Toward minimal bacterial cells: evolution vs. design.

Andrés Moya 1,2,3 , Rosario Gil 1,2,3 , Amparo Latorre 1,2,3 , Juli Peretó 1,2,4 , Maria Pilar Garcillán-Barcia 5 & Fernando de la Cruz 5
1 Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, València, Spain; 2 CIBER de Epidemiología y Salud Pública, Madrid, Spain; 3 Departament de Genètica, Universitat de València, València, Spain; 4 Departament de Bioquímica i Biologia Molecular, Universitat de València, València, Spain; and 5 Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain

Recent technical and conceptual advances in the biological sciences opened the possibility of the construction of newly designed cells. In this paper we review the state of the art of cell engineering in the context of genome research, paying particular attention to what we can learn on naturally reduced genomes from either symbiotic or free living bacteria. Different minimal hypothetically viable cells can be defined on the basis of several computational and experimental approaches. Projects aiming at simplifying living cells converge with efforts to make synthetic genomes for minimal cells. The panorama of this particular view of synthetic biology lead us to consider the use of defined minimal cells to be applied in biomedical, bioremediation, or bioenergy application by taking advantage of existing naturally minimized cells.

http://dx.doi.org/10.1111/j.1574-6976.2008.00151.x

Coprinus cinereus rad50 Mutants Reveal an Essential Structural Role for Rad50 in Axial Element and Synaptonemal Complex Formation, Homolog Pairing and

Sonia N. Acharya*,1, Alexander M. Many*,1,2, Andrew P. Schroeder*, Felicia M. Kennedy*, Oleksandr P. Savytskyy*, Jennifer T. Grubb*, Jack A. Vincent*, Elizabeth A. Friedle*, Martina Celerin*, Daniel S. Maillet*, Heather J. Palmerini*, Megan A. Greischar*, Gabriel Moncalian,3, R. Scott Williams, John A. Tainer and Miriam E. Zolan*,4
* Department of Biology, Indiana University, Bloomington, Indiana 47405 and The Scripps Research Institute, La Jolla, California 92037

The Mre11/Rad50/Nbs1 (MRN) complex is required for eukaryotic DNA double-strand break (DSB) repair and meiotic recombination. We cloned the Coprinus cinereus rad50 gene and showed that it corresponds to the complementation group previously named rad12, identified mutations in 15 rad50 alleles, and mapped two of the mutations onto molecular models of Rad50 structure. We found that C. cinereus rad50 and mre11 mutants arrest in meiosis and that this arrest is Spo11 dependent. In addition, some rad50 alleles form inducible, Spo11-dependent Rad51 foci and therefore must be forming meiotic DSBs. Thus, we think it likely that arrest in both mre11-1 and the collection of rad50 mutants is the result of unrepaired or improperly processed DSBs in the genome and that Rad50 and Mre11 are dispensable in C. cinereus for DSB formation, but required for appropriate DSB processing. We found that the ability of rad50 mutant strains to form Rad51 foci correlates with their ability to promote synaptonemal complex formation and with levels of stable meiotic pairing and that partial pairing, recombination initiation, and synapsis occur in the absence of wild-type Rad50 catalytic domains. Examination of single- and double-mutant strains showed that a spo11 mutation that prevents DSB formation enhances axial element (AE) formation for rad50-4, an allele predicted to encode a protein with intact hook region and hook-proximal coiled coils, but not for rad50-1, an allele predicted to encode a severely truncated protein, or for rad50-5, which encodes a protein whose hook-proximal coiled-coil region is disrupted. Therefore, Rad50 has an essential structural role in the formation of AEs, separate from the DSB-processing activity of the MRN complex.

http://dx.doi.org/10.1534/genetics.108.092775%20

Analysis of ColE1 MbeC unveils an extended ribbon-helix-helix family of nicking-accessory proteins

Athanasia Varsaki, Gabriel Moncalián, Maria del Pilar Garcillán-Barcia, Constantin Drainas, and Fernando de la Cruz*
MbeC is a 13 kDa ColE1-encoded protein required for efficient mobilization of ColE1, a plasmid widely used in cloning vector technology. MbeC protein was purified and used for in vitro DNA-binding, which showed that it binds specifically dsDNA containing the ColE1 oriT. Amino-acid sequence comparison and secondary structure prediction imply that MbeC is related to the ribbon-helix-helix (RHH) protein family. Alignment with RHH members pointed a conserved arginine (R13 in MbeC) which was mutated to alanine. The mutant MbeC(R13A) was unable to bind either ssDNA or dsDNA. Limited proteolysis fragmented MbeC in two stable folding domains: the N-terminal domain, which contains the RHH motif and the C-terminal domain, which comprises a signature shared by nicking-accessory proteins. Results indicate that MbeC plays a similar role in conjugation as TraY and TrwA of plasmids F and R388, respectively. Thus, it appears that an extended, possibly universal mechanism of DNA conjugative processing exists, in which oriT-processing is carried out by relaxases assisted by homologous nicking-accessory proteins. This mechanism seems to be shared by all major conjugative systems analyzed so far.
http://dx.doi.org/10.1128/JB.01342-08

Reply to The binding stoichiometry of CIN85 SH3 domain A and Cbl-b

Daniela Jozic, Nayra Cardenes, Yonathan Lissanu Deribe, Gabriel Moncalian, Daniela Hoeller, Yvonne Groemping, Ivan Dikic, Katrin Rittinger, Jeronimo Bravo
Nature Structural & Molecular Biology 15, 891 - 892 (01 Sep 2008), doi: http://dx.doi.org/10.1038/nsmb0908-891, Correspondence