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W R Strohl

Publications and source records attributed to W R Strohl.

At least 19 recordsLinked to original sources
PubMed1992

Compilation and analysis of DNA sequences associated with apparent streptomycete promoters.

The DNA sequences associated with 139 apparent streptomycete transcriptional start sites are compiled and compared. Of these, 29 promoters appeared to belong to a group which are similar to those recognized by eubacterial RNA polymerases containing sigma 70-like subunits. The other 110 putative promoter regions contain a wide diversity of sequences; several of these promoters have obvious sequence similarities in the -10 and/or -35 regions. The apparent Shine-Dalgarno regions of 44 streptomycete genes are also examined and compared. These were found to have a wide range of degree of complementarity to the 3' end of streptomycete 16S rRNA. Eleven streptomycete genes are described and compared in which transcription and translation are proposed to be initiated from the same or nearby nucleotide. An updated consensus sequence for the E sigma 70-like promoters is proposed and a potential group of promoter sequences containing guanine-rich -35 regions also is identified.

Actinomycetales↗
PubMed1992

High cell density and high-productivity microbial fermentation.

Several interesting approaches to high cell density systems, molecular strategies coupled with fermentation technology, and updated traditional strategies have been used in the past year to obtain high productivity in the formation of important products. The most significant advances include new strategies for controlling high cell density fermentation reactors and expression of heterologous proteins in different strains of yeasts.

Bacteria↗
PubMed1992

Purification and characterization of TDP-D-glucose 4,6-dehydratase from anthracycline-producing streptomycetes.

TDP-D-glucose 4,6-dehydratase, which converts TDP-D-glucose to TDP-D-4-keto-6-deoxyglucose, was purified to near-homogeneity from the daunorubicin and baumycin-producing organism Streptomyces sp. C5 (968-fold purification with a 41% recovery), and from the daunorubicin producer Streptomyces peucetius ATCC 29050 (1000-fold purification with a 37% recovery). The TDP-D-glucose 4,6-dehydratases from Streptomyces sp. C5 and S. peucetius were determined by SDS-PAGE and HPLC gel filtration to be homodimers with subunit relative molecular masses of 39,000 and 36,000, respectively. For the enzymes from both organisms, negligible activity was observed in the absence of added NAD+, or when ADP-glucose, ADP-mannose, GDP-mannose, UDP-glucose or UDP-galactose was substituted for TDP-D-glucose as substrate. For the enzyme from Streptomyces sp. C5, the K'm values for NAD+ and TDP-D-glucose were 19.2 microM and 31.3 microM, respectively. The V'max for TDP-D-glucose was 309 nmol min-1 (mg protein)-1. For the S. peucetius enzyme, the K'm values for NAD+ and TDP-D-glucose were 20.1 microM and 34.7 microM, respectively. V'max values were 180 nmol min-1 (mg protein)-1 for NAD+ and 201 nmol min-1 (mg protein)-1 for TDP-D-glucose. TDP was a good inhibitor of TDP-D-glucose 4,6-dehydratase from both organisms. The N-terminal amino acid sequence of the TDP-D-glucose 4,6-dehydratase from S. peucetius and from the erythromycin producer, Saccharopolyspora erythraea, were similar, whereas the enzyme from Streptomyces sp. C5 contained a different N-terminal amino acid sequence from either of the other two enzymes.

Amino Acid Sequence↗
PubMed1992

Significance of anthraquinone formation resulting from the cloning of actinorhodin genes in heterologous streptomycetes.

This review explores the underlying biochemical and genetic principles leading to the formation of hybrid anthraquinones by recombinant anthracycline-producing streptomycetes transformed with genes encoding the early steps in actinorhodin biosynthesis. Experiments indicate that simple aromatic polyketides are probably synthesized using very similar mechanisms, allowing for the interspecies cloning of polyketide synthase genes for the potential production of novel aromatic polyketide structures.

Anthraquinones↗
PubMed1992

Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326.

The gene encoding a novel milk protein-hydrolyzing proteinase was cloned on a 6.56-kb SstI fragment from Streptomyces sp. strain C5 genomic DNA into Streptomyces lividans 1326 by using the plasmid vector pIJ702. The gene encoding the small neutral proteinase (snpA) was located within a 2.6-kb BamHI-SstI restriction fragment that was partially sequenced. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 15,740, which corresponds very closely with the relative molecular mass of the purified protein (15,500) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified neutral proteinase was determined, and the DNA encoding this sequence was found to be located within the sequenced DNA. The deduced amino acid sequence contains a conserved zinc binding site, although secondary ligand binding and active sites typical of thermolysinlike metalloproteinases are absent. The combination of its small size, deduced amino acid sequence, and substrate and inhibition profile indicate that snpA encodes a novel neutral proteinase.

Amino Acid Sequence↗
PubMed1991

Transcriptional organization and regulation of the nosiheptide resistance gene in Streptomyces actuosus.

The nosiheptide resistance gene (nshR) and a putative regulatory gene (nshA) are found together on a 2326 bp BamHI-PstI DNA fragment isolated from Streptomyces actuosus ATCC 25421. The putative regulatory gene, nshA, situated upstream from the nosiheptide resistance gene in the 2326 bp DNA fragment, contains apparent DNA-binding and RNA-binding domains. Interruption of nshA in the chromosome of S. actuosus alters nosiheptide production, suggesting that nshA is involved in regulation of nosiheptide biosynthesis. Two transcription initiation sites were found upstream of nshA as demonstrated by high-resolution S1 nuclease mapping. A weak transcription start site for nshR was found which initiated transcription from the first nucleotide of the open reading frame. Although a stem-loop structure with apparent termination activity was found between nshA and nshR, readthrough of transcription between nshA and nshR was demonstrated by S1 nuclease mapping of the 3' terminus of the nshA transcript. Time-course S1 experiments of the three promoters (nshA-pl, nshA-p2, nshR-p) indicated highly regulated differential expression of the promoters. nshA-p2 is a strong, constitutive promoter whereas 30% of the total nshA-p1/p2 transcript reads through the terminator and into the nshR gene, accounting for more than half of the total steady-state nshR transcript. The implications of the regulation of nshA and nshR gene expression, as well as the expression of two other linked genes, are presented.

Amino Acid Sequence↗
PubMed1991

Expression of polyketide biosynthesis and regulatory genes in heterologous streptomycetes.

There are now several examples showing that hybrid secondary metabolites can be produced as a result of interspecies cloning of antibiotic biosynthesis genes in streptomycetes. This paper reviews examples of hybrid secondary metabolite production, and examines the underlying biochemical and regulatory principles leading to the formation of hybrid anthraquinones by recombinant anthracycline-producing streptomycetes carrying actinorhodin biosynthesis genes. An anthraquinone, aloesaponarin II, was produced by cloning the actI, actIII, actIV, and actVII genes (pANT12) of actinorhodin biosynthesis pathway from Streptomyces coelicolor in anthracycline producing streptomycetes. Streptomyces galilaeus strains 31 133 and 31 671, aclacinomycin and 2-hydroxyaklavinone producers, respectively, formed aloesaponarin II as their major polyketide product when transformed with pANT12. Subcloning experiments indicated that a 2.8-kb XhoI fragment containing only the actI and actVII loci was necessary for aloesaponarin II biosynthesis by S. galilaeus 31 133. When S. galilaeus 31 671 was transformed with the actI, actVII, and actIV genes, however, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin and 1-O-methyldesoxyerythrolaccin. When S. galilaeus 31 671 was transformed with only the intact actIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for the actIII gene, which is the reduction of the keto group at C-9 from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The effects of three regulatory loci, dauG, dnrR1, and asaA, on the production of natural and hybrid polyketides were also shown.

Anti-Bacterial Agents↗
PubMed1991

A predictive and feedback control algorithm maintains a constant glucose concentration in fed-batch fermentations.

A combined predictive and feedback control algorithm based on measurements of the concentration of glucose on-line has been developed to control fed-batch fermentations of Escherichia coli. The predictive control algorithm was based on the on-line calculation of glucose demand by the culture and plotting a linear regression to the next datum point to obtain a predicted glucose demand. This provided a predictive "coarse" control for the glucose-based nutrient feed. A direct feedback control using a proportional controller, based on glucose measurements every 2 min, fine-tuned the feed rate. These combined control schemes were used to maintain glucose concentrations in fed-batch fermentations as tight as 0.49 +/- 0.04 g/liter during growth of E. coli to high cell densities.

Acetates↗
PubMed1991

Glucose-stat, a glucose-controlled continuous culture.

A predictive and feedback proportional control algorithm, developed for fed-batch fermentations and described in a companion paper (G. L. Kleman, J. J. Chalmers, G. W. Luli, and W. R. Strohl, Appl. Environ. Microbiol. 57:910-917, 1991), was used in this work to control a continuous culture on the basis of the soluble-glucose concentration (called the glucose-stat). This glucose-controlled continuous-culture system was found to reach and maintain steady state for 11 to 24 residence times when four different background glucose concentrations (0.27, 0.50, 0.7, and 1.5 g/liter) were used. The predictive-plus-feedback control system yielded very tight control of the continuous nutristat cultures; glucose concentrations were maintained at the set points with less than 0.003 standard error. Acetate production by Escherichia coli B in glucose-stats was found not to be correlated with the level of steady-state soluble-glucose concentration.

Acetates↗
PubMed1990

Nucleotide sequence and transcriptional analysis of the nosiheptide-resistance gene from Streptomyces actuosus.

The nucleotide (nt) sequence of a 2326-bp BamHI-PstI DNA fragment previously isolated from Streptomyces actuosus ATCC25421 that confers resistance to the thiopeptide antibiotics, nosiheptide (Nh) and thiostrepton (Ts) upon Streptomyces lividans 1326 was determined. Two open reading frames (ORFs) were found in this 2326-bp sequence; one containing 699 nt and another of 822 nt, both reading in the same direction. The Nh-resistance gene determinant (nsh) is encoded by orf822, as determined by the 74% identity of the deduced amino acid sequence of its gene product to that of the 23S rRNA methylase encoded by the Ts-resistance gene (tsr) of Streptomyces azureus. (The respective sequences had a 72% homology.) ORF699, encoded by a gene situated upstream from orf822, contained an apparent alpha-helix-beta-turn-alpha-helix configuration which is common to DNA-binding proteins and suggests that ORF699 may be a regulatory protein. Two transcription start points (tsp) were found upstream from orf699 as demonstrated by high-resolution S1 nuclease mapping. There was also a weak tsp for the nsh gene at the first nt of ORF. Moreover, transcription was observed to read through a stem-loop structure separating the orf699 and nsh genes, as demonstrated by S1 nuclease mapping of the 3' terminus of the orf699 gene, suggesting an antitermination mechanism for regulation of nsh transcription.

Amino Acid Sequence↗
PubMed1990

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations.

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.

Acetates↗
PubMed1990

Biosynthesis of anthraquinones by interspecies cloning of actinorhodin biosynthesis genes in streptomycetes: clarification of actinorhodin gene functions.

Streptomyces galilaeus ATCC 31133 and ATCC 31671, producers of the anthracyclines aclacinomycin A and 2-hydroxyaklavinone, respectively, formed an anthraquinone, aloesaponarin II, when they were transformed with DNA from Streptomyces coelicolor containing four genetic loci, actI, actIII, actIV, and actVII, encoding early reactions in the actinorhodin biosynthesis pathway. Subcloning experiments indicated that a 2.8-kilobase-pair XhoI fragment containing only the actI and actVII loci was necessary for aloesaponarin II biosynthesis by S. galilaeus ATCC 31133. Aloesaponarin II was synthesized via the condensation of 8 acetyl coenzyme A equivalents, followed by a decarboxylation reaction as demonstrated by [1,2-13C2]acetate feeding experiments. S. coelicolor B22 and B159, actVI blocked mutants, also formed aloesaponarin II as an apparent shunt product. Mutants of S. coelicolor blocked in several other steps in actinorhodin biosynthesis did not synthesize aloesaponarin II or other detectable anthraquinones. When S. galilaeus ATCC 31671 was transformed with the DNA carrying the actI, actIII, and actVII loci, the recombinant strain produced both aloesaponarin II and aklavinone, suggesting that the actinorhodin biosynthesis DNA encoded a function able to deoxygenate 2-hydroxyaklavinone to aklavinone. When S. galilaeus ATCC 31671 was transformed with a plasmid carrying only the intact actIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for the actIII gene, which is the reduction of the keto group at C-9 from the carboxy terminus of the assembled polyketide to the corresponding secondary alcohol. In the presence of the actIII gene, anthraquinones or anthracyclines formed as a result of dehydration and aromatization lack an oxygen function on the carbon on which the keto reductase operated. When S. galilaeus ATCC 31671 was transformed with the DNA carrying the actI, actVII, and actIV loci, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin, the 3-hydroxy analog of aloesaponarin II, and 1-O-methyldesoxyerythrolaccin. The results obtained in these experiments together with earlier data suggest a pathway for the biosynthesis of actinorhodin and related compounds by S. coelicolor.

Anthraquinones↗
PubMed1988

Molecular cloning of the nosiheptide resistance gene from Streptomyces actuosus ATCC 25421.

An 8.5 kb BamHI DNA fragment conferring resistance to nosiheptide, a peptide antibiotic of the 'thiostrepton group', was cloned from Streptomyces actuosus ATCC 25421 in Streptomyces lividans 1326. Two BamHI fragments of S. actuosus, the 8.5 kb fragment and an additional 3.0 kb fragment, hybridized with a thiostrepton resistance gene probe (pIJ30). The 8.5 kb fragment showed a relatively low degree of homology with the thiostrepton resistance gene. The restriction map of the nosiheptide resistance gene isolated here was significantly different from the map of the thiostrepton resistance gene previously published.

Anti-Bacterial Agents↗
PubMed1988

Physiological regulation of protease activity in Streptomyces peucetius.

Streptomyces peucetius ATCC 29050, a producer of anthracycline antineoplastic agents, was investigated for the expression of intracellular and extracellular azocaseinase activities as a function of growth and medium conditions. When cultures were grown in either nitrate-containing defined medium or glucose-yeast extract complex medium, the intracellular proteolytic activity was greatest during early to mid stationary phase, whereas the extracellular proteolytic activity was produced in late stationary phase. All of the proteolytic activity detected against azocasein was of a serine-type protease activity. These late-occurring proteases may have some function in cellular turnover associated with secondary metabolism and (or) morphogenesis.

Caseins↗
PubMed1988

Biosynthesis of epsilon-rhodomycinone from glucose by Streptomyces C5 and comparison with intermediary metabolism of other polyketide-producing streptomycetes.

The catabolism of glucose by Streptomyces C5, a producer of anthracycline antibiotics, was investigated to determine the pathways that supply precursors for anthracycline biosynthesis. Carbons for the biosynthesis of epsilon-rhodomycinone, an anthracycline aglycone, from radiolabelled glucose were derived primarily from the Embden-Meyerhof-Parnas pathway, with a minor contribution from the pentose phosphate pathway. Furthermore, the anthracycline-producing strain, Streptomyces C5, as well as Streptomyces aureofaciens and Streptomyces lividans, strains that produce nonanthracycline polyketide antibiotics, displayed enzyme activities indicative of the Embden-Meyerhof-Parnas and pentose phosphate glycolytic pathways. As determined from labelling patterns, Streptomyces C5 apparently has a complete tricarboxylic acid cycle, but does not have a glyoxylate bypass pathway.

Anthracyclines↗
PubMed1988

Activity of phosphoenolpyruvate carboxylase of an anthracycline-producing streptomycete.

During fermantation studies on the production of anthracycline antibiotics by Streptomyces C5, it was observed that among the intermediate metabolism enzymes tested, only phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) increased significantly in specific activity during stationary phase. The specific activity of the Streptomyces C5 PEPCase increased ca. 3-fold during antibiotic production phase from the logarithmic phase levels. To characterize the regulation of the enzyme further, the Streptomyces C5 PEPCase was purified 150-fold from crude extracts. Acetyl-CoA and Mg2+ were shown to be required for PEPCase activity. The activity of the partially purified PEPCase was stimulated slightly by fructose 1,6-bisphosphate and AMP, and was inhibited severely by oxaloacetate, aspartate, malate, succinate, ATP, citrate, and CoASH.

Allosteric Regulation↗
PubMed1987

Isolation and characterization of a temperate bacteriophage from Streptomyces galilaeus.

A new temperate actinophage from Streptomyces galilaeus ATCC 31133 was purified after that strain was crossed with S. peucetius ATCC 29050. Sensitive hosts became lysogenized and yielded turbid plaques of 2 to 3 mm in diameter. Host-range analysis indicated that 16 of 27 Streptomyces strains tested were sensitive to infection on solid medium. S. lividans and S. coelicolor A3(2) were among those not infected by this new actinophage. The new actinophage, designated phi SPK1, belongs to the Bradley group B morphological type, the pH optimum for infection is 6.75 to 7.0, it is not efficiently induced by mitomycin C or UV irradiation, it has a circular chromosome of 35.8 +/- 0.5 kilobase pairs in length containing overlapping (cohesive) ends, and the G+C content of its DNA was calculated from the buoyant density of 1.7240 to be 69 mol%. The DNA of phage phi SPK1 was cleaved by the restriction endonucleases ApaI, AluII, EcoRI, PvuII, and SalI, but, in all cases except that with EcoRI, treatment yielded greater than 20 restriction fragments. No sites were detected for BamHI, BclI, BglII, ClaI, HindIII, MluI, PstI, SmaI, SphI, SstI, XbaI, or XhoI.

Bacteriophages↗