scholarly journals Novel Toxin-Antitoxin System Composed of Serine Protease and AAA-ATPase Homologues Determines the High Level of Stability and Incompatibility of the Tumor-Inducing Plasmid pTiC58

2009 ◽  
Vol 191 (14) ◽  
pp. 4656-4666 ◽  
Author(s):  
Shinji Yamamoto ◽  
Kazuya Kiyokawa ◽  
Katsuyuki Tanaka ◽  
Kazuki Moriguchi ◽  
Katsunori Suzuki
Keyword(s):  
Serine Proteases ◽  
Binding Activity ◽  
Open Reading Frames ◽  
Aaa Atpase ◽  
Plant Tumor ◽  
Ti Plasmids ◽  
High Level ◽  
Plasmid Stabilization ◽  
Aaa Atpases ◽  
Reading Frames

ABSTRACT Stability of plant tumor-inducing (Ti) plasmids differs among strains. A high level of stability prevents basic and applied studies including the development of useful strains. The nopaline type Ti plasmid pTiC58 significantly reduces the transconjugant efficiency for incoming incompatible plasmids relative to the other type, such as octopine-type plasmids. In this study we identified a region that increases the incompatibility and stability of the plasmid. This region was located on a 4.3-kbp segment about 38 kbp downstream of the replication locus, repABC. We named two open reading frames in the segment, ietA and ietS, both of which were essential for the high level of incompatibility and stability. Plasmid stabilization by ietAS was accomplished by a toxin-antitoxin (TA) mechanism, where IetS is the toxin and IetA is the antitoxin. A database search revealed that putative IetA and IetS proteins are highly similar to AAA-ATPases and subtilisin-like serine proteases, respectively. Amino acid substitution experiments in each of the highly conserved characteristic residues, in both putative enzymes, suggested that the protease activity is essential and that ATP binding activity is important for the operation of the TA system. The ietAS-containing repABC plasmids expelled Ti plasmids even in strains which were tolerant to conventional Ti-curing treatments.

scholarly journals Heterochromatic Stellate Gene Cluster in Drosophila melanogaster: Structure and Molecular Evolution

Genetics ◽  
1997 ◽  
Vol 146 (1) ◽  
pp. 253-262 ◽  
Author(s):  
Alexei V Tulin ◽  
Galina L Kogan ◽  
Dominik Filipp ◽  
Maria D Balakireva ◽  
Vladimir A Gvozdev
Keyword(s):  
Drosophila Melanogaster ◽  
Molecular Evolution ◽  
Protein Kinase Ck2 ◽  
Unknown Origin ◽  
Open Reading Frames ◽  
Β Subunit ◽  
Retrotransposon Insertion ◽  
High Level ◽  
Kinase Ck2 ◽  
Reading Frames

The 30-kb cluster comprising close to 20 copies of tandemly repeated Stellate genes was localized in the distal heterochromatin of the X chromosome. Of 10 sequenced genes, nine contain undamaged open reading frames with extensive similarity to protein kinase CK2 β-subunit; one gene is interrupted by an insertion. The heterochromatic array of Stellate repeats is divided into three regions by a 4.5-kb DNA segment of unknown origin and a retrotransposon insertion: the A region (∼14 Stellate genes), the adjacent B region (approximately three Stellate genes), and the C region (about four Stellate genes). The sequencing of Stellate copies located along the discontinuous cluster revealed a complex pattern of diversification. The lowest level of divergence was detected in nearby Stellate repeats. The marginal copies of the A region, truncated or interrupted by an insertion, escaped homogenization and demonstrated high levels of divergence. Comparison of copies in the B and C regions, which are separated by a retrotransposon insertion, revealed a high level of diversification. These observations suggest that homogenization takes place in the Stellate cluster, but that inserted sequences may impede this process.

scholarly journals Differences in editing of mitochondrial nad3 transcripts from CMS and fertile carrots.

2001 ◽  
Vol 48 (3) ◽  
pp. 711-717 ◽  
Author(s):  
M Rurek ◽  
M Szklarczyk ◽  
N Adamczyk ◽  
B Michalik ◽  
H Augustyniak
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
Sequence Conservation ◽  
Open Reading Frames ◽  
Male Sterile ◽  
Nucleotide Substitutions ◽  
Cytoplasmic Male Sterile ◽  
Fertile Line ◽  
High Level ◽  
Reading Frames

A high level of the nucleotide sequence conservation was found for mitochondrial nad3 gene of carrot. Three silent nucleotide substitutions differentiate nad3 open reading frames from cytoplasmic male sterile and male fertile carrots. All these differences are preserved on the RNA level. Partial and silent editing also distinguished both carrots. Three of the C to U conversions were specific to the fertile line. In the two examined carrot lines editing did not affect the mode of alteration of encoded amino acids.

scholarly journals Synthesis of a Klebsiella pneumoniae O-Antigen Heteropolysaccharide (O12) Requires an ABC 2 Transporter

2003 ◽  
Vol 185 (5) ◽  
pp. 1634-1641 ◽  
Author(s):  
Luis Izquierdo ◽  
Susana Merino ◽  
Miguel Regué ◽  
Florencia Rodriguez ◽  
Juan M. Tomás
Keyword(s):  
Klebsiella Pneumoniae ◽  
Genomic Library ◽  
Gene Clusters ◽  
Open Reading Frames ◽  
Complete Analysis ◽  
E Coli ◽  
O Antigen ◽  
High Level ◽  
K 12 ◽  
Reading Frames

ABSTRACT A recombinant clone encoding enzymes for Klebsiella pneumoniae O12-antigen lipopolysaccharide (LPS) was found when we screened for serum resistance of a cosmid-based genomic library of K. pneumoniae KT776 (O12:K80) introduced into Escherichia coli DH5α. A total of eight open reading frames (ORFs) (wb O12 gene cluster) were necessary to produce K. pneumoniae O12-antigen LPS in E. coli K-12. A complete analysis of the K. pneumoniae wb O12 cluster revealed an interesting coincidence with the wb O4 cluster of Serratia marcescens from ORF5 to ORF8 (or WbbL to WbbA). This prompted us to generate mutants of K. pneumoniae strain KT776 (O12) and to study complementation between the two enterobacterial wb clusters using mutants of S. marcescens N28b (O4) obtained previously. Both wb gene clusters are examples of ABC 2 transporter-dependent pathways for O-antigen heteropolysaccharides. The wzm-wzt genes and the wbbA or wbbB genes were not interchangeable between the two gene clusters despite their high level of similarity. However, introduction of three cognate genes (wzm-wzt-wbbA or wzm-wzt-wbbB) into mutants unable to produce O antigen allowed production of the specific O antigen. The K. pneumoniae O12 WbbL protein performs the same function as WbbL from S. marcescens O4 in either the S. marcescens O4 or E. coli K-12 genetic background.

scholarly journals Genotypic and Phenotypic Evaluation of the Evolution of High-Level Daptomycin Nonsusceptibility in Vancomycin-Resistant Enterococcus faecium

2012 ◽  
Vol 56 (11) ◽  
pp. 6051-6053 ◽  
Author(s):  
Romney M. Humphries ◽  
Theodoros Kelesidis ◽  
Ryan Tewhey ◽  
Warren E. Rose ◽  
Nicholas Schork ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Open Reading Frames ◽  
Vancomycin Resistant Enterococcus ◽  
Single Nucleotide Variants ◽  
Phosphotransferase System ◽  
Single Nucleotide ◽  
Content Type ◽  
Vancomycin Resistant ◽  
High Level ◽  
Reading Frames

ABSTRACTWhole-genome sequencing and cell membrane studies of three clonalEnterococcus faeciumstrains with daptomycin MICs of 4, 32, and 192 μg/ml were performed, revealing nonsynonymous single nucleotide variants in eight open reading frames, including those predicted to encode a phosphoenolpyruvate-dependent, mannose-specific phosphotransferase system, cardiolipin synthetase, and EzrA. Membrane studies revealed a higher net surface charge among the daptomycin-nonsusceptible isolates and increased septum formation in the isolate with a daptomycin MIC of 192 μg/ml.

scholarly journals c-Type Cytochromes in Pelobacter carbinolicus

2006 ◽  
Vol 72 (11) ◽  
pp. 6980-6985 ◽  
Author(s):  
Shelley A. Haveman ◽  
Dawn E. Holmes ◽  
Yan-Huai R. Ding ◽  
Joy E. Ward ◽  
Raymond J. DiDonato ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Geobacter Sulfurreducens ◽  
Open Reading Frames ◽  
Genetic Studies ◽  
Close Relatives ◽  
High Level ◽  
Reading Frames

ABSTRACT Previous studies failed to detect c-type cytochromes in Pelobacter species despite the fact that other close relatives in the Geobacteraceae, such as Geobacter and Desulfuromonas species, have abundant c-type cytochromes. Analysis of the recently completed genome sequence of Pelobacter carbinolicus revealed 14 open reading frames that could encode c-type cytochromes. Transcripts for all but one of these open reading frames were detected in acetoin-fermenting and/or Fe(III)-reducing cells. Three putative c-type cytochrome genes were expressed specifically during Fe(III) reduction, suggesting that the encoded proteins may participate in electron transfer to Fe(III). One of these proteins was a periplasmic triheme cytochrome with a high level of similarity to PpcA, which has a role in Fe(III) reduction in Geobacter sulfurreducens. Genes for heme biosynthesis and system II cytochrome c biogenesis were identified in the genome and shown to be expressed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of protein extracted from acetoin-fermenting P. carbinolicus cells contained three heme-staining bands which were confirmed by mass spectrometry to be among the 14 predicted c-type cytochromes. The number of cytochrome genes, the predicted amount of heme c per protein, and the ratio of heme-stained protein to total protein were much smaller in P. carbinolicus than in G. sulfurreducens. Furthermore, many of the c-type cytochromes that genetic studies have indicated are required for optimal Fe(III) reduction in G. sulfurreducens were not present in the P. carbinolicus genome. These results suggest that further evaluation of the functions of c-type cytochromes in the Geobacteraceae is warranted.

scholarly journals Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control

1994 ◽  
Vol 14 (1) ◽  
pp. 606-618
Author(s):  
C M Grant ◽  
A G Hinnebusch
Keyword(s):  
Translational Control ◽  
Stop Codon ◽  
Inhibitory Effect ◽  
Open Reading Frames ◽  
Sequence Context ◽  
The Third ◽  
Hybrid Element ◽  
Low Efficiency ◽  
High Level ◽  
Reading Frames

Translational control of the GCN4 gene involves two short open reading frames in the mRNA leader (uORF1 and uORF4) that differ greatly in the ability to allow reinitiation at GCN4 following their own translation. The low efficiency of reinitiation characteristic of uORF4 can be reconstituted in a hybrid element in which the last codon of uORF1 and 10 nucleotides 3' to its stop codon (the termination region) are substituted with the corresponding nucleotides from uORF4. To define the features of these 13 nucleotides that determine their effects on reinitiation, we separately randomized the sequence of the third codon and termination region of the uORF1-uORF4 hybrid and selected mutant alleles with the high-level reinitiation that is characteristic of uORF1. The results indicate that many different A+U-rich triplets present at the third codon of uORF1 can overcome the inhibitory effect of the termination region derived from uORF4 on the efficiency of reinitiation at GCN4. Efficient reinitiation is not associated with codons specifying a particular amino acid or isoacceptor tRNA. Similarly, we found that a diverse collection of A+U-rich sequences present in the termination region of uORF1 could restore efficient reinitiation at GCN4 in the presence of the third codon derived from uORF4. To explain these results, we propose that reinitiation can be impaired by stable base pairing between nucleotides flanking the uORF1 stop codon and either the tRNA which pairs with the third codon, the rRNA, or sequences located elsewhere in GCN4 mRNA. We suggest that these interactions delay the resumption of scanning following peptide chain termination at the uORF and thereby lead to ribosome dissociation from the mRNA.

scholarly journals Complete Nucleotide Sequence of Klebsiella pneumoniae Multiresistance Plasmid pJHCMW1

2002 ◽  
Vol 46 (11) ◽  
pp. 3422-3427 ◽  
Author(s):  
Renee Sarno ◽  
Glen McGillivary ◽  
David J. Sherratt ◽  
Luis A. Actis ◽  
Marcelo E. Tolmasky
Keyword(s):  
Klebsiella Pneumoniae ◽  
Single Gene ◽  
Antibiotic Resistance Genes ◽  
Gene Cassette ◽  
Open Reading Frames ◽  
The Other ◽  
Gene Encoding ◽  
The Subject ◽  
Plasmid Stabilization ◽  
Reading Frames

ABSTRACT The multiresistance plasmid pJHCMW1, harbored by a clinical Klebsiella pneumoniae strain isolated from a neonate with meningitis, was sequenced. A circular sequence of 11,354 bp was generated, of which 7,993 bp make up Tn1331, a transposon including the antibiotic resistance genes aac(6′)-Ib, aadA1, bla OXA-9, and bla TEM-1. The gene aac(6′)-Ib is included in a gene cassette, and both aadA1 and bla OXA-9 are included in a single-gene cassette that may have arisen as a consequence of a recombination event involving two integrons. The pJHCMW1 plasmid replicates through a ColE1-like RNA-regulated mechanism, includes a functional oriT, and two loci with similarity to XerCD site-specific recombination target sites involved in plasmid stabilization by the resolution of multimers. One of these two loci, mwr, is active and has been the subject of previous studies, and the other, dxs, is not functional but binds the recombinase XerD with low affinity. Two additional open reading frames were identified, one with low similarity to two hypothetical membrane proteins from Mycobacterium tuberculosis and Mycobacterium leprae and the other with low similarity to psiB, a gene encoding a function that facilitates the establishment of the transferring plasmid in the recipient bacterial cell during the process of conjugation.

scholarly journals Acquisition of a Plasmid-Borne blaOXA-58 Gene with an Upstream IS1008 Insertion Conferring a High Level of Carbapenem Resistance to Acinetobacter baumannii

2008 ◽  
Vol 52 (7) ◽  
pp. 2573-2580 ◽  
Author(s):  
Te-Li Chen ◽  
Roy Chen-Chih Wu ◽  
Men-Fang Shaio ◽  
Chang-Phone Fung ◽  
Wen-Long Cho
Keyword(s):  
Acinetobacter Baumannii ◽  
Clinical Isolate ◽  
Shuttle Vector ◽  
Carbapenem Resistance ◽  
Open Reading Frames ◽  
Transcription Control ◽  
Genetic Construct ◽  
Fold Reduction ◽  
High Level ◽  
Reading Frames

ABSTRACT The oxacillinase gene was reported to confer limited resistance to carbapenem in Acinetobacter baumannii. In this study, we have demonstrated that an A. baumannii clinical isolate harboring a plasmid, pTVICU53, has 11,037 bp encoding 13 open reading frames. A bla OXA-58 gene with an upstream insertion of truncated ISAba3 (ΔISAba3) and IS1008 was found in this plasmid. ΔISAba3and IS1008 provided two independent promoters for the transcription control of the bla OXA-58 gene. The transformation of pTVICU53 or a shuttle vector bearing IS1008-ΔISAba3-bla OXA-58 to different A. baumannii recipients can increase their MICs of carbapenem 64- to 256-fold. The deletion of promoters provided by IS1008 resulted in dramatic decreases in bla OXA-58 transcription and a 32- to 64-fold reduction in the carbapenem MIC. These findings highlight that A. baumannii might develop carbapenem resistance with a single transformation step, taking up a plasmid containing a genetic construct with a potentially high level of transcription of the bla OXA-58 gene.

scholarly journals Duplication of Hemolysin Genes in a Virulent Isolate of Vibrio harveyi

2001 ◽  
Vol 67 (7) ◽  
pp. 3161-3167 ◽  
Author(s):  
X.-H. Zhang ◽  
P. G. Meaden ◽  
B. Austin
Keyword(s):  
Hemolytic Activity ◽  
Vibrio Harveyi ◽  
High Titer ◽  
Open Reading Frames ◽  
Amino Acid Residues ◽  
E Coli ◽  
Native Promoter ◽  
Hemolysin Gene ◽  
High Level ◽  
Reading Frames

ABSTRACT Vibrio harveyi VIB 645, which is very pathogenic towards salmonids and produces extracellular product with a high titer of hemolytic activity towards fish erythrocytes, was found to contain two closely related hemolysin genes (designated vhhA andvhhB), whereas the majority of strains examined (11 of 13) carried only a single hemolysin gene. Both genes from VIB 645 were cloned and sequenced. The open reading frames (ORFs) ofvhhA and vhhB shared a high level of identity (98.8%) and were predicted to encode identical polypeptides comprising 418 amino acid residues. The VHH protein shows homology to the lecithinase of V. mimicus and V. cholerae. Transformants of Escherichia coli containing the ORF of either vhhA or vhhB displayed weak hemolytic activity in rainbow trout blood agar. The hemolytic activity was very high when the ORF of vhhB was cloned in E. colitogether with the native promoter. Surprisingly, the level ofvhh-specific RNA transcript produced by VIB 645 was found to be very low. We conclude that the hemolytic phenotype of VIB 645 is not due to increased expression of one or both copies of thevhh gene.

scholarly journals Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control.

1994 ◽  
Vol 14 (1) ◽  
pp. 606-618 ◽  
Author(s):  
C M Grant ◽  
A G Hinnebusch
Keyword(s):  
Translational Control ◽  
Stop Codon ◽  
Inhibitory Effect ◽  
Open Reading Frames ◽  
Sequence Context ◽  
The Third ◽  
Hybrid Element ◽  
Low Efficiency ◽  
High Level ◽  
Reading Frames

Translational control of the GCN4 gene involves two short open reading frames in the mRNA leader (uORF1 and uORF4) that differ greatly in the ability to allow reinitiation at GCN4 following their own translation. The low efficiency of reinitiation characteristic of uORF4 can be reconstituted in a hybrid element in which the last codon of uORF1 and 10 nucleotides 3' to its stop codon (the termination region) are substituted with the corresponding nucleotides from uORF4. To define the features of these 13 nucleotides that determine their effects on reinitiation, we separately randomized the sequence of the third codon and termination region of the uORF1-uORF4 hybrid and selected mutant alleles with the high-level reinitiation that is characteristic of uORF1. The results indicate that many different A+U-rich triplets present at the third codon of uORF1 can overcome the inhibitory effect of the termination region derived from uORF4 on the efficiency of reinitiation at GCN4. Efficient reinitiation is not associated with codons specifying a particular amino acid or isoacceptor tRNA. Similarly, we found that a diverse collection of A+U-rich sequences present in the termination region of uORF1 could restore efficient reinitiation at GCN4 in the presence of the third codon derived from uORF4. To explain these results, we propose that reinitiation can be impaired by stable base pairing between nucleotides flanking the uORF1 stop codon and either the tRNA which pairs with the third codon, the rRNA, or sequences located elsewhere in GCN4 mRNA. We suggest that these interactions delay the resumption of scanning following peptide chain termination at the uORF and thereby lead to ribosome dissociation from the mRNA.

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