scholarly journals Organization and Expression of the Polynucleotide Phosphorylase Gene (pnp) of Streptomyces: Processing of pnp Transcripts in Streptomyces antibioticus

2004 ◽  
Vol 186 (10) ◽  
pp. 3160-3172 ◽  
Author(s):  
Patricia Bralley ◽  
George H. Jones
Keyword(s):  
Intergenic Region ◽  
Transcription Initiation ◽  
Streptomyces Coelicolor ◽  
Open Reading Frames ◽  
Streptomyces Avermitilis ◽  
Polynucleotide Phosphorylase ◽  
Rnase Iii ◽  
Stem Loop ◽  
Streptomyces Antibioticus ◽  
E Coli

ABSTRACT We have examined the expression of pnp encoding the 3′-5′-exoribonuclease, polynucleotide phosphorylase, in Streptomyces antibioticus. We show that the rpsO-pnp operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both pnp and the gene for ribosomal protein S15 (rpsO) and a second, Ppnp, located in the rpsO-pnp intergenic region. Unlike the situation in Escherichia coli, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type S. antibioticus mycelia. The Ppnp transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5′ ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The Ppnp sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in rspO-pnp structure and function between S. antibioticus and E. coli. In E. coli, pnp autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an rpsO-pnp intergenic hairpin. Computer modeling of the secondary structure of the S. antibioticus readthrough transcript predicts a stem-loop structure analogous to that in E. coli. However, our analysis suggests that while the readthrough transcript observed in S. antibioticus may be processed by an RNase III-like activity, transcripts originating from Ppnp are not. Furthermore, the S. antibioticus rpsO-pnp intergenic region contains two open reading frames. The larger of these, orfA, may be a pseudogene. The smaller open reading frame, orfX, also observed in Streptomyces coelicolor and Streptomyces avermitilis, may be translationally coupled to pnp and the gene downstream from pnp, a putative protease.

scholarly journals Kinetics of Polynucleotide Phosphorylase: Comparison of Enzymes from Streptomyces and Escherichia coli and Effects of Nucleoside Diphosphates

2007 ◽  
Vol 190 (1) ◽  
pp. 98-106 ◽  
Author(s):  
Samantha A. Chang ◽  
Madeline Cozad ◽  
George A. Mackie ◽  
George H. Jones
Keyword(s):  
Escherichia Coli ◽  
Streptomyces Coelicolor ◽  
Loop Structure ◽  
Polynucleotide Phosphorylase ◽  
Mobility Shift ◽  
Stem Loop ◽  
Streptomyces Antibioticus ◽  
E Coli ◽  
Stem Loop Structure ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT We examined the activity of polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli in phosphorolysis using substrates derived from the rpsO-pnp operon of S. coelicolor. The Streptomyces and E. coli enzymes were both able to digest a substrate with a 3′ single-stranded tail although E. coli PNPase was more effective in digesting this substrate than were the Streptomyces enzymes. The k cat for the E. coli enzyme was ca. twofold higher than that observed with the S. coelicolor enzyme. S. coelicolor PNPase was more effective than its E. coli counterpart in digesting a substrate possessing a 3′ stem-loop structure, and the Km for the E. coli enzyme was ca. twice that of the S. coelicolor enzyme. Electrophoretic mobility shift assays revealed an increased affinity of S. coelicolor PNPase for the substrate possessing a 3′ stem-loop structure compared with the E. coli enzyme. We observed an effect of nucleoside diphosphates on the activity of the S. coelicolor PNPase but not the E. coli enzyme. In the presence of a mixture of 20 μM ADP, CDP, GDP, and UDP, the Km for the phosphorolysis of the substrate with the 3′ stem-loop was some fivefold lower than the value observed in the absence of nucleoside diphosphates. No effect of nucleoside diphosphates on the phosphorolytic activity of E. coli PNPase was observed. To our knowledge, this is the first demonstration of an effect of nucleoside diphosphates, the normal substrates for polymerization by PNPase, on the phosphorolytic activity of that enzyme.

scholarly journals (p)ppGpp Inhibits Polynucleotide Phosphorylase from Streptomyces but Not from Escherichia coli and Increases the Stability of Bulk mRNA in Streptomyces coelicolor

2010 ◽  
Vol 192 (17) ◽  
pp. 4275-4280 ◽  
Author(s):  
Marcha L. Gatewood ◽  
George H. Jones
Keyword(s):  
Escherichia Coli ◽  
Chemical Stability ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Polynucleotide Phosphorylase ◽  
Streptomyces Antibioticus ◽  
E Coli ◽  
The Stability

ABSTRACT ppGpp regulates gene expression in a variety of bacteria and in plants. We proposed previously that ppGpp or its precursor, pppGpp [referred to collectively as (p)ppGpp], or both might regulate the activity of the enzyme polynucleotide phosphorylase in Streptomyces species. We have examined the effects of (p)ppGpp on the polymerization and phosphorolysis activities of PNPase from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli. We have shown that (p)ppGpp inhibits the activities of both Streptomyces PNPases but not the E. coli enzyme. The inhibition kinetics for polymerization using the Streptomyces enzymes are of the mixed noncompetitive type, suggesting that (p)ppGpp binds to a region other than the active site of the enzyme. ppGpp also inhibited the phosphorolysis of a model RNA substrate derived from the rpsO-pnp operon of S. coelicolor. We have shown further that the chemical stability of mRNA increases during the stationary phase in S. coelicolor and that induction of a plasmid-borne copy of relA in a relA-null mutant increases the chemical stability of bulk mRNA as well. We speculate that the observed inhibition in vitro may reflect a role of ppGpp in the regulation of antibiotic production in vivo.

scholarly journals RNase III Is Required for Actinomycin Production in Streptomyces antibioticus

2013 ◽  
Vol 79 (20) ◽  
pp. 6447-6451 ◽  
Author(s):  
Jung-Hoon Lee ◽  
Marcha L. Gatewood ◽  
George H. Jones
Keyword(s):  
Parental Strain ◽  
Insertional Mutagenesis ◽  
Southern Blotting ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Production Medium ◽  
Wild Type ◽  
Rnase Iii ◽  
Content Type ◽  
Streptomyces Antibioticus

ABSTRACTUsing insertional mutagenesis, we have disrupted the RNase III gene,rnc, of the actinomycin-producing streptomycete,Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of therncdisruption with the wild-typerncgene fromS. antibioticusrestored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case inStreptomyces coelicolor, RNase III is required for antibiotic production inS. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of theS. antibioticus rncmutant and its parental strain.

scholarly journals A Streptomyces coelicolor Antibiotic Regulatory Gene, absB, Encodes an RNase III Homolog

1999 ◽  
Vol 181 (19) ◽  
pp. 6142-6151 ◽  
Author(s):  
Brenda Price ◽  
Trifon Adamidis ◽  
Renqui Kong ◽  
Wendy Champness
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Open Reading Frame ◽  
Dependent Manner ◽  
Rnase Iii ◽  
Reading Frame ◽  
E Coli ◽  
Lines Of Evidence ◽  
Global Defect

ABSTRACT Streptomyces coelicolor produces four genetically and structurally distinct antibiotics in a growth-phase-dependent manner.S. coelicolor mutants globally deficient in antibiotic production (Abs− phenotype) have previously been isolated, and some of these were found to define the absB locus. In this study, we isolated absB-complementing DNA and show that it encodes the S. coelicolor homolog of RNase III (rnc). Several lines of evidence indicate that theabsB mutant global defect in antibiotic synthesis is due to a deficiency in RNase III. In marker exchange experiments, the S. coelicolor rnc gene rescued absB mutants, restoring antibiotic production. Sequencing the DNA of absB mutants confirmed that the absB mutations lay in thernc open reading frame. Constructed disruptions ofrnc in both S. coelicolor 1501 andStreptomyces lividans 1326 caused an Abs−phenotype. An absB mutation caused accumulation of 30S rRNA precursors, as had previously been reported for E. coli rncmutants. The absB gene is widely conserved in streptomycetes. We speculate on why an RNase III deficiency could globally affect the synthesis of antibiotics.

scholarly journals Genome Sequence of the Streptomycin-Producing Microorganism Streptomyces griseus IFO 13350

2008 ◽  
Vol 190 (11) ◽  
pp. 4050-4060 ◽  
Author(s):  
Yasuo Ohnishi ◽  
Jun Ishikawa ◽  
Hirofumi Hara ◽  
Hirokazu Suzuki ◽  
Miwa Ikenoya ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Streptomyces Coelicolor ◽  
Streptomyces Griseus ◽  
Aminoglycoside Antibiotic ◽  
Gene Clusters ◽  
Open Reading Frames ◽  
Streptomyces Avermitilis ◽  
Trna Genes ◽  
Biosynthesis Gene Cluster ◽  
Regulatory Cascade

ABSTRACT We determined the complete genome sequence of Streptomyces griseus IFO 13350, a soil bacterium producing an antituberculosis agent, streptomycin, which is the first aminoglycoside antibiotic, discovered more than 60 years ago. The linear chromosome consists of 8,545,929 base pairs (bp), with an average G+C content of 72.2%, predicting 7,138 open reading frames, six rRNA operons (16S-23S-5S), and 66 tRNA genes. It contains extremely long terminal inverted repeats (TIRs) of 132,910 bp each. The telomere's nucleotide sequence and secondary structure, consisting of several palindromes with a loop sequence of 5′-GGA-3′, are different from those of typical telomeres conserved among other Streptomyces species. In accordance with the difference, the chromosome has pseudogenes for a conserved terminal protein (Tpg) and a telomere-associated protein (Tap), and a novel pair of Tpg and Tap proteins is instead encoded by the TIRs. Comparisons with the genomes of two related species, Streptomyces coelicolor A3(2) and Streptomyces avermitilis, clarified not only the characteristics of the S. griseus genome but also the existence of 24 Streptomyces-specific proteins. The S. griseus genome contains 34 gene clusters or genes for the biosynthesis of known or unknown secondary metabolites. Transcriptome analysis using a DNA microarray showed that at least four of these clusters, in addition to the streptomycin biosynthesis gene cluster, were activated directly or indirectly by AdpA, which is a central transcriptional activator for secondary metabolism and morphogenesis in the A-factor (a γ-butyrolactone signaling molecule) regulatory cascade in S. griseus.

scholarly journals Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase Expression Revisited

2009 ◽  
Vol 191 (6) ◽  
pp. 1738-1748 ◽  
Author(s):  
Thomas Carzaniga ◽  
Federica Briani ◽  
Sandro Zangrossi ◽  
Giuseppe Merlino ◽  
Paolo Marchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Current Model ◽  
Dependent Manner ◽  
Rnase E ◽  
Polynucleotide Phosphorylase ◽  
Rnase Iii ◽  
Independent Manner ◽  
Stem Loop ◽  
Autogenous Regulation ◽  
Additional Support

ABSTRACT The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5′-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III-processed pnp mRNA, thus exposing the transcript to degradative pathways. More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus destroying the double-stranded structure at the 5′ end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5′ double-stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double-stranded structure at its 5′ end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus, the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5′ fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript.

scholarly journals Autoregulation of AbsB (RNase III) Expression in Streptomyces coelicolor by Endoribonucleolytic Cleavage of absB Operon Transcripts

2008 ◽  
Vol 190 (15) ◽  
pp. 5526-5530 ◽  
Author(s):  
Weijing Xu ◽  
Jianqiang Huang ◽  
Stanley N. Cohen
Keyword(s):  
Streptomyces Coelicolor ◽  
Regulatory Role ◽  
Antibiotic Biosynthesis ◽  
Rnase Iii ◽  
Stem Loop ◽  
Polycistronic Transcript

ABSTRACT The Streptomyces coelicolor absB gene encodes an RNase III family endoribonuclease and is normally essential for antibiotic biosynthesis. Here we report that AbsB controls its own expression by sequentially and site specifically cleaving stem-loop segments of its polycistronic transcript. Our results demonstrate a ribonucleolytic regulatory role for AbsB in vivo.

CHARACTERIZATION OF THE AUXIN SYNTHESIS GENES OF ERWINIA HERBICOLA PV. GYPSOPHILAE

1997 ◽  
Vol 45 (4) ◽  
pp. 279-284 ◽  
Author(s):  
Yedidya Gafni ◽  
Shulamit Manulis ◽  
Talya Kunik ◽  
Amnon Lichter ◽  
Isaac Barash ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Unit ◽  
Open Reading Frames ◽  
Virulence Plasmid ◽  
Base Pairs ◽  
Erwinia Herbicola ◽  
E Coli ◽  
Auxin Synthesis ◽  
Translation Initiation Codon

We present the DNA sequence of some 3590 base pairs from the virulence plasmid of the crown-gall-forming bacteriumErwinia herbicolapv.gypsophilaestrain PD713. This region includes the entire transcription unit of an operon encompassing the IAA synthesis genes. These genes, designatediaaMandiaaH, reside on a 78-MDa native plasmid. The sequence analysis indicates that the operon contains two open reading frames of 562 (iaaM) and 460 amino acids (iaaH), corresponding to proteins with molecular weight of 62,473 and 49,300 daltons, respectively. When cloned and expressed in minicell-producingE. coli, this sequence encodes production of two proteins, 49 and 62 kDa. Both genes show moderate to high homology to IAA synthesis genes from other plant-associated bacteria. Analysis by primer extension of the transcription initiation site located it 63 bases upstream of the translation initiation codon.

scholarly journals Complex Regulation of the Organic Hydroperoxide Resistance Gene (ohr) from XanthomonasInvolves OhrR, a Novel Organic Peroxide-Inducible Negative Regulator, and Posttranscriptional Modifications

2001 ◽  
Vol 183 (15) ◽  
pp. 4405-4412 ◽  
Author(s):  
Rojana Sukchawalit ◽  
Suvit Loprasert ◽  
Sopapan Atichartpongkul ◽  
Skorn Mongkolsuk
Keyword(s):  
Secondary Structure ◽  
Organic Peroxide ◽  
Primer Extension ◽  
Negative Regulator ◽  
Rnase Iii ◽  
Stem Loop ◽  
Strong Activity ◽  
Reading Frame ◽  
Bicistronic Mrna

ABSTRACT Analysis of the sequence immediate upstream of ohrrevealed an open reading frame, designated ohrR, with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression. ohrR was transcribed as bicistronic mRNA with ohr, while ohr mRNA was found to be 95% monocistronic and 5% bicistronic with ohrR. Expression of both genes was induced by tert-butyl hydroperoxide (tBOOH) treatment. High-level expression ofohrR negatively regulated ohr expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the ohrR promoter (P1) has organic peroxide-inducible, strong activity, while the ohrpromoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR. ohr primer extension results revealed three major primer extension products corresponding to the 5′ ends ofohr mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical Xanthomonas promoter. Instead, the regions can form a stem-loop secondary structure with the 5′ ends ofohr mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the ohrR-ohr operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of ohr mRNA, while ohrR mRNA is rapidly degraded.

pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

1998 ◽  
Vol 44 (12) ◽  
pp. 1186-1192
Author(s):  
Guy Daxhelet ◽  
Philippe Gilot ◽  
Etienne Nyssen ◽  
Philippe Hoet
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Ribosome Binding Site ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

Sign in / Sign up

Export Citation Format

Share Document