Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

High resolution landscape of an antibiotic binding site

ABSTRACT: Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations, however their study is limited by positive selection. Using multiplex genome engineering1 to overcome this constraint, we generate and characterize a collection of 760 single residue mutants encompassing the entire rifampicin binding site of E. coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations dramatically enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication-dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP. Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics, and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE236920 | GEO | 2023/08/01

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Targeting the fitness cost of antibiotic resistance via the non-essential genome

Project description:Mutations in the rifampicin (Rif)-binding site of RNA polymerase (RNAP) impart antibiotic resistance and inextricably affect transcription initiation, elongation, and termination properties as well. At each step of the transcription cycle, RNAP responds to non-essential transcription factors, signaling molecules, and substrate availability. As such, the non- essential genome and its impact on fitness cost potentially represent an untapped resource for new combination therapies. Using transposon sequencing (Tn-seq), we present a genome- wide analysis of resistance cost in a clinically common rpoB H526Y mutant. Our data show that cost-compounding genes include factors that promote high transcription elongation rate, whereas cost-mitigating genes function in cell wall synthesis and division. We demonstrate that cell wall synthesis and division defects in rpoB H526Y are a consequence of an abnormally high transcription elongation rate, which is further exacerbated by superfluous activity of the uracil salvage pathway and indifference of the mutant RNAP to alarmone ppGpp. Leveraging on this knowledge, we identified drugs that are highly potent against rpoB H526Y and other RifR alleles from the same phenotypic class. Thus, genome-wide analysis of fitness cost of antibiotic resistant mutants should expedite discovery of new combination therapies and delineate cellular pathways that underlie molecular mechanisms of cost.

2021-11-25 | PXD027810 | Pride

Biological cost of rifampicin resistance in Helicobacter pylori

Project description:The present study was aimed at analyzing (i) the biological cost of RNA polymerase (rpoB) mutations conferring rifampin resistance on H.pylori, (ii) the relationship between the cost of rpoB mutations and the chromosomal mutaion, (iii) the relationship between the cost of rpoB mutations and the transcription profile of sensitive and resistantrif strains of H.pylori (iv) and rpoB mutations in view of the possible fitness burden associated with resistance to another antibiotics. H.pylori reference strain 26695 was routinely maintained on Columbia agar plates and H. pylori-selective antibiotic mix Dent. Liquid culture was grown in BHI broth. Both plates and broth cultures were incubated at 37C under atmosphere enriched with 5% CO2 for 2-3 days . Mutant strains were selected by culturing H. pylori 26695 on selective plates containing rifampicin. In 5 days resistant colonies were picked up and passed under rifampicin pressure. RNA isolated was reverse transcribed and used to probe H. pylori home-made arrays

2010-11-30 | E-GEOD-25333 | biostudies-arrayexpress

Identifying the fitness cost of rifampicin-resistance in Mycobacterium tuberculosis through RNAseq profiling.

Project description:The experiment was designed to infer the fitness cost of rifampicin-resistance in Mycobacterium tuberculosis through expression analysis. The approach relied on: 1. Tracking the expression changes occurring as a result of the rifampicin-resistance conferring mutation Ser450Leu in RpoB and subsequent gain of compensatory mutation Leu516Pro in RpoC. The hypothesis was that any cost-incurring expressional changes would be reversed in the presence of compensatory mutations. The strains in this set were described before here: PMID 22179134. 2. Comparing the impact of the same rifampicin-resistance mutation (RpoB Ser450Leu) in five different genetic backgrounds. Here the comparison was solely between RpoB Ser450Leu and their cognate drug susceptible wild type ancestor. One of the strain pairs is the same as in point 1. above.

2018-11-18 | E-MTAB-7359 | biostudies-arrayexpress

The DUF1013 protein TrcR tracks with RNA polymerase to control bacterial cell cycle and protection against antibiotics (RNA-seq dataset)

Project description:The regulatory roles of proteins associating with DNA-dependent RNA polymerase (RNAP) during transcription elongation are poorly characterized in bacteria. A forward genetic selection for Caulobacter crescentus cell cycle mutants revealed the uncharacterized DUF1013 protein (TrcR, transcriptional cell cycle regulator). TrcR associates with promoters and coding sequences (CDSs) and tracks with active RNAP. Loss of TrcR causes a cell division cycle and growth defect and an insufficiency in the essential cell cycle regulator CtrA. TrcR also protects cells from the quinolone antibiotic nalidixic acid that induces a multi-drug efflux pump and from the RNAP inhibitor rifampicin (Rif) that prevents transcription elongation. TrcR interacts biochemically and genetically with RNAP and no longer associates with chromatin when RNAP is inhibited with Rif. We show that these TrcR functions and its RNAP-dependent chromatin recruitment are conserved in symbiotic Sinorhizobium sp. and pathogenic Brucella sp., indicating that TrcR represents a new antibiotic target.

2021-02-03 | GSE148653 | GEO

The DUF1013 protein TrcR tracks with RNA polymerase to control bacterial cell cycle and protection against antibiotics (ChIP-seq dataset)

2021-02-03 | GSE148652 | GEO

Measuring gene expression in serially-evolved E. coli BW25113 strains in M9+glucose and M9+glucose+rifampicin

Project description:We measured steady-state E. coli transcript levels in 1) a pykF(C8Y) mutant, 2) a rpoB(T1037P) mutant, and 3) five rifampicin-resistant rpoB mutant strains selected from each of these single mutants, in the presence and absence of rifampicin.

2020-10-23 | GSE136977 | GEO

Identification and characterization of pleiotropic high-persistence mutations in the beta subunit of the bacterial RNA polymerase

Project description:We report identification and characterization of antibiotic persister mutants carrying characteristic mutations in the Escherichia coli rpoB gene

2021-01-26 | GSE165446 | GEO

ChIP-chip of RNA Polymerase (RNAP) and RpoD in Salmonella enterica serovar Typhimurium strain SL1344

Project description:ChIP-on-chip analysis of RNAP and RpoD binding to the Salmonella enterica serovar Typhimurium chromosome demonstrated a high degree of overlap between RNAP and RpoD binding and provided us with important insights into the global distribution of these factors. Furthermore this data was correlated with information on the location of 1873 transcription start sites identified by RNA-Seq technology, thereby providing a detailed transcriptional map of Salmonella Typhimurium. Analysis of RNAP, RNAP-Rifampicin and and RpoD binding in Luria Broth (LB)

2012-02-16 | E-GEOD-35827 | biostudies-arrayexpress

ChIP-chip of MG1655 with antibody against E. coli RNAP beta subunit under various conditions

Project description:We integrated RNAP binding regions (RBRs) and mRNA transcript abundance to determine segments of contiguous transcription originating from promoter regions. To measure RBRs at a genome scale, we employed a ChIP-chip method to E. coli K-12 MG1655 grown in the presence or absence of rifampicin under multiple growth conditions using antibody against E. coli RNAP beta subunit.

2009-11-04 | GSE15588 | GEO

Downregulation of miR-760 Causes Human Intervertebral Disc Degeneration by Targeting the MyD88/Nuclear Factor-Kappa B Signaling Pathway

Project description:Dysregulation of microRNAs (miRNAs) plays a critical role in the development of intervertebral disc degeneration (IDD). In this study, we present evidence from in vitro and in vivo research to elucidate the mechanism underlying the role of miR-760 in IDD. miRNA microarray and quantitative reverse transcription-polymerase chain reaction were used to determine the miRNA profiles in patients with IDD. Functional analysis was performed to evaluate the role of miR-760 in the pathogenesis of IDD. Luciferase reporter and western blotting assays were used to confirm the miRNA targets. The expression of miR-760 was significantly decreased in degenerative nucleus pulposus (NP) cells and negatively correlated with disc degeneration grade. Functional assays demonstrated that miR-760 delivery significantly increased NP cell proliferation and promoted the expression of collagen II and aggrecan. Moreover, MyD88 was identified as a target gene of miR-760. miR-760 effectively suppressed MyD88 expression by interacting with the 3′-untranslated region, which was abolished by miR-760 binding site mutations. An in vivo experiment using an IDD mouse model showed that the upregulation of miR-760 could effectively suspend IDD. Therefore, miR-760 was found to play an important role in IDD and can be used as a promising therapeutic target for the treatment of patients with IDD.

2022-03-17 | E-MTAB-11554 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data