Host factor titration by chromosomal R-loops as a mechanism for runaway plasmid replication in transcription termination-defective mutants of Escherichia coli

Two Escherichia coli genes, rnhA and recG, encode products that disrupt R-loops by hydrolysis and unwinding, respectively. It is known that the propensity for R-loop formation in vivo is increased during growth at 21 °C. We have identified several links between rnhA, recG, and R-loop-dependent plasmid replication on the one hand, and genes rho and nusG involved in factor-dependent transcription termination on the other. A novel nusG-G146D mutation phenocopied a rho-A243E mutation in conferring global deficiency in transcription termination, and both mutants were killed at 21 °C following overexpression of rnhA+. Mutant combinations rnhA-nusG or recG-rho were synthetically lethal at 21 °C, with the former being suppressed by recG+ overexpression. rho and nusG mutants were killed following transformation with plasmids such as pACYC184 or pUC19 (which have R-loop replication intermediates) even at 30 °C or 37 °C, and the lethality was correlated with greatly increased content of supercoiled monomer species of these and other co-resident R-loop-dependent plasmids. Plasmid-mediated lethality in the mutants was suppressed by overexpression of rnhA+ or recG+. Two additional categories of trans-acting suppressors of the plasmid-mediated lethality were identified whose primary effects were, respectively, a reduction in plasmid copy number even in the wild-type strain, and a restoration of the proficiency of in vivo transcription termination in the nusG and rho mutant strains. The former category of suppressors included rom+, and mutations in rpoB(Q513L), pcnB, and polA, whereas the latter included a mutation in rho (R221C) and several non-null mutations (E74K, L26P, and Δ64-137) in the gene encoding the nucleoid protein H-NS. We propose that an increased occurrence of chromosomal R-loops in the rho and nusG mutants leads to titration of a cyloplasmic host factor(s) that negatively modulates the stability of plasmid R-loop replication intermediates and consequently to runaway plasmid replication

A dnaC mutation in Escherichia coli that affects copy number of ColE1-like plasmids and the PriA-PriB (but Not Rep-PriC) pathway of chromosomal replication restart

Escherichia coli nusG and rho mutants, which are defective in transcription termination, are killed following transformation with several ColE1-like plasmids that lack the plasmid-encoded copy-number regulator gene rom because of uncontrolled plasmid replication within the cells. In this study, a mutation [dnaC1331(A84T)] in the dnaC gene encoding the replicative helicase-loading protein was characterized as a suppressor of this plasmid-mediated lethality phenotype. The mutation also reduced the copy number of the plasmids in otherwise wild-type strains. In comparison with the isogenic dnaC+ strain, the dnaC mutant was largely unaffected for (i) growth on rich or minimal medium, (ii) tolerance to UV irradiation, or (iii) survival in the absence of the PriA, RecA, or RecB proteins. However, it was moderately SOS-induced and was absolutely dependent on both the Rep helicase and the PriC protein for its viability. A dnaC1331(A84T) dam mutant, but not its mutH derivative, exhibited sensitivity to growth on rich medium, suggestive of a reduced capacity in the dnaC1331(A84T) strains to survive chromosomal double-strand breaks. We propose that DnaC-A84T is proficient in the assembly of replication forks for both initiation of chromosome replication (at oriC) and replication restart via the Rep-PriC pathway, but that it is specifically defective for replication restart via the PriA-PriB pathway (and consequently also for replication of the Rom- ColE1-like plasmids)

Why is transcription coupled to translation in bacteria?

Active mechanisms exist to prevent transcription that is uncoupled from translation in the protein-coding genes of bacteria, as exemplified by the phenomenon of nonsense polarity. Bacterial transcription-translation coupling may be viewed as one among several co-transcriptional processes, including those for mRNA processing and export in the eukaryotes, that operate in the various life forms to render the nascent transcript unavailable for formation of otherwise deleterious R-loops in the genome

Revolutionizing physics: a comprehensive survey of machine learning applications

In the context of the 21st century and the fourth industrial revolution, the substantial proliferation of data has established it as a valuable resource, fostering enhanced computational capabilities across scientific disciplines, including physics. The integration of Machine Learning stands as a prominent solution to unravel the intricacies inherent to scientific data. While diverse machine learning algorithms find utility in various branches of physics, there exists a need for a systematic framework for the application of Machine Learning to the field. This review offers a comprehensive exploration of the fundamental principles and algorithms of Machine Learning, with a focus on their implementation within distinct domains of physics. The review delves into the contemporary trends of Machine Learning application in condensed matter physics, biophysics, astrophysics, material science, and addresses emerging challenges. The potential for Machine Learning to revolutionize the comprehension of intricate physical phenomena is underscored. Nevertheless, persisting challenges in the form of more efficient and precise algorithm development are acknowledged within this review

Transcription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors

Poly(A) polymerase I (PAP I), encoded by the pcnB gene, is a major enzyme responsible for RNA polyadenylation in Escherichia coli, a process involved in the global control of gene expression in this bacterium through influencing the rate of transcript degradation. Recent studies have suggested a complicated regulation of pcnB expression, including a complex promoter region, a control at the level of translation initiation and dependence on bacterial growth rate. In this report, studies on transcription regulation of the pcnB gene are described. Results of in vivo and in vitro experiments indicated that (a) there are three σ70-dependent (p1, pB, and p2) and two σS-dependent (pS1 and pS2) promoters of the pcnB gene, (b) guanosine tetraphosphate (ppGpp) and DksA directly inhibit transcription from pB, pS1 and pS2, and (c) pB activity is drastically impaired at the stationary phase of growth. These results indicate that regulation of the pcnB gene transcription is a complex process, which involves several factors acting to ensure precise control of PAP I production. Moreover, inhibition of activities of pS1 and pS2 by ppGpp and DksA suggests that regulation of transcription from promoters requiring alternative σ factors by these effectors of the stringent response might occur according to both passive and active models

Loss of Genetic Redundancy in Reductive Genome Evolution

Biological systems evolved to be functionally robust in uncertain environments, but also highly adaptable. Such robustness is partly achieved by genetic redundancy, where the failure of a specific component through mutation or environmental challenge can be compensated by duplicate components capable of performing, to a limited extent, the same function. Highly variable environments require very robust systems. Conversely, predictable environments should not place a high selective value on robustness. Here we test this hypothesis by investigating the evolutionary dynamics of genetic redundancy in extremely reduced genomes, found mostly in intracellular parasites and endosymbionts. By combining data analysis with simulations of genome evolution we show that in the extensive gene loss suffered by reduced genomes there is a selective drive to keep the diversity of protein families while sacrificing paralogy. We show that this is not a by-product of the known drivers of genome reduction and that there is very limited convergence to a common core of families, indicating that the repertoire of protein families in reduced genomes is the result of historical contingency and niche-specific adaptations. We propose that our observations reflect a loss of genetic redundancy due to a decreased selection for robustness in a predictable environment

Impact Factor: 2.1506 (UIF) TOTAL GLOBAL DOMINATION IN FUZZY GRAPH

ABSTRACT:Let G be a fuzzy graph without isolated nodes. A subset D of V is said to be a total dominating set if every node in V is dominated by a node in D. A total dominating set D of a graph G = (V,E) is a total global fuzzy dominating set (t.g.f.d. set) if D is also a total dominating set of G. The total global fuzzy domination number γtgf(G) of G is the minimum cardinality of a t.g.f.d. set for some classes of fuzzy graphs and obtain bounds for the same

Cellular stress created by intermediary metabolite imbalances

Small molecules generally activate or inhibit gene transcription as externally added substrates or as internally accumulated end-products, respectively. Rarely has a connection been made that links an intracellular intermediary metabolite as a signal of gene expression. We report that a perturbation in the critical step of a metabolic pathway—the D-galactose amphibolic pathway—changes the dynamics of the pathways leading to accumulation of the intermediary metabolite UDP-galactose. This accumulation causes cell stress and transduces signals that alter gene expression so as to cope with the stress by restoring balance in the metabolite pool. This underscores the importance of studying the global effects of alterations in the level of intermediary metabolites in causing stress and coping with it by transducing signals to genes to reach a stable state of equilibrium (homeostasis). Such studies are an essential component in the integration of metabolomics, proteomics, and transcriptomics

Rho directs widespread termination of intragenic and stable RNA transcription

The transcription termination factor Rho is a global regulator of RNA polymerase (RNAP). Although individual Rho-dependent terminators have been studied extensively, less is known about the sites of RNAP regulation by Rho on a genome-wide scale. Using chromatin immunoprecipitation and microarrays (ChIP-chip), we examined changes in the distribution of Escherichia coli RNAP in response to the Rho-specific inhibitor bicyclomycin (BCM). We found ≈200 Rho-terminated loci that were divided evenly into 2 classes: intergenic (at the ends of genes) and intragenic (within genes). The intergenic class contained noncoding RNAs such as small RNAs (sRNAs) and transfer RNAs (tRNAs), establishing a previously unappreciated role of Rho in termination of stable RNA synthesis. The intragenic class of terminators included a previously uncharacterized set of short antisense transcripts, as judged by a shift in the distribution of RNAP in BCM-treated cells that was opposite to the direction of the corresponding gene. These Rho-terminated antisense transcripts point to a role of noncoding transcription in E. coli gene regulation that may resemble the ubiquitous noncoding transcription recently found to play myriad roles in eukaryotic gene regulation