scholarly journals Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii.

1995 ◽  
Vol 131 (2) ◽  
pp. 427-440 ◽  
Author(s):  
G J Pazour ◽  
O A Sineshchekov ◽  
G B Witman
Keyword(s):  
Signal Transduction ◽  
Chlamydomonas Reinhardtii ◽  
Insertional Mutagenesis ◽  
Mutational Analysis ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Normal Manner ◽  
Normal Light ◽  
Phototransduction Pathway

Chlamydomonas has two photobehavioral responses, phototaxis and photoshock. Rhodopsin is the photoreceptor for these responses and the signal transduction process involves transmembrane Ca2+ fluxes. This causes transient changes in flagellar beating, ultimately resulting in phototaxis or photoshock. To identify components that make up this signal transduction pathway, we generated nonphototactic strains by insertional mutagenesis. Seven new phototaxis genes were identified (ptx2-ptx8); alleles of six of these are tagged by the transforming DNA and therefore should be easily cloned. To order the mutants in the pathway, we characterized them electrophysiologically, behaviorally, and structurally, ptx5, ptx6, and ptx7 have normal light-induced photoreceptor currents (PRC) and flagellar currents (FC) but their pattern of swimming does not change in the normal manner when the intraflagellar Ca2+ concentration is decreased, suggesting that they have defects in the ability of their axonemes to respond to changes in Ca2+ concentration. ptx2 and ptx8 lack the FC but have normal PRCs, suggesting that they are defective in the flagellar Ca2+ channel or some factor that regulates it. ptx4 mutants have multiple eye-spots. ptx3 mutants are defective in a component essential for phototaxis but bypassed during photoshock; this component appears to be located downstream of the PRC but upstream of the axoneme.

Analysis of Chlamydomonas mutants with abnormal expression of CO2 and HCO3- uptake systems

2002 ◽  
Vol 29 (3) ◽  
pp. 251 ◽  
Author(s):  
Christoph Thyssen ◽  
Eddy van Hunnik ◽  
Marie Teresa Navarro ◽  
Emilio Fernández ◽  
Aurora Galván ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Insertional Mutagenesis ◽  
Signal Transduction Pathway ◽  
Transport Systems ◽  
Carbonic Anhydrases ◽  
Transduction Pathway ◽  
Membrane Inlet Mass Spectrometry ◽  
Whole Cells ◽  
High Affinity ◽  
Uptake Data

Eukaryotic microalgae such as Chlamydomonas reinhardtii possess an inducible CO2 concentrating mechanism that operates as a very close interaction between pyrenoid-based Rubisco, various carbonic anhydrases (CAs), and inorganic carbon (Ci) transport systems. While external and internal CAs have been characterised to the molecular level, the biochemistry and molecular biology of Ci uptake mechanisms have not been elucidated. Both Ci species, CO2 and HCO3-, are taken up by the cells and chloroplasts during steady-state photosynthesis. After acclimation to limiting Ci, CO2 and HCO3- transport, measured in whole cells or chloroplasts, change their kinetic characteristics from a constitutive low-affinity state to an inducible high-affinity state. In order to learn more about the genes involved in the signal transduction pathway and in the Ci transport systems, we performed insertional mutagenesis using the arg7 gene as a selectable marker. Application of aqueous membrane inlet mass spectrometry allowed discrimination between CO2 and HCO3- uptake. Data is presented on two mutants, M46 and M21, which show severe damage to the constitutive Ci uptake systems and which are unable to induce a high-affinity state. The mutations might be either in the signal transduction pathway or in the transporters themselves. In addition, we present data that shows a very close connection between high-affinity HCO3- uptake and high-affinity NO3- uptake in cells of C. reinhardtii.

scholarly journals Copper Response Element and Crr1-Dependent Ni2+-Responsive Promoter for Induced, Reversible Gene Expression in Chlamydomonas reinhardtii

2003 ◽  
Vol 2 (5) ◽  
pp. 995-1002 ◽  
Author(s):  
Jeanette M. Quinn ◽  
Janette Kropat ◽  
Sabeeha Merchant
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Chlamydomonas Reinhardtii ◽  
Target Genes ◽  
Response Regulator ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Nickel Ions ◽  
Cobalt Ions ◽  
Inducible Gene

ABSTRACT The Cpx1 and Cyc6 genes of Chlamydomonas reinhardtii are activated in copper-deficient cells via a signal transduction pathway that requires copper response elements (CuREs) and a copper response regulator defined by the CRR1 locus. The two genes can also be activated by provision of nickel or cobalt ions in the medium. The response to nickel ions requires at least one CuRE and also CRR1 function, suggesting that nickel interferes with a component in the nutritional copper signal transduction pathway. Nickel does not act by preventing copper uptake/utilization because (i) holoplastocyanin formation is unaffected in Ni2+-treated cells and (ii) provision of excess copper cannot reverse the Ni-dependent activation of the target genes. The CuRE is sufficient for conferring Ni-responsive expression to a reporter gene, which suggests that the system has practical application as a vehicle for inducible gene expression. The inducer can be removed either by replacing the medium or by chelating the inducer with excess EDTA, either of which treatments reverses the activation of the target genes.

scholarly journals Mutational Analysis of a Bifunctional Ferrisiderophore Receptor and Signal-Transducing Protein from Pseudomonas aeruginosa

2005 ◽  
Vol 187 (13) ◽  
pp. 4514-4520 ◽  
Author(s):  
H. Ellen James ◽  
Paul A. Beare ◽  
Lois W. Martin ◽  
Iain L. Lamont
Keyword(s):  
Signal Transduction ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Mutational Analysis ◽  
Signal Transduction Pathway ◽  
Insertion Mutagenesis ◽  
Transduction Pathway ◽  
Loss Of Function ◽  
Signaling Function ◽  
Linker Insertion Mutagenesis

ABSTRACT The FpvA protein of Pseudomonas aeruginosa strain PAO1 mediates uptake of a siderophore, ferripyoverdine. It is also a component of a signal transduction pathway that controls production of an exotoxin, a protease, pyoverdine, and FpvA itself. The purpose of the research described here was to dissect these different functions of FpvA. Signaling involves an N-terminal domain of FpvA, and it was shown that this domain is probably located in the periplasm, as expected. Short peptides were inserted at 36 sites within FpvA by linker insertion mutagenesis. The effects of these mutations on the presence of FpvA in the outer membrane, on FpvA-mediated uptake of ferripyoverdine, and on pyoverdine synthesis and gene expression were determined. Five of the mutations resulted in the absence of FpvA from the outer membrane of the bacteria. All of the remaining mutations eliminated either the transport or signaling function of FpvA and most affected both functions. Three mutations prevented transport of ferripyoverdine but had no effect on the signal transduction pathway showing that transport of ferripyoverdine is not required for the trans-membrane signaling process. Conversely, eight mutations affected pyoverdine-mediated signaling but had no effect on transport of ferripyoverdine. These data show that insertions throughout FpvA resulted in loss of function and that signaling and transport are separate and discrete functions of FpvA.

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