RNA silencing of hydrogenase(-like) genes and investigation of their physiological roles in the green alga Chlamydomonas reinhardtii

2010 ◽  
Vol 431 (3) ◽  
pp. 345-352 ◽  
Author(s):  
James E. Godman ◽  
Attila Molnár ◽  
David C. Baulcombe ◽  
Janneke Balk
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Rna Silencing ◽  
Aldehyde Oxidase ◽  
Eukaryotic Cell ◽  
Unique Combination ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Production Cell

The genome of the green alga Chlamydomonas reinhardtii encodes two [FeFe]-hydrogenases, HydA1 and HydA2, and the hydrogenase-like protein HYD3. The unique combination of these proteins in one eukaryotic cell allows for direct comparison of their in vivo functions, which have not been established for HydA2 and HYD3. Using an artificial microRNA silencing method developed recently, the expression of HydA1, HydA2 and HYD3 was specifically down-regulated. Silencing of HydA1 resulted in 4-fold lower hydrogenase protein and activity under anaerobic conditions. In contrast, silencing of HydA2 or HYD3 did not affect hydrogen production. Cell lines with strongly (>90%) decreased HYD3 transcript levels grew more slowly than wild-type. The activity of aldehyde oxidase, a cytosolic Fe-S enzyme, was decreased in HYD3-knockdown lines, whereas Fe-S dependent activities in the chloroplast and mitochondria were unaffected. In addition, the HYD3-knockdown lines grew poorly on hypoxanthine, indicating impaired function of xanthine dehydrogenase, another cytosolic Fe-S enzyme. The expression levels of selected genes in response to hypoxia were unaltered upon HYD3 silencing. Together, our results clearly distinguish the cellular roles of HydA1 and HYD3, and indicate that HYD3, like its yeast and human homologues, has an evolutionary conserved role in the biogenesis or maintenance of cytosolic Fe-S proteins.

Identification of genes required for hydrogenase activity in Chlamydomonas reinhardtii

2005 ◽  
Vol 33 (1) ◽  
pp. 102-104 ◽  
Author(s):  
M.C. Posewitz ◽  
P.W. King ◽  
S.L. Smolinski ◽  
R. Davis Smith ◽  
A.R. Ginley ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Transcriptional Response ◽  
H2 Production ◽  
Hydrogenase Activity ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
H2 Photoproduction ◽  
H2 Sensor

The eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by an [FeFe]-hydrogenase. To identify genes that influence H2 production in C. reinhardtii, a library of 6000 colonies on agar plates was screened with sensitive chemochromic H2-sensor films for clones defective in H2 production. Two mutants of particular interest were fully characterized. One mutant, hydEF-1, is unable to assemble an active [FeFe]-hydrogenase. This is the first reported C. reinhardtii mutant that is not capable of producing any H2. The second mutant, sta7-10, is not able to accumulate insoluble starch and has significantly lowered H2-photoproduction rates in comparison with the wild-type. In hydEF-1, anaerobiosis induces transcription of the two reported C. reinhardtii hydrogenase genes, HydA1 and HydA2, indicating a normal transcriptional response to anaerobiosis. In contrast, the transcription of both hydrogenase genes in sta7-10 is significantly attenuated.

scholarly journals A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c550 and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd1 Nitrite Reductase

2003 ◽  
Vol 185 (21) ◽  
pp. 6308-6315 ◽  
Author(s):  
Isobel V. Pearson ◽  
M. Dudley Page ◽  
Rob J. M. van Spanning ◽  
Stuart J. Ferguson
Keyword(s):  
Nitrite Reductase ◽  
Paracoccus Denitrificans ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Electron Mediator ◽  
Intact Cells ◽  
Cytochrome Cd1 ◽  
Membrane Bound ◽  
Electron Carriers

ABSTRACT In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc 1 complex to the periplasmic nitrite reductase, cytochrome cd 1. The periplasmic protein cytochrome c 550 has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells. Here, the hypothesis that cytochrome c 550 and pseudoazurin are alternative electron carriers from the cytochrome bc 1 complex to the nitrite reductase was tested by construction of mutants of P. denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome c 550. The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium. Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome c 550 or both these proteins. We concluded that pseudoazurin and cytochrome c 550 are the alternative electron mediator proteins between the cytochrome bc 1 complex and the cytochrome cd 1-type nitrite reductase. We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP.

scholarly journals Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii.

1984 ◽  
Vol 98 (1) ◽  
pp. 229-236 ◽  
Author(s):  
S K Dutcher ◽  
B Huang ◽  
D J Luck
Keyword(s):  
Molecular Weight ◽  
Chlamydomonas Reinhardtii ◽  
Gene Product ◽  
Structural Gene ◽  
Chemical Extraction ◽  
Genetic Dissection ◽  
Wild Type ◽  
Central Pair ◽  
The Stability

Mutations at two loci, which cause an altered mobility of the flagella, affected the central pair microtubule complex of Chlamydomonas reinhardtii flagella. The mutations at both loci primarily affected the C1 microtubule of the complex. Three alleles at the PF16 locus affected the stability of the C1 microtubule in isolated axonemes. This phenotype has allowed us to determine that at least ten polypeptides of the central pair complex are unique to the C1 microtubule. The motility defect was correlated with the failure to assemble three of these ten polypeptides in vivo. The structural gene product of the PF16 locus was a polypeptide with molecular weight 57,000 as shown by analysis of five intragenic revertants and by analysis of axonemes from dikaryon rescue experiments. Three alleles at the PF6 locus affected the assembly of one of the two projections of the C1 microtubule and this projection was formed by at least three polypeptide components, which are a subset of polypeptides missing in isolated pf16 axonemes. No structural gene product has been identified for the PF6 locus. The gene product is probably not one of the identified projection constituents as shown by analysis of dikaryon rescue experiments. Chemical extraction of isolated wild-type axonemes suggests that at least seven polypeptide components are unique to the C2 microtubule.

scholarly journals The 5' leader of a chloroplast mRNA mediates the translational requirements for two nucleus-encoded functions in Chlamydomonas reinhardtii.

1994 ◽  
Vol 14 (8) ◽  
pp. 5268-5277 ◽  
Author(s):  
W Zerges ◽  
J D Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Rna Binding ◽  
Mrna Translation ◽  
Binding Activity ◽  
Leader Sequence ◽  
Sequence Motif ◽  
Wild Type ◽  
Chloroplast Mrna

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previously shown to abolish the synthesis of the photosystem II core polypeptide subunit P6, which is encoded by the chloroplast psbC gene. In this report the functions encoded by F34 and F64 are shown to be required for translation of the psbC mRNA, on the basis of the finding that the expression of a heterologous reporter gene fused to the psbC 5' nontranslated leader sequence requires wild-type F34 and F64 alleles in vivo. Moreover, a point mutation in the psbC 5' nontranslated leader sequence suppresses this requirement for wild-type F34 function. In vitro RNA-protein cross-linking studies reveal that chloroplast protein extracts from strains carrying the F64 mutation contain an approximately 46-kDa RNA-binding protein. The absence of the RNA-binding activity of this protein in chloroplast extracts of wild-type strains suggests that it is related to the role of the F64-encoded function for psbC mRNA translation. The binding specificity of this protein appears to be for an AU-rich RNA sequence motif.

scholarly journals The Cytochrome c Gene from the Green Alga Chlamydomonas reinhardtii. Structure and Expression in Wild-Type Cells and in Obligate Photoautotrophic (dk) Mutants

2000 ◽  
Vol 41 (10) ◽  
pp. 1149-1156 ◽  
Author(s):  
Silvina A. Felitti ◽  
Raquel L. Chan ◽  
Manuel González Sierra ◽  
Daniel H. Gonzalez
Keyword(s):  
Cytochrome C ◽  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Wild Type

Structural and functional consequences of the replacement of proximal residues Cys172 and Cys192 in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

2008 ◽  
Vol 411 (2) ◽  
pp. 241-247 ◽  
Author(s):  
María-Jesús García-Murria ◽  
Saeid Karkehabadi ◽  
Julia Marín-Navarro ◽  
Sriram Satagopan ◽  
Inger Andersson ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Large Subunit ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Bisphosphate Carboxylase ◽  
Specificity Factor

Proximal Cys172 and Cys192 in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys172 has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys172 and Cys192 by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The Vc (Vmax for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the Km for CO2 and O2 was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 °C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 °C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of β-strand 1 of the α/β-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.

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