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

2021 ◽  
Author(s):  
Lev Ostrer ◽  
Yinduo Ji ◽  
Arkady Khodursky
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Survival Rates ◽  
Beta Subunit ◽  
High Survival ◽  
Resistance Mutations ◽  
Switch Region ◽  
E Coli ◽  
Bacterial Rna ◽  
Survival Frequency

Mutations conferring resistance to bactericidal antibiotics reduce average susceptibility of the mutant populations. It is unknown, however, how those mutations affect survival of individual bacteria. Since surviving bacteria can be a reservoir for recurring infections, it is important to know how survival rates may be affected by resistance mutations and by the choice of antibiotics. Here we present the evidence that: i) Escherichia coli mutants with 100-1000 times increased frequency of survival in ciprofloxacin, an archetypal fluoroquinolone antibiotic, can be readily obtained in a stepwise selection; ii) the high survival frequency is conferred by mutations in the switch region of the beta subunit of the RNA polymerase; iii) the switch-region mutations are (p)ppGpp mimics, partially analogous to rpoB stringent mutations; iv) the stringent and switch-region rpoB mutations frequently occur in clinical isolates of E. coli , Acinetobacter baumannii , Mycobacterium tuberculosis and Staphylococcus aureus , and at least one of them, RpoB S488L, which is a common rifampicin-resistance mutations, dramatically increases survival of a clinical MRSA strain in ampicillin; v) the RpoB-associated high-survival phenotype can be reversed by sub-inhibitory concentrations of chloramphenicol.

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

2020 ◽  
Author(s):  
Lev Ostrer ◽  
Yinduo Ji ◽  
Arkady Khodursky
Keyword(s):  
Antibiotic Resistance ◽  
Rna Polymerase ◽  
Low Frequency ◽  
Beta Subunit ◽  
Resistance Mutations ◽  
Chronic Infections ◽  
Gene Encoding ◽  
Bacterial Populations ◽  
Bacterial Rna ◽  
Transcriptional Changes

AbstractIndividual bacteria can escape killing by bactericidal antibiotics by becoming dormant. Such cells, also known as persisters, naturally occur in bacterial populations at a low frequency. Here we present the finding that antibiotic-resistance mutations in the rpoB gene, encoding the beta subunit of RNA polymerase, increase the frequency of persisters by orders of magnitude. Furthermore, we show that: i) the persistent state depends on the (p)ppGpp transcriptional program and not on (p)ppGpp itself; ii) the high persistence (hip) is associated with increased populational heterogeneity in transcription; iii) indole overproduction, caused by transcriptional changes in the hip mutants, explains 50-80% of the hip phenotype. We report that the analogous rpoB mutations occur frequently in clinical isolates of Acinetobacter baumannii, Mycobacterium tuberculosis and Staphylococcus aureus, and we demonstrate that one of those rpoB mutations causes high persistence in MRSA. We also show that the RpoB-associated hip phenotype can be reversed by inhibiting protein synthesis.ImportancePersistence is an inevitable consequence of antibiotic usage. Although persistence is not a genetically heritable trait, here we demonstrate for the first time that antibiotic resistance, which is heritable, can promote persistence formation. Our finding that resistance to one antibiotic, rifampicin, can boost persistence to other antibiotics, such as ciprofloxacin and ampicillin, may help explain why certain chronic infections are particularly recalcitrant to antibiotic therapies. Out results also emphasize the need to assess the effects of combination antibiotic therapies on persistence.

scholarly journals New target for inhibition of bacterial RNA polymerase: ‘switch region’

2011 ◽  
Vol 14 (5) ◽  
pp. 532-543 ◽  
Author(s):  
Aashish Srivastava ◽  
Meliza Talaue ◽  
Shuang Liu ◽  
David Degen ◽  
Richard Y Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Switch Region ◽  
Bacterial Rna

scholarly journals Predicting Rifampicin Resistance Mutations in Bacterial RNA Polymerase Subunit Beta Based on Machine Learning Algorithms

2020 ◽  
Author(s):  
Qing Ning ◽  
Dali Wang ◽  
Fei Cheng ◽  
Yuheng Zhong ◽  
Qi Ding ◽  
...  
Keyword(s):  
Machine Learning ◽  
Antibiotic Resistance ◽  
Rna Polymerase ◽  
Binding Energies ◽  
Machine Learning Algorithms ◽  
New Drugs ◽  
Structural Basis ◽  
Resistance Mutations ◽  
Bacterial Rna ◽  
Majority Consensus

Abstract BackgroundMutations in an enzyme target are one of the most common mechanisms whereby antibiotic resistance arises. Identification of the resistance mutations in bacteria is essential for understanding the structural basis of antibiotic resistance and design of new drugs. However, the traditionally used experimental approaches to identify resistance mutations were usually labor-intensive and costly. ResultsWe present a machine learning (ML)-based classifier for predicting rifampicin (Rif) resistance mutations in bacterial RNA Polymerase subunit β (RpoB). A total of 66 resistance mutations were gathered from the literature to form positive dataset, while 53 residue variations of RpoB among a series of naturally occurring species were obtained as negative database. The features of the mutated RpoB and their binding energies with Rif were calculated through computational methods, and used as the mutation attributes for modelling. Classifiers based on four ML algorithms, i.e. decision tree, k nearest neighbors, naïve Bayes and supporting vector machine, were developed, which showed accuracy ranging from 0.69 to 0.76. A majority consensus approach was then used to obtain a new classifier based on the classifications of the four individual ML algorithms. The majority consensus classifier significantly improved the predictive performance, with accuracy, precision, recall and specificity of 0.83, 0.84, 0.86 and 0.83, respectively. ConclusionThe majority consensus classifier provides an alternative methodology for rapid identification of resistance mutations in bacteria, which may help with early detection of antibiotic resistance and new drug discovery.

INTRAGENIC PROMOTOR-LIKE SITES IN THE GENOME OFESCHERICHIA COLIDISCOVERY AND FUNCTIONAL IMPLICATION

2007 ◽  
Vol 05 (02b) ◽  
pp. 549-560 ◽  
Author(s):  
MARIA N. TUTUKINA ◽  
KONSTANTIN S. SHAVKUNOV ◽  
IRINA S. MASULIS ◽  
OLGA N. OZOLINE
Keyword(s):  
Rna Polymerase ◽  
Antisense Transcription ◽  
Antisense Strand ◽  
Entire Genome ◽  
Coding Sequences ◽  
E Coli ◽  
Chip Technology ◽  
Bacterial Rna ◽  
Small Regulatory Rnas ◽  
Polymerase Binding

Mapping of putative promoters within the entire genome of Escherichia coli (E. coli) by means of pattern-recognition software PlatProm revealed several thousand of sites having high probability to perform promoter function. Along with the expected promoters located upstream of coding sequences, PlatProm identified more than a thousand potential promoters for antisense transcription and several hundred very similar signals within coding sequences having the same direction with the genes. Since recently developed ChIP–chip technology also testified the presence of intragenic RNA polymerase binding sites, such distribution of putative promoters is likely to be a general biological phenomenon reflecting yet undiscovered regulatory events. Here, we provide experimental evidences that two internal promoters are recognized by bacterial RNA polymerase. One of them is located within the hns coding sequence and may initiate synthesis of RNA from the antisense strand. Another one is found within the overlapping genes htgA/yaaW and may control the production of a shortened mRNA or an RNA-product complementary to mRNA of yaaW. Both RNA-products can form secondary structures with free energies of folding close to those of small regulatory RNAs (sRNAs) of the same length. Folding propensity of known sRNAs was further compared with that of antisense RNAs (aRNAs), predicted in E. coli as well as in Salmonella typhimurium (S. typhimurium). Slightly lower stability observed for aRNAs assumes that their structural compactness may be less significant for biological function.

scholarly journals Mechanism of Eukaryotic RNA Polymerase III Transcription Termination

Science ◽  
2013 ◽  
Vol 340 (6140) ◽  
pp. 1577-1580 ◽  
Author(s):  
Soren Nielsen ◽  
Yulia Yuzenkova ◽  
Nikolay Zenkin
Keyword(s):  
Rna Polymerase ◽  
Rna Secondary Structure ◽  
Common Ancestor ◽  
Rna Polymerase Iii ◽  
Rna Molecules ◽  
Polymerase Iii ◽  
Bacterial Rna ◽  
Catalytic Inactivation ◽  
The Common ◽  
Pol Iii

Gene expression in organisms involves many factors and is tightly controlled. Although much is known about the initial phase of transcription by RNA polymerase III (Pol III), the enzyme that synthesizes the majority of RNA molecules in eukaryotic cells, termination is poorly understood. Here, we show that the extensive structure of Pol III–synthesized transcripts dictates the release of elongation complexes at the end of genes. The poly-T termination signal, which does not cause termination in itself, causes catalytic inactivation and backtracking of Pol III, thus committing the enzyme to termination and transporting it to the nearest RNA secondary structure, which facilitates Pol III release. Similarity between termination mechanisms of Pol III and bacterial RNA polymerase suggests that hairpin-dependent termination may date back to the common ancestor of multisubunit RNA polymerases.

scholarly journals Predicting rifampicin resistance mutations in bacterial RNA polymerase subunit beta based on majority consensus

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Qing Ning ◽  
Dali Wang ◽  
Fei Cheng ◽  
Yuheng Zhong ◽  
Qi Ding ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Rna Polymerase ◽  
New Drugs ◽  
Structural Basis ◽  
Support Vector ◽  
Resistance Mutations ◽  
Training Set ◽  
Test Set ◽  
Bacterial Rna ◽  
Majority Consensus

Abstract Background Mutations in an enzyme target are one of the most common mechanisms whereby antibiotic resistance arises. Identification of the resistance mutations in bacteria is essential for understanding the structural basis of antibiotic resistance and design of new drugs. However, the traditionally used experimental approaches to identify resistance mutations were usually labor-intensive and costly. Results We present a machine learning (ML)-based classifier for predicting rifampicin (Rif) resistance mutations in bacterial RNA Polymerase subunit β (RpoB). A total of 186 mutations were gathered from the literature for developing the classifier, using 80% of the data as the training set and the rest as the test set. The features of the mutated RpoB and their binding energies with Rif were calculated through computational methods, and used as the mutation attributes for modeling. Classifiers based on five ML algorithms, i.e. decision tree, k nearest neighbors, naïve Bayes, probabilistic neural network and support vector machine, were first built, and a majority consensus (MC) approach was then used to obtain a new classifier based on the classifications of the five individual ML algorithms. The MC classifier comprehensively improved the predictive performance, with accuracy, F-measure and AUC of 0.78, 0.83 and 0.81for training set whilst 0.84, 0.87 and 0.83 for test set, respectively. Conclusion The MC classifier provides an alternative methodology for rapid identification of resistance mutations in bacteria, which may help with early detection of antibiotic resistance and new drug discovery.

scholarly journals A T7 phage factor required for managing RpoS inEscherichia coli

2018 ◽  
Author(s):  
Aline Tabib-Salazar ◽  
Bing Liu ◽  
Declan Barker ◽  
Lynn Burchell ◽  
Udi Qimron ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Stationary Phases ◽  
Specific Inhibitor ◽  
E Coli ◽  
Bacterial Transcription ◽  
Bacterial Rna ◽  
Phage T7 ◽  
T7 Phage

T7 development inEscherichia colirequires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. While the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit EσS, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growingE. colias a consequence of buildup of guanosine pentaphosphate ((p)ppGpp) during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development inE. colicells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.Significance statementVirus that infect bacteria (phages) represent the most abundant living entities on the planet and many aspects of our fundamental knowledge of phage-bacteria relationships have been derived in the context of exponentially growing bacteria. In the case of the prototypicalEscherichia coliphage T7, specific inhibition of the housekeeping form of the RNA polymerase (Eσ70) by a T7 protein, called Gp2, is essential for the development of viral progeny. We now reveal that T7 uses a second specific inhibitor that selectively inhibits the stationary phase RNAP (EσS), which enables T7 to develop well in exponentially growing and stationary phase bacteria. The results have broad implications for our understanding of phage-bacteria relationships and therapeutic application of phages.

scholarly journals Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein

2021 ◽  
Vol 8 ◽  
Author(s):  
Virtu Solano-Collado ◽  
Sofía Ruiz-Cruz ◽  
Fabián Lorenzo-Díaz ◽  
Radoslaw Pluta ◽  
Manuel Espinosa ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Sigma Factor ◽  
Regulation Of Gene Expression ◽  
E Coli ◽  
Core Enzyme ◽  
Protein Architecture ◽  
Bacterial Rna ◽  
Promoter Recognition ◽  
Rna Polymerase Holoenzyme

Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158.

Cibacron Blue inhibition of prokaryotic and eukaryotic DNA-dependent RNA polymerases

1990 ◽  
Vol 55 (11) ◽  
pp. 2769-2780
Author(s):  
Aleš Cvekl ◽  
Květa Horská
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Rna Polymerase Iii ◽  
Enzymic Activity ◽  
Rna Polymerases ◽  
Cibacron Blue ◽  
E Coli ◽  
Polymer Formation ◽  
Calf Thymus

A comparison was drawn between the action of Cibacron Blue F3GA on the enzymic activity of DNA-dependent RNA polymerases from different sources, e.g. Escherichia coli, calf thymus and wheat germ (polymerase II). Sensitivity towards this inhibitor was determined for polymer formation and primed abortive synthesis of trinucleotide UpApU. In case of E. coli polymerase and wheat germ polymerase II the dye inhibits both polymer formation and abortive synthesis. Calf thymus polymerase II is inhibited only in the polymerisation step. The primed initiation reaction was found to be resistant towards the dye. In case of E. coli polymerase and wheat germ polymerase II the sensitive step is the formation of internucleotide bond whereas in case of calf thymus polymerase II the translocation of the enzyme is influenced. An analysis of kinetic data indicates more than one binding site for the dye on RNA polymerase II from calf thymus and wheat germ. Cibacron blue does not inhibit specific transcription catalyzed by RNA polymerase III from human HeLa cells and mouse leukemia L1210 cells.

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