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M Bibb

Publications and source records attributed to M Bibb.

16 recordsLinked to original sources
PubMed2002

Carbon flux distribution in antibiotic-producing chemostat cultures of Streptomyces lividans.

The carbon metabolism of derivatives of Streptomyces lividans growing under phosphate limitation in chemostat cultures and producing the antibiotics actinorhodin and undecylprodigiosin was investigated. By applying metabolic flux analysis to a stoichiometric model, the relationship between antibiotic production, biomass accumulation, and carbon flux through the major carbon metabolic pathways (the Embden Meyerhoff Parnas and pentose-phosphate pathways) was analyzed. Distribution of carbon flux through the catabolic pathways was shown to be dependent on growth rate, as well as on the carbon and energy source (glucose or gluconate) used. Increasing growth rates promoted an increase in the flux of carbon through glycolysis and the pentose-phosphate pathway. The synthesis of both actinorhodin and undecylprodigiosin was found to be inversely related to flux through the pentose-phosphate pathway.

Anthraquinones↗
PubMed2001

Induction of ppGpp synthesis in Streptomyces coelicolor A3(2) grown under conditions of nutritional sufficiency elicits actII-ORF4 transcription and actinorhodin biosynthesis.

Production of ppGpp in Streptomyces coelicolor A3(2) was achieved independently of amino acid limitation by placing N-terminal segments of the ppGpp synthetase gene, relA, under the control of a thiostrepton-inducible promoter (tipAp). S1 nuclease protection experiments indicated that induced ppGpp concentrations of 6-12 pmol mg(-1) dry weight in late-exponential phase cultures caused activation of transcription of actII-ORF4, the pathway-specific activator gene for actinorhodin production. This level of ppGpp had no effect on growth rate, implying a causal role for ppGpp in activating actII-ORF4 transcription. No effect was observed on the transcription of the corresponding and homologous activator gene for undecylprodigiosin production, redD, reflecting a requirement for additional regulatory factors for activation of its transcription. This work provides the most compelling evidence yet for the activation of an antibiotic biosynthetic pathway by the stringent factor ppGpp.

Alcohol Oxidoreductases↗
PubMed2001

Functional analysis of relA and rshA, two relA/spoT homologues of Streptomyces coelicolor A3(2).

Deletion of the (p)ppGpp synthetase gene, relA, of Streptomyces coelicolor A3(2) results in loss of production of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) and delayed morphological differentiation when the mutant is grown under conditions of nitrogen limitation. To analyze the role of (p)ppGpp as an intracellular signaling molecule for the initiation of antibiotic production, several C-terminally deleted derivatives of S. coelicolor relA that could potentially function in the absence of ribosome activation were placed under the control of the thiostrepton-inducible tipA promoter. While 0.82- and 1.28-kb N-terminal segments failed to restore (p)ppGpp and antibiotic production upon induction in a relA null mutant, 1.46- and 2.07-kb segments did. Under conditions of phosphate limitation, deletion of relA had little or no effect on Act or Red synthesis, potentially reflecting an alternative mechanism for ppGpp synthesis. A second S. coelicolor RelA homologue (RshA, with 42% identity to S. coelicolor RelA) was identified in the genome sequence. However, deletion of rshA had no effect on the ability of the relA mutant to make Act and Red when grown under conditions of phosphate limitation. While high-level induction of tipAp::rshA in the relA mutant resulted in growth inhibition, low-level induction restored antibiotic production and sporulation. In neither case, nor in the relA mutant that was grown under phosphate limitation and producing Act and Red, could (p)ppGpp synthesis be detected. Thus, a ppGpp-independent mechanism exists to activate antibiotic production under conditions of phosphate limitation that can be mimicked by overexpression of rshA.

Adenosine↗
PubMed2000

Purification and structural determination of SCB1, a gamma-butyrolactone that elicits antibiotic production in Streptomyces coelicolor A3(2).

Early stationary phase culture supernatants of Streptomyces coelicolor A3(2) contained at least four small diffusible signaling molecules that could elicit precocious antibiotic synthesis in the producing strain. The compounds were not detected in exponentially growing cultures. One of these compounds, SCB1, was purified to homogeneity and shown to be a gamma-butyrolactone of structure (2R, 3R,1'R)-2-(1'-hydroxy-6-methylheptyl)-3-hydroxymethylbutanolide . Bioassays of chemically synthesized SCB1, and of its purified stereoisomers, suggest that SCB1 acts in a highly specific manner to elicit the production of both actinorhodin and undecylprodigiosin, the two pigmented antibiotics made by S. coelicolor.

4-Butyrolactone↗
PubMed1999

The linear plasmid SCP1 of Streptomyces coelicolor A3(2) possesses a centrally located replication origin and shows significant homology to the transposon Tn4811.

The linear plasmid SCP1 of Streptomyces coelicolor A3(2) is one of the genetically more studied linear streptomycete replicons. Although the genetics of SCP1 and its interaction with the host chromosome have been analyzed for nearly three decades no information exists on its replication. With the help of an ordered cosmid contig for the complete 360-kb element, we have localized a 5439-bp fragment from the central region that confers autonomous replication in Streptomyces lividans. The minimal origin contains two overlapping ORFs which are separated from an AT-rich region which might correspond to the replication start point. ORF1 revealed intensive similarity to a class of DNA-primase/helicases of actinophages and archael plasmids. In addition, we have identified a region in both terminal inverted repeats of SCP1 that shows significant homology to the transposable element Tn4811 located near the ends of the S. lividans 66 chromosome.

Amino Acid Sequence↗
PubMed1998

Actinorhodin and undecylprodigiosin production in wild-type and relA mutant strains of Streptomyces coelicolor A3(2) grown in continuous culture.

The effects of growth rate and nutrient feed rate on the production of actinorhodin (Act) and undecylprodigiosin (Red) were determined in Streptomyces coelicolor A3(2) and in a congenic relA null-mutant known to be deficient in ppGpp synthesis and antibiotic production under conditions of nitrogen limitation. In the relA+ strain, Act production was inversely related to specific growth rate in continuous cultures limited by glucose, ammonium, or phosphate, while Red biosynthesis was optimal at 0.05 h-1 regardless of the specific nutrient limitation. Production of Act and Red in the relA mutant was lower than that of the parental strain, particularly under conditions of glucose- and ammonium-limitation, indicating an important and general role for ppGpp in determining the onset of the antibiotic biosynthesis under conditions of nutrient limitation. At constant growth rate, but with varying nutrient feed rates, the specific rate of Act production was adversely influenced by increasing levels of glucose, ammonium, and phosphate, with phosphate having the greatest inhibitory effect. Under the same conditions, the specific rate of Red production was stimulated by increasing glucose levels, but markedly decreased by increased levels of phosphate.

Anthraquinones↗
PubMed1997

The ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2) plays a conditional role in antibiotic production and morphological differentiation.

Deletion of most of the coding region of the ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2) resulted in loss of ppGpp synthesis, both upon entry into stationary phase under conditions of nitrogen limitation and following amino acid starvation during exponential growth, but had no effect on growth rate. The relA mutant, which showed continued rRNA synthesis upon amino acid depletion (the relaxed response), failed to produce the antibiotics undecylprodigiosin (Red) and actinorhodin (Act) under conditions of nitrogen limitation. The latter appears to reflect diminished transcription of pathway-specific regulatory genes for Red and Act production, redD and actII-ORF4, respectively. In addition to the changes in secondary metabolism, the relA mutant showed a marked delay in the onset and extent of morphological differentiation, resulting in a conspicuously altered colony morphology.

Anthraquinones↗
PubMed1997

bldA dependence of undecylprodigiosin production in Streptomyces coelicolor A3(2) involves a pathway-specific regulatory cascade.

The production of the red-pigmented tripyrrole antibiotic undecylprodigiosin (Red) by Streptomyces coelicolor A3(2) depends on two pathway-specific regulatory genes, redD and redZ. RedD is homologous to several other proteins that regulate antibiotic production in streptomycetes; RedZ is a member of the response regulator family. redZ transcripts were detected during exponential growth and increased in amount during transition and stationary phases; transcription of redD was confined to the two latter stages of growth. Whereas mutation of redD had no effect on redZ transcription, transcription of redD was highly dependent on redZ, suggesting that RedZ is a transcriptional activator of redD. bldA, which encodes the only tRNA of S. coelicolor that can efficiently translate the rare leucine codon UUA, is required for Red production at higher phosphate concentrations. While the redD transcript contains no UUA codons, the redZ mRNA contains one. Transcription of redZ appeared to be unaffected in a bldA mutant; in contrast, redD transcription was undetectable, consistent with the translational dependence of redZ on bldA and the transcriptional dependence of redD on redZ. Red production in a bldA mutant was restored by multiple copies of redZ, presumably reflecting a low level of mistranslation of the redZ UUA codon, while multiple copies of redD had no effect, presumably a consequence of the severe dependence of redD transcription on RedZ. Transcription of redZ appears to be negatively autoregulated.

Anti-Bacterial Agents↗
PubMed1996

Cloning, characterization and disruption of a (p)ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2).

An internal segment of the (p)ppGpp synthetase gene, relA, of Streptomyces coelicolor A3(2) was amplified from genomic DNA using the polymerase chain reaction and used as a hybridization probe to isolate the complete gene from a cosmid library. relA lies downstream of a gene (apt) that apparently encodes adenine phosphoribosyltransferase and is transcribed from two promoters, relAp1 and relAp2, and by transcriptional readthrough from apt. While the level of relAp2 transcripts remained relatively constant, relAp1 activity apparently peaked during transition phase, following a decline in readthrough transcription from apt. Disruption of relA using an att- derivative of the temperate phage phi C31 abolished ppGpp synthesis on amino acid depletion. When grown on agar, the disruptants grew more slowly than a control lysogen made with an att+ phage vector and gave smaller colonies that sporulated normally. The relA mutation had no consistent or marked effect on actinorhodin production in either liquid- or agar-grown cultures, indicating that elevated levels of (p)ppGpp are not essential for triggering the onset of antibiotic production.

Amino Acid Sequence↗
PubMed1996

afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2).

The N-terminal region of AfsR, a putative pleiotropic regulatory protein for antibiotic production in Streptomyces coelicolor A3(2), is homologous to RedD and Actil-ORF4, pathway-specific regulatory proteins required for the production of the antibiotics undecylprodigiosin (Red) and actinorhodin (Act), respectively. The recent identification of afsS, which lies immediately 3' of afsR and which stimulates antibiotic production when cloned at high copy number, questioned whether afsR was a pleiotropic regulatory gene. In this study we demonstrate that multiple copies of afsR can stimulate both Act and Red production and that, despite its homology, it cannot substitute for the pathway-specific regulatory genes. Moreover, an in-frame deletion that removed most of the afsR coding sequence resulted in loss of Act and Red production, and a marked reduction in the synthesis of the calcium-dependent antibiotic (CDA), but only under some (non-permissive) nutritional conditions. Although additional copies of afsR resulted in elevated levels of the actII-ORF4 and redD transcripts, transcription of the pathway-specific regulatory genes under non-permissive conditions was unaffected by deletion of afsR. While afsR may operate independently of the pathway-specific regulatory proteins to influence antibiotic production, the activity of ActII-ORF4 and of RedD under non-permissive conditions could depend on interaction with, or modification by, AfsR.

Amino Acid Sequence↗
PubMed1994

Transcriptional regulation of the four promoters of the agarase gene (dagA) of Streptomyces coelicolor A3(2).

The agarase gene (dagA) of Streptomyces coelicolor A3(2) is transcribed from four promoters that are recognized by at least three, and probably four, different RNA polymerase holoenzymes, each containing a different sigma factor. S1 nuclease protection studies revealed that transcription from all four promoters is induced by the products of agar hydrolysis and strongly repressed by glucose. Mutants deficient in glucose kinase activity were defective in glucose repression of all four promoters. Mutants were isolated or identified in which transcription from all four promoters had become inducer-independent (i.e. constitutive), establishing the existence of a repressor gene for dagA that does not appear to be located within 9 kb of the structural gene. The cloned dagA gene was also constitutively expressed in the closely related strain Streptomyces lividans, which does not normally make agarase and which presumably lacks the repressor gene. Glucose was still able to repress dagA transcription even under conditions of constitutive expression, suggesting that glucose kinase does not mediate its effect via inducer exclusion. Relative differences in the use of the four promoters were not detected during different stages of growth of surface-grown cultures, although dagA transcription appeared to peak during the production of aerial mycelium.

Base Sequence↗
PubMed1985

The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product.

The sequence of a 1.56-kb DNA fragment containing the tyrosinase gene (mel) from Streptomyces antibioticus was determined and the Mr (30612) and amino acid (aa) sequence of the protein were deduced from the nucleotide (nt) sequence. Intracellular and extracellular tyrosinase from S. antibioticus, transformed with pIJ702 (containing mel), were purified to homogeneity; the Mr (29 500), as determined by Sephadex G-75 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was consistent with the value derived from the nt sequence. Edman degradation established that the N-terminal sequence of both the intracellular and extracellular forms of tyrosinase are identical and correspond to the aa sequence derived from the structural gene. In addition, this sequence exhibits striking homology to the N-terminal region of the intracellular and extracellular enzyme purified from Streptomyces glaucescens (Crameri et al., 1982). An additional open reading frame (ORF438) upstream of the mel gene, was also identified that appears to code for a protein (Mr = 14 754) with a putative signal sequence.

Amino Acid Sequence↗
PubMed1984

Thymoma: an immunohistochemical study.

Four thymomas, two from patients with myasthenia gravis, were studied using a panel of monoclonal antibodies. Almost all the associated lymphocytes were T-cells with the common thymic lymphocyte phenotype, suggesting that the neoplastic epithelium can provide a microenvironment that retains or perhaps attracts immature T-cells. HLA-Dr antigens were expressed by some of the epithelial cells and many of the lymphoid cells in the perivascular space. The Leu-7 antigen was present on a subset of epithelial cells and had a highly variable expression in different thymomas. The percentage of lymphocytes bearing the OKT8-defined antigen appeared to be lower in patients with myasthenia gravis, while the expression of other markers did not appear to be consistently or significantly different. There did not appear to be marked differences in the lymphocyte subpopulations of thymomas with different morphology, and there was no evidence of differentiation of the thymomas into cortical and medullary areas.

Adult↗