scholarly journals Characterization of Replication and Conjugation of Streptomyces Circular Plasmids pFP1 and pFP11 and Their Ability To Propagate in Linear Mode with Artificially Attached Telomeres

2008 ◽  
Vol 74 (11) ◽  
pp. 3368-3376 ◽  
Author(s):  
Ran Zhang ◽  
Ana Zeng ◽  
Ping Fang ◽  
Zhongjun Qin
Keyword(s):  
Plasmid Transfer ◽  
Dna Transfer ◽  
Linear Plasmids ◽  
Gene Encoding ◽  
Gram Negative ◽  
Linear Mode ◽  
Dna Translocase ◽  
Stable Linear

ABSTRACT Many Streptomyces species harbor circular plasmids (8 to 31 kb) as well as linear plasmids (12 to 1,700 kb). We report the characterization of two newly detected circular plasmids, pFP11 (35,139 bp) and pFP1 (39,360 bp). As on linear plasmids, their replication loci comprise repA genes and adjacent iterons, to which RepA proteins bind specifically in vitro. Plasmids containing the minimal iterons plus the repA locus of pFP11 were inherited extremely unstably; par and additional loci were required for stable inheritance. Surprisingly, plasmids containing replication loci from pFP11 or Streptomyces circular plasmid SCP2 but not from pFP1, SLP1, or pIJ101 propagated in a stable linear mode when the telomeres of a linear plasmid were attached. These results indicate bidirectional replication for pFP11 and SCP2. Both pFP11 and pFP1 contain, for plasmid transfer, a major functional traB gene (encoding a DNA translocase typical for Streptomyces plasmids) as well as, surprisingly, a putative traA gene (encoding a DNA nickase, characteristic of single-stranded DNA transfer of gram-negative plasmids), but this did not appear to be functional, at least in isolation.

scholarly journals Two Internal Origins of Replication in Streptomyces Linear Plasmid pFRL1

2010 ◽  
Vol 76 (17) ◽  
pp. 5676-5683 ◽  
Author(s):  
Ran Zhang ◽  
Shiyuan Peng ◽  
Zhongjun Qin
Keyword(s):  
Reverse Transcription ◽  
Northern Hybridization ◽  
Linear Plasmids ◽  
Time Points ◽  
Linear Mode ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr ◽  
Origins Of Replication ◽  
Additional Locus

ABSTRACT Previous reports showed that Streptomyces linear plasmids usually contain one internal replication locus. Here, we identified two new replication loci on pFRL1, one (rep1A-ncs1) next to a telomere and another (rep2A-ncs2) ∼10 kb from it. The rep1A-ncs1 locus was able to direct replication independently in both linear and circular modes, whereas rep2A-ncs2 required an additional locus, rlrA-rorA, in order to direct propagation in linear mode. Rep1A protein bound to ncs1 in vitro. By quantitative reverse transcription-PCR and Northern hybridization, we showed that transcription of rep1A and rep2A varied during development and that each dominated at different time points. pFRL1-derived linear plasmids were inherited through spores more stably than circular plasmids and were more stable with pSLA2 telomeres than with pFRL1 telomeres in Streptomyces lividans.

scholarly journals Characterization of the tyramine-producing pathway in Sporolactobacillus sp. P3J

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1841-1849 ◽  
Author(s):  
Monika Coton ◽  
María Fernández ◽  
Hein Trip ◽  
Victor Ladero ◽  
Niels L. Mulder ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Cloning And Expression ◽  
Clear Correlation ◽  
Tyrosine Decarboxylase ◽  
Gene Encoding ◽  
Polycistronic Mrna ◽  
Tyrosine Concentration ◽  
Putative Phage

A sporulated lactic acid bacterium (LAB) isolated from cider must was shown to harbour the tdc gene encoding tyrosine decarboxylase. The isolate belonged to the Sporolactobacillus genus and may correspond to a novel species. The ability of the tdc-positive strain, Sporolactobacillus sp. strain P3J, to produce tyramine in vitro was demonstrated by using HPLC. A 7535 bp nucleotide sequence harbouring the putative tdc gene was determined. Analysis of the obtained sequence showed that four tyramine production-associated genes [tyrosyl-tRNA synthetase (tyrS), tyrosine decarboxylase (tdc), tyrosine permease (tyrP) and Na+/H+ antiporter (nhaC)] were present and were organized as already described in other tyramine-producing LAB. This operon was surrounded by genes showing the highest identities with mobile elements: a putative phage terminase and a putative transposase (downstream and upstream, respectively), suggesting that the tyramine-forming trait was acquired through horizontal gene transfer. Transcription analyses of the tdc gene cluster suggested that tyrS and nhaC are expressed as monocistronic genes while tdc would be part of a polycistronic mRNA together with tyrP. The presence of tyrosine in the culture medium induced the expression of all genes except for tyrS. A clear correlation was observed between initial tyrosine concentration and tyramine production combined with an increase in the final pH reached by the culture. Finally, cloning and expression of the tyrP gene in Lactococcus lactis demonstrated that its product catalyses the exchange of tyrosine and tyramine.

scholarly journals Characterization of the Exoglucanase-Encoding Gene PaEXG2 and Study of Its Role in the Biocontrol Activity of Pichia anomala Strain K

2003 ◽  
Vol 93 (9) ◽  
pp. 1145-1152 ◽  
Author(s):  
Cathy Grevesse ◽  
Philippe Lepoivre ◽  
Mohamed Haïssam Jijakli
Keyword(s):  
Marker Gene ◽  
Glycosylation Site ◽  
Pichia Anomala ◽  
Gene Encoding ◽  
Biocontrol Activity ◽  
Monophosphate Decarboxylase ◽  
Uracil Auxotroph

The PaEXG2 gene, encoding an exo-β-1,3-glucanase, was isolated from the biocontrol agent Pichia anomala strain K. PaEXG2 has the capacity for coding an acidic protein of 427 amino acids with a predicted molecular weight of 45.7 kDa, a calculated pI of 4.7, and one potential N-glycosylation site. PaEXG2 was disrupted by the insertion of the URA3 marker gene, encoding orotidine monophosphate decarboxylase in strain KU1, a uracil auxotroph derived from strain K. Strain KU1 showed inferior biocontrol activity and colonization of wounds on apples, compared to the prototrophic strain. Antagonism and colonization were recovered after the restoration of prototrophy by transformation with the URA3 gene. Integrative transformation was shown to be mostly ectopic in strain K descendants (only 4% of integration by homologous recombination). PaEXG2 disruption abolished all detectable extracellular exo-β-1,3-glucanase activity in vitro and in situ but did not affect biocontrol of Botrytis cinerea on wounded apples.

scholarly journals Characterization of the Genetic Components of Streptomyces lividans Linear Plasmid SLP2 for Replication in Circular and Linear Modes

2006 ◽  
Vol 188 (19) ◽  
pp. 6851-6857 ◽  
Author(s):  
Mingxuan Xu ◽  
Yingmin Zhu ◽  
Ran Zhang ◽  
Meijuan Shen ◽  
Weihong Jiang ◽  
...  
Keyword(s):  
Streptomyces Lividans ◽  
Linear Plasmid ◽  
Replication Proteins ◽  
Linear Mode ◽  
Genetic Components ◽  
Efficient Replication ◽  
Telomere Replication ◽  
In Vitro Interactions

ABSTRACT The nucleotide sequence of Streptomyces lividans linear plasmid SLP2 consists of 50,410 bp (C. H. Huang, C. Y. Chen, H. H. Tsai, C. Chen, Y. S. Lin, and C. W. Chen, Mol. Microbiol. 47:1563-1576, 2003). Here we report that the basic SLP2 locus for plasmid replication in circular mode resembles that of Streptomyces linear plasmids pSLA2 and SCP1 and comprises iteronsSLP2 and the adjacent rep SLP2 gene. More efficient replication additionally required the 47-bp sequence between bp 581 and 628 upstream of the iterons. Replacement of either the iterons or the rep gene of SLP2 by the corresponding genes of pSLA2 or SCP1 still allows propagation in Streptomyces, although the transformation frequencies were 3 orders of magnitude lower than the original plasmids, suggesting that these plasmids share similar replication mechanisms. To replicate SLP2 in linear mode, additional SLP2 loci—either mtap SLP2 /tpg SLP2 or mtap SLP2 /ilrA SLP2 —were required. IlrASLP2 protein binds specifically to the iteronsSLP2 in vitro. Interactions were detected between these SLP2-borne replication proteins (MtapSLP2, TpgSLP2, and IlrASLP2) and the telomeric replication proteins (TpgL, TapL, and TpgL) of the S. lividans chromosome, respectively, but the SLP2 proteins failed to interact. These results suggest that SLP2 recruits chromosomally encoded replication proteins for its telomere replication.

scholarly journals Release of Nonstop Ribosomes Is Essential

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Heather A. Feaga ◽  
Patrick H. Viollier ◽  
Kenneth C. Keiler
Keyword(s):  
Messenger Rna ◽  
Caulobacter Crescentus ◽  
Stop Codon ◽  
Selective Advantage ◽  
Gene Encoding ◽  
Content Type ◽  
Bacterial Phyla ◽  
Almost All

ABSTRACTBacterial ribosomes frequently translate to the 3′ end of an mRNA without terminating at a stop codon. Almost all bacteria use the transfer-messenger RNA (tmRNA)-basedtrans-translation pathway to release these “nonstop” ribosomes and maintain protein synthesis capacity.trans-translation is essential in some species, but in others, such asCaulobacter crescentus,trans-translation can be inactivated. To determine whytrans-translation is dispensable inC. crescentus, a Tn-seq screen was used to identify genes that specifically alter growth in cells lackingssrA, the gene encoding tmRNA. One of these genes,CC1214, was essential in ΔssrAcells. Purified CC1214 protein could release nonstop ribosomesin vitro. CC1214 is a homolog of theEscherichia coliArfB protein, and using the CC1214 sequence, ArfB homologs were identified in the majority of bacterial phyla. Most species in whichssrAhas been deleted contain an ArfB homolog, suggesting that release of nonstop ribosomes may be essential in most or all bacteria.IMPORTANCEGenes that are conserved across large phylogenetic distances are expected to confer a selective advantage. The genes required fortrans-translation,ssrAandsmpB, have been found in >99% of sequenced bacterial genomes, suggesting that they are broadly important. However, these genes can be deleted in some species without loss of viability. The identification and characterization ofC. crescentusArfB reveals whytrans-translation is not essential inC. crescentusand suggests that many other bacteria are likely to use ArfB to survive whentrans-translation is compromised.

scholarly journals Identification and Characterization of the Bacillus thuringiensis phaZ Gene, Encoding New Intracellular Poly-3-Hydroxybutyrate Depolymerase

2006 ◽  
Vol 188 (21) ◽  
pp. 7592-7599 ◽  
Author(s):  
Chi-Ling Tseng ◽  
Hui-Ju Chen ◽  
Gwo-Chyuan Shaw
Keyword(s):  
Bacillus Thuringiensis ◽  
Wild Type ◽  
Gene Encoding ◽  
Significant Similarity ◽  
Phb Depolymerase ◽  
New Type ◽  
Identification And Characterization

ABSTRACTA gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome ofBacillus thuringiensissubsp.israelensisATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designatedphaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD ofPseudomonas putidawas unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. TheB. thuringiensisPhaZ was inactive againstp-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-typeB. thuringiensiscells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from aphaZmutant lost this activity. ThephaZmutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S102-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate thatB. thuringiensisharbors a new type of intracellular PHB depolymerase.

Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Biochemical Features and Therapeutic Approaches

2007 ◽  
Vol 27 (1-3) ◽  
pp. 151-163 ◽  
Author(s):  
M. C. Lara ◽  
M. L. Valentino ◽  
J. Torres-Torronteras ◽  
M. Hirano ◽  
R. Martí
Keyword(s):  
Molecular Genetic ◽  
In Vivo Studies ◽  
Gene Encoding ◽  
Mitochondrial Neurogastrointestinal Encephalomyopathy ◽  
Mtdna Replication ◽  
Biochemical Features

Over the last 15 years, important research has expanded our knowledge of the clinical, molecular genetic, and biochemical features of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). The characterization of mitochondrial involvement in this disorder and the seminal determination of its genetic cause, have opened new possibilities for more detailed and deeper studies on the pathomechanisms in this progressive and fatal disease. It has been established that MNGIE is caused by mutations in the gene encoding thymidine phosphorylase (TP), which lead to absolute or nearly complete loss of its catalytic activity, producing systemic accumulations of its substrates, thymidine (dThd) and deoxyuridine (dUrd). Findings obtained from in vitro and in vivo studies indicate that the biochemical imbalances specifically impair mitochondrial DNA (mtDNA) replication, repair, or both leading to mitochondrial dysfunction. We have proposed that therapy for MNGIE should be aimed at reducing the concentrations of these toxic nucleosides to normal or nearly normal levels. The first treatment, allogeneic stem-cell transplantation (alloSCT) reported in 2006, produced a nearly full biochemical correction of the dThd and dUrd imbalances in blood. Clinical follow-up of this and other patients receiving alloSCT is necessary to determine whether this and other therapies based on a permanent restoration of TP will be effective treatment for MNGIE.

scholarly journals Development and Characterization of Novel Cellulose Composites Obtained in 1-Ethyl-3-methylimidazolium Chloride Used as Drug Delivery Systems

Polymers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 2176
Author(s):  
Iuliana Spiridon ◽  
Iuliana-Marilena Andrei ◽  
Narcis Anghel ◽  
Maria Valentina Dinu ◽  
Bianca-Iulia Ciubotaru
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Compressive Strength ◽  
Drug Delivery Systems ◽  
Gram Negative ◽  
Cellulose Composites ◽  
The Matrix ◽  
Mechanical Performances

Two polysaccharides (cellulose and chitosan) and polyurethane dissolved in 1-ethyl-3-methylimidazolium chloride represented the matrix for the obtainment of new composite formulations comprised of lignin, ferrite–lignin hybrid and ketoconazole. The mechanical performances (Young’s modulus and compressive strength) increased with the filler addition. The nature of the filler used in the studied formulations influenced both bioadhesion and mucoadhesion parameters. It was found that the incorporation of lignin and ferrite–lignin hybrid into the matrix has influenced the in vitro rate of ketoconazole release, which is described by the Korsmeyer–Peppas model. All materials exhibited activity against Gram positive (Staphylococcus aureus ATCC 25923) and Gram negative (Escherichia coli ATCC 25922) bacteria.

scholarly journals Antibacterial Activities and Characterization of Novel Inhibitors of LpxC

2002 ◽  
Vol 46 (6) ◽  
pp. 1793-1799 ◽  
Author(s):  
John M. Clements ◽  
Fanny Coignard ◽  
Ian Johnson ◽  
Stephen Chandler ◽  
Shilpa Palan ◽  
...  
Keyword(s):  
Burkholderia Cepacia ◽  
Lipid A ◽  
Antibacterial Activities ◽  
Vitro Assay ◽  
Gram Negative ◽  
E Coli ◽  
Antibacterial Target ◽  
Derivatives Of

ABSTRACT Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) and forms the major lipid component of the outer monolayer of the outer membrane of gram-negative bacteria. Lipid A is required for bacterial growth and virulence, and inhibition of its biosynthesis is lethal to bacteria. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a metalloenzyme that catalyzes the second step in the biosynthesis of lipid A. Inhibitors of LpxC have previously been shown to have antibiotic activities. We have screened a metalloenzyme inhibitor library for antibacterial activities against an Escherichia coli strain with reduced LpxC activity. From this screen, a series of sulfonamide derivatives of the α-(R)-amino hydroxamic acids, exemplified by BB-78484 and BB-78485, have been identified as having potent inhibitory activities against LpxC in an in vitro assay. Leads from this series showed gram-negative selective activities against members of the Enterobacteriaceae, Serratia marcescens, Morganella morganii, Haemophilus influenzae, Moraxella catarrhalis, and Burkholderia cepacia. BB-78484 was bactericidal against E. coli, achieving 3-log killing in 4 h at a concentration 4 times above the MIC, as would be predicted for an inhibitor of lipid A biosynthesis. E. coli mutants with decreased susceptibility to BB-78484 were selected. Analysis of these mutants revealed that resistance arose as a consequence of mutations in the fabZ or lpxC genes. These data confirm the antibacterial target of BB-78484 and BB-78485 and validate LpxC as a target for gram-negative selective antibacterials.

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