Branched-chain amino acid catabolism provides precursors for the Type II polyketide antibiotic, actinorhodin, via pathways that are nutrient dependent.

Polyketide antibiotics are among the most important therapeutics used in human and animal health care. Type II polyketides are composed primarily of acetate-derived thioesters, and the subunits for the PKS are contained in a single module that includes a ketosynthase, acyl carrier protein, chain-length factor and sometimes a keto-reductase, aromatase, cyclase and modifying enzymes, such as glycosylases or hydroxylases. While the enzyme complexes that make up the PKS have been the focus of intense study (Khosla in Chem Rev 7:2577-2590, 1997), the pathways for precursor synthesis have not been established and predictions are complicated by the fact that acetate may be derived from a number of metabolic pathways.

Large-scale gene expression analysis of cholesterol dependence in NS0 cells.

NS0, a nonsecreting mouse myeloma cell, is a major host line used for recombinant antibody production. These cells have a cholesterol-dependent phenotype and rely on an exogenous supply of cholesterol for their survival and growth. To better understand the physiology underlying cholesterol dependence, we compared NS0 cells, cultivated under standard cholesterol-dependent growth conditions (NS0), to cells adapted to cholesterol-independent conditions (NS0 revertant, NS0_r).

The bkdR gene of Streptomyces coelicolor is required for morphogenesis and antibiotic production and encodes a transcriptional regulator of a branched-chain amino acid dehydrogenase complex.

Products from the degradation of the branched-chain amino acids valine, leucine, and isoleucine contribute to the production of a number of important cellular metabolites, including branched-chain fatty acids, ATP and other energy production, cell-cell signaling for morphological development, and the synthesis of precursors for polyketide antibiotics. The first nonreversible reactions in the degradation of all three amino acids are catalyzed by the same branched-chain alpha-keto acid dehydrogenase (BCDH) complex. Actinomycetes are apparently unique among bacteria in that they contain two separate gene clusters, each of which encodes a BCDH enzyme complex.

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.

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.

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.

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.

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).

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.

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.

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.

Mono- and dimethylating activities and kinetic studies of the ermC 23 S rRNA methyltransferase.

The ermC 23 S rRNA methyltransferase converts a single adenine residue to N6,N6-dimethyladenine, both in vivo and in vitro. The ermC methyltransferase was demonstrated to produce both N6-mono and N6,N6-dimethylated adenine residues in Bacillus subtilis 23 S rRNA during the course of the reaction in vitro. An almost total conversion of monomethylated intermediates into dimethylated products was observed upon completion of the reaction.

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.

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.

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.

Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid from Bacillus subtilis.

We initiated a study of pIM13, a multicopy, macrolide-lincosamide-streptogramin B (MLS) plasmid first isolated from a strain of Bacillus subtilis and described by Mahler and Halvorson (J. Gen. Microbiol.

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.

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.