We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.

Salmonella enterica serovar Typhimurium produces two Cu/Zn cofactored periplasmic superoxide dismutases, SodCI and SodCII. While mutations in sodCI attenuate virulence eightfold, loss of SodCII does not confer a virulence phenotype, nor does it enhance the defect observed in a sodCI background. Despite this in vivo phenotype, SodCI and SodCII are expressed at similar levels in vitro during the stationary phase of growth.By exchanging the open reading frames of sodCI and sodCII, we found that SodCI contributes to virulence when placed under the control of the sodCII promoter. In contrast, SodCII does not contribute to virulence even when expressed from the sodCI promoter. Thus, the disparity in virulence phenotypes is due primarily to some physical difference between the two enzymes. In an attempt to identify the unique property of SodCI, we have tested factors that might affect enzyme activity inside a phagosome. We found no significant difference between SodCI and SodCII in their resistance to acid, resistance to hydrogen peroxide, or ability to obtain copper in a copper-limiting environment. Both enzymes are synthesized as apoenzymes in the absence of copper and can be fully remetallated when copper is added. The one striking difference that we noted is that, whereas SodCII is released normally by an osmotic shock, SodCI is "tethered" within the periplasm by an apparently noncovalent interaction. We propose that this novel property of SodCI is crucial to its ability to contribute to virulence in serovar Typhimurium.Superoxide dismutases (SODs) use metal cofactors to dismutate superoxide (O 2 Ϫ ) to hydrogen peroxide (H 2 O 2 ) and molecular oxygen: O 2 Ϫ ϩ O 2 Ϫ ϩ 2 H ϩ 3 H 2 O 2 ϩ O 2 . Superoxide is generated in bacterial cytoplasms as an adventitious by-product of normal metabolism (15,16,22). Because this O 2 Ϫ can damage cytoplasmic targets-notably, the [4Fe-4S] clusters of dehydratases (14-16)-virtually all bacteria synthesize manganese-or iron-cofactored cytoplasmic SODs to scavenge it. Mutants that lack these SODs exhibit growth defects due to enzyme inactivation, and they also exhibit high rates of oxidative DNA damage as an indirect consequence of the iron that is released from the degraded clusters (4, 24).Many gram-negative bacteria also export copper-containing SODs to their periplasm (reference 1 and reference 26 and references therein). The presence of SODs in the periplasm of intracellular pathogens has led to the hypothesis that these enzymes protect bacteria against macrophage-derived superoxide (1). Bacteria internalized in macrophage phagosomes are exposed to a variety of reactive oxygen and nitrogen species: notably O 2 Ϫ , formed by the phagocytic NADPH oxidase (Phox), and nitric oxide, formed by the inducible nitric oxide synthase (32). Periplasmic SODs could plausibly protect periplasmic targets in the captive bacteria from O 2 Ϫ . Further, because O 2 Ϫ could be protonated to HO 2 • in the acidic interior of the phagolysosome, periplasmic SOD could prevent this neutral species from pen...

BackgroundInfections by pan-drug resistant Acinetobacter baumannii plague military and civilian healthcare systems. Previous A. baumannii pan-genomic studies used modest sample sizes of low diversity and comparisons to a single reference genome, limiting our understanding of gene order and content. A consensus representation of multiple genomes will provide a better framework for comparison. A large-scale comparative study will identify genomic determinants associated with their diversity and adaptation as a successful pathogen.ResultsWe determine draft-level genomic sequence of 50 diverse military isolates and conduct the largest bacterial pan-genome analysis of 249 genomes. The pan-genome of A. baumannii is open when the input genomes are normalized for diversity with 1867 core proteins and a paralog-collapsed pan-genome size of 11,694 proteins. We developed a novel graph-based algorithm and use it to assemble the first consensus pan-chromosome, identifying both the order and orientation of core genes and flexible genomic regions. Comparative genome analyses demonstrate the existence of novel resistance islands and isolates with increased numbers of resistance island insertions over time, from single insertions in the 1950s to triple insertions in 2011. Gene clusters responsible for carbon utilization, siderophore production, and pilus assembly demonstrate frequent gain or loss among isolates.ConclusionsThe highly variable and dynamic nature of the A. baumannii genome may be the result of its success in rapidly adapting to both abiotic and biotic environments through the gain and loss of gene clusters controlling fitness. Importantly, some archaic adaptation mechanisms appear to have reemerged among recent isolates.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0701-6) contains supplementary material, which is available to authorized users.

Salmonella enterica strains survive and propagate in macrophages by both circumventing and resisting the antibacterial effectors normally delivered to the phagosome. An important aspect of Salmonella resistance is the production of periplasmic superoxide dismutase to combat phagocytic superoxide. S. enterica serovar Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Both enzymes are produced during infection, but only SodCI contributes to virulence in the animal. Although 60% identical to SodCII at the amino acid level with very similar enzymatic properties, SodCI is dimeric, protease resistant, and tethered within the periplasm via a noncovalent interaction. In contrast, SodCII is monomeric and protease sensitive and is released from the periplasm normally by osmotic shock. We have constructed an enzymatically active monomeric SodCI enzyme by site-directed mutagenesis. The resulting protein was released by osmotic shock and sensitive to protease and could not complement the loss of wild-type dimeric SodCI during infection. To distinguish which property is most critical during infection, we cloned and characterized related SodC proteins from a variety of bacteria. Brucella abortus SodC was monomeric and released by osmotic shock but was protease resistant and could complement SodCI in the animal. These data suggest that protease resistance is a critical property that allows SodCI to function in the harsh environment of the phagosome to combat phagocytic superoxide. We propose a model to account for the various properties of SodCI and how they contribute to bacterial survival in the phagosome.Cu/Zn superoxide dismutases (SODs) are metalloproteins that dismute toxic superoxide radicals to H 2 O 2 and O 2 through the alternate oxidation and reduction of the copper(II) ion in the active site. Among bacteria, Cu/Zn SODs (referred to as SodCs) are located in the periplasms of certain gram-negative bacteria (2, 25) and anchored to the surfaces of some grampositive bacteria (13). Salmonella enterica serovar Typhimurium strain 14028 produces two Cu/Zn SODs-SodCI and SodCII. SodCII is the ortholog of Escherichia coli SodC, while SodCI is encoded by the Gifsy-2 prophage. The ability of these two enzymes to contribute to virulence has been studied extensively (12,14,15,24,34,36). Uzzau et al. (36) showed that only SodCI is required for the full virulence of serovar Typhimurium and that SodCII does not contribute to virulence even in the absence of SodCI. Our previous work has confirmed this result, and we have shown that this disparity in virulence phenotypes is due mainly to some differences at the protein level rather than in the regulation of the genes (24). Indeed, SodCI under the control of the more weakly in vivo-induced sodCII promoter (20) was fully capable of complementing wild-type SodCI in an animal infection. These and other data suggested that any differences in enzymatic activity or stability of the active site were insufficient to explain the differential roles of SodC...

Sense codon recoding is the basis for genetic code expansion with more than two different noncanonical amino acids. It requires an unused or rarely used codon, and an orthogonal tRNA synthetase:tRNA pair with the complementary anticodon. Mycoplasma capricolum contains only 6 CGG arginine codons without a dedicated tRNAArg. We wanted to reassign this codon to pyrrolysine by providing M. capricolum with pyrrolysyl-tRNA synthetase, a synthetic tRNA with a CCG anticodon (tRNAPylCCG), and the genes for pyrrolysine biosynthesis. Here we show that tRNAPylCCG is efficiently recognized by the endogenous arginyl-tRNA synthetase, presumably at the anticodon. Mass spectrometry reveals that in the presence of tRNAPylCCG, CGG codons are translated as arginine. This result is not unexpected as most tRNA synthetases use the anticodon as a recognition element. The data suggest that tRNA misidentification by endogenous aminoacyl-tRNA synthetases needs to be overcome for sense codon recoding.