Toward a Predictive Understanding of Earth's Microbiomes to Address 21st Century Challenges.

Microorganisms have shaped our planet and its inhabitants for over 3.5 billion years. Humankind has had a profound influence on the biosphere, manifested as global climate and land use changes, and extensive urbanization in response to a growing population. The challenges we face to supply food, energy, and clean water while maintaining and improving the health of our population and ecosystems are significant. Given the extensive influence of microorganisms across our biosphere, we propose that a coordinated, cross-disciplinary effort is required to understand, predict, and harness microbiome function. From the parallelization of gene function testing to precision manipulation of genes, communities, and model ecosystems and development of novel analytical and simulation approaches, we outline strategies to move microbiome research into an era of causality. These efforts will improve prediction of ecosystem response and enable the development of new, responsible, microbiome-based solutions to significant challenges of our time.

Effective killing of the human pathogen Candida albicans by a specific inhibitor of non-essential mitotic kinesin Kip1p.

Kinesins from the bipolar (Kinesin-5) family are conserved in eukaryotic organisms and play critical roles during the earliest stages of mitosis to mediate spindle pole body separation and formation of a bipolar mitotic spindle. To date, genes encoding bipolar kinesins have been reported to be essential in all organisms studied. We report the characterization of CaKip1p, the sole member of this family in the human pathogenic yeast Candida albicans. C. albicans Kip1p appears to localize to the mitotic spindle and loss of CaKip1p function interferes with normal progression through mitosis. Inducible excision of CaKIP1 revealed phenotypes unique to C. albicans, including viable homozygous Cakip1 mutants and an aberrant spindle morphology in which multiple spindle poles accumulate in close proximity to each other. Expression of the C. albicans Kip1 motor domain in Escherichia coli produced a protein with microtubule-stimulated ATPase activity that was inhibited by an aminobenzothiazole (ABT) compound in an ATP-competitive fashion. This inhibition results in 'rigor-like', tight association with microtubules in vitro. Upon treatment of C. albicans cells with the ABT compound, cells were killed, and terminal phenotype analysis revealed an aberrant spindle morphology similar to that induced by loss of the CaKIP1 gene. The ABT compound discovered is the first example of a fungal spindle inhibitor targeted to a mitotic kinesin. Our results also show that the non-essential nature and implementation of the bipolar motor in C. albicans differs from that seen in other organisms, and suggest that inhibitors of a non-essential mitotic kinesin may offer promise as cidal agents for antifungal drug discovery.

Nuclear fusion occurs during mating in Candida albicans and is dependent on the KAR3 gene.

It is now well established that mating can occur between diploid a and alpha cells of Candida albicans. There is, however, controversy over when, and with what efficiency, nuclear fusion follows cell fusion to create stable tetraploid a/alpha cells. In this study, we have analysed the mating process between C. albicans strains using both cytological and genetic approaches. Using strains derived from SC5314, we used a number of techniques, including time-lapse microscopy, to demonstrate that efficient nuclear fusion occurs in the zygote before formation of the first daughter cell. Consistent with these observations, zygotes micromanipulated from mating mixes gave rise to mononuclear tetraploid cells, even when no selection for successful mating was applied to them. Mating between different clinical isolates of C. albicans revealed that while all isolates could undergo nuclear fusion, the efficiency of nuclear fusion varied in different crosses. We also show that nuclear fusion in C. albicans requires the Kar3 microtubule motor protein. Deletion of the CaKAR3 gene from both mating partners had little or no effect on zygote formation but reduced the formation of stable tetraploids more than 600-fold, as determined by quantitative mating assays. These findings demonstrate that nuclear fusion is an active process that can occur in C. albicans at high frequency to produce stable, mononucleate mating products.

Integrating transcriptional and metabolite profiles to direct the engineering of lovastatin-producing fungal strains.

We describe a method to decipher the complex inter-relationships between metabolite production trends and gene expression events, and show how information gleaned from such studies can be applied to yield improved production strains. Genomic fragment microarrays were constructed for the Aspergillus terreus genome, and transcriptional profiles were generated from strains engineered to produce varying amounts of the medically significant natural product lovastatin. Metabolite detection methods were employed to quantify the polyketide-derived secondary metabolites lovastatin and (+)-geodin in broths from fermentations of the same strains. Association analysis of the resulting transcriptional and metabolic data sets provides mechanistic insight into the genetic and physiological control of lovastatin and (+)-geodin biosynthesis, and identifies novel components involved in the production of (+)-geodin, as well as other secondary metabolites. Furthermore, this analysis identifies specific tools, including promoters for reporter-based selection systems, that we employed to improve lovastatin production by A. terreus.