We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species, Escherichia coli, by directed evolution. Four populations of Escherichia coli K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D 37 values for strains isolated from two of the populations approached that exhibited by Deinococcus radiodurans. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the recA gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.

In the absence of sulfur (S), Chlamydomonas reinhardtii increases the abundance of several transcripts encoding proteins associated with S acquisition and assimilation, conserves S amino acids, and acclimates to suboptimal growth conditions. A positive regulator, SAC1 (for sulfur acclimation protein 1), and a negative regulator, SAC3, were shown to participate in the control of these processes. In this study, we investigated two allelic mutants (ars11 and ars44) affected in a gene encoding a SNRK2 (for SNF1-related protein kinase 2) kinase designated SNRK2.1. Like the sac1 mutant, both snrk2.1 mutants were deficient in the expression of S-responsive genes. Furthermore, the mutant cells bleached more rapidly than wild-type cells during S deprivation, although the phenotypes of ars11 and ars44 were not identical: ars11 exhibited a more severe phenotype than either ars44 or sac1. The phenotypic differences between the ars11 and ars44 mutants reflected distinct alterations of SNRK2.1 mRNA splicing caused by insertion of the marker gene. The ars11 phenotype could be rescued by complementation with SNRK2.1 cDNA. In contrast to the nonepistatic relationship between SAC3 and SAC1, characterization of the sac3 ars11 double mutant showed that SNRK2.1 is epistatic to SAC3. These data reveal the crucial regulatory role of SNRK2.1 in the signaling cascade critical for eliciting S deprivation responses in Chlamydomonas. The phylogenetic relationships and structures of the eight members of the SNRK2 family in Chlamydomonas are discussed.

This report describes a Chlamydomonas reinhardtii mutant that lacks Rubisco activase (Rca). Using the Ble R (bleomycin resistance) gene as a positive selectable marker for nuclear transformation, an insertional mutagenesis screen was performed to select for cells that required a high-CO 2 atmosphere for optimal growth. The DNA flanking the Ble R insert of one of the high-CO 2 -requiring strains was cloned using thermal asymmetric interlaced-polymerase chain reaction and inverse polymerase chain reaction and sequenced. The flanking sequence matched the C. reinhardtii Rca cDNA sequence previously deposited in the National Center for Biotechnology Information database. The loss of a functional Rca in the strain was confirmed by the absence of Rca mRNA and protein. The open reading frame for Rca was cloned and expressed in pSL18, a C. reinhardtii expression vector conferring paromomycin resistance. This construct partially complemented the mutant phenotype, supporting the hypothesis that the loss of Rca was the reason the mutant grew poorly in a low-CO 2 atmosphere. Sequencing of the C. reinhardtii Rca gene revealed that it contains 10 exons ranging in size from 18 to 470 bp. Low-CO 2 -grown rca1 cultures had a growth rate and maximum rate of photosynthesis 60% of wild-type cells. Results obtained from experiments on a cia5 rca1 double mutant also suggest that the CO 2 -concentrating mechanism partially compensates for the absence of an active Rca in the green alga C. reinhardtii.The green alga Chlamydomonas reinhardtii is an excellent model to study photosynthetic processes. Although it is very difficult to maintain higher plant photosynthetic mutants, C. reinhardtii cells that are unable to perform photosynthesis can be grown heterotrophically on acetate. Furthermore, the C. reinhardtii nuclear, mitochondrial, and chloroplastic genomes can be genetically manipulated to produce mutant phenotypes (Lefebvre and Silflow, 1999; Rochaix, 2002). A random insertional mutagenesis screen was performed to generate C. reinhardtii mutants that were unable to grow optimally in a low-CO 2 atmosphere (Colombo et al., 2002). From the mutants generated, some exhibited a high fluorescence phenotype, whereas others were obligate heterotrophs that died in the light. Approximately onehalf of the selected transformants required high CO 2 for optimal growth and grew slowly in a low-CO 2 atmosphere. In the higher plant Arabidopsis, Somerville and Ogren (1982) performed a similar screen where they isolated mutants that required high levels of atmospheric CO 2 for growth. Several Arabidopsis mutants with defects in photorespiratory carbon and nitrogen metabolism were isolated. One Arabidopsis mutant isolated in that screen exhibited a reduced affinity of the carboxylation reaction for CO 2 and a much lower in vivo activity of ribulose 1,5-bisphosphate (RuBP) carboxylase . Later studies determined that had isolated a Rubisco activase (Rca) mutant that contained a guanine to adenine transition at the 5Ј splice junction of intron three (Salv...

During sulfur deprivation, the photosynthetic green alga Chlamydomonas reinhardtii develops a high-affinity sulfate uptake system and increases the expression of genes encoding proteins involved in sulfur assimilation. Although two regulatory elements, SAC1 and SAC3, have been shown to be required for normal acclimation of C. reinhardtii to sulfur deprivation, a number of other regulatory elements appear to also be involved. The molecular mechanisms by which these regulatory elements function are largely unknown. This manuscript presents our current knowledge of sulfur deprivation responses and the regulation of these responses in C. reinhardtii. In addition, we present preliminary results of a sub-saturation screen for novel sulfur acclimation mutants of C. reinhardtii. A speculative model, incorporating the activities of established regulatory elements with putative novel components of the signal transduction pathway(s) is discussed.

The unicellular green alga Chlamydomonas reinhardtii acclimates to a low-CO2 environment by modifying the expression of a number of messages. Many of the genes that increase in abundance during acclimation to low-CO2 are under the control of the putative transcription factor Cia5. C. reinhardtii mutants null for cia5 do not express several of the known low-CO2 inducible genes and do not grow in a low-CO2 environment. Two of the genes under the control of Cia5, Ccp1 and Ccp2 , encode polypeptides that are localized to the chloroplast envelope and have a high degree of similarity to members of the mitochondrial carrier family of proteins. Since their discovery, Ccp1/2 have been candidates for bicarbonate uptake proteins of the chloroplast envelope membrane. In this report, RNA interference was successful in dramatically decreasing the abundance of the mRNAs for Ccp1 and Ccp2 . The abundance of the Ccp1 and Ccp2 proteins were also reduced in the RNAi strains. The RNAi strains grew slower than WT in a low-CO2 environment, but did not exhibit a mutant carbon concentrating phenotype as determined by the cells' apparent affinity for dissolved inorganic carbon. Possible explanations of this RNAi phenotype are discussed.

During sulfur (S) deprivation, the unicellular alga Chlamydomonas reinhardtii exhibits increased expression of numerous genes. These genes encode proteins associated with sulfate (SO 4 22 ) acquisition and assimilation, alterations in cellular metabolism, and internal S recycling. Administration of the cytoplasmic translational inhibitor cycloheximide prevents S deprivationtriggered accumulation of transcripts encoding arylsulfatases (ARS), an extracellular polypeptide that may be important for cell wall biosynthesis (ECP76), a light-harvesting protein (LHCBM9), the selenium-binding protein, and the haloperoxidase (HAP2). In contrast, the rapid accumulation of transcripts encoding high-affinity SO 4 22 transporters is not affected. These results suggest that there are two tiers of transcriptional regulation associated with S deprivation responses: the first is protein synthesis independent, while the second requires de novo protein synthesis. A mutant designated ars73a exhibited low ARS activity and failed to show increases in ECP76, LHCBM9, and HAP2 transcripts (among others) in response to S deprivation; increases in transcripts encoding the SO 4 22 transporters were not affected. These results suggest that the ARS73a protein, which has no known activity but might be a transcriptional regulator, is required for the expression of genes associated with the second tier of transcriptional regulation. Analysis of the ars73a strain has helped us generate a model that incorporates a number of complexities associated with S deprivation responses in C. reinhardtii.

The availability of genome sequences makes it possible to develop microarrays that can be used for profiling gene expression over developmental time, as organisms respond to environmental challenges, and for comparison between wild-type and mutant strains under various conditions. The desired characteristics of microarrays (intense signals, hybridization specificity and extensive coverage of the transcriptome) were not fully met by the previous Chlamydomonas reinhardtii microarray: probes derived from cDNA sequences (approximately 300 bp) were prone to some nonspecific cross-hybridization and coverage of the transcriptome was only approximately 20%. The near completion of the C. reinhardtii nuclear genome sequence and the availability of extensive cDNA information have made it feasible to improve upon these aspects. After developing a protocol for selecting a high-quality unigene set representing all known expressed sequences, oligonucleotides were designed and a microarray with approximately 10,000 unique array elements (approximately 70 bp) covering 87% of the known transcriptome was developed. This microarray will enable researchers to generate a global view of gene expression in C. reinhardtii. Furthermore, the detailed description of the protocol for selecting a unigene set and the design of oligonucleotides may be of interest for laboratories interested in developing microarrays for organisms whose genome sequences are not yet completed (but are nearing completion).

Chlamydomonas reinhardtii possesses a CO 2 -concentrating mechanism (CCM) that allows the alga to grow at low CO 2 concentrations. One common feature seen in photosynthetic organisms possessing a CCM is the tight packaging of Rubisco within the cell. In many eukaryotic algae, Rubisco is localized to the pyrenoid, an electron-dense structure within the chloroplast. In order to identify genes required for a functional CCM, insertional Bleomycin resistance (Ble R ) mutants were generated and screened for growth on minimal medium under high CO 2 conditions (5% CO 2 in air) but only slow or no growth under very low CO 2 conditions (0.01% CO 2 in air). One mutant identified from this screen was named cia6. Physiological studies established that cia6 grows poorly on low levels of CO 2 and has an impaired ability to accumulate inorganic carbon. The inserted Ble R disrupted a gene encoding a protein with sequence similarity to proteins containing SET domain methyltransferase, although experiments using overexpressed CIA6 failed to demonstrate the methyltransferase activity. Electron microscopy revealed that the pyrenoid of cia6 mutant cells is highly disorganized. Complementation of the mutant restored the pyrenoid, the ability to grow under low-CO 2 conditions, and the ability to concentrate inorganic carbon. Quantitative reverse transcriptionpolymerase chain reaction data from a low-CO 2 induction time-course experiment demonstrated that the up-regulation of several CCM components is slower in cia6 compared with the wild type. This slow induction was further confirmed at the protein level using western blots. These results indicated that CIA6 is required for the formation of the pyrenoid and further supported the notion that the pyrenoid is required for a functional CCM in C. reinhardtii.