The Occurrence of Deacetoxy-Cephalosporin C in Fungi and Streptomycetes

Higgens, C. E.; Hamill, Robert L.; Sands, T. H.; Hoehn, Marvin M.; Davis, Norman E.; Nagarajan, Ramaswamy; Boeck, L. D.

doi:10.7164/antibiotics.27.298

Cited by 76 publications

(28 citation statements)

References 6 publications

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“…S. coelicolor strains M145 (Kieser et al, 2000) and INB101 (DphoP) were manipulated according to standard procedures (Kieser et al, 2000). TBO medium (Higgens et al, 1974) was used to obtain spore preparations. E. coli DH5a was the general cloning host.…”

Section: Methodsmentioning

confidence: 99%

Phosphate-dependent regulation of the low- and high-affinity transport systems in the model actinomycete Streptomyces coelicolor

et al. 2008

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The transport of inorganic phosphate (P i ) is essential for the growth of all organisms. The metabolism of soil-dwelling Streptomyces species, and their ability to produce antibiotics and other secondary metabolites, are strongly influenced by the availability of phosphate. The transcriptional regulation of the SCO4138 and SCO1845 genes of Streptomyces coelicolor was studied. These genes encode the two putative low-affinity P i transporters PitH1 and PitH2, respectively. Expression of these genes and that of the high-affinity transport system pstSCAB follows a sequential pattern in response to phosphate deprivation, as shown by coupling their promoters to a luciferase reporter gene. Expression of pitH2, but not that of pap-pitH1 (a bicistronic transcript), is dependent upon the response regulator PhoP. PhoP binds to specific sequences consisting of direct repeats of 11 nt in the promoter of pitH2, but does not bind to the pap-pitH1 promoter, which lacks these direct repeats for PhoP recognition. The transcription start point of the pitH2 promoter was identified by primer extension analyses, and the structure of the regulatory sequences in the PhoP-protected DNA region was established. It consists of four central direct repeats flanked by two other less conserved repeats. A model for PhoP regulation of this promoter is proposed based on the four promoter DNA-PhoP complexes detected by electrophoretic mobility shift assays and footprinting studies.

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Section: Methodsmentioning

confidence: 99%

Phosphate-dependent regulation of the low- and high-affinity transport systems in the model actinomycete Streptomyces coelicolor

et al. 2008

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“…Spores were produced as reported (16) and kept frozen at Ϫ20°C in 20% w/v glycerol (17). DNA manipulations and sequence analysis were carried out as reported previously (18).…”

Section: Methodsmentioning

confidence: 99%

A New Class of Glutamate Dehydrogenases (GDH)

Miñambres

Olivera

Jensen³

et al. 2000

Journal of Biological Chemistry

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A new class of glutamate dehydrogenase (GDH) is reported. The GDH of Streptomyces clavuligerus was purified to homogeneity and characterized. It has a native molecular mass of 1,100 kDa and exists as an ␣ 6 oligomeric structure composed of 183-kDa subunits. GDH, which requires AMP as an essential activator, shows a maximal rate of catalysis in 100 mM phosphate buffer, pH 7.0, at 30°C. Under these conditions, GDH displayed hyperbolic behavior toward ammonia (K m , 33 mM) and sigmoidal responses to changes in ␣-ketoglutarate (S 0.5 1.3 mM; n H 1.50) and NADH (S 0.5 20 M; n H 1.52) concentrations. Aspartate and asparagine were found to be allosteric activators. This enzyme is inhibited by an excess of NADH or NH 4 ؉ , by some tricarboxylic acid cycle intermediates and by ATP. This GDH seems to be a catabolic enzyme as indicated by the following: (i) it is NAD-specific; (ii) it shows a high value of K m for ammonia; and (iii) when S. clavuligerus was cultured in minimal medium containing glutamate as the sole source of carbon and nitrogen, a 5-fold increase in specific activity of GDH was detected compared with cultures provided with glycerol and ammonia. GDH has 1,651 amino acids, and it is encoded by a DNA fragment of 4,953 base pairs (gdh gene). It shows strong sequence similarity to proteins encoded by unidentified open reading frames present in the genomes of species belonging to the genera Mycobacterium, Rickettsia, Pseudomonas, Vibrio, Shewanella, and Caulobacter, suggesting that it has a broad distribution. The GDH of S. clavuligerus is the first member of a class of GDHs included in a subfamily of GDHs (large GDHs) whose catalytic requirements and evolutionary implications are described and discussed.

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“…Streptomyces coelicolor strains M145 (18) and INB201 (⌬phoP) (40) and gmk::Tn5062 and ⌬rpoZ mutants (this study) were manipulated according to standard procedures (18). TBO medium (17) Complementation of ⌬rpoZ strains. A 550-bp fragment of S. coelicolor M145 that contains the rpoZ gene with its promoter and terminator sequences was amplified with primers RPOZ-Cu1 (5Ј-GGGCCTCTAGATAAGTCAGCGCA) and RPOZ-PD (5Ј-ACGAGCTGGATCCGCAGGCTCA), which had been modified with XbaI and BamHI restriction sites, respectively.…”

Section: Methodsmentioning

confidence: 99%

The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

Santos-Beneit

Barriuso-Iglesias

Fernández-Martínez

et al. 2011

Appl Environ Microbiol

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The RNA polymerase (RNAP) omega factor () forms a complex with the ␣ 2 ␤␤ core of this enzyme in bacteria. We have characterized the rpoZ gene of Streptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of the rpoZ gene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ⌬rpoZ strain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ⌬rpoZ mutant with the wild-type rpoZ allele restored both phenotype and antibiotic production. Interestingly, the rpoZ gene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed that rpoZ promoter activity was increased in a ⌬phoP background, it can be concluded that rpoZ is controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation in Streptomyces.In bacteria, the RNA polymerase (RNAP) complex plays a central role in transcription and is a target for regulation of primary metabolism (6,7,44). The rpoZ gene encodes the RNAP omega () subunit, which forms a complex with the ␣ 2 ␤␤Ј core of this enzyme. The subunit has been identified in the RNAPs of most free-living bacteria. This protein is functionally homologous to the RpoK subunit of the archaeal RNA polymerase complex and the RPB6 subunit of the eukaryotic RNA polymerases I, II, and III (32). In Escherichia coli the subunit interacts with the ␤Ј subunit and promotes assembly of the RNA polymerase complex (14, 32), although it is not essential for survival in this bacterium (13).Streptomyces spp. are soil-dwelling bacteria that are notorious for their ability to produce thousands of antibiotics, pigments, antitumor agents, immunomodulators, and a variety of other bioactive secondary metabolites (1, 2, 8). Differential expression of secondary metabolism genes occurs following nutrient depletion (34), but the transcriptional control mechanisms that govern the onset of secondary metabolites are still obscure (29). The rpoZ gene of Streptomyces kasugaensis has been shown to be required for antibiotic production and morphological differentiation but is not essential for growth (21). A DNA fragment containing the rpoZ gene was shown to complement an S. kasugaensis pleiotropic mutant deficient in aerial mycelium formation and kasugamycin biosynthesis. Although sigma factors in Streptomyces have received considerable attention in relation to the expression of antibiotic biosynthetic genes (9,19,20), the role of the RNAP subunit is still obscure.The expression of many genes involved in antibiotic biosynthesis is negatively controlled by the phosphate concentration in the medium (reviewed in references 28 and 30). Limitati...

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The Occurrence of Deacetoxy-Cephalosporin C in Fungi and Streptomycetes

Cited by 76 publications

References 6 publications

Phosphate-dependent regulation of the low- and high-affinity transport systems in the model actinomycete Streptomyces coelicolor

Phosphate-dependent regulation of the low- and high-affinity transport systems in the model actinomycete Streptomyces coelicolor

A New Class of Glutamate Dehydrogenases (GDH)

The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

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