scholarly journals The Unusual 23S rRNA Gene of Coxiella burnetii: Two Self-Splicing Group I Introns Flank a 34-Base-Pair Exon, and One Element Lacks the Canonical ΩG

2007 ◽  
Vol 189 (18) ◽  
pp. 6572-6579 ◽  
Author(s):  
Rahul Raghavan ◽  
Scott R. Miller ◽  
Linda D. Hicks ◽  
Michael F. Minnick
Keyword(s):  
Coxiella Burnetii ◽  
Acanthamoeba Castellanii ◽  
23S Rrna ◽  
Rrna Gene ◽  
Group I Introns ◽  
Group I ◽  
23S Rrna Gene ◽  
Self Splicing

ABSTRACT We describe the presence and characteristics of two self-splicing group I introns in the sole 23S rRNA gene of Coxiella burnetii. The two group I introns, Cbu.L1917 and Cbu.L1951, are inserted at sites 1917 and 1951 (Escherichia coli numbering), respectively, in the 23S rRNA gene of C. burnetii. Both introns were found to be self-splicing in vivo and in vitro even though the terminal nucleotide of Cbu.L1917 is adenine and not the canonical conserved guanine, termed ΩG, found in Cbu.L1951 and all other group I introns described to date. Predicted secondary structures for both introns were constructed and revealed that Cbu.L1917 and Cbu.L1951 were group IB2 and group IA3 introns, respectively. We analyzed strains belonging to eight genomic groups of C. burnetii to determine sequence variation and the presence or absence of the elements and found both introns to be highly conserved (≥99%) among them. Although phylogenetic analysis did not identify the specific identities of donors, it indicates that the introns were likely acquired independently; Cbu.L1917 was acquired from other bacteria like Thermotoga subterranea and Cbu.L1951 from lower eukaryotes like Acanthamoeba castellanii. We also confirmed the fragmented nature of mature 23S rRNA in C. burnetii due to the presence of an intervening sequence. The presence of three selfish elements in C. burnetii's 23S rRNA gene is very unusual for an obligate intracellular bacterium and suggests a recent shift to its current lifestyle from a previous niche with greater opportunities for lateral gene transfer.

scholarly journals A Unique Group I Intron in Coxiella burnetii Is a Natural Splice Mutant

2009 ◽  
Vol 191 (12) ◽  
pp. 4044-4046 ◽  
Author(s):  
Rahul Raghavan ◽  
Linda D. Hicks ◽  
Michael F. Minnick
Keyword(s):  
Coxiella Burnetii ◽  
Group I Intron ◽  
23S Rrna ◽  
Rrna Gene ◽  
Group I Introns ◽  
Group I ◽  
23S Rrna Gene ◽  
Self Splicing

ABSTRACT Cbu.L1917, a group I intron present in the 23S rRNA gene of Coxiella burnetii, possesses a unique 3′-terminal adenine in place of a conserved guanine. Here, we show that, unlike all other group I introns, Cbu.L1917 utilizes a different cofactor for each splicing step and has a decreased self-splicing rate in vitro.

scholarly journals Toxic Introns and Parasitic Intein in Coxiella burnetii: Legacies of a Promiscuous Past

2008 ◽  
Vol 190 (17) ◽  
pp. 5934-5943 ◽  
Author(s):  
Rahul Raghavan ◽  
Linda D. Hicks ◽  
Michael F. Minnick
Keyword(s):  
Coxiella Burnetii ◽  
Growth Cycle ◽  
Host Protein ◽  
23S Rrna ◽  
Rrna Gene ◽  
Group I Introns ◽  
Bacterial Growth Rate ◽  
Group I ◽  
Halorhodospira Halophila

ABSTRACT The genome of the obligate intracellular pathogen Coxiella burnetii contains a large number of selfish genetic elements, including two group I introns (Cbu.L1917 and Cbu.L1951) and an intervening sequence that interrupts the 23S rRNA gene, an intein (Cbu.DnaB) within dnaB and 29 insertion sequences. Here, we describe the ability of the intron-encoded RNAs (ribozymes) to retard bacterial growth rate (toxicity) and examine the functionality and phylogenetic history of Cbu.DnaB. When expressed in Escherichia coli, both introns repressed growth, with Cbu.L1917 being more inhibitory. Both ribozymes were found to associate with ribosomes of Coxiella and E. coli. In addition, ribozymes significantly reduced in vitro luciferase translation, again with Cbu.L1917 being more inhibitory. We analyzed the relative quantities of ribozymes and genomes throughout a 14-day growth cycle of C. burnetii and found that they were inversely correlated, suggesting that the ribozymes have a negative effect on Coxiella's growth. We determined possible sites for ribozyme associations with 23S rRNA that could explain the observed toxicities. Further research is needed to determine whether the introns are being positively selected because they promote bacterial persistence or whether they were fixed in the population due to genetic drift. The intein, Cbu.DnaB, is able to self-splice, leaving the host protein intact and presumably functional. Similar inteins have been found in two extremophilic bacteria (Alkalilimnicola ehrlichei and Halorhodospira halophila) that are distantly related to Coxiella, making it difficult to determine whether the intein was acquired by horizontal gene transfer or was vertically inherited from a common ancestor.

scholarly journals A Self-Splicing Group I Intron in DNA Polymerase Genes of T7-Like Bacteriophages

2004 ◽  
Vol 186 (23) ◽  
pp. 8153-8155 ◽  
Author(s):  
Richard P. Bonocora ◽  
David A. Shub
Keyword(s):  
Dna Polymerase ◽  
Group I Intron ◽  
Group I Introns ◽  
Homing Endonucleases ◽  
Structural Element ◽  
Group I ◽  
Close Relatives ◽  
Self Splicing

ABSTRACT Group I introns are inserted into genes of a wide variety of bacteriophages of gram-positive bacteria. However, among the phages of enteric and other gram-negative proteobacteria, introns have been encountered only in phage T4 and several of its close relatives. Here we report the insertion of a self-splicing group I intron in the coding sequence of the DNA polymerase genes of ΦI and W31, phages that are closely related to T7. The introns belong to subgroup IA2 and both contain an open reading frame, inserted into structural element P6a, encoding a protein belonging to the HNH family of homing endonucleases. The introns splice efficiently in vivo and self-splice in vitro under mild conditions of ionic strength and temperature. We conclude that there is no barrier for maintenance of group I introns in phages of proteobacteria.

scholarly journals Effect of Macrolide Usage on Emergence of Erythromycin-Resistant Campylobacter Isolates in Chickens

2007 ◽  
Vol 51 (5) ◽  
pp. 1678-1686 ◽  
Author(s):  
Jun Lin ◽  
Meiguan Yan ◽  
Orhan Sahin ◽  
Sonia Pereira ◽  
Yun-Juan Chang ◽  
...  
Keyword(s):  
Prolonged Exposure ◽  
Efflux Pump ◽  
Spontaneous Mutation ◽  
23S Rrna ◽  
Rrna Gene ◽  
Campylobacter Coli ◽  
Growth Promoter ◽  
23S Rrna Gene

ABSTRACT In this work we conducted both in vitro and in vivo experiments to examine the development and mechanisms of erythromycin (Ery) resistance in Campylobacter jejuni and Campylobacter coli. In vitro plating revealed that both Campylobacter species had similar but low spontaneous mutation frequencies (3 × 10−9 to <5.41 × 10−10) for Ery resistance. Chickens infected with C. jejuni or C. coli were subjected to single or multiple treatments with medicated water containing tylosin (0.53 g/liter), which transiently reduced the level of Campylobacter colonization but did not select for Ery-resistant (Eryr) mutants in the treated birds. However, when tylosin was given to the chickens in feed at a growth-promoting dose (0.05 g/kg feed), Eryr mutants emerged in the birds after prolonged exposure to the antibiotic. The vast majority of the in vitro- and in vivo-selected Campylobacter mutants with Ery MICs of 8 to 256 μg/ml lacked the known resistance-associated mutations in the 23S rRNA gene, while the highly resistant mutants (Ery MIC > 512 μg/ml) had the A2074G mutation in the 23S rRNA gene. Inactivation of CmeABC, a multidrug efflux pump, dramatically reduced the Ery MIC in all of the examined mutants regardless of the presence of the A2074G mutation. Together, these results reveal distinct features associated with Ery resistance development in Campylobacter, demonstrate the significant role of CmeABC in Ery resistance, and suggest that long-term use of a macrolide as a growth promoter selects for the emergence of Eryr Campylobacter in animal reservoirs.

Autocatalytic activities of intron 5 of the cob gene of yeast mitochondria

1988 ◽  
Vol 8 (6) ◽  
pp. 2562-2571
Author(s):  
S Partono ◽  
A S Lewin
Keyword(s):  
Group I Introns ◽  
Yeast Mitochondria ◽  
High Concentration ◽  
Functional Protein ◽  
Full Spectrum ◽  
Group I ◽  
Intron Boundary ◽  
Monovalent Ion

The terminal intron of the mitochondrial cob gene of Saccharomyces cerevisiae can undergo autocatalytic splicing in vitro. Efficient splicing of this intron required a high concentration of monovalent ion (1 M). We found that at a high salt concentration this intron was very active and performed many of the reactions described for other group I introns. The rate of the splicing reaction was dependent on the choice of the monovalent ion; the reaction intermediate, the intron-3' exon molecule, accumulated in NH4Cl but not in KCl. In addition, the intron was more reactive in KCl, accumulating in two different circular forms: one cyclized at the 5' intron boundary and the other at 236 nucleotides from the 5' end. These circular forms were able to undergo the opening and recyclization reactions previously described for the Tetrahymena rRNA intron. Cleavage of the 5' exon-intron boundary by the addition of GTP did not require the 3' terminus of the intron and the downstream exon. An anomalous guanosine addition at the 3' exon and at the middle of the intron was also detected. Hence, this intron, which requires a functional protein to splice in vivo, demonstrated a full spectrum of characteristic reactions in the absence of proteins.

scholarly journals In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA.

1987 ◽  
Vol 7 (7) ◽  
pp. 2545-2551 ◽  
Author(s):  
A Gampel ◽  
A Tzagoloff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Cytochrome B Gene ◽  
Group I Introns ◽  
Vitro Assay ◽  
Group I ◽  
Mitochondrial Cytochrome B ◽  
Nuclease Mapping

A region of the Saccharomyces cerevisiae mitochondrial cytochrome b gene encompassing the entire terminal intron plus flanking exonic sequences has been cloned in an SP6 vector. A runoff transcript prepared from this construct as well as the native cytochrome b pre-mRNA containing the terminal intervening sequence were found to act as substrates for the autocatalytic excision of the intervening sequence in vitro. This reaction proceeds under conditions previously shown by Cech and co-workers to promote protein-independent excision of the Tetrahymena rRNA intervening sequence. The 5' and 3' termini of the excised intervening sequence, determined by S1 nuclease mapping and sequence analysis, are consistent with the known sequence of the cytochrome b mRNA. The same region of the cytochrome b gene from a yeast mutant, defective in splicing due to a mutation in a critical sequence inside the terminal intron, has also been cloned in an SP6 vector. The mutant transcript fails to self-splice in the in vitro assay. These observations provide strong presumptive evidence that in vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA occurs by an autocatalytic mechanism analogous to that shown for other group I introns. In vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA, however, exhibits complete dependence on a protein factor previously shown to be encoded by the nuclear gene CBP2.

scholarly journals Two New Point Mutations at A2062 Associated with Resistance to 16-Membered Macrolide Antibiotics in Mutant Strains ofMycoplasma hominis

2001 ◽  
Vol 45 (10) ◽  
pp. 2958-2960 ◽  
Author(s):  
Pio Maria Furneri ◽  
Giancarlo Rappazzo ◽  
Maria Pia Musumarra ◽  
Patrizia Di Pietro ◽  
Lucrezia S. Catania ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Mycoplasma Hominis ◽  
23S Rrna ◽  
Macrolide Antibiotics ◽  
Rrna Gene ◽  
In Vitro Exposure ◽  
Mutant Strains ◽  
23S Rrna Gene

ABSTRACT We describe two mutants of Mycoplasma hominis PG-21 which show resistance to 16-membered macrolides but susceptibility to lincosamides, obtained by in vitro exposure to increasing doses of josamycin. The 23S rRNA gene showed that each had a mutation (A2062G and A2062T) corresponding to nucleotide 2062 in Escherichia coli, which was associated with the acquired phenotype.

scholarly journals Atypical Mutation in Neisseria gonorrhoeae 23S rRNA Associated with High-Level Azithromycin Resistance.

2020 ◽  
Author(s):  
Cau D. Pham ◽  
Evelyn Nash ◽  
Hsi Liu ◽  
Matthew W. Schmerer ◽  
Samera Sharpe ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Genetic Analysis ◽  
Neisseria Gonorrhoeae ◽  
23S Rrna ◽  
Rrna Gene ◽  
Neisseria Gonorrhoea ◽  
23S Rrna Gene ◽  
High Level ◽  
Azithromycin Resistance

A2059G mutation in the 23S rRNA gene is the only reported mechanism conferring high-level azithromycin resistance (HL-AZMR) in Neisseria gonorrhoea. Through U.S. gonococcal antimicrobial resistance surveillance projects, we identified four HL-AZMR gonococcal isolates lacking this mutational genotype. Genetic analysis revealed an A2058G mutation of 23S rRNA alleles in all four isolates. In vitro selected gonococcal strains with homozygous A2058G recapitulated the HL-AZMR phenotype. Taken together, we postulate that A2058G mutation confers HL-AZMR in N. gonorrhoeae.

scholarly journals Mutations in a 23S rRNA Gene of Chlamydia trachomatis Associated with Resistance to Macrolides

2004 ◽  
Vol 48 (4) ◽  
pp. 1347-1349 ◽  
Author(s):  
O. Y. Misyurina ◽  
E. V. Chipitsyna ◽  
Y. P. Finashutina ◽  
V. N. Lazarev ◽  
T. A. Akopian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chlamydia Trachomatis ◽  
Gene Mutations ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Rrna Gene ◽  
In Vitro Susceptibility ◽  
23S Rrna Gene ◽  
Mixed Populations

ABSTRACT For six clinical isolates of Chlamydia trachomatis, in vitro susceptibility to erythromycin, azithromycin, and josamycin has been determined. Four isolates were resistant to all the antibiotics and had the mutations A2058C and T2611C (Escherichia coli numbering) in the 23S rRNA gene. All the isolates had mixed populations of bacteria that did and did not carry 23S rRNA gene mutations.

scholarly journals Self-Splicing of the Bacteriophage T4 Group I Introns Requires Efficient Translation of the Pre-mRNA In Vivo and Correlates with the Growth State of the Infected Bacterium

2006 ◽  
Vol 189 (3) ◽  
pp. 980-990 ◽  
Author(s):  
Linus Sandegren ◽  
Britt-Marie Sjöberg
Keyword(s):  
Stationary Phase ◽  
De Novo ◽  
Bacteriophage T4 ◽  
Group I Introns ◽  
Growth State ◽  
Group I ◽  
Splicing Efficiency ◽  
Self Splicing ◽  
Upstream Exon

ABSTRACT Bacteriophage T4 contains three self-splicing group I introns in genes in de novo deoxyribonucleotide biosynthesis (in td, coding for thymidylate synthase and in nrdB and nrdD, coding for ribonucleotide reductase). Their presence in these genes has fueled speculations that the introns are retained within the phage genome due to a possible regulatory role in the control of de novo deoxyribonucleotide synthesis. To study whether sequences in the upstream exon interfere with proper intron folding and splicing, we inhibited translation in T4-infected bacteria as well as in bacteria containing recombinant plasmids carrying the nrdB intron. Splicing was strongly reduced for all three T4 introns after the addition of chloramphenicol during phage infection, suggesting that the need for translating ribosomes is a general trait for unperturbed splicing. The splicing of the cloned nrdB intron was markedly reduced in the presence of chloramphenicol or when translation was hindered by stop codons inserted in the upstream exon. Several exon regions capable of forming putative interactions with nrdB intron sequences were identified, and the removal or mutation of these exon regions restored splicing efficiency in the absence of translation. Interestingly, splicing of the cloned nrdB intron was also reduced as cells entered stationary phase and splicing of all three introns was reduced upon the T4 infection of stationary-phase bacteria. Our results imply that conditions likely to be frequently encountered by natural phage populations may limit the self-splicing efficiency of group I introns. This is the first time that environmental effects on bacterial growth have been linked to the regulation of splicing of phage introns.

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