N
-acetylcytidine (ac
C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA
. However, the distribution, ...dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac
C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac
C at single-nucleotide resolution. In human and yeast mRNAs, ac
C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac
C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac
C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac
C and its potential thermoadaptive role. Our studies quantitatively define the ac
C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease
.
Genetic manipulation is an essential tool to investigate complex microbiological phenomena. In this chapter we describe the techniques required to transform the model hyperthermophilic, anaerobic ...archaeon Thermococcus kodakarensis. T. kodakarensis can support two modes of genetic manipulation, dependent either on homologous recombination into the genome or through retention of autonomously replicating plasmids. The robust genetic system developed in T. kodakarensis offers a variety of selectable and counterselectable markers for complex, accurate and iterative genetic manipulations offering greater flexibility to probe gene function in vivo.
The sole unifying feature of Archaea is the use of isoprenoid-based glycerol lipid ethers to compose cellular membranes. The branched hydrocarbon tails of archaeal lipids are synthesized via the ...polymerization of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but many questions still surround the pathway(s) that result in production of IPP and DMAPP in archaeal species. Isotopic-labeling strategies argue for multiple biological routes for production of mevalonate, but biochemical and bioinformatic studies support only a linear pathway for mevalonate production. Here, we use a combination of genetic and biochemical assays to detail the production of mevalonate in the model archaeon
Thermococcus kodakarensis
. We demonstrate that a single, linear pathway to mevalonate biosynthesis is essential and that alternative routes of mevalonate production, if present, are not biologically sufficient to support growth in the absence of the classical mevalonate pathway resulting in IPP production from acetyl-CoA. Archaeal species provide an ideal platform for production of high-value isoprenoids in large quantities, and the results obtained provide avenues to further increase the production of mevalonate to drive isoprenoid production in archaeal hosts.
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