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...