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Aline Tabib-Salazar, Bing Liu, Declan Barker, Lynn Burchell, Udi Qimron, Steve J Matthews, Sivaramesh Wigneshweraraj
T7 development in Escherichia coli requires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70 , by two T7 proteins: Gp2 and Gp5.7. Although 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 growing E. coli as a consequence of the buildup of guanosine pentaphosphate [(p)ppGpp] during T7 development...
May 22, 2018: Proceedings of the National Academy of Sciences of the United States of America
Madeline E Sherlock, Narasimhan Sudarsan, Ronald R Breaker
Riboswitches are noncoding portions of certain mRNAs that bind metabolite, coenzyme, signaling molecule, or inorganic ion ligands and regulate gene expression. Most known riboswitches sense derivatives of RNA monomers. This bias in ligand chemical composition is consistent with the hypothesis that widespread riboswitch classes first emerged during the RNA World, which is proposed to have existed before proteins were present. Here we report the discovery and biochemical validation of a natural riboswitch class that selectively binds guanosine tetraphosphate (ppGpp), a widespread signaling molecule and bacterial "alarmone" derived from the ribonucleotide GTP...
May 21, 2018: Proceedings of the National Academy of Sciences of the United States of America
Sukanya Iyer, Dai Le, Bo Ryoung Park, Minsu Kim
Bacteria adapt to environmental stress by producing proteins that provide stress protection. However, stress can severely perturb the kinetics of gene expression, disrupting protein production. Here, we characterized how Escherichia coli mitigates such perturbations under nutrient stress through the kinetic coordination of transcription and translation. We observed that, when translation became limiting under nitrogen starvation, transcription elongation slowed accordingly. This slowdown was mediated by (p)ppGpp, the alarmone whose primary role is thought to be promoter regulation...
May 14, 2018: Nature Microbiology
Jia Chen, Yanni Huang, Xiaoyan Yang, Haijuan Zhang, Zhan Li, Bo Qin, Xingguo Chen, Hongdeng Qiu
Guanosine 3'-diphosphate-5'-di(tri)phosphate (ppGpp) plays a crucial role in the gene expression, metabolism, growth, and other significant processes of microorganisms. In this work, a facile sensitive and visual strategy for the detection of ppGpp has been established by developing a colorimetric probe of copper ions (Cu2+ )-mediated 4-mercaptobenzoic acid (4-MBA) modified gold nanoparticles (AuNPs). The sensing process was characterized by transmission electron microscopy (TEM), zeta potential, dynamic light scattering (DLS) and UV-vis spectroscopy...
September 6, 2018: Analytica Chimica Acta
Mário Hüttener, Alejandro Prieto, Joan Espelt, Manuel Bernabeu, Antonio Juárez
Virulence expression in the enteroaggregative Escherichia coli strain 042 requires the transcriptional activator AggR. We show in this report that, as reported for other virulence factors, the nucleotide second messenger (p)ppGpp is needed for a high expression level of AggR. As expected from these findings, expression of AggR-activated genes such as the AafA pilin subunit is downregulated in the absence of (p)ppGpp. Considering the fact that biofilm formation in strain 042 requires the AafA protein, biofilm development in strain 042 is impaired in derivatives that lack either the AggR protein, the virulence plasmid that encodes AggR (pAA2) or the ability to synthesize (p)ppGpp...
2018: Frontiers in Microbiology
Kristoffer Skovbo Winther, Mohammad Roghanian, Kenn Gerdes
RelA/SpoT homologs (RSHs) are ubiquitous bacterial enzymes that synthesize and hydrolyze (p)ppGpp in response to environmental challenges. Bacteria cannot survive in hosts and produce infection without activating the (p)ppGpp-mediated stringent response, but it is not yet understood how the enzymatic activities of RSHs are controlled. Using UV crosslinking and deep sequencing, we show that Escherichia coli RelA ((p)ppGpp synthetase I) interacts with uncharged tRNA without being activated. Amino acid starvation leads to loading of cognate tRNA⋅RelA complexes at vacant ribosomal A-sites...
April 5, 2018: Molecular Cell
Anirban Ghosh, Özden Baltekin, Marcus Wäneskog, Dina Elkhalifa, Disa L Hammarlöf, Johan Elf, Sanna Koskiniemi
Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity...
March 23, 2018: EMBO Journal
George H Jones
Polynucleotide phosphorylase (PNPase) is a 3'-5'-exoribnuclease that is found in most bacteria and in some eukaryotic organelles. The enzyme plays a key role in RNA decay in these systems. PNPase structure and function have been studied extensively in Escherichia coli , but there are several important aspects of PNPase function in Streptomyces that differ from what is observed in E. coli and other bacterial genera. This review highlights several of those differences: (1) the organization and expression of the PNPase gene in Streptomyces ; (2) the possible function of PNPase as an RNA 3'-polyribonucleotide polymerase in Streptomyces ; (3) the function of PNPase as both an exoribonuclease and as an RNA 3'-polyribonucleotide polymerase in Streptomyces ; (4) the function of (p)ppGpp as a PNPase effector in Streptomyces ...
March 18, 2018: Antibiotics
Neerupma Bhardwaj, Kirtimaan Syal, Dipankar Chatterji
ppGpp, an alarmone for stringent response, plays an important role in the reprogramming of the transcription complex at the time of stress. In Escherichia coli, ppGpp mediates its action by binding to at least two different sites on RNA polymerase (RNAP). One of the sites to which ppGpp binds to RNAP is at the β'-ω interface; however, the underlying molecular mechanism and the physiological relevance of ppGpp binding to this site remain unclear. In this study, we have performed UV cross-linking experiments using 32 P azido-labeled ppGpp to probe its association with RNAP in the absence and presence of ω, and observed weaker binding of ppGpp to the RNAP without ω...
May 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Hwa Young Kim, Sang-Mi Yu, Sang Chul Jeong, Sang Sun Yoon, Young Taek Oh
The stringent response (SR), which is activated by accumulation of (p)ppGpp under conditions of growth-inhibiting stresses, plays an important role ongrowth and virulence in Vibrio cholerae . Herein, we carried out a genome-wide screen using transposon random mutagenesis to identify genes controlled by SR in (p)ppGpp-overproducing mutant strain. One of the identified SR target genes was the flaC encoding flagellin. Genetic studies using flaC and SR mutants demonstrated that FlaC involved in bacterial growth, toxin production, and normal flagellum function under conditions of high (p)ppGpp levels, suggesting FlaC plays an important role in SR-induced pathogenicityin V...
March 15, 2018: Journal of Microbiology and Biotechnology
Yong Zhang, Eva Zborníková, Dominik Rejman, Kenn Gerdes
The alarmone (p)ppGpp plays pivotal roles in basic bacterial stress responses by increasing tolerance of various nutritional limitations and chemical insults, including antibiotics. Despite intensive studies since (p)ppGpp was discovered over 4 decades ago, (p)ppGpp binding proteins have not been systematically identified in Escherichia coli We applied DRaCALA (<u>d</u>ifferential <u>ra</u>dial <u>c</u>apillary <u>a</u>ction of <u>l</u>igand <u>a</u>ssay) to identify (p)ppGpp-protein interactions...
March 6, 2018: MBio
Sophie E Irving, Rebecca M Corrigan
The stringent response is a conserved bacterial stress response mechanism that allows bacteria to respond to nutritional challenges. It is mediated by the alarmones pppGpp and ppGpp, nucleotides that are synthesized and hydrolyzed by members of the RSH superfamily. Whilst there are key differences in the binding targets for (p)ppGpp between Gram-negative and Gram-positive bacterial species, the transient accumulation of (p)ppGpp caused by nutritional stresses results in a global change in gene expression in all species...
March 2018: Microbiology
Bin Bin Chen, Meng Li Liu, Lei Zhan, Chun Mei Li, Cheng Zhi Huang
Highly selective and sensitive detection of guanosine 3'-diphosphate-5'-diphosphate (ppGpp), namely, the stringent in plants or microorganisms responding to strict or extreme environmental conditions such as stress and starvation, which plays an important role in gene expression, rRNA and antibiotics production, regulations of virulence of bacteria, and growth of plants, faces a great challenge owing to its extreme similarity to normal nucleotides. By modifying the surface groups of a facile two-step hydrothermal route prepared carbon dots (CDs) with terbium ions (Tb3+ ) in this contribution, a novel fluorescent probe with excellent properties such as highly physical and chemical stability, narrow emission and excitation wavelength-independent emission was prepared...
March 20, 2018: Analytical Chemistry
Vadim Molodtsov, Elena Sineva, Lu Zhang, Xuhui Huang, Michael Cashel, Sarah E Ades, Katsuhiko S Murakami
DksA and ppGpp are the central players in the stringent response and mediate a complete reprogramming of the transcriptome. A major component of the response is a reduction in ribosome synthesis, which is accomplished by the synergistic action of DksA and ppGpp bound to RNA polymerase (RNAP) inhibiting transcription of rRNAs. Here, we report the X-ray crystal structures of Escherichia coli RNAP in complex with DksA alone and with ppGpp. The structures show that DksA accesses the template strand at the active site and the downstream DNA binding site of RNAP simultaneously and reveal that binding of the allosteric effector ppGpp reshapes the RNAP-DksA complex...
March 1, 2018: Molecular Cell
Galina V Smirnova, Aleksey V Tyulenev, Nadezda G Muzyka, Oleg N Oktyabrsky
Amino acid starvation causes an RelA-dependent increase in the regulatory nucleotide (p)ppGpp that leads to pleiotropic changes in Escherichia coli metabolism, but the role of (p)ppGpp in regulation of respiration remains unclear. Here we demonstrate that amino acid starvation is accompanied by sharp RelA-dependent inhibition of respiration. The sharp phase of inhibition is absent in relA mutants, and can be prevented by translation inhibitors chloramphenicol and tetracycline, which abolish accumulation of (p)ppGpp...
April 2018: Research in Microbiology
Hwa Young Kim, Junhyeok Go, Kang-Mu Lee, Young Taek Oh, Sang Sun Yoon
The pathogen Vibrio cholerae is the causative agent of cholera. Emergence of antibiotic-resistant V. cholerae strains is increasing, but the underlying mechanisms remain unclear. Herein, we report that the stringent response regulator and stress alarmone guanosine tetra- and pentaphosphate ((p)ppGpp) significantly contributes to antibiotic tolerance in V. cholerae We found that N16961, a pandemic V. cholerae strain, and its isogenic (p)ppGpp-overexpressing mutant Δ relA Δ spoT are both more antibiotic-resistant than (p)ppGpp0 (Δ relA Δ relV Δ spoT ) and Δ dksA mutants, which cannot produce or utilize (p)ppGpp, respectively...
April 13, 2018: Journal of Biological Chemistry
Etienne Maisonneuve, Manuela Castro-Camargo, Kenn Gerdes
No abstract text is available yet for this article.
February 22, 2018: Cell
Kaneyoshi Yamamoto, Yuki Yamanaka, Tomohiro Shimada, Paramita Sarkar, Myu Yoshida, Neerupma Bhardwaj, Hiroki Watanabe, Yuki Taira, Dipankar Chatterji, Akira Ishihama
The RNA polymerase (RNAP) of Escherichia coli K-12 is a complex enzyme consisting of the core enzyme with the subunit structure α2 ββ'ω and one of the σ subunits with promoter recognition properties. The smallest subunit, omega (the rpoZ gene product), participates in subunit assembly by supporting the folding of the largest subunit, β', but its functional role remains unsolved except for its involvement in ppGpp binding and stringent response. As an initial approach for elucidation of its functional role, we performed in this study ChIP-chip (chromatin immunoprecipitation with microarray technology) analysis of wild-type and rpoZ -defective mutant strains...
January 2018: MSystems
Shanshan Zhang, Shuang Liu, Nan Wu, Youhua Yuan, Wenhong Zhang, Ying Zhang
As dormant phenotypic variants of bacteria, persisters account for many chronic infections affecting human health. Despite numerous studies, the role of small non-coding RNA (sRNA) in bacterial persistence has not been reported. To investigate the role of Hfq-interacting sRNA in persistence, we constructed the deletion mutants of 20 Hfq-interacting sRNAs (RyhB, GcvB, MgrR, RybB, MicF, SgrS, RprA, DicF, SsrS, FnrS, GadY, DsrA, OmrB, ArcZ, RyeB, RydC, OmrA, MicA, MicC, and ChiX) to assess their persistence capacity in uropathogenic Escherichia coli strain UTI89 and identified a new sRNA RyhB being involved in persister formation...
2018: Frontiers in Microbiology
Sousuke Imamura, Yuhta Nomura, Tokiaki Takemura, Imran Pancha, Keiko Taki, Kazuki Toguchi, Yuzuru Tozawa, Kan Tanaka
Chloroplasts are plant organelles that carry out oxygenic photosynthesis. Chloroplast biogenesis depends upon chloroplast ribosomes and their translational activity. However, regulation of chloroplast ribosome biogenesis remains an important unanswered question. In this study, we found that inhibition of target of rapamycin (TOR), a general eukaryotic checkpoint kinase, results in a decline in chloroplast ribosomal RNA (rRNA) transcription in the unicellular red alga, Cyanidioschyzon merolae. Upon TOR inhibition, transcriptomics and other analyses revealed increased expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) gene (CmRSH4b), which encodes a homolog of the guanosine 3'-diphosphate 5'-diphosphate (ppGpp) synthetases that modulate rRNA synthesis in bacteria...
April 2018: Plant Journal: for Cell and Molecular Biology
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