High tolerance to self-targeting of the genome by the endogenous CRISPR-Cas system in an archaeon.

CRISPR-Cas systems allow bacteria and archaea to acquire sequence-specific immunity against selfish genetic elements such as viruses and plasmids, by specific degradation of invader DNA or RNA. However, this involves the risk of autoimmunity if immune memory against host DNA is mistakenly acquired. Such autoimmunity has been shown to be highly toxic in several bacteria and is believed to be one of the major costs of maintaining these defense systems. Here we generated an experimental system in which a non-essential gene, required for pigment production and the reddish colony color, is targeted by the CRISPR-Cas I-B system of the halophilic archaeon Haloferax volcanii. We show that under native conditions, where both the self-targeting and native crRNAs are expressed, self-targeting by CRISPR-Cas causes no reduction in transformation efficiency of the plasmid encoding the self-targeting crRNA. Furthermore, under such conditions, no effect on organismal growth rate or loss of the reddish colony phenotype due to mutations in the targeted region could be observed. In contrast, in cells deleted for the pre-crRNA processing gene cas6, where only the self-targeting crRNA exists as mature crRNA, self-targeting leads to moderate toxicity and the emergence of deletion mutants. Sequencing of the deletions caused by CRISPR-Cas self targeting indicated DNA repair via microhomology-mediated end joining.

Differences in homologous recombination and maintenance of heteropolyploidy between Haloferax volcanii and Haloferax mediterranei.

Polyploidy, the phenomenon of having more than one copy of the genome in an organism, is common among haloarchaea. While providing short-term benefits for DNA repair, polyploidy is generally regarded as an "evolutionary trap" that by the notion of the Muller's ratchet will inevitably conclude in the species' decline or even extinction due to a gradual reduction in fitness. In most reported cases of polyploidy in archaea, the genetic state of the organism is considered as homoploidy i.e. all copies of the genome are identical. Here we demonstrate that while this is indeed the prevalent genetic status in the halophilic archaeon Haloferax volcanii, its close relative H. mediterranei maintains a prolonged heteroploidy state in a nonselective environment once a second allele is introduced. Moreover, a strong genetic linkage was observed between two distant loci in H. mediterranei indicating a low rate of homologous recombination while almost no such linkage was shown in H. volcanii indicating a high rate of recombination in the latter species. We suggest that H. volcanii escapes Muller's ratchet by means of an effective chromosome-equalizing gene-conversion mechanism facilitated by highly active homologous recombination, whereas H. mediterranei must elude the ratchet via a different, yet to be elucidated mechanism.

Maize Transposable Elements Ac/Ds as Insertion Mutagenesis Tools in Candida albicans.

In nonmodel systems, genetic research is often limited by the lack of techniques for the generation and identification of gene mutations. One approach to overcome this bottleneck is the application of transposons for gene tagging. We have established a two-element transposon tagging system, based on the transposable elements Activator (Ac)/Dissociation (Ds) from maize, for in vivo insertion mutagenesis in the fungal human pathogen Candida albicans A nonautonomous Ds transposon carrying a selectable marker was constructed into the ADE2 promoter on chromosome 3 and a codon usage-adapted Ac transposase gene was inserted into the neutral NEUT5L locus on chromosome 5. In C. albicans cells expressing the transposase, the Ds element efficiently excised and reintegrated elsewhere in the genome, which makes the Ac/Ds transposons promising tools for saturating insertion mutagenesis in clinical strains of C. albicans.

Maximal sum of metabolic exchange fluxes outperforms biomass yield as a predictor of growth rate of microorganisms.

Growth rate has long been considered one of the most valuable phenotypes that can be measured in cells. Aside from being highly accessible and informative in laboratory cultures, maximal growth rate is often a prime determinant of cellular fitness, and predicting phenotypes that underlie fitness is key to both understanding and manipulating life. Despite this, current methods for predicting microbial fitness typically focus on yields [e.g., predictions of biomass yield using GEnome-scale metabolic Models (GEMs)] or notably require many empirical kinetic constants or substrate uptake rates, which render these methods ineffective in cases where fitness derives most directly from growth rate. Here we present a new method for predicting cellular growth rate, termed SUMEX, which does not require any empirical variables apart from a metabolic network (i.e., a GEM) and the growth medium. SUMEX is calculated by maximizing the SUM of molar EXchange fluxes (hence SUMEX) in a genome-scale metabolic model. SUMEX successfully predicts relative microbial growth rates across species, environments, and genetic conditions, outperforming traditional cellular objectives (most notably, the convention assuming biomass maximization). The success of SUMEX suggests that the ability of a cell to catabolize substrates and produce a strong proton gradient enables fast cell growth. Easily applicable heuristics for predicting growth rate, such as what we demonstrate with SUMEX, may contribute to numerous medical and biotechnological goals, ranging from the engineering of faster-growing industrial strains, modeling of mixed ecological communities, and the inhibition of cancer growth.