The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Judith P Armitage - One of the best experts on this subject based on the ideXlab platform.

  • inducible expression plasmid for Rhodobacter Sphaeroides and paracoccus denitrificans
    Applied and Environmental Microbiology, 2009
    Co-Authors: Alice C. Ind, Mostyn T Brown, Steven L. Porter, Elaine D. Byles, Jennifer A. De Beyer, Scott A. Godfrey, Judith P Armitage
    Abstract:

    We have developed a stable isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible-expression plasmid, pIND4, which allows graduated levels of protein expression in the alphaproteobacteria Rhodobacter Sphaeroides and Paracoccus denitrificans. pIND4 confers kanamycin resistance and combines the stable replicon of pMG160 with the lacI(q) gene from pYanni3 and the lac promoter, P(A1/04/03), from pJBA24.

  • a molecular brake not a clutch stops the Rhodobacter Sphaeroides flagellar motor
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Teuta Pilizota, Mostyn T Brown, Mark C Leake, Richard W Branch, Richard M Berry, Judith P Armitage
    Abstract:

    Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter Sphaeroides employs a single stop-start flagellar motor. Here, we asked, how does the binding of CheY-P stop the motor in R. Sphaeroides—using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. Sphaeroides motor is stopped using a brake. The motor stops at 27–28 discrete angles, locked in place by a relatively high torque, approximately 2–3 times its stall torque.

  • Rhodobacter Sphaeroides complexity in chemotactic signalling
    Trends in Microbiology, 2008
    Co-Authors: Steven L. Porter, George H Wadhams, Judith P Armitage
    Abstract:

    Most bacteria have much more complex chemosensory systems than those of the extensively studied Escherichia coli. Rhodobacter Sphaeroides, for example, has multiple homologues of the E. coli chemosensory proteins. The roles of these homologues have been extensively investigated using a combination of deletion, subcellular localization and phosphorylation assays. These studies have shown that the homologues have specific roles in the sensory pathway, and they differ in their cellular localization and interactions with other components of the pathway. The presence of multiple chemosensory pathways might enable bacteria to tune their tactic responses to different environmental conditions.

  • the cheys of Rhodobacter Sphaeroides
    Journal of Biological Chemistry, 2006
    Co-Authors: Steven L. Porter, Elaine D. Byles, Angela Martin, George H Wadhams, David E Lancaster, Judith P Armitage
    Abstract:

    Abstract The Escherichia coli two-component chemosensory pathway has been extensively studied, and its response regulator, CheY, has become a paradigm for response regulators. However, unlike E. coli, most chemotactic nonenteric bacteria have multiple CheY homologues. The roles and cellular localization of the CheYs in Rhodobacter Sphaeroides were determined. Only two CheYs were required for chemotaxis, CheY6 and either CheY3 or CheY4. These CheYs were partially localized to either of the two chemotaxis signaling clusters, with the remaining protein delocalized. Interestingly, mutation of the CheY6 phosphorylatable aspartate to asparagine produced a stopped motor, caused by phosphorylation on alternative site Ser-83 by CheA. Extensive mutagenesis of E. coli CheY has identified a number of activating mutations, which have been extrapolated to other response regulators (D13K, Y106W, and I95V). Analogous mutations in R. Sphaeroides CheYs did not cause activation. These results suggest that although the R. Sphaeroides and E. coli CheYs are similar in that they require phosphorylation for activation, they may differ in both the nature of the phosphorylation-induced conformational change and their subsequent interactions with the flagellar motor. Caution should therefore be used when projecting from E. coli CheY onto novel response regulators.

  • fine tuning bacterial chemotaxis analysis of Rhodobacter Sphaeroides behaviour under aerobic and anaerobic conditions by mutation of the major chemotaxis operons and chey genes
    The EMBO Journal, 2000
    Co-Authors: Deepan S H Shah, Steven L. Porter, Angela Martin, Paul A Hamblin, Judith P Armitage
    Abstract:

    Rhodobacter Sphaeroides chemotaxis is significantly more complex than that of enteric bacteria. Rhodobacter Sphaeroides has multiple copies of chemotaxis genes (two cheA, one cheB, two cheR, three cheW, five cheY but no cheZ), controlling a single ‘stop–start’ flagellum. The growth environment controls the level of expression of different groups of genes. Tethered cell analysis of mutants suggests that CheY4 and CheY5 are the motor-binding response regulators. The histidine protein kinase CheA2 mediates an attractant (‘normal’) response via CheY4, while CheA1 and CheY5 appear to mediate a repellent (‘inverted’) response. CheY3 facilitates signal termination, possibly acting as a phosphate sink, although CheY1 and CheY2 can substitute. The normal and inverted responses may be initiated by separate sets of chemoreceptors with their relative strength dependent on growth conditions. Rhodobacter Sphaeroides may use antagonistic responses through two chemosensory pathways, expressed at different levels in different environments, to maintain their position in a currently optimum environment. Complex chemotaxis systems are increasingly being identified and the strategy adopted by R.Sphaeroides may be common in the bacterial kingdom.

Gabriele Klug - One of the best experts on this subject based on the ideXlab platform.

  • adaptation of the alphaproteobacterium Rhodobacter Sphaeroides to stationary phase
    Environmental Microbiology, 2019
    Co-Authors: Matthew Mcintosh, Katrin M H Eisenhardt, Bernhard Remes, Anne Konzer, Gabriele Klug
    Abstract:

    Exhaustion of nutritional resources stimulates bacterial populations to adapt their growth behaviour. General mechanisms are known to facilitate this adaptation by sensing the environmental change and coordinating gene expression. However, the existence of such mechanisms among the Alphaproteobacteria remains unclear. This study focusses on global changes in transcript levels during growth under carbon-limiting conditions in a model Alphaproteobacterium, Rhodobacter Sphaeroides, a metabolically diverse organism capable of multiple modes of growth including aerobic and anaerobic respiration, anaerobic anoxygenic photosynthesis and fermentation. We identified genes that showed changed transcript levels independently of oxygen levels during the adaptation to stationary phase. We selected a subset of these genes and subjected them to mutational analysis, including genes predicted to be involved in manganese uptake, polyhydroxybutyrate production and quorum sensing and an alternative sigma factor. Although these genes have not been previously associated with the adaptation to stationary phase, we found that all were important to varying degrees. We conclude that while R. Sphaeroides appears to lack a rpoS-like master regulator of stationary phase adaptation, this adaptation is nonetheless enabled through the impact of multiple genes, each responding to environmental conditions and contributing to the adaptation to stationary phase.

  • rnase e cleavage shapes the transcriptome of Rhodobacter Sphaeroides and strongly impacts phototrophic growth
    Life Science Alliance, 2018
    Co-Authors: Carina M Reuscher, Konrad U Forstner, Kerstin Haberzettl, Lennart Weber, Gabriele Klug
    Abstract:

    Bacteria adapt to changing environmental conditions by rapid changes in their transcriptome. This is achieved not only by adjusting rates of transcription but also by processing and degradation of RNAs. We applied TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) for the transcriptome-wide identification of RNase E cleavage sites and of 5' RNA ends, which are enriched when RNase E activity is reduced in Rhodobacter Sphaeroides. These results reveal the importance of RNase E for the maturation and turnover of mRNAs, rRNAs, and sRNAs in this guanine-cytosine-rich α-proteobacterium, some of the latter have well-described functions in the oxidative stress response. In agreement with this, a role of RNase E in the oxidative stress response is demonstrated. A remarkably strong phenotype of a mutant with reduced RNase E activity was observed regarding the formation of photosynthetic complexes and phototrophic growth, whereas there was no effect on chemotrophic growth.

  • The PhyR homolog RSP_1274 of Rhodobacter Sphaeroides is involved in defense of membrane stress and has a moderate effect on RpoE (RSP_1092) activity.
    BMC microbiology, 2018
    Co-Authors: Tao Peng, Gabriele Klug
    Abstract:

    A major role of the PhyR-NepR-σ(EcfG) cascade in the general stress response was demonstrated for some bacterial species and considered as conserved in Alphaproteobacteria. The σ(EcfG) factor activates its target genes in response to diverse stresses and NepR represents its anti-sigma factor. PhyR comprises a response regulator domain and a sigma factor domain and acts as anti-sigma factor antagonist. The facultative phototrophic alphaproteobacterium Rhodobacter Sphaeroides harbours a PhyR homolog in the same genomic context as found in other members of this class. Our study reveals increased expression of the phyR gene in response to superoxide, singlet oxygen, and diamide and also an effect of PhyR on rpoE expression. RpoE has a central role in mounting the response to singlet oxygen in R. Sphaeroides. Despite these findings a mutant lacking PhyR was not significantly impeded in resistance to oxidative stress, heat stress or osmotic stress. However a role of PhyR in membrane stress is demonstrated. These results support the view that the effect of the PhyR-NepR-σ(EcfG) cascade on diverse stress responses varies among members of the Alphaproteobacteria. In the facultative phototroph Rhodobacter Sphaeroides PhyR plays no major role in the general stress or the oxidative stress response but rather has a more specialized role in defense of membrane stress.

  • cryb from Rhodobacter Sphaeroides a unique class of cryptochromes with new cofactors
    EMBO Reports, 2012
    Co-Authors: Yann Geisselbrecht, Sebastian Fruhwirth, Claudia Schroeder, Antonio J Pierik, Gabriele Klug, Lars-oliver Essen
    Abstract:

    Cryptochromes and photolyases are structurally related but have different biological functions in signalling and DNA repair. Proteobacteria and cyanobacteria harbour a new class of cryptochromes, called CryPro. We have solved the 2.7 A structure of one of its members, cryptochrome B from Rhodobacter Sphaeroides, which is a regulator of photosynthesis gene expression. The structure reveals that, in addition to the photolyase-like fold, CryB contains two cofactors only conserved in the CryPro subfamily: 6,7-dimethyl-8-ribityl-lumazine in the antenna-binding domain and a [4Fe-4S] cluster within the catalytic domain. The latter closely resembles the iron–sulphur cluster harbouring the large primase subunit PriL, indicating that PriL is evolutionarily related to the CryPro class of cryptochromes.

  • photooxidative stress induced and abundant small rnas in Rhodobacter Sphaeroides
    Molecular Microbiology, 2009
    Co-Authors: Bork A Berghoff, Jens Glaeser, Cynthia M Sharma, Jorg Vogel, Gabriele Klug
    Abstract:

    Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen ( 1 O 2 ) in Rhodobacter Sphaeroides. Our study reports the genome-wide search for small RNAs (sRNAs) involved in the regulatory response to 1 O 2 . By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. Sphaeroides aerobic cultures or following treatment with 1 O 2 or superoxide (O ― 2 ). One sRNA was specifically induced by 1 O 2 and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by 1 O 2 and O ― 2 were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoH I and RpoH II . The most abundant sRNA was processed in the presence of 1 O 2 but not by O ― 2 . From this and a second sRNA a conserved 3'-segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half-lives, abundance and processing of 1 O 2 -affected sRNAs. Orthologues of three sRNA genes are present in different alpha-proteobacteria, but the majority was unique to R. Sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by 1 O 2 exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.

Daria Esyunina - One of the best experts on this subject based on the ideXlab platform.

  • Recognition of double-stranded DNA by the Rhodobacter Sphaeroides Argonaute protein.
    Biochemical and biophysical research communications, 2020
    Co-Authors: Lidia Lisitskaya, Alexei A. Aravin, Daria Esyunina, Ivan Petushkov, Andrey Kulbachinskiy
    Abstract:

    In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter Sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.

  • accommodation of helical imperfections in Rhodobacter Sphaeroides argonaute ternary complexes with guide rna and target dna
    Cell Reports, 2018
    Co-Authors: M Teplova, Alexei A. Aravin, Daria Esyunina, Andrey Kulbachinskiy, Ivan Olovnikov, Dinshaw J. Patel
    Abstract:

    Prokaryotic Argonaute (Ago) proteins were recently shown to target foreign genetic elements, thus making them a perfect model for studies of interference mechanisms. Here, we study interactions of Rhodobacter Sphaeroides Ago (RsAgo) with guide RNA (gRNA) and fully complementary or imperfect target DNA (tDNA) using biochemical and structural approaches. We show that RsAgo can specifically recognize both the first nucleotide in gRNA and complementary nucleotide in tDNA, and both interactions contribute to nucleic acid binding. Non-canonical pairs and bulges on the target strand can be accommodated by RsAgo with minimal perturbation of the duplex but significantly reduce RsAgo affinity to tDNA. Surprisingly, mismatches between gRNA and tDNA induce dissociation of the guide-target duplex from RsAgo. Our results reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, show how this might affect the efficiency of RNA silencing, and suggest a potential mechanism for guide release and Ago recycling.

  • Accommodation of Helical Imperfections in Rhodobacter Sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA
    Elsevier, 2018
    Co-Authors: Yiwei Liu, M Teplova, Alexei A. Aravin, Daria Esyunina, Andrey Kulbachinskiy, Ivan Olovnikov, Dinshaw J. Patel
    Abstract:

    Summary: Prokaryotic Argonaute (Ago) proteins were recently shown to target foreign genetic elements, thus making them a perfect model for studies of interference mechanisms. Here, we study interactions of Rhodobacter Sphaeroides Ago (RsAgo) with guide RNA (gRNA) and fully complementary or imperfect target DNA (tDNA) using biochemical and structural approaches. We show that RsAgo can specifically recognize both the first nucleotide in gRNA and complementary nucleotide in tDNA, and both interactions contribute to nucleic acid binding. Non-canonical pairs and bulges on the target strand can be accommodated by RsAgo with minimal perturbation of the duplex but significantly reduce RsAgo affinity to tDNA. Surprisingly, mismatches between gRNA and tDNA induce dissociation of the guide-target duplex from RsAgo. Our results reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, show how this might affect the efficiency of RNA silencing, and suggest a potential mechanism for guide release and Ago recycling. : Prokaryotic Argonaute proteins (pAgos) target foreign genetic elements. Liu et al. structurally and functionally characterize interactions of pAgo with guide RNA and fully complementary or imperfectly targeted DNA, reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, and suggest a potential mechanism for guide release. Keywords: Rhodobacter Sphaeroides Argonaute, RsAgo, guide RNA, target DNA, RNA-DNA heteroduplex, non-canonical base pairs and bulge

Andrey Kulbachinskiy - One of the best experts on this subject based on the ideXlab platform.

  • Recognition of double-stranded DNA by the Rhodobacter Sphaeroides Argonaute protein.
    Biochemical and biophysical research communications, 2020
    Co-Authors: Lidia Lisitskaya, Alexei A. Aravin, Daria Esyunina, Ivan Petushkov, Andrey Kulbachinskiy
    Abstract:

    In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter Sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.

  • accommodation of helical imperfections in Rhodobacter Sphaeroides argonaute ternary complexes with guide rna and target dna
    Cell Reports, 2018
    Co-Authors: M Teplova, Alexei A. Aravin, Daria Esyunina, Andrey Kulbachinskiy, Ivan Olovnikov, Dinshaw J. Patel
    Abstract:

    Prokaryotic Argonaute (Ago) proteins were recently shown to target foreign genetic elements, thus making them a perfect model for studies of interference mechanisms. Here, we study interactions of Rhodobacter Sphaeroides Ago (RsAgo) with guide RNA (gRNA) and fully complementary or imperfect target DNA (tDNA) using biochemical and structural approaches. We show that RsAgo can specifically recognize both the first nucleotide in gRNA and complementary nucleotide in tDNA, and both interactions contribute to nucleic acid binding. Non-canonical pairs and bulges on the target strand can be accommodated by RsAgo with minimal perturbation of the duplex but significantly reduce RsAgo affinity to tDNA. Surprisingly, mismatches between gRNA and tDNA induce dissociation of the guide-target duplex from RsAgo. Our results reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, show how this might affect the efficiency of RNA silencing, and suggest a potential mechanism for guide release and Ago recycling.

  • Accommodation of Helical Imperfections in Rhodobacter Sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA
    Elsevier, 2018
    Co-Authors: Yiwei Liu, M Teplova, Alexei A. Aravin, Daria Esyunina, Andrey Kulbachinskiy, Ivan Olovnikov, Dinshaw J. Patel
    Abstract:

    Summary: Prokaryotic Argonaute (Ago) proteins were recently shown to target foreign genetic elements, thus making them a perfect model for studies of interference mechanisms. Here, we study interactions of Rhodobacter Sphaeroides Ago (RsAgo) with guide RNA (gRNA) and fully complementary or imperfect target DNA (tDNA) using biochemical and structural approaches. We show that RsAgo can specifically recognize both the first nucleotide in gRNA and complementary nucleotide in tDNA, and both interactions contribute to nucleic acid binding. Non-canonical pairs and bulges on the target strand can be accommodated by RsAgo with minimal perturbation of the duplex but significantly reduce RsAgo affinity to tDNA. Surprisingly, mismatches between gRNA and tDNA induce dissociation of the guide-target duplex from RsAgo. Our results reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, show how this might affect the efficiency of RNA silencing, and suggest a potential mechanism for guide release and Ago recycling. : Prokaryotic Argonaute proteins (pAgos) target foreign genetic elements. Liu et al. structurally and functionally characterize interactions of pAgo with guide RNA and fully complementary or imperfectly targeted DNA, reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, and suggest a potential mechanism for guide release. Keywords: Rhodobacter Sphaeroides Argonaute, RsAgo, guide RNA, target DNA, RNA-DNA heteroduplex, non-canonical base pairs and bulge

Wolfgang Lubitz - One of the best experts on this subject based on the ideXlab platform.

  • a conformational change of the photoactive bacteriopheophytin in reaction centers from Rhodobacter Sphaeroides
    Biochemistry, 1998
    Co-Authors: Joann Williams, James P Allen, Wolfgang Lubitz
    Abstract:

    It is demonstrated by ENDOR and Special TRIPLE spectroscopy that two distinct radical anion states of the intermediate electron acceptor (I), a bacteriopheophytin, can be freeze-trapped in isolated photosynthetic reaction centers of Rhodobacter Sphaeroides. The formation of these states depends on the illumination time prior to freezing and the temperature. The first state, I1•-, is metastable and relaxes irreversibly at T ≈ 160 K to the second state, I2•-. Experiments on quinone depleted as well as mutant reaction centers help to exclude the possibility that other cofactors besides the bacteriopheophytin in the A-branch, ΦA, are reduced during the trapping procedure. In particular, two mutants are investigated, in which the hydrogen bonds to ΦA that exist in the wild type are removed. These mutants are EL(L104), in which Glu at position L104 near the 131-keto group of ΦA is replaced by Leu, and WF(L100), in which Trp at position L100 near the 132-methyl ester of ΦA is replaced by Phe. Both mutations have...

  • effects of hydrogen bonding to a bacteriochlorophyll bacteriopheophytin dimer in reaction centers from Rhodobacter Sphaeroides
    Biochemistry, 1996
    Co-Authors: Jim Allen, Joann Williams, Katie Artz, Delphine Albouy, Alexandra Fetsch, Tony A Mattioli, Matthias Kuhn, Anabella Ivancich, Wolfgang Lubitz
    Abstract:

    The properties of the primary electron donor in reaction centers from Rhodobacter Sphaeroides have been investigated in mutants containing a bacteriochlorophyll (BChl)−bacteriopheophytin (BPhe) dimer with and without hydrogen bonds to the conjugated carbonyl groups. The heterodimer mutation His M202 to Leu was combined with each of the following mutations:  His L168 to Phe, which should remove an existing hydrogen bond to the BChl molecule; Leu L131 to His, which should add a hydrogen bond to the BChl molecule; and Leu M160 to His and Phe M197 to His, each of which should add a hydrogen bond to the BPhe molecule [Rautter, J., Lendzian, F., Schulz, C., Fetsch, A., Kuhn, M., Lin, X., Williams, J. C., Allen, J. P., & Lubitz, W. (1995) Biochemistry 34, 8130−8143]. Pigment extractions and Fourier transform Raman spectra confirm that all of the mutants contain a heterodimer. The bands in the resonance Raman spectra arising from the BPhe molecule, which is selectively enhanced, exhibit the shifts expected for th...