Fatty-acid biosynthesis in a branched-chain alpha-keto acid dehydrogenase mutant of Streptomyces avermitilis.

Fatty-acid biosynthesis by a branched-chain alpha-keto acid dehydrogenase (bkd) mutant of Streptomyces avermitilis was analyzed. This mutant is unable to produce the appropriate precursors of branched-chain fatty acid (BCFA) biosynthesis, but unlike the comparable Bacillus subtilis mutant, was shown not to have an obligate growth requirement for these precursors. The bkd mutant produced only straight-chain fatty acids (SCFAs) with membrane fluidity provided entirely by unsaturated fatty acids (UFAs), the levels of which increased dramatically compared to the wild-type strain. The levels of UFAs increased in both the wild-type and bkd mutant strains as the growth temperature was lowered from 37 degrees C to 24 degrees C, suggesting that a regulatory mechanism exists to alter the proportion of UFAs in response either to a loss of BCFA biosynthesis, or a decreased growth temperature. No evidence of a regulatory mechanism for BCFAs was observed, as the types of these fatty acids, which contribute significantly to membrane fluidity, did not alter when the wild-type S. avermitilis was grown at different temperatures. The principal UFA produced by S. avermitilis was shown to be delta 9-hexadecenoate, the same fatty acid produced by Escherichia coli. This observation, and the inability of S. avermitilis to convert exogenous labeled palmitate to the corresponding UFA, was shown to be consistent with an anaerobic pathway for UFA biosynthesis. Incorporation studies with the S. avermitilis bkd mutant demonstrated that the fatty acid synthase has a remarkably broad substrate specificity and is able to process a wide range of exogenous branched chain carboxylic acids into unusual BCFAs.

Cloning, expression, and characterization of a type II 3-dehydroquinate dehydratase gene from Streptomyces hygroscopicus.

A gene encoding dehydroquinate dehydratase (DHQase) was cloned from Streptomyces hygroscopicus var. ascomyceticus. The 528-bp open reading frame specified a primary translation product of 175 amino acids with a calculated Mr of 18,789. The predicted amino acid sequence of the DHQase showed similarities to bacterial and fungal type II DHQases. Overexpression of the dhq gene was accomplished in Escherichia coli using a gene fusion technique in which a malE, the gene encoding the maltose binding protein (MBP), was fused via a short oligonucleotide region to the beginning of dhq. The recombinant MBP-DHQase fusion protein was purified by affinity chromatography and cleaved using thrombin. The resulting DHQase, separated from the MBP, demonstrated typical properties of a type II DHQase: a relatively high Km for the dehydroquinate substrate (650 microM) and extreme thermal stability. The subunit Mr estimated by SDS-PAGE was 19,000, and the native Mr estimated by gel-exclusion chromatography and sucrose-density centrifugation was 130,000, suggesting that the enzyme is a homoheptamer (type II DHQases are typically homododecamers). The MBP-DHQase complex also adopted a heptameric structure and was a thermostable, fully active DHQase, indicating that the N-terminus is not involved in formation of protomer-protomer complexes. Previous analyses have supported positioning the N-terminus of type II DHQases close to the active site and a conformational change in this region coincident with ligand binding. Nonetheless, the Km and relative kcat obtained for MBP-DHQase were indistinguishable from those observed for DHQase. Inactivation data of the DHQase from S. hygroscopicus with the arginine-specific reagent phenylglyoxal showed that a modified Arg residue(s) is likely close to the N-terminus and active site of DHQase, but does not play an essential role in catalysis.

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.

A second branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH) from Streptomyces avermitilis: its relationship to avermectin biosynthesis and the construction of a bkdF mutant suitable for the production of novel antiparasitic avermectins.

A second cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microorganism which produces anthelmintic avermectins. Open reading frame 1 (ORF1) (bkdF, encoding E1 alpha), would encode a polypeptide of 44,394 Da (406 amino acids). The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1 beta) is located 83 bp downstream from the end of ORF1. The deduced amino acid sequence of bkdF resembled the corresponding E1 alpha subunit of several prokaryotic and eukaryotic BCDH complexes. An S. avermitilis bkd mutant constructed by deletion of a genomic region comprising the 5' end of bkdF is also described. The mutant exhibited a typical Bkd- phenotype: it lacked E1 BCDH activity and had lost the ability to grow on solid minimal medium containing isoleucine, leucine, and valine as sole carbon sources. Since BCDH provides an alpha-branched-chain fatty acid starter unit, either S(+)-alpha-methylbutyryl coenzyme A or isobutyryl coenzyme A, which is essential to initiate the synthesis of the avermectin polyketide backbone in S. avermitilis, the disrupted mutant cannot make the natural avermectins in a medium lacking both S(+)-alpha-methylbutyrate and isobutyrate. Supplementation with either one of these compounds restores production of the corresponding natural avermectins, while supplementation of the medium with alternative fatty acids results in the formation of novel avermectins. These results verify that the BCDH-catalyzed reaction of branched-chain amino acid catabolism constitutes a crucial step to provide fatty acid precursors for antibiotic biosynthesis in S. avermitilis.

Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli.

A cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH) of Streptomyces avermitilis has been cloned and sequenced. Open reading frame 1 (ORF1) (E1 alpha), 1,146 nucleotides long, would encode a polypeptide of 40,969 Da (381 amino acids). ORF2 (E1 beta), 1,005 nucleotides long, would encode a polypeptide of 35,577 Da (334 amino acids). The intergenic distance between ORF1 and ORF2 is 73 bp. The putative ATG start codon of the incomplete ORF3 (E2) overlaps the stop codon of ORF2. Computer-aided searches showed that the deduced products of ORF1 and ORF2 resembled the corresponding E1 subunit (alpha or beta) of several prokaryotic and eukaryotic BCDH complexes. When these ORFs were overexpressed in Escherichia coli, proteins of about 41 and 34 kDa, which are the approximate masses of the predicted S. avermitilis ORF1 and ORF2 products, respectively, were detected. In addition, specific E1 [alpha beta] BCDH activity was detected in E. coli cells carrying the S. avermitilis ORF1 (E1 alpha) and ORF2 (E1 beta) coexpressed under the control of the T7 promoter.

A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli.

A 1.5-kb genomic fragment isolated from Streptomyces avermitilis that directs the synthesis of a brown pigment in Escherichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototype of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients. Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitilis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.

A simple DNA polymerase chain reaction method to locate and define orientation of specific sequences in cloned bacterial genomic fragments.

A simple DNA polymerase chain reaction (PCR) method, to rapidly locate and define the orientation of a particular sequence within a cloned bacterial genomic fragment several kilobases (kb) long, is described. The technique is particularly useful when cloning (by DNA PCR amplification) a specific sequence of a conserved gene from several microorganisms following an homology probing approach. The method requires two universal primers derived from the vector, two specific primers derived from each end of the specific sequence in inverted tail-to-tail directions, and a single round of PCR. In addition, PCR conditions applicable to DNA inserts having a G + C content up to 75% (e.g. Pseudomonas and actinomycete genomic fragments), and allowing efficient amplification of DNA fragments up to 7 kb long, are described and discussed.

Cloning of a DNA fragment involved in pigment production in Streptomyces avermitilis.

Streptomyces avermitilis has the ability to synthesize a diffusible, brown, melanin-like pigment, a common property among many Streptomyces species. A region of the S. avermitilis chromosome involved in the production of this pigment was cloned in Escherichia coli. Production of the brown pigment was attained in E. coli, and is optimal when medium is supplemented with copper ions, tyrosine and IPTG. The cloned S. avermitilis pigment-producing DNA fragment is under the control of the lac promoter carried in the E. coli vector. The gene involved in pigment production could be used as a tool to analyse gene expression in S. avermitilis, and as an alternative cloning marker in Streptomyces-Escherichia coli vectors.

Bacterial adhesion to cerebrospinal fluid shunts.

Bacterial adherence to cerebrospinal fluid (CSF) shunts was analyzed in vivo and in vitro. Scanning electron micrographs (SEM's) of catheters removed from pediatric patients with shunts infected by Staphylococcus aureus or Klebsiella pneumoniae revealed numerous bacterial cells and microcolonies, leukocytes, and erythrocytes attached to the CSF catheters' inner walls, as well as the existence of surface irregularities, such as fissures, rugosities, and holes. Permeability analyses and SEM's demonstrated that catheters develop physical alterations over the period of implantation. Different bacterial strains presented a different in vitro adherence to CSF shunts, suggesting that this attachment may be affected by specific properties of the outer structures of each strain. The attachment of microbial pathogens to CSF shunts seems to contribute to the persistence of bacterial cells within a catheter and the onset of recurrent shunt infection. This study demonstrated that some bacteria can remain attached within shunts in vitro despite a CSF flow at rates up to 200 times higher than those normally demonstrated in vivo. Furthermore, surface irregularities found throughout this study may help to anchor and hide bacterial microcolonies. Based on these findings, it seems advisable to remove an infected shunt and to replace it with a new one after proper antimicrobial therapy, in order to prevent recurrent infections.

Site and substrate specificity of the ermC 23S rRNA methyltransferase.

The purified ermC methyltransferase described here incorporates two methyl groups per Bacillus subtilis 23S rRNA molecule in vitro. The Km for S-adenosyl-L-methionine was 12 microM, and for B. subtilis 23S rRNA the Km was 375 nM. In vivo methylation specified by several related resistance determinants prevented in vitro methylation by the ermC enzyme, suggesting that methylation specified by all of these determinants occurs at homologous sites. Since methyl groups were incorporated in protein-free 23S rRNA molecules, the structure of rRNA alone must contain sufficient information to specify the methylation site.

Purificación parcial de interferón humano tipo alfa para su uttilización en productos farmacéuticos / Partial purification of Hu-IFN-* for pharmaceutical purposes

La purificación parcial del Hu-IFN-* se logra en un solo paso por adsorción y elución sobre ácido silícico. La recuperación de la actividad antiviral es del 80-100 por ciento, con un aumento de la actividad específica de aproximadamente 20-80 veces. El producto obtenido por este método es estable y apropiado para ser utilizado en terapéuticas locales

Translational autoregulation of ermC 23S rRNA methyltransferase expression in Bacillus subtilis.

ermC specifies an rRNA methyltransferase that confers resistance to erythromycin. The expression of this determinant is induced by the addition of erythromycin. The induction mechanism has been shown to operate posttranscriptionally, and its mechanism has been elucidated. We now show that synthesis of the ermC gene product in Bacillus subtilis is also autoregulated by a mechanism operating on the level of translation. The synthesis of methyltransferase was shown to be gene dosage compensated by Western blot analysis. Several mutants were analyzed that specify altered ermC gene products and are deregulated. Analysis of mutants and of the wild-type strain by Northern blotting demonstrated that autoregulation is posttranscriptional. We suggest a translational repression model in which the ermC methyltransferase binds to its own mRNA, at a region that resembles the methylation target site on 23S rRNA. The overall control of ermC expression is discussed in light of these multiple regulatory mechanisms.

Adherence of multiresistant strains of Klebsiella pneumoniae to cerebrospinal fluid shunts: correlation with plasmid content.

A nosocomial multiresistant Klebsiella pneumoniae strain (KMD01) isolated from a patient with an infected ventriculoperitoneal (V-P) shunt was found to contain three plasmids of mol. wts (10(6)) c. 85, 50 and 2.4. A derivative isogenic strain (KMD11) carrying only the plasmids of mol. wts (10(6)) 50 and 2.4 was obtained spontaneously by plating the parent strain. The absence of the plasmid of mol. wt 85 X 10(6) in strain KMD11 correlated with an increased adherence to V-P catheters and glass surfaces, as well as autoagglutination in minimal medium. Bacterial cells containing the whole set of plasmids (strain KMD01) also showed the incorporation into the outer membrane of a new polypeptide (mol. wt, c. 41 X 10(3)), when grown in minimal medium. The presence of this polypeptide correlated with absence of autoagglutination, as shown by strain KMD01 under these cultural conditions. These data suggest that the cell-surface characteristics in K. pneumoniae may be affected by the plasmid content of the strain. Since nosocomial strains of K. pneumoniae usually contain one or more plasmids, and strains easily exchange these extrachromosomal elements, it seems reasonable to speculate that new variants with higher V-P shunt colonisation effectiveness, like the one described in this work, may also evolve in nature.

Producción de un polipéptido con actividad antiviral por fusión del gen interferón-alpha 2 humano al gen lacZ del bacteriófago M13mp8 / Production of a polypeptide with antiviral activity by fusion of the alpha 2 IFN gen to the lacZ gen in the M13mp8 bacteriophage

Un gen de IFN-* recientemente aislado en nuestro laboratorio a partir de una librería de ADN crosomal humano, fue genéticamente manipulado in vitro para poder acortar la secuencia señal y mejorar el rendimiento de IFN en cultivos bacterianos. Primero, la secuencia señal fue acortada en una extensión equivalente a nueve codones, usando la enzima de restricción HacIII y la secuencia remanente fue fusionada en fase con el codón iniciador del gen lacZ presente en el bacteriófago M13mp8. Uno de los clones recombinantes fue analizado en detalle. Este clon mostró una alta inestabilidad del inserto y una baja producción de actividad IFN, con un rendimiento similar al obtenido con el gen pre-IFN-*2 completo. El gen fusionado lacZ/IFN-* fue entonces transferido por clonaje mplecular al plásmido pBR322. Uno de los clones obtenidos mostró una buena estabilidad del plásmido híbrido, y aunque el rendimiento de actividad de IFN fue bajo (3x 10 a la 5 unidades/ml de extracto bacteriano crudo) la producción se mantuvo aún después de más de 20 pases. Finalmente, la secuencia señal recombinante presente en este clon fue nuevamente acortada en una extensión equivalente a 4 codones y se encontró que uno de los nuevos clones incrementaba aproximadamente en 20 veces el nivel de producción de actividad IFN. A pesar de que el polipéptido sintetizado constituye un producto de fusión, la actividad biológica antiviral de IFN parece quedar conservada

Deletions in Klebsiella pneumoniae R plasmids induced by growth in the presence of acridine orange at high temperature.

The generation in vivo of plasmids deleted at specific sites in strains of Klebsiella pneumoniae containing R plasmids, by treatment with high concentrations of acridine orange (1.2 mg/ml) at 42 degrees C are reported. These deletions seem to be site specific because loss of specific restriction fragments after digestion with restriction enzymes was demonstrated.