scholarly journals Mechanism of Signalling and Adaptation through the Rhodobacter sphaeroides Cytoplasmic Chemoreceptor Cluster

2019 ◽  
Vol 20 (20) ◽  
pp. 5095
Author(s):  
Beyer ◽  
Szöllössi ◽  
Byles ◽  
Fischer ◽  
Armitage
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Tandem Mass ◽  
Wild Type ◽  
Hybrid Analysis ◽  
Phosphatase Domain ◽  
E Coli ◽  
Tandem Mass Spectroscopy ◽  
Membrane Spanning ◽  
State Concentration ◽  
Two Hybrid

Rhodobacter sphaeroides has two chemotaxis clusters, an Escherichia coli-like cluster with membrane-spanning chemoreceptors and a less-understood cytoplasmic cluster. The cytoplasmic CheA is split into CheA4, a kinase, and CheA3, a His-domain phosphorylated by CheA4 and a phosphatase domain, which together phosphorylate and dephosphorylate motor-stopping CheY6. In bacterial two-hybrid analysis, one major cytoplasmic chemoreceptor, TlpT, interacted with CheA4, while the other, TlpC, interacted with CheA3. Both clusters have associated adaptation proteins. Deleting their methyltransferases and methylesterases singly and together removed chemotaxis, but with opposite effects. The cytoplasmic cluster signal overrode the membrane cluster signal. Methylation and demethylation of specific chemoreceptor glutamates controls adaptation. Tandem mass spectroscopy and bioinformatics identified four putative sites on TlpT, three glutamates and a glutamine. Mutating each glutamate to alanine resulted in smooth swimming and loss of chemotaxis, unlike similar mutations in E. coli chemoreceptors. Cells with two mutated glutamates were more stoppy than wild-type and responded and adapted to attractant addition, not removal. Mutating all four sites amplified the effect. Cells were non-motile, began smooth swimming on attractant addition, and rapidly adapted back to non-motile before attractant removal. We propose that TlpT responds and adapts to the cell’s metabolic state, generating the steady-state concentration of motor-stopping CheY6~P. Membrane-cluster signalling produces a pulse of CheY3/CheY4~P that displaces CheY6~P and allows flagellar rotation and smooth swimming before both clusters adapt.

scholarly journals ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery

2008 ◽  
Vol 183 (2) ◽  
pp. 313-322 ◽  
Author(s):  
Noveera T. Ahmed ◽  
Chunlei Gao ◽  
Ben F. Lucker ◽  
Douglas G. Cole ◽  
David R. Mitchell
Keyword(s):  
Binding Site ◽  
Intraflagellar Transport ◽  
Site Formation ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
B Subunit ◽  
Dynein Arms ◽  
Two Hybrid

Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

scholarly journals Gonococcal MinD Affects Cell Division inNeisseria gonorrhoeae and Escherichia coli and Exhibits a Novel Self-Interaction

2001 ◽  
Vol 183 (21) ◽  
pp. 6253-6264 ◽  
Author(s):  
Jason Szeto ◽  
Sandra Ramirez-Arcos ◽  
Claude Raymond ◽  
Leslie D. Hicks ◽  
Cyril M. Kay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Morphological Changes ◽  
Shuttle Vector ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
E Coli ◽  
Two Hybrid

ABSTRACT The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus Neisseria gonorrhoeae contains a min operon, and the present study investigates the role of minD from this operon. A gonococcal minD insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from N. gonorrhoeae(MinDNg) in this mutant. Hence, MinDNg is required for maintaining proper cell division and growth in N. gonorrhoeae. Immunoblotting of soluble and insoluble gonococcal cell fractions revealed that MinDNg is both cytosolic and associated with the insoluble membrane fraction. The joint overexpression of MinCNg and MinDNg from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed with plasmids encoding either MinCNg or MinDNg alone did not display noticeable morphological changes. These studies suggest that MinDNg is involved in inhibiting gonococcal cell division, likely in conjunction with MinCNg. The alignment of MinD sequences from various bacteria showed that the proteins are highly conserved and share several regions of identity, including a conserved ATP-binding cassette. The overexpression of MinDNg in wild-type Escherichia coli led to cell filamentation, while overexpression in an E. coli minD mutant restored a wild-type morphology to the majority of cells; therefore, gonococcal MinD is functional across species. Yeast two-hybrid studies and gel-filtration and sedimentation equilibrium analyses of purified His-tagged MinDNg revealed a novel MinDNgself-interaction. We have also shown by yeast two-hybrid analysis that MinD from E. coli interacts with itself and with MinDNg. These results indicate that MinDNg is required for maintaining proper cell division and growth in N. gonorrhoeae and suggests that the self-interaction of MinD may be important for cell division site selection across species.

scholarly journals Metabolic Roles of a Rhodobacter sphaeroides Member of the ς32 Family

1998 ◽  
Vol 180 (1) ◽  
pp. 10-19 ◽  
Author(s):  
Russell K. Karls ◽  
Jacqueline Brooks ◽  
Peter Rossmeissl ◽  
Janelle Luedke ◽  
Timothy J. Donohue
Keyword(s):  
Heat Shock ◽  
Temperature Profile ◽  
Rhodobacter Sphaeroides ◽  
Growth Temperature ◽  
Heat Shock Response ◽  
Anaerobic Respiration ◽  
Wild Type ◽  
Lower Growth ◽  
E Coli ◽  
Shock Response

ABSTRACT We report the role of a gene (rpoH) from the facultative phototroph Rhodobacter sphaeroides that encodes a protein (ς37) similar to Escherichia coliς32 and other members of the heat shock family of eubacterial sigma factors. R. sphaeroides ς37controls genes that function during environmental stress, since anR. sphaeroides ΔRpoH mutant is ∼30-fold more sensitive to the toxic oxyanion tellurite than wild-type cells. However, the ΔRpoH mutant lacks several phenotypes characteristic of E. coli cells lacking ς32. For example, anR. sphaeroides ΔRpoH mutant is not generally defective in phage morphogenesis, since it plates the lytic virus RS1, as well as its wild-type parent. In characterizing the response ofR. sphaeroides to heat, we found that its growth temperature profile is different when cells generate energy by aerobic respiration, anaerobic respiration, or photosynthesis. However, growth of the ΔRpoH mutant is comparable to that of a wild-type strain under each of these conditions. The ΔRpoH mutant mounted a heat shock response when aerobically grown cells were shifted from 30 to 42°C, but it exhibited altered induction kinetics of ∼120-, 85-, 75-, and 65-kDa proteins. There was also reduced accumulation of several presumed heat shock transcripts (rpoD PHS,groESL 1, etc.) when aerobically grown ΔRpoH cells were placed at 42°C. Under aerobic conditions, it appears that another sigma factor enables the ΔRpoH mutant to mount a heat shock response, since either RNA polymerase preparations from an ΔRpoH mutant, reconstituted Eς37, or a holoenzyme containing a 38-kDa protein (ς38) each transcribed E. coliEς32-dependent promoters. The lower growth temperature profile of photosynthetic cells is correlated with a difference in heat-inducible gene expression, since neither wild-type cells or the ΔRpoH mutant mount a typical heat shock response after such cultures were shifted from 30 to 37°C.

scholarly journals DOT4 Links Silencing and Cell Growth inSaccharomyces cerevisiae

1999 ◽  
Vol 19 (10) ◽  
pp. 6608-6620 ◽  
Author(s):  
Alon Kahana ◽  
Daniel E. Gottschling
Keyword(s):  
Slow Growth ◽  
26S Proteasome ◽  
Growth Defect ◽  
Wild Type ◽  
Hybrid Analysis ◽  
Amino Terminal ◽  
Sir Proteins ◽  
A Subunit ◽  
Processing Protease ◽  
Two Hybrid

ABSTRACTTranscriptional silencing inSaccharomyces cerevisiaeoccurs at specific loci and is mediated by a multiprotein complex that includes Rap1p and the Sir proteins. We studied the function of a recently identified gene,DOT4, that disrupts silencing when overexpressed.DOT4encodes an ubiquitin processing protease (hydrolase) that is primarily located in the nucleus. By two-hybrid analysis, the amino-terminal third of Dot4p interacts with the silencing protein Sir4p. Cells lackingDOT4exhibited reduced silencing and a corresponding decrease in the level of Sir4p. Together, these findings suggest that Dot4p regulates silencing by acting on Sir4p. In strains with several auxotrophic markers, loss ofDOT4ubiquitin hydrolase activity also results in a slow-growth defect. The defect can be partially suppressed by mutations in a subunit of the 26S proteasome, suggesting that Dot4p has the ability to prevent ubiquitin-mediated degradation. Furthermore, wild-typeSIR2,SIR3, andSIR4are required for full manifestation of the growth defect in adot4strain, indicating that the growth defect is caused in part by a silencing-related mechanism. We propose that Dot4p helps to restrict the location of silencing proteins to a limited set of genomic loci.

scholarly journals The Default State of the Membrane-Localized Histidine Kinase PrrB of Rhodobacter sphaeroides 2.4.1 Is in the Kinase-Positive Mode

2001 ◽  
Vol 183 (23) ◽  
pp. 6807-6814 ◽  
Author(s):  
Jeong-Il Oh ◽  
In-Jeong Ko ◽  
Samuel Kaplan
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Rhodobacter Sphaeroides ◽  
Histidine Kinase ◽  
Transmembrane Domain ◽  
Dominant Mode ◽  
Phosphatase Domain ◽  
Positive Mode ◽  
Default State ◽  
Membrane Spanning

ABSTRACT The PrrBA two-component activation system of Rhodobacter sphaeroides plays a major role in the induction of photosynthesis gene expression under oxygen-limiting or anaerobic conditions. The PrrB histidine kinase is composed of two structurally identifiable regions, the conserved C-terminal kinase/phosphatase domain and the N-terminal membrane-spanning domain with six transmembrane helices framing three periplasmic and two cytoplasmic loops. Using a set of PrrB mutants with lesions in the transmembrane domain, we demonstrate that the central portion of the PrrB transmembrane domain including the second periplasmic loop plays an important role in both sensing and signal transduction. Signal transduction via the transmembrane domain is ultimately manifested by controlling the activity of the C-terminal kinase/phosphatase domain. The extent of signal transduction is determined by the ability of the transmembrane domain to sense the strength of the inhibitory signal received from the cbb 3 terminal oxidase (J.-I Oh, and S. Kaplan, EMBO J. 19:4237–4247, 2000). Therefore, the intrinsic (“default”) state of PrrB is in the kinase-dominant mode. It is also demonstrated that the extent ofprrB gene expression is subject to the negative autoregulation of the PrrBA system.

scholarly journals YgfY Contributes to Stress Tolerance in Shewanella oneidensis Neither as an Antitoxin Nor as a Flavinylation Factor of Succinate Dehydrogenase

2021 ◽  
Vol 9 (11) ◽  
pp. 2316
Author(s):  
Ming-Xing Zhang ◽  
Kai-Li Zheng ◽  
Ai-Guo Tang ◽  
Xiao-Xia Hu ◽  
Xin-Xin Guo ◽  
...  
Keyword(s):  
Low Temperature ◽  
Succinate Dehydrogenase ◽  
Wild Type Strain ◽  
Shewanella Oneidensis ◽  
Growth Defect ◽  
Wild Type ◽  
E Coli ◽  
Growth Defects ◽  
Type Iv ◽  
Two Hybrid

YgfY(SdhE/CptB) is highly conserved while has controversial functions in bacteria. It works as an antitoxin and composes a type IV toxin–antitoxin system with YgfX(CptA) typically in Escherichia coli, while functions as an flavinylation factor of succinate dehydrogenase and fumarate reductase typically in Serratia sp. In this study, we report the contribution of YgfY in Shewanella oneidensis MR-1 to tolerance of low temperature and nitrite. YgfY deficiency causes several growth defects of S. oneidensis MR-1 at low temperature, while YgfX do not cause a growth defect or morphological change of S. oneidensis MR1-1 and E. coli. YgfY do not interact with FtsZ and MreB nor with YgfX examined by bacterial two-hybrid assay. YgfY effect on growth under low temperature is not attributed to succinate dehydrogenase (SDH) because a mutant without SDH grows comparably with the wild-type strain in the presence of succinate. The ygfY mutant shows impaired tolerance to nitrite. Transcription of nitrite reductase and most ribosome proteins is significantly decreased in the ygfY mutant, which is consistent with the phenotypes detected above. Effects of YgfY on growth and nitrite tolerance are closely related to the RGXXE motif in YgfY. In summary, this study demonstrates pleiotropic impacts of YgfY in S. oneidensis MR-1, and sheds a light on the physiological versatility of YgfY in bacteria.

scholarly journals The Tomato Metallocarboxypeptidase Inhibitor I, which Interacts with a Heavy Metal-Associated Isoprenylated Protein, Is Implicated in Plant Response to Cadmium

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 700 ◽  
Author(s):  
Anna Manara ◽  
Elisa Fasani ◽  
Barbara Molesini ◽  
Giovanni DalCorso ◽  
Federica Pennisi ◽  
...  
Keyword(s):  
Heavy Metal ◽  
Active Site ◽  
Abiotic Stresses ◽  
Lower Amount ◽  
Interacting Proteins ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Model Species ◽  
Two Hybrid

Metallocarboxypeptidases are metal-dependent enzymes, whose biological activity is regulated by inhibitors directed on the metal-containing active site. Some metallocarboxypeptidase inhibitors are induced under stress conditions and have a role in defense against pests. This paper is aimed at investigating the response of the tomato metallocarboxypeptidase inhibitor (TCMP)-1 to Cd and other abiotic stresses. To this aim, the tomato TCMP-1 was ectopically expressed in the model species Arabidopsis thaliana, and a yeast two-hybrid analysis was performed to identify interacting proteins. We demonstrate that TCMP-1 is responsive to Cd, NaCl, and abscisic acid (ABA) and interacts with the tomato heavy metal-associated isoprenylated plant protein (HIPP)26. A. thaliana plants overexpressing TCMP-1 accumulate lower amount of Cd in shoots, display an increased expression of AtHIPP26 in comparison with wild-type plants, and are characterized by a modulation in the expression of antioxidant enzymes. Overall, these results suggest a possible role for the TCMP-1/HIPP26 complex in Cd response and compartmentalization.

Current Trends of Liquid Chromatography Tandem Mass Spectroscopy Use in Clinical Gynecology

2019 ◽  
Vol 70 (6) ◽  
pp. 2021-2025
Author(s):  
Aida Petca ◽  
Dan Cristian Radu ◽  
Razvan Cosmin Petca ◽  
Claudia Mehedintu ◽  
Ramona Ileana Barac ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Mass Spectroscopy ◽  
Tandem Mass ◽  
Quality Healthcare ◽  
Current Trends ◽  
Healthcare It ◽  
Liquid Chromatography Tandem Mass ◽  
Tandem Mass Spectroscopy ◽  
Technical Innovations ◽  
Hormonal Milieu

In the present environment of staggering technical innovations and increasing expectations of quality healthcare it is evident that we need to fine tune our diagnostic abilities in order to fulfil patients� demands for more efficient therapies and augmented quality of life. We are looking for current trends in clinical gynecology that make use of Liquid chromatography tandem mass spectroscopy, technology not yet employed in Romanian laboratories for the clinical practice but that is rapidly becoming the worldwide method of choice for accurate characterization of the hormonal milieu essential for the requirements of women healthcare.

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