RESUMEN
BldD is a transcription factor required for aerial hyphae formation in the filamentous bacterium Streptomyces coelicolor. Three targets of BldD regulation were discovered by a number of means, including examination of bld gene interdependence, selective enrichment of chromosomal DNA fragments bound by BldD and searching the promoter regions of known developmental genes for matches to a previously characterized BldD binding site. The three BldD targets identified were the developmental sigma factor genes, whiG and bldN, and a previously uncharacterized gene, designated bdtA, encoding a putative transcription factor. In each target gene, the sequences bound by BldD were characterized by electrophoretic mobility shift and DNase I footprinting assays, and their alignment suggested AGTgA (n)m TCACc as a consensus BldD operator. The in vivo effect of mutation in bldD on the expression of these three target genes was assessed using S1 nuclease protection assays. In each case, target gene expression was upregulated during early colony development in the bldD background, suggesting that, in the wild type, BldD acts to repress premature expression of whiG, bldN and bdtA during vegetative growth.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes Bacterianos , Streptomyces/genética , Factores de Transcripción , Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano , Datos de Secuencia Molecular , Unión Proteica , Factor sigma/genética , Factor sigma/metabolismoRESUMEN
Gel mobility shift assays with His-tagged BldD isolated from Escherichia coli have illustrated that BldD is capable of specifically recognizing its own promoter region. DNase I and hydroxyl radical footprinting assays have served to delimit the BldD binding site, revealing that BldD recognizes and binds to a site just upstream from, and overlapping with, the -10 region of the promoter. How BldD binds to its promoter and the effect this binding has on the expression of BldD are discussed.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Streptomyces/genética , Factores de Transcripción , Secuencia de Bases , Sitios de Unión , ADN Bacteriano/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Regiones Promotoras Genéticas , Streptomyces/metabolismoRESUMEN
Previous studies showed that benzothiophene and 3- and 5-methylbenzothiophenes are oxidized by some bacteria to yield their corresponding sulfones, which were not subsequently degraded. In this study, a filamentous bacterium was isolated, which grew on each of these three sulfones as its sole carbon, sulfur, and energy source. Based on 16S rRNA gene sequencing and scanning electron microscopy, the isolate was found to belong to the genus Pseudonocardia and assigned the strain designation DB1. Benzothiophene sulfone and 3-methylbenzothiophene sulfone were more readily biodegraded than 5-methylbenzothiophene sulfone, and growth on these three compounds resulted in the release of 57, 62, and 28% of the substrate carbon as CO2, respectively. The thiophene ring was also cleaved, and between 44 and 88% of the sulfur from the consumed substrate was found as sulfate and (or) sulfite. Strain DB1 grew on benzoate, dibenzothiophene sulfone, and hexadecanoic acid, but it could not grow on benzofuran, dibenzothiophene, dibenzothiophene sulfoxide, hexadecane, indole, naphthalene, phenol, 2-sulfobenzoic acid, sulfolane, benzothiophene, or 3- or 5-methylbenzothiophenes. In addition, it did not oxidize the latter three compounds to their sulfones.
Asunto(s)
Actinomycetales/metabolismo , Sulfonas/metabolismo , Actinomycetales/aislamiento & purificación , Actinomycetales/ultraestructura , Benzoatos/metabolismo , Biodegradación Ambiental , Cromatografía de Gases , Microscopía Electrónica , Datos de Secuencia Molecular , Ácido Palmítico/metabolismo , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Sulfatos/análisis , Sulfitos/análisisRESUMEN
Antidote/toxin gene pairs known as "addiction modules" can maintain plasmids in bacterial populations by means of post-segregational killing. However, several chromosome-encoded addiction modules may provide an entirely distinct function in the programmed cell death of moribund subpopulations under starvation conditions. We now report a novel chromosomal bacteriolytic module of Escherichia coli called the entericidin locus, which is activated in stationary phase under high osmolarity conditions by sigmaS and simultaneously repressed by the osmoregulatory EnvZ/OmpR signal transduction pathway. The entericidin locus encodes tandem paralogous genes (ecnAB) and directs the synthesis of two small cell-envelope lipoproteins. An attenuator precedes ecnA and an ompR-sensitive sigmaS promoter governs expression of ecnB. The entericidin A lipoprotein is an antidote to the bacteriolytic lipoprotein entericidin B. The entericidins are predicted to adopt amphipathic alpha-helical structures and to reciprocally modulate membrane stability. The entericidin locus is not present on any known plasmids, but is conserved in the homologous region of the Citrobacter freundii chromosome. Although the cloned C. freundii entericidin locus is expressed in E. coli independently of ompR, it carries an additional ompR-like gene called ecnR. The organization of the entericidin locus as a chromosomal antidote/toxin gene pair, which is regulated by both positive and negative osmotic signals during starvation, is consistent with an emerging paradigm of programmed bacterial cell death.
Asunto(s)
Apoptosis/genética , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Lipoproteínas/genética , Secuencia de Aminoácidos , Bacteriólisis/genética , Secuencia de Bases , Lipoproteínas/química , Lipoproteínas/fisiología , Datos de Secuencia Molecular , Conformación Proteica , Alineación de SecuenciaRESUMEN
Polymerase chain reaction (PCR) was used to amplify a fragment of DNA encoding a tRNA that recognizes the abundant CUC leucine codon from the chromosome of Streptomyces coelicolor. Sequence analysis of the gene, designated leuU, indicated that it codes for a tRNA 88 nucleotides in length that shares 75% identity with the Escherichia coli tRNA(Leu)CUC, while it shares only 65% identity with the only other sequenced leucyl tRNA from S. coelicolor, the bldA encoded tRNA(Leu)UUA. Accumulation of the leuU tRNA was examined by Northern blot analysis and shown to be present at constant levels throughout growth in contrast to the bldA-encoded tRNA which shows a temporal pattern of accumulation [Leskiw et al., 1993. J. Bacteriol., 175, 1995-2005].
Asunto(s)
Proteínas Bacterianas , Genes Bacterianos , Reacción en Cadena de la Polimerasa/métodos , ARN Bacteriano/genética , ARN de Transferencia de Leucina/genética , Streptomyces/genética , Secuencia de Bases , Cartilla de ADN , Código Genético , Datos de Secuencia Molecular , ARN Bacteriano/aislamiento & purificación , ARN de Transferencia de Leucina/aislamiento & purificación , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Streptomyces/crecimiento & desarrolloRESUMEN
The crisis in antibiotic resistance has resulted in an increasing fear of the emergence of untreatable organisms. Resistance to the glycopeptide antibiotic vancomycin in the enterococci, and the spread of these pathogens throughout the environment, has shown that this scenario is a matter of fact rather than fiction. The basis for vancomycin resistance is the manufacture of the depsipeptide D-Ala-D-lactate, which is incorporated into the peptidoglycan cell wall in place of the vancomycin target D-Ala-D-Ala. Pivotal to the resistance mechanism is the production of a D-Ala-D-Ala ligase capable of ester formation. Two highly efficient depsipeptide ligases have been cloned from vancomycin-resistant enterococci: VanA and VanB. These ligases show high amino acid sequence similarity to each other ( approximately 75%), but less so to other D-Ala-D-X ligases (<30%). We have cloned ddls from two glycopeptide-producing organisms, the vancomycin producer Amycolatopsis orientalis and the A47934 producer Streptomyces toyocaensis. These ligases show strong predicted amino acid homology to VanA and VanB (>60%) but not to other D-Ala-D-X ligases (<35%). The D-Ala-D-Ala ligase from S. toyocaensis shows D-Ala-D-lactate synthase activity in cell-free extracts of S. lividans transformed with the ddl gene and confirms the predicted enzymatic activity. These results imply a close evolutionary relationship between resistance mechanisms in the clinics and in drug-producing bacteria.
Asunto(s)
Actinobacteria/genética , Proteínas Bacterianas/química , Ligasas de Carbono-Oxígeno , Ligasas/química , Péptido Sintasas/química , Streptomyces/genética , Actinobacteria/enzimología , Secuencia de Aminoácidos , Antibacterianos/biosíntesis , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Evolución Molecular , Glicopéptidos/biosíntesis , Ligasas/biosíntesis , Ligasas/genética , Datos de Secuencia Molecular , Péptido Sintasas/biosíntesis , Péptido Sintasas/genética , Filogenia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Streptomyces/enzimologíaRESUMEN
Isopenicillin N synthase (IPNS) from Streptomyces clavuligerus catalyses the oxidative cyclization of the acyclic tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine into isopenicillin N. All four of the cysteine residues found in this enzyme were mutated individually into serine residues, either by the polymerase chain reaction or by single-strand site-directed mutagenesis. Functional analysis of these single mutants showed that the C104S mutant lost more than 96% of its activity, while the remaining C37S, C142S, and C251S mutants each lost 30-50% of their activity. Treatment with the thiol-group-specific reagent N-ethylmaleimide confirmed the importance of the cysteine 104 residue. Activity analysis of an IPNS triple mutant (C37S, C142S, and C251S), prepared by recombining fragments of the IPNS-encoding pcbC gene from each of the three single mutants, showed that it had lost more than 90% of its activity. Conformational analysis by circular dichroism spectroscopy indicated that the IPNS triple mutant was structurally different from the wild type, suggesting that the loss of activity may be due to conformational changes rather than active site modifications.
Asunto(s)
Oxidorreductasas/genética , Streptomyces/genética , Secuencia de Bases , Cisteína/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oxidorreductasas/metabolismo , Streptomyces/enzimología , Relación Estructura-ActividadRESUMEN
Transcription of bli, the gene encoding beta-lactamase (Bla) inhibitor protein (BLIP) of Streptomyces clavuligerus, was analyzed by promoter-probe studies, Northern hybridization and high-resolution S1 nuclease mapping. The 1-kb SalI DNA fragment immediately upstream from the bli open reading frame (ORF) showed promoter activity when tested using the xylE-based promoter-probe vector, pIJ4083. The promoter activity was approx. 36-fold higher in S. clavuligerus than in S. lividans. Northern hybridization analysis of S. clavuligerus RNA revealed that bli was expressed as a 0.7-kb monocistronic transcript. High-resolution S1 nuclease mapping identified the transcription start point as an A residue 47 bp upstream from the bli start codon. When the bli ORF, along with 111 bp of upstream sequence including the promoter, was introduced into S. lividans, the transformants produced BLIP, but in amounts approx. 12-fold lower than that produced by S. clavuligerus. Involvement of some additional regulatory element that is present in S. clavuligerus, but absent in S. lividans, could explain the difference in the promoter activities and therefore the difference in the overall expression of bli in the two hosts.
Asunto(s)
Proteínas Bacterianas/genética , Streptomyces/genética , Transcripción Genética , Inhibidores de beta-Lactamasas , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Bacteriano , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Bacteriano , Streptomyces/metabolismoRESUMEN
Isopenicillin-N synthase (IPNS) of Streptomyces clavuligerus is encoded by the pcbC gene which is found within the cephamycin biosynthetic gene cluster. pcbC is located directly downstream from lat and pcbAB, which encode the enzymes, lysine epsilon-amino transferase and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, respectively. These enzymes act prior to IPNS in the biosynthetic pathway, and the three genes are transcribed in the same direction. Previous pcbC transcriptional studies involving recombinant promoter probe plasmids, Northern analysis and 5' primer extension indicated the presence of a monocistronic 1.2-kb transcript that initiated within pcbAB, 92-bp upstream from the pcbC start codon. S1 nuclease mapping studies have now shown, not only the transcript initiating 92 bp upstream from pcbC, but also a transcript initiating further upstream, possibly including the entire pcbAB gene. Promoter probe analysis and S1 nuclease mapping failed to detect promoter activity or a transcription start point (tsp) directly upstream from pcbAB, suggesting that pcbAB transcripts initiated within or upstream from lat. Northern analysis, to search for a pcbAB transcript, showed no distinct transcript and indicated severely degraded mRNA. Similar results were obtained when Northern analysis was used to search for lat transcripts. Promoter probe analysis to locate the lat promoter indicated that a sequence promoting transcription was present in a 330-bp DNA fragment that extended from 227-bp upstream from the lat structural gene to 103 bp inside the gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Cefamicinas/biosíntesis , Genes Bacterianos , Oxidorreductasas , Péptido Sintasas/genética , Streptomyces/genética , Transaminasas/genética , Transcripción Genética , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , ADN Bacteriano , L-Lisina 6-Transaminasa , Datos de Secuencia Molecular , Familia de Multigenes , Péptido Sintasas/metabolismo , Regiones Promotoras Genéticas , Streptomyces/enzimología , Transaminasas/metabolismoRESUMEN
Deletion of the bldA gene of Streptomyces coelicolor A3(2), which encodes the only tRNA for the rare UUA codon, had no obvious effects on primary growth but interfered with aerial mycelium formation and antibiotic production. To investigate the possible regulatory role of bldA, its transcription start point was identified, and time courses were determined for the appearance of its primary transcript, the processing of the primary transcript to give a mature 5' end, and the apparent efficiency of translation of ampC mRNA, which contains multiple UUA codons. The bldA promoter was active at all times, but processing of the 5' end of the primary transcript was comparatively inefficient in young cultures. This may perhaps involve an antisense RNA, evidence of which was provided by promoter probing and in vitro transcription. The presence of low levels of the processed form of the tRNA in young cultures followed by increased abundance in older cultures contrasted with the pattern observed for accumulation of a different, presumably typical tRNA which was approximately equally abundant throughout growth. The increased accumulation of the 5' processed form of bldA tRNA coincided with more-efficient translation of ampC mRNA in older cultures, supporting the hypothesis that in at least some physiological conditions, bldA may have a regulatory influence on events late in growth, such as morphological differentiation and antibiotic production.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Morfogénesis/genética , ARN de Transferencia de Leucina/genética , Streptomyces/genética , Antibacterianos/biosíntesis , Secuencia de Bases , Codón/genética , Medios de Cultivo/metabolismo , Eliminación de Gen , Expresión Génica , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas , Precursores del ARN/genética , ARN Mensajero/genética , ARN de Transferencia de Leucina/biosíntesis , ARN de Transferencia de Lisina/biosíntesis , Factores de TiempoRESUMEN
A range of circumstantial evidence suggests that in Streptomyces spp., genes required for vegetative growth do not contain the leucine codon TTA. Instead, the codon seems to be confined to a few genes necessary during differentiation, when the colonies begin to produce aerial hyphae and antibiotics. Thus, mutations in bldA, the structural gene for tRNATTALeu, do not retard vegetative growth, but they prevent normal aerial mycelium and antibiotic production. Most of the known TTA-containing genes specify regulatory or resistance proteins associated with antibiotic-production clusters. Possibly the ability to translate the UUA codons in mRNA from such genes is confined to late stages of colony development. Factors that might have contributed to the evolution of this unusual situation are discussed.
Asunto(s)
Diferenciación Celular/genética , Codón , Morfogénesis/genética , Streptomyces/genética , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN de Transferencia de Leucina/genéticaRESUMEN
In Streptomyces coelicolor A3(2) and the related species Streptomyces lividans 66, aerial mycelium formation and antibiotic production are blocked by mutations in bldA, which specifies a tRNA(Leu)-like gene product which would recognize the UUA codon. Here we show that phenotypic expression of three disparate genes (carB, lacZ, and ampC) containing TTA codons depends strongly on bldA. Site-directed mutagenesis of carB, changing its two TTA codons to CTC (leucine) codons, resulted in bldA-independent expression; hence the bldA product is the principal tRNA for the UUA codon. Two other genes (hyg and aad) containing TTA codons show a medium-dependent reduction in phenotypic expression (hygromycin resistance and spectinomycin resistance, respectively) in bldA mutants. For hyg, evidence is presented that the UUA codon is probably being translated by a tRNA with an imperfectly matched anticodon, giving very low levels of gene product but relatively high resistance to hygromycin. It is proposed that TTA codons may be generally absent from genes expressed during vegetative growth and from the structural genes for differentiation and antibiotic production but present in some regulatory and resistance genes associated with the latter processes. The codon may therefore play a role in developmental regulation.
Asunto(s)
Antibacterianos/farmacología , Cinamatos , Codón/genética , Farmacorresistencia Microbiana/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , ARN de Transferencia/genética , Streptomyces/genética , Secuencia de Bases , Higromicina B/análogos & derivados , Higromicina B/farmacología , Leucina , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Espectinomicina/farmacología , Streptomyces/efectos de los fármacos , Streptomyces/enzimología , beta-Galactosidasa/genética , beta-Lactamasas/genéticaRESUMEN
Culture filtrates of Streptomyces clavuligerus contain a proteinaceous beta-lactamase inhibitor (BLIP) in addition to a variety of beta-lactam compounds. BLIP was first detected by its ability to inhibit Bactopenase, a penicillinase derived from Bacillus cereus, but it has also been shown to inhibit the plasmid pUC- and chromosomally mediated beta-lactamases of Escherichia coli. BLIP showed no inhibitory effect against Enterobacter cloacae beta-lactamase, and it also showed no activity against an alternative source of B. cereus penicillinase. BLIP was purified to homogeneity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a size estimate for BLIP of 16,900 to 18,000. The interaction between purified BLIP and the E. coli(pUC) beta-lactamase was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determined to be noncovalent, with an estimated 1:1 molar stoichiometry. The BLIP gene was isolated on a 13.5-kilobase fragment of S. clavuligerus chromosomal DNA which did not overlap a 40-kilobase region of DNA known to contain genes for beta-lactam antibiotic biosynthesis. The gene encoded a mature protein with a deduced amino acid sequence of 165 residues (calculated molecular weight of 17,523) and also encoded a 36-amino-acid signal sequence. No significant sequence similarity to BLIP was found by pairwise comparisons using various protein and nucleotide sequence data banks or by hybridization experiments, and no BLIP activity was detected in the culture supernatants of other Streptomyces spp.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Genes Bacterianos , Streptomyces/genética , Inhibidores de beta-Lactamasas , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular/métodos , ADN Recombinante/metabolismo , Escherichia coli/enzimología , Cinética , Datos de Secuencia Molecular , Peso Molecular , Sondas de Oligonucleótidos , Señales de Clasificación de Proteína/genética , Mapeo Restrictivo , Streptomyces/crecimiento & desarrollo , Streptomyces/metabolismoRESUMEN
We have identified an insertion sequence, IS116, present in Streptomyces clavuligerus at one copy per genome. The element was discovered as a 1.4 kb insertion into the multicopy plasmid pIJ702 after propagation in S. clavuligerus. The nucleotide sequence of IS116 and the flanking sequences from pIJ702 have been determined. The junctions with pIJ702 show no target site duplication and there are no inverted repeats at the ends of the element. One putative coding open reading frame of 1197 bp was identified which would code for a protein product of 399 amino acids. This protein resembles deduced integrase/transposase proteins specified by three other transposable elements of actinomycetes: IS110 and the mini-circle from Streptomyces coelicolor A3(2), and--most particularly--IS900 of Mycobacterium paratuberculosis. Two regions that are relatively conserved among these gene products show features found in similar positions in many reverse transcriptases. IS116 and IS900 are also closely similar in their general organization and (apparently) in their insertion site specificity, whereas IS110 and the mini-circle are quite different in these features.
Asunto(s)
Actinomycetales/genética , Elementos Transponibles de ADN , Streptomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Genes Bacterianos , Datos de Secuencia Molecular , Mycobacterium/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Sistemas de Lectura Abierta , Plásmidos , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Homología de Secuencia de Ácido Nucleico , TransposasasRESUMEN
Streptomyces clavuligerus isopenicillin N synthase (IPNS) gene expression was achieved in Escherichia coli by the construction of two-cistron expression systems formed in the high copy number plasmid vector pUC119. These two-cistron constructions were composed of the IPNS gene and its flanking sequences which encoded an upstream open reading frame (ORF), the IPNS ribosome binding site and a putative transcription terminator. No E. coli- like Streptomyces promoter motif was present upstream of the IPNS gene therefore transcriptional regulation of the two-cistron system was provided by the lac promoter of pUC119. Enzymatically active IPNS was detected in E. coli cells harboring the recombinant plasmids thereby providing evidence for the activity of the IPNS ORF and for the feasibility of production of S. clavuligerus IPNS in E. coli. These results indicate that simple two-cistron constructions involving foreign gene flanking sequences may be used to express foreign proteins in E. coli.
Asunto(s)
Expresión Génica , Genes Bacterianos , Oxidorreductasas/biosíntesis , Streptomyces/enzimología , Secuencia de Bases , Clonación Molecular/métodos , ADN Bacteriano/genética , Escherichia coli/genética , Genes , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Oxidorreductasas/genética , Oxidorreductasas/aislamiento & purificación , Mapeo RestrictivoRESUMEN
The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.
Asunto(s)
Proteínas Bacterianas/genética , Enzimas/genética , Genes Bacterianos , Oxidorreductasas , Streptomyces/genética , Acremonium/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Fúngicas/genética , Genes , Datos de Secuencia Molecular , Penicillium chrysogenum/genética , Proteínas Recombinantes/genética , Homología de Secuencia de Ácido Nucleico , Streptomyces/enzimologíaRESUMEN
Isopenicillin N synthetase was purified from Streptomyces clavuligerus by sequential salt precipitation, ion-exchange and gel-filtration chromatography using both conventional open column and high-performance liquid chromatographic techniques. Material from the final purification step had a specific activity of 204.1 X 10(-3) units/mg of protein which represented a 130-fold purification over the cell-free extract. The purified isopenicillin N synthetase was determined to have a molecular weight of 33,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have a Km of 0.32 mM with respect to its substrate delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine. The enzyme showed a sensitivity to thiol-specific inhibitors with N-ethylmaleimide giving the strongest inhibitory effect.