scholarly journals Helcococcus kunzii methyltransferase Erm(47) responsible for MLSB resistance is induced by diverse ribosome-targeting antibiotics

2019 ◽  
Author(s):  
François Guerin ◽  
Simon Rose ◽  
Vincent Cattoir ◽  
Stephen Douthwaite
Keyword(s):  
Binding Site ◽  
Clinical Isolate ◽  
Ribosomal Rna ◽  
Methylation Status ◽  
Ribosomal Subunit ◽  
Opportunistic Pathogen ◽  
Induction Mechanism ◽  
Wide Range ◽  
30S Subunit ◽  
Subinhibitory Concentrations

Abstract Objectives To determine the mechanism of induction of erm(47) and its atypical expression in the Gram-positive opportunistic pathogen Helcococcus kunzii, where it confers resistance to a subset of clinically important macrolide, lincosamide and streptogramin B (MLSB) antibiotics. Methods The resistant H. kunzii clinical isolate UCN99 was challenged with subinhibitory concentrations of a wide range of ribosome-targeting drugs. The methylation status of the H. kunzii ribosomal RNA at the MLSB binding site was then determined using an MS approach and was correlated with any increase in resistance to the drugs. Results The H. kunzii erm(47) gene encodes a monomethyltransferase. Expression is induced by subinhibitory concentrations of the macrolide erythromycin, as is common for many erm genes, and surprisingly also by 16-membered macrolide, lincosamide, streptogramin, ketolide, chloramphenicol and linezolid antibiotics, all of which target the 50S ribosomal subunit. No induction was detected with spectinomycin, which targets the 30S subunit. Conclusions The structure of the erm(47) leader sequence functions as a hair trigger for the induction mechanism that expresses resistance. Consequently, translation of the erm(47) mRNA is tripped by MLSB compounds and also by drugs that target the 50S ribosomal subunit outside the MLSB site. Expression of erm(47) thus extends previous assumptions about how erm genes can be induced.

scholarly journals The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

2021 ◽  
Author(s):  
Haina Huang ◽  
Melissa Parker ◽  
Katrin Karbstein
Keyword(s):  
Quality Control ◽  
Binding Site ◽  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Kinase Activity ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Yeast Strains

AbstractRibosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

scholarly journals Assessing anaerobic gut fungal (Neocalliamstigomycota) diversity using PacBio D1/D2 LSU rRNA amplicon sequencing and multi-year isolation

2020 ◽  
Author(s):  
Radwa A. Hanafy ◽  
Britny Johnson ◽  
Noha H. Youssef ◽  
Mostafa S. Elshahed
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Subunit ◽  
Amplicon Sequencing ◽  
Sequencing Technology ◽  
Its1 Region ◽  
5.8S Rrna ◽  
Wide Range ◽  
Culture Independent ◽  
Long Read ◽  
Pacbio Smrt Sequencing

AbstractThe anaerobic gut fungi (AGF, Neocallimastigomycota) reside in the alimentary tracts of herbivores where they play a central role in the breakdown of ingested plant material. Accurate assessment of AGF diversity has been hampered by inherent deficiencies of the internal transcribed spacer1 (ITS1) region as a phylogenetic marker. Here, we report on the development and implementation of the D1/D2 region of the large ribosomal subunit (D1/D2 LSU) as a novel marker for assessing AGF diversity in culture-independent surveys. Sequencing a 1.4-1.5 Kbp amplicon encompassing the ITS1-5.8S rRNA-ITS2-D1/D2 LSU region in the ribosomal RNA locus from fungal strains and environmental samples generated a reference D1/D2 LSU database for all cultured AGF genera, as well as the majority of candidate genera encountered in prior ITS1-based diversity surveys. Subsequently, a D1/D2 LSU-based diversity survey using long read PacBio SMRT sequencing technology was conducted on fecal samples from 21 wild and domesticated herbivores. Twenty-eight genera and candidate genera were identified in the 17.7 K sequences obtained, including multiple novel lineages that were predominantly, but not exclusively, identified in wild herbivores. Association between certain AGF genera and animal lifestyles, or animal host family was observed. Finally, to address the current paucity of AGF isolates, concurrent isolation efforts utilizing multiple approaches to maximize recovery yielded 216 isolates belonging to twelve different genera, several of which have no prior cultured-representatives. Our results establish the utility of D1/D2 LSU and PacBio sequencing for AGF diversity surveys, and the culturability of a wide range of AGF taxa, and demonstrate that wild herbivores represent a yet-untapped reservoir of AGF diversity.

scholarly journals The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

RNA ◽  
2022 ◽  
pp. rna.078994.121
Author(s):  
Haina Huang ◽  
Melissa D Parker ◽  
Katrin Karbstein
Keyword(s):  
Quality Control ◽  
Binding Site ◽  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Kinase Activity ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Yeast Strains

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

scholarly journals Polyamine Metabolism and Gene Methylation in Conjunction with One-Carbon Metabolism

2018 ◽  
Vol 19 (10) ◽  
pp. 3106 ◽  
Author(s):  
Kuniyasu Soda
Keyword(s):  
Dna Methylation ◽  
Carbon Metabolism ◽  
Methylation Status ◽  
Significant Risk ◽  
Significant Risk Factor ◽  
Dna Methyltransferases ◽  
Polyamine Metabolism ◽  
Methyl Group ◽  
Wide Range ◽  
One Carbon Metabolism

Recent investigations have revealed that changes in DNA methylation status play an important role in aging-associated pathologies and lifespan. The methylation of DNA is regulated by DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) in the presence of S-adenosylmethionine (SAM), which serves as a methyl group donor. Increased availability of SAM enhances DNMT activity, while its metabolites, S-adenosyl-l-homocysteine (SAH) and decarboxylated S-adenosylmethionine (dcSAM), act to inhibit DNMT activity. SAH, which is converted from SAM by adding a methyl group to cytosine residues in DNA, is an intermediate precursor of homocysteine. dcSAM, converted from SAM by the enzymatic activity of adenosylmethionine decarboxylase, provides an aminopropyl group to synthesize the polyamines spermine and spermidine. Increased homocysteine levels are a significant risk factor for the development of a wide range of conditions, including cardiovascular diseases. However, successful homocysteine-lowering treatment by vitamins (B6, B12, and folate) failed to improve these conditions. Long-term increased polyamine intake elevated blood spermine levels and inhibited aging-associated pathologies in mice and humans. Spermine reversed changes (increased dcSAM, decreased DNMT activity, aberrant DNA methylation, and proinflammatory status) induced by the inhibition of ornithine decarboxylase. The relation between polyamine metabolism, one-carbon metabolism, DNA methylation, and the biological mechanism of spermine-induced lifespan extension is discussed.

scholarly journals Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

2010 ◽  
Vol 189 (7) ◽  
pp. 1079-1086 ◽  
Author(s):  
Jayati Sengupta ◽  
Cyril Bussiere ◽  
Jesper Pallesen ◽  
Matthew West ◽  
Arlen W. Johnson ◽  
...  
Keyword(s):  
Binding Site ◽  
Nuclear Export ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Adaptor Protein ◽  
Structural Data ◽  
Release Mechanism ◽  
Nuclear Pore ◽  
Subunit Joining

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.

scholarly journals Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria

2017 ◽  
Vol 114 (35) ◽  
pp. E7226-E7235 ◽  
Author(s):  
Philip H. Choi ◽  
Thu Minh Ngoc Vu ◽  
Huong Thi Pham ◽  
Joshua J. Woodward ◽  
Mark S. Turner ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Pyruvate Carboxylase ◽  
Adenosine Monophosphate ◽  
Binding Mode ◽  
Functional Studies ◽  
Cellular Processes ◽  
Wide Range ◽  
A Site ◽  
Amp Inhibition

Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism inListeria monocytogenesby inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition ofLactococcus lactisPC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. InL. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool inL. lactisis negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP–insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

scholarly journals Structural basis of RNA polymerase III transcription repression by Maf1

2019 ◽  
Author(s):  
Matthias K. Vorländer ◽  
Florence Baudin ◽  
Robyn D. Moir ◽  
René Wetzel ◽  
Wim J. H. Hagen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Structural Basis ◽  
Metabolic Efficiency ◽  
Transcription Repression ◽  
Polymerase Iii ◽  
Wide Range ◽  
Pol Iii

ABSTRACTMaf1 is a highly conserved central regulator of transcription by RNA polymerase III (Pol III), and Maf1 activity influences a wide range of phenotypes from metabolic efficiency to lifespan. Here, we present a 3.3 Å cryo-EM structure of yeast Maf1 bound to Pol III, which establishes how Maf1 achieves transcription repression. In the Maf1-bound state, Pol III elements that are involved in transcription initiation are sequestered, and the active site is sealed off due to ordering of the mobile C34 winged helix 2 domain. Specifically, the Maf1 binding site overlaps with the binding site of the Pol III transcription factor TFIIIB and DNA in the pre-initiation complex, rationalizing that binding of Maf1 and TFIIIB to Pol III are mutually exclusive. We validate our structure using variants of Maf1 with impaired transcription-inhibition activity. These results reveal the exact mechanism of Pol III inhibition by Maf1, and rationalize previous biochemical data.

scholarly journals Cefazolin and Imipenem Enhance AmpC Expression and Resistance in NagZ-Dependent Manner in Enterobacter Cloacae

2021 ◽  
Author(s):  
Xianggui Yang ◽  
Zhenguo Wang ◽  
Xuejing Yu ◽  
Yuanxiu Zhong ◽  
Fuying Wang ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Target Genes ◽  
Ectopic Expression ◽  
Enterobacter Cloacae ◽  
Opportunistic Pathogen ◽  
Resistance Mechanisms ◽  
Induction Effect ◽  
Dependent Manner ◽  
Critical Determinant ◽  
2Nd Generation

Abstract Background: Enterobacter cloacae (EC) is a commonly occurring opportunistic pathogen and is responsible for causing various infections in humans. Owing to its inducible chromosomal AmpC β-lactamase (AmpC), EC is inherently resistant to the 1st- and 2nd- generation cephalosporins. However, whether β-lactams antibiotics enhance EC resistance remains unclear.Results: In this study, we found that subinhibitory concentrations (SICs) of cefazolin (CFZ) and imipenem (IMP) are able to advance the expression of AmpC and improve its resistance towards β - lactams through NagZ in EC clinical isolate. Our work indicate that AmpC manifested a substantial upregulation in EC in response to SICs of CFZ and IMP. In nagZ knockout EC (ΔnagZ), we found that the resistance to β - lactam antibiotics was rather weakened and the effect of CFZ and IMP on induction of AmpC was completely abrogated. Ectopic expression of NagZ can rescue the induction effect of CFZ and IMP on AmpC and enhance resistance in ΔnagZ. More importantly, CFZ and IMP have the potential to bring about the target genes expressions of AmpR in a NagZ-dependent manner.Conclusions: Our findings show that NagZ is a critical determinant for CFZ and IMP to promote AmpC expression and improve resistance and that CFZ and IMP should be used with caution since they may aggravate EC resistance. At the same time, this study further improves our understanding of resistance mechanisms in EC.

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