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 Actinomycin Production Persists in a Strain ofStreptomyces antibioticus Lacking Phenoxazinone Synthase

2000 ◽  
Vol 44 (5) ◽  
pp. 1322-1327 ◽  
Author(s):  
George H. Jones
Keyword(s):  
Southern Blotting ◽  
Wild Type Strain ◽  
Antibiotic Production ◽  
Conjugative Plasmid ◽  
Wild Type ◽  
Genetic Changes ◽  
Streptomyces Antibioticus ◽  
Phenoxazinone Synthase ◽  
Wild Type Parent ◽  
Production Strain

ABSTRACT Truncated fragments of the phenoxazinone synthase gene,phsA, were prepared by the PCR. The resulting fragments were cloned into conjugative plasmid pKC1132 and transferred toStreptomyces antibioticus by conjugation fromEscherichia coli. Two of the resulting constructs were integrated into the S. antibioticus chromosome by homologous recombination, and each of the resulting strains, designated 3720/pJSE173 and 3720/pJSE174, contained a disrupted phsAgene. Strain 3720/pJSE173 grew poorly, and Southern blotting suggested that genetic changes other than the disruption of the phsAgene might have occurred during the construction of that strain. Strain 3720/pJSE174 sporulated well and grew normally on the medium used to prepare inocula for antibiotic production. Strain 3720/pJSE174 also grew as well as the wild-type strain on antibiotic production medium containing either 1 or 5.7 mM phosphate. Strain 3720/pJSE174 was shown to be devoid of phenoxazinone synthase (PHS) activity, and PHS protein was undetectable in this strain by Western blotting. Despite the absence of detectable PHS activity, strain 3720/pJSE174 produced slightly more actinomycin than did the wild-type parent strain in medium containing 1 or 5.7 mM phosphate. The observation that strain 3720/pJSE174, lacking detectable PHS protein or enzyme activity, retained the ability to produce actinomycin supports the conclusion that PHS is not required for actinomycin biosynthesis in S. antibioticus.

scholarly journals relA Is Required for Actinomycin Production in Streptomyces antibioticus

1999 ◽  
Vol 181 (12) ◽  
pp. 3824-3829 ◽  
Author(s):  
Shannan Hoyt ◽  
George H. Jones
Keyword(s):  
Biosynthetic Pathway ◽  
Wild Type Strain ◽  
Streptomyces Coelicolor ◽  
Production Medium ◽  
Wild Type ◽  
Streptomyces Antibioticus ◽  
Phenoxazinone Synthase ◽  
Key Enzyme ◽  
In The Wild ◽  
Phosphate Medium

ABSTRACT The relA gene from Streptomyces antibioticus has been cloned and sequenced. The gene encodes a protein with an M r of 93,653, which is 91% identical to the corresponding protein from Streptomyces coelicolor. Disruption of S. antibioticus relAproduces a strain which grows significantly more slowly on actinomycin production medium than the wild type or a disruptant to which the intact relA gene was restored. Moreover, the disruptant was unable to accumulate ppGpp to the levels observed during the normal course of growth and actinomycin production in the wild type. The strain containing the disrupted relA gene did not produce actinomycin and contained significantly lower levels of the enzyme phenoxazinone synthase than the wild-type strain. Actinomycin synthetase I, a key enzyme in the actinomycin biosynthetic pathway, was undetectable in the relA disruptant. Growth of the disruptant on low-phosphate medium did not restore actinomycin production.

scholarly journals Substrate Inhibition of VanA by d-Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner

2016 ◽  
Vol 60 (8) ◽  
pp. 4930-4939 ◽  
Author(s):  
Lizah T. van der Aart ◽  
Nicole Lemmens ◽  
Willem J. van Wamel ◽  
Gilles P. van Wezel
Keyword(s):  
Cell Wall ◽  
Streptomyces Coelicolor ◽  
Substrate Inhibition ◽  
Model Organism ◽  
Vancomycin Resistance ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
Lipid Ii ◽  
Vancomycin Resistant

ABSTRACTThe increasing resistance of clinical pathogens against the glycopeptide antibiotic vancomycin, a last-resort drug against infections with Gram-positive pathogens, is a major problem in the nosocomial environment. Vancomycin inhibits peptidoglycan synthesis by binding to thed-Ala–d-Ala terminal dipeptide moiety of the cell wall precursor lipid II. Plasmid-transferable resistance is conferred by modification of the terminal dipeptide into the vancomycin-insensitive variantd-Ala–d-Lac, which is produced by VanA. Here we show that exogenousd-Ala competes withd-Lac as a substrate for VanA, increasing the ratio of wild-type to mutant dipeptide, an effect that was augmented by several orders of magnitude in the absence of thed-Ala–d-Ala peptidase VanX. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that high concentrations ofd-Ala led to the production of a significant amount of wild-type cell wall precursors, whilevanX-null mutants produced primarily wild-type precursors. This enhanced the efficacy of vancomycin in the vancomycin-resistant model organismStreptomyces coelicolor, and the susceptibility of vancomycin-resistant clinical isolates ofEnterococcus faecium(VRE) increased by up to 100-fold. The enhanced vancomycin sensitivity ofS. coelicolorcells correlated directly to increased binding of the antibiotic to the cell wall. Our work offers new perspectives for the treatment of diseases associated with vancomycin-resistant pathogens and for the development of drugs that target vancomycin resistance.

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

2011 ◽  
Vol 77 (21) ◽  
pp. 7586-7594 ◽  
Author(s):  
Fernando Santos-Beneit ◽  
Mónica Barriuso-Iglesias ◽  
Lorena T. Fernández-Martínez ◽  
Miriam Martínez-Castro ◽  
Alberto Sola-Landa ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Response Regulator ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Luciferase Reporter ◽  
Antibiotic Biosynthesis ◽  
Binding Assays ◽  
Content Type ◽  
Phosphate Depletion

ABSTRACTThe RNA polymerase (RNAP) omega factor (ω) forms a complex with the α2ββ′ core of this enzyme in bacteria. We have characterized therpoZgene ofStreptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of therpoZgene 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 ΔrpoZstrain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ΔrpoZmutant with the wild-typerpoZallele restored both phenotype and antibiotic production. Interestingly, therpoZgene 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 thatrpoZpromoter activity was increased in a ΔphoPbackground, it can be concluded thatrpoZis controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation inStreptomyces.

scholarly journals Influence of the Protein Kinase C Activator Phorbol Myristate Acetate on the Intracellular Activity of Antibiotics against Hemin- and Menadione-Auxotrophic Small-Colony Variant Mutants of Staphylococcus aureus and Their Wild-Type Parental Strain in Human THP-1 Cells

2012 ◽  
Vol 56 (12) ◽  
pp. 6166-6174 ◽  
Author(s):  
Laetitia G. Garcia ◽  
Sandrine Lemaire ◽  
Barbara C. Kahl ◽  
Karsten Becker ◽  
Richard A. Proctor ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Parental Strain ◽  
Wild Type ◽  
Content Type ◽  
Small Colony Variant ◽  
Intracellular Activity ◽  
Kinase C ◽  
Pharmacodynamic Profile ◽  
Small Colony ◽  
Intracellular Fate

ABSTRACTIn a previous study (L. G. Garcia et al., Antimicrob. Agents Chemother. 56:3700–3711, 2012), we evaluated the intracellular fate ofmenDandhemBmutants (corresponding to menadione- and hemin-dependent small-colony variants, respectively) of the parental COL methicillin-resistantStaphylococcus aureusstrain and the pharmacodynamic profile of the intracellular activity of a series of antibiotics in human THP-1 monocytes. We have now examined the phagocytosis and intracellular persistence of the same strains in THP-1 cells activated by phorbol 12-myristate 13-acetate (PMA) and measured the intracellular activity of gentamicin, moxifloxacin, and oritavancin in these cells. Postphagocytosis intracellular counts and intracellular survival were lower in PMA-activated cells, probably due to their higher killing capacities. Gentamicin and moxifloxacin showed a 5- to 7-fold higher potency (lower static concentrations) against the parental strain, itshemBmutant, and the genetically complemented strain in PMA-activated cells and against themenDstrain in both activated and nonactivated cells. This effect was inhibited when cells were incubated withN-acetylcysteine (a scavenger of oxidant species). In parallel, we observed that the MICs of these drugs were markedly reduced if bacteria had been preexposed to H2O2. In contrast, the intracellular potency of oritavancin was not different in activated and nonactivated cells and was not decreased by the addition ofN-acetylcysteine, regardless of the phenotype of the strains. The oritavancin MIC was also unaffected by preincubation of the bacteria with H2O2. Thus, activation of THP-1 cells by PMA may increase the intracellular potency of certain antibiotics (probably due to synergy with reactive oxygen species), but this effect cannot be generalized to all antibiotics.

scholarly journals γ-Glutamyl Spermine Synthetase PauA2 as a Potential Target of Antibiotic Development against Pseudomonas aeruginosa

2012 ◽  
Vol 56 (10) ◽  
pp. 5309-5314 ◽  
Author(s):  
Xiangyu Yao ◽  
Congran Li ◽  
Jianmei Zhang ◽  
Chung-Dar Lu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Human Serum ◽  
Parental Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Strong Support ◽  
Uptake System ◽  
Wild Type ◽  
Content Type ◽  
Antibiotic Development

ABSTRACTPolyamines are absolute requirements for cell growth. When in excess,Pseudomonas aeruginosapossesses six γ-glutamylpolyamine synthetases (GPSs) encoded by thepauA1-pauA7genes to initiate polyamine catabolism. Recently, thepauA2mutant was reported to lose the capability to grow on spermine (Spm) and spermidine (Spd) as sole carbon and nitrogen sources. Although this mutant grew normally in defined minimal medium and LB broth, growth was completely abolished by the addition of Spm or Spd. These two compounds exert a bactericidal effect (Spm > Spd) on the mutants as demonstrated by MIC measurements (over 500-fold reduction) and time-killing curves. Spm toxicity in thepauA2mutant was attenuated when the major uptake system was further deleted from the strain, suggesting cytoplasmic targets of toxicity. In addition, the synergistic effect of Spm and carbenicillin in the wild-type strain PAO1 was diminished in mutants without functional PauA2. Furthermore, Spm MIC was reduced by 8-fold when the Spm uptake system was deleted from the wild-type strain, suggesting a second target of Spm toxicity in the periplasm. Experiments were also conducted to test the hypothesis that native Spm and Spd in human serum may be sufficient to kill thepauA2mutant. Growth of the mutant was completely inhibited by 40% (vol/vol) human serum, whereas the parental strain required 80%. Colony counts indicated that the mutant but not the parent was in fact killed by human plasma. In addition, carbenicillin MIC against the mutant was reduced by 16-fold in the presence of 20% human serum while that of the parental strain remained unchanged. Taking PauA2 as the template, sequence comparison indicates that putative PauA2 homologues are widespread in a variety of Gram-negative bacteria. In summary, this study reveals the importance of GPS in alleviation of polyamine toxicity when in excess, and it provides strong support to the feasibility of GPS as a molecular target for new antibiotic development.

scholarly journals EshA Accentuates ppGpp Accumulation and Is Conditionally Required for Antibiotic Production in Streptomyces coelicolor A3(2)

2006 ◽  
Vol 188 (13) ◽  
pp. 4952-4961 ◽  
Author(s):  
Natsumi Saito ◽  
Jun Xu ◽  
Takeshi Hosaka ◽  
Susumu Okamoto ◽  
Hiroyuki Aoki ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Molecular Signaling ◽  
Wild Type ◽  
Nucleotide Binding Domain ◽  
In The Wild ◽  
Actinorhodin Production ◽  
Cyclic Nucleotide Binding Domain

ABSTRACT Disruption of eshA, which encodes a 52-kDa protein that is produced late during the growth of Streptomyces coelicolor A3(2), resulted in elimination of actinorhodin production. In contrast, disruption of eshB, a close homologue of eshA, had no effect on antibiotic production. The eshA disruptant accumulated lower levels of ppGpp than the wild-type strain accumulated. The loss of actinorhodin production in the eshA disruptant was restored by expression of a truncated relA gene, which increased the ppGpp level to the level in the wild-type strain, indicating that the reduced ppGpp accumulation in the eshA mutant was solely responsible for the loss of antibiotic production. Antibiotic production was also restored in the eshA mutant by introducing mutations into rpoB (encoding the RNA polymerase β subunit) that bypassed the requirement for ppGpp, which is consistent with a role for EshA in modulating ppGpp levels. EshA contains a cyclic nucleotide-binding domain that is essential for its role in triggering actinorhodin production. EshA may provide new insights and opportunities to unravel the molecular signaling events that occur during physiological differentiation in streptomycetes.

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 Identification of Three New Genes Involved in Morphogenesis and Antibiotic Production in Streptomyces coelicolor

2003 ◽  
Vol 185 (20) ◽  
pp. 6147-6157 ◽  
Author(s):  
Ondrej Sprusansky ◽  
Liqin Zhou ◽  
Sarah Jordan ◽  
Jared White ◽  
Janet Westpheling
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Transcriptional Regulators ◽  
Protein Product ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Prolonged Incubation ◽  
New Genes ◽  
Rich Media

ABSTRACT We report the isolation and partial characterization of three new mutants of Streptomyces coelicolor that are defective in morphogenesis and antibiotic production. The genes identified by the mutations were located and cloned by using a combination of Tn5 in vitro mutagenesis, cotransformation, and genetic complementation. Mutant SE69 produces lower amounts of antibiotics than the wild type produces, produces spores only after prolonged incubation on rich media, and identifies a gene whose predicted protein product is similar to the GntR family of transcriptional regulators; also, production of aerial mycelia on both rich and poor media is significantly delayed in this mutant. Mutant SE293 is defective in morphogenesis, overproduces antibiotics on rich media, fails to grow on minimal media, and identifies a gene whose predicted protein product is similar to the TetR family of transcriptional regulators. Preliminary evidence suggests that the SE293 gene product may control a molybdopterin binding protein located immediately adjacent to it. Mutant SJ175 sporulates sooner and more abundantly than the wild type and overproduces antibiotics on rich media, and it identifies a gene whose predicted protein product contains regions of predominantly hydrophobic residues similar to those of integral membrane proteins.

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