We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.

SummaryExpression of genes for Bacillus anthracis toxin and capsule virulence factors are dependent upon the AtxA transcription factor. The mechanism by which AtxA regulates the transcription of its target genes is unknown. Here we report that bioinformatic analyses suggested the presence in AtxA of two PTS (phosphenolpyruvate : sugar phosphotransferase system) regulation domains (PRD) generally regulated by phosphorylation/dephosphorylation at conserved histidine residues. By means of amino acid substitutions that mimic the phosphorylated (H to D) or the unphosphorylated (H to A) state of the protein, we showed that phosphorylation of H199 of PRD1 is likely to be necessary for AtxA activation while phosphorylation of H379 in PRD2 is inhibitory to toxin gene transcription. In vivo labelling experiments with radioactive phosphate allowed us to propose that H199 and H379 are AtxA residues subject to regulated phosphorylation. In support to these notions, we also show that deletion of ptsHI, encoding the HPr intermediate and the EI enzymes of PTS, or growth in the presence of glucose affect positively and negatively, respectively, the activity of AtxA. Our results link virulence factor production in B. anthracis to carbohydrate metabolism and, for the first time, provide a mechanistic explanation for AtxA transcriptional activity.

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Tunicamycin is a reversible inhibitor of polyprenolphosphate: N-acetylhexosamine-1-phosphate translocases and is produced by several Streptomyces species. We have examined tunicamycin biosynthesis, an important but poorly characterized biosynthetic pathway. Biosynthetic precursors have been identified by incorporating radioactive and stable isotopes, and by determining the labeling pattern using electrospray ionization-collision induced dissociation-mass spectrometry (ESI-CID-MS), and proton, deuterium, and C-13 nuclear magnetic resonance ( Tunicamycins are nucleotide antibiotics produced by several Streptomyces species. They are potent inhibitors of the UDPGlcNAc:polyprenol phosphate GlcNAc-1-P translocase family and are often used to block protein N-glycosylation. The structures are highly unusual but well characterized (1, 2, 3) and are composed of uracil, N-acetylglucosamine (GlcNAc), a unique 11-carbon 2-aminodialdose sugar called tunicamine, and an amide-linked fatty acid. The ␣␤1,1Ј-glycosidic linkage between tunicamine and the GlcNAc substituent is also unique to the tunicamycin family of compounds. Tunicamycin structural variants occur that differ only in the nature of the N-linked acyl chain. We have recently introduced a structure-based naming system that identifies each tunicamycin by its signature fatty acid, i.e. Tun 13:1-Tun 18:1 (4).Although a great deal is known about tunicamycin structure and function, no previous analysis of tunicamycin biosynthesis has been reported. The key to understanding the biosynthesis of tunicamycin is the origin of the 11-carbon tunicamine dialdose sugar and the kinetics for the formation of the ␣,␤-1ЈЈ,11Ј-glycosidic bond. A large number of natural products of Streptomyces origin are synthesized from 2-carbon units via a polyketide-type reaction sequence (5). However, other long chain sugars such as sialic acids, ketodeoxyoctulosonate (KDO) and ketodeoxyheptulosonate are synthesized from aldol condensation of lower sugars with phosphoenolpyruvate (PEP) 1 (6). In addition, the biosynthesis of similar nucleoside antibiotics, polyoxins and nikkomycins, occurs by ligation of PEP and uridine-5-aldehyde, generating 8-carbon octofuranuloseuronic acid nucleoside as an intermediate (7,8,9).Here, metabolic radiolabeling experiments and stable isotope incorporations have been applied to unravel the metabolic origin of the 11-carbon dialdose sugar, tunicamine. We report that [2-14 C]uridine and [1-14 C]glucosamine are efficiently incorporated into tunicamycin by resting cells of Streptomyces chartreusis and that the [1-14 C]glucosamine feeds into both the 11-carbon tunicamine and the attached ␣-1ЈЈ-GlcNAc residue. Stable isotope incorporations using 2 H-or 13 C-labeled glucose and competitive metabolic experiments were monitored by LC-ESI-CID-MS and NMR (H-1, C-13, and HSQC) spectroscopy. The isotopic labeling patterns were consistent with carboncarbon bond formation between a 5-carbon precursor derived from uridine and a 6-carbon hexose intermediate, the latter most probably de...