The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover lightsensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.As with other model organisms, the availability of genome sequence data is revolutionizing and revitalizing research into the biology of the unicellular green alga Chlamydomonas reinhardtii Ledford et al., 2005). Over the past four decades, many fundamental insights into the structure, function, assembly, and regulation of the photosynthetic apparatus have come from studies of Chlamydomonas, which offer several advantages for the genetic dissection of eukaryotic photosynthesis (for review, see Davies and Grossman, 1998; Hippler et al., 1998;Grossman, 2000;Rochaix, 2001). First and foremost, photosynthesis is fully dispensable in Chlamydomonas, as cells can grow heterotrophically in the dark using acetate as a sole carbon source. Cells grown in the dark, however, still synthesize and assemble a fully functional photosynthetic apparatus. This allows the isolation and analysis of mutants that are unable to perform photosynthesis, and lightsensitive mutants can be maintained in complete darkness. Because Chlamydomonas is predominantly maintained in a haploid form, it is not necessary to generate homozygous nuclear mutants, and mutants affecting photosynthesis can be screened immediately following mutagenesis. Chlamydomonas has an easily controlled and rapid sexual cycle (approximately 2 weeks) with the possibility of tetrad analysis, which facilitates genetic analysis. Its rapid cell-doubling time (approximately 10 h) and microbial lifestyle mean that it is easy to grow homogeneous cultures on any scale, simplifying physiological and biochemical characterization in comparison to multicellular land plants (Ledford et al., 2005). By way of example, the application of inhibitors and generators of various types of reactive oxygen species results in uniform uptake of the chemical by each cell. In land plants, multicellularity leads to differential ...

Intracellular pathogens subvert the host cell cytoskeleton to promote their own survival, replication, and dissemination. Study of these microbes has led to many discoveries about host cell biology, including the identification of cytoskeletal proteins, regulatory pathways, and mechanisms of cytoskeletal function. Actin is a common target of bacterial pathogens, but recent work also highlights the use of microtubules, cytoskeletal motors, intermediate filaments, and septins. The study of pathogen interactions with the cytoskeleton has illuminated key cellular processes such as phagocytosis, macropinocytosis, membrane trafficking, motility, autophagy, and signal transduction.

Diverse intracellular pathogens subvert the host actin polymerization machinery to drive movement within and between cells during infection. Rickettsia in the spotted fever group (SFG) are Gram-negative, obligate intracellular bacterial pathogens that undergo actin-based motility and assemble distinctive ‘comet tails’ that consist of long, unbranched actin filaments1,2. Despite this distinct organization, it was proposed that actin in Rickettsia comet tails is nucleated by the host Arp2/3 complex and the bacterial protein RickA, which assemble branched actin networks3,4. However, a second bacterial gene, sca2, was recently implicated in actin tail formation by R. rickettsii5. Here, we demonstrate that Sca2 is a bacterial actin-assembly factor that functionally mimics eukaryotic formin proteins. Sca2 nucleates unbranched actin filaments, processively associates with growing barbed ends, requires profilin for efficient elongation, and inhibits the activity of capping protein, all properties shared with formins. Sca2 localizes to the Rickettsia surface and is sufficient to promote the assembly of actin filaments in cytoplasmic extract. These results suggest that Sca2 mimics formins to determine the unique organization of actin filaments in Rickettsia tails and drive bacterial motility, independently of host nucleators.

SUMMARYChlamydomonas reinhardtii is a unicellular green alga that is a key model organism in the study of photosynthesis and oxidative stress. Here we describe the large-scale generation of a population of insertional mutants that have been screened for phenotypes related to photosynthesis and the isolation of 459 flanking sequence tags from 439 mutants. Recent phylogenomic analysis has identified a core set of genes, named GreenCut2, that are conserved in green algae and plants. Many of these genes are likely to be central to the process of photosynthesis, and they are over-represented by sixfold among the screened insertional mutants, with insertion events isolated in or adjacent to 68 of 597 GreenCut2 genes. This enrichment thus provides experimental support for functional assignments based on previous bioinformatic analysis. To illustrate one of the uses of the population, a candidate gene approach based on genome position of the flanking sequence of the insertional mutant CAL027_01_20 was used to identify the molecular basis of the classical C. reinhardtii mutation ac17. These mutations were shown to affect the gene PDH2, which encodes a subunit of the plastid pyruvate dehydrogenase complex. The mutants and associated flanking sequence data described here are publicly available to the research community, and they represent one of the largest phenotyped collections of algal insertional mutants to date.