Proteínas , RNA de Transferência de Leucina/genética , Streptomyces/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Cefamicinas/biossíntese , Ácido Clavulânico/biossíntese , Códon , Dados de Sequência Molecular , RNA Bacteriano , RNA de Transferência de Leucina/metabolismo , Análise de Sequência de DNA , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismo
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