Gene Transfer from Bacteria and Archaea Facilitated Evolution of an
Extremophilic Eukaryote

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and Wildlife Foundation, the National Geographic Society, Gene Transfer from Bacteria and lead to expansion of existing gene families (8). Incontrast, archaea and bacteria commonly adaptthrough horizontal gene transfer (HGT) from other Archaea Facilitated Evolution of an lineages (9). HGT has also been observed insome unicellular eukaryotes (10); however, to Extremophilic Eukaryote our knowledge, horizontally acquired genes havenot been linked to fitness-relevant traits in free-living eukaryotes (11). Phylogenetic analyses of Gerald Schönknecht,1,2*† Wei-Hua Chen,3,4† Chad M. Ternes,1† Guillaume G. Barbier,5†‡ G. sulphuraria genes using highly stringent crite- Roshan P. Shrestha,5†§ Mario Stanke,6 Andrea Bräutigam,2 Brett J. Baker,7 Jillian F. Banfield,8 ria indicate at least 75 separate gene acquisi- R. Michael Garavito,9 Kevin Carr,10 Curtis Wilkerson,5,10 Stefan A. Rensing,11 David Gagneul,12 tions from archaea and bacteria (supplementary Nicholas E. Dickenson,13 Christine Oesterhelt,14 Martin J. Lercher,3,15 Andreas P. M. Weber2,5,15* materials). The origin of these G. sulphurariagenes from HGT is supported by the finding Some microbial eukaryotes, such as the extremophilic red alga Galdieria sulphuraria, live in that compared to the genomic average, they have hot, toxic metal-rich, acidic environments. To elucidate the underlying molecular mechanisms ofadaptation, we sequenced the 13.7-megabase genome of G. sulphuraria. This alga shows an 1Department of Botany, Oklahoma State University, Stillwater, enormous metabolic flexibility, growing either photoautotrophically or heterotrophically on more OK 74078, USA. 2Institute of Plant Biochemistry, Heinrich- than 50 carbon sources. Environmental adaptation seems to have been facilitated by horizontal Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany.
3 gene transfer from various bacteria and archaea, often followed by gene family expansion. At least Institute for Computer Science, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany. 4European Molecular 5% of protein-coding genes of G. sulphuraria were probably acquired horizontally. These proteins Biology Laboratory (EMBL) Heidelberg, EMBL, Meyerhofstrasse are involved in ecologically important processes ranging from heavy-metal detoxification to 1, 69117 Heidelberg, Germany. 5Department of Plant Biology, glycerol uptake and metabolism. Thus, our findings show that a pan-domain gene pool has 612 Wilson Road, Michigan State University, East Lansing, MI facilitated environmental adaptation in this unicellular eukaryote.
48824, USA. 6Institut für Mathematik und Informatik, ErnstMoritz Arndt Universität Greifswald, Walther-Rathenau-Straße47, 17487 Greifswald, Germany. 7Department of Earth and Envi- Althoughbacteriaandarchaeausuallydom- (6). The only member of the Cyanidiophyceae ronmentalSciences,4011CCLittleBuilding,1100NorthUni- inate extreme environments, hot and ex- for which a genome sequence was previously versity Avenue, University of Michigan, Ann Arbor, MI 48109, tremely acidic habitats are typically devoid available, Cyanidioschyzon merolae (7), diverged USA. 8Department of Earth and Planetary Science, Departmentof Environmental Science, Policy, and Management, University of photosynthetic bacteria. Instead, eukaryotic from G. sulphuraria about 1 billion years ago, of California, Berkeley CA 94720–4767, USA. 9Department of unicellular red algae of the Cyanidiophyceae are which approximates the evolutionary distance be- Biochemistry and Molecular Biology, 603 Wilson Road, Michigan the principal photosynthetic organisms in these tween fruit flies and humans (see fig. S1 and State University, East Lansing, MI 48824, USA. 10Research Tech- ecological niches (1). Cyanidiophyceae can grow supplementary materials). C. merolae maintains nology Support Facility, Plant Biology Laboratories, 612 WilsonRoad, Michigan State University, East Lansing, MI 48824, USA.
at pH 0 to 4 and temperatures up to 56°C, close a strictly photoautotrophic lifestyle and does not 11Faculty of Biology and BIOSS Centre for Biological Signalling to the upper temperature limit for eukaryotic life tolerate high salt or metal concentrations; it dif- Studies, University of Freiburg, Schänzlestrasse 1, 79104 Freiburg, (2). Galdieria sulphuraria is a unique member of fers markedly from G. sulphuraria in ecology, cell Germany. 12UMR USTL-INRA 1281 "Stress Abiotiques et Dif- the Cyanidiophyceae, displaying high salt and biology, and physiology. Accordingly, we find férenciation des Végétaux cultivés," Université de Lille 1, 59650Villeneuve d'Ascq Cédex, France. 13Department of Microbiology metal tolerance and exhibiting extensive meta- orthologs for only 42% of the 6623 G. sulphuraria and Molecular Genetics, Oklahoma State University, Stillwater, bolic versatility (3, 4). G. sulphuraria naturally proteins in C. merolae, and only 25% of both ge- OK 74078, USA. 14CyanoBiofuels GmbH, Magnusstrasse 11, inhabits volcanic hot sulfur springs, solfatara soils, nomes constitute syntenic blocks (fig. S2). Coding 12489 Berlin, Germany. 15Cluster of Excellence on Plant Sci- and anthropogenic hostile environments. In habi- sequences make up 77.5% of the G. sulphuraria ences (CEPLAS), Heinrich-Heine-Universität Düsseldorf, 40225 tats with high concentrations of arsenic, alumi- genome, resulting in a median intergenic distance num, cadmium, mercury, and other toxic metals, of 20 base pairs (bp) (fig. S3). Protein-coding *To whom correspondence should be addressed. (G.S.); G. sulphuraria frequently represents up to 90% genes contain on average two introns (fig. S4), of total biomass and almost all the eukaryotic with median lengths of 55 bp (fig. S5). Thus, the †These authors contributed equally to this work.
biomass (1, 5).
G. sulphuraria genome is highly condensed by ‡Permanent address: Novozymes, Inc, 1445 Drew Avenue, To understand the molecular mechanisms comparison with that of C. merolae and most Davis, CA 95618, USA.
§Permanent address: Scripps Institution of Oceanography, underlying G. sulphuraria's extremophilic and other eukaryotes.
University of California, San Diego, CA 92037, USA.
metabolically flexible lifestyle (Fig. 1), we deter- Eukaryotic innovations usually arise through Permanent address: Faculty of Biology, Philipps-University mined its genome sequence (13.7 Mb; table S1) gene duplications and neofunctionalizations, which Marburg, 35032 Marburg, Germany. SCIENCE VOL 339 8 MARCH 2013 significantly fewer introns (mean 0.8 versus 2.06, archaeal ATPases (adenosine triphosphatases) relation between ATPase gene copy number and P = 0.0012, Mann-Whitney test; fig. S6), slightly (Fig. 2A). These soluble ATPases have not been optimal growth temperature across thermophilic higher GC content (40.6% versus 39.9%, P = observed in other eukaryotes. Phylogenetic analy- and hyperthermophilic archaea (Fig. 2C). These 0.0030, Student's t test; fig. S7), and deviating oli- ses indicate that G. sulphuraria acquired an an- findings suggest that G. sulphuraria's adaptation gonucleotide usage (P = 0.00034, Mann-Whitney cestral ATPase gene from archaea, followed by to heat may have been facilitated by the acqui- test; fig. S7). Gene transfers can be traced to a duplications and diversification into separate sition and subsequent expansion of an archaeal broad range of donor taxa (fig. S8 and table S4), families (fig. S11). Genes encoding the different ATPase gene family.
with a significant enrichment from extremophile families cluster in pairs on the G. sulphuraria G. sulphuraria's tolerance to high salinity is bacteria (P = 7.8 × 10−9, Fisher's exact test). The genome (Fig. 2B). Pairs of homologous ATPase also likely to have been facilitated by HGT. Re- genome of G. sulphuraria thus shows notable genes that are transcribed together are observed sistance to salt stress requires the removal of Na+ contributions from a pan-domain gene pool.
in archaeal genomes (12). Although their physio- from the cytosol and an increase of osmolarity The two largest G. sulphuraria protein families logical function is unknown (13), archaeal ATPases using compatible solutes in the cytosol. In addi- form a monophyletic branch within the so-called may contribute to heat tolerance. We found a cor- tion to several Na+:H+ antiporters of eukaryoticorigin, G. sulphuraria encodes two monovalentcation:proton antiporters that appear to have Fig. 1. Photoautotrophic been acquired from bacteria (fig. S13). Further- (left) and heterotrophic more, genes encoding sarcosine dimethylglycine (right) G. sulphuraria cells.
methyltransferase (SDMT) appear to originate Cell cultures (top) and from halophilic cyanobacteria (fig. S14). These light microscopic images enzymes allow the production of the compatible (bottom; bar represents solute betaine from glycine (14), which indeed 10 mm) of G. sulphuraria accumulates in G. sulphuraria under salt stress cells grown under contin- uous illumination in the absence of glucose (left) G. sulphuraria maintain near-neutral cytosolic pH against a 106-fold H+ gradient across or in darkness in the pres- its plasma membrane ( ence of 200 mM glucose 15, 16)? There is no evi- dence for an enhanced capacity to pump H+ outof the cytosol (which would be an energeticallyintense strategy). Yet, there are indications of areduced H+ permeability of the plasma mem-brane. For G. sulphuraria, one voltage-gated ionchannel gene was identified in the genome, com-pared to three in C. merolae (table S3) and 16 ormore in other unicellular algae. Voltage-gatedion channels allow single-file diffusion of water, and therefore have a very high conductance forprotons. A plasma membrane devoid of proton-conducting voltage-gated ion channels probably Fig. 2. Archaeal ATPases in G. sulphuraria. (A) The phylogeny of archaealATPases indicates HGT into G. sulphuraria. The unrooted Bayesian tree isshown with posterior probabilities (full tree and details in fig. S9, align-ment of ATPase domains in fig. S10, and detailed tree of families #1a,#1b, #2, and #19 in fig. S11). Major phylogenetic groups are color-coded(Archaea, gray; Cyanobacteria, teal; other Bacteria, olive; Rhodophyta,red; Amoebozoa, purple) and labeled according to Leipe et al. (20). (B)Genes encoding archaeal ATPases in G. sulphuraria form unidirectionalclusters in specific combinations; pairs are always formed from different(sub)families, with #1b genes always at the 5′ end. (C) Copy number ofarchaeal ATPase genes in genomes of thermophilic and hyperthermophilicarchaea is correlated with optimum growth temperature (for details, seefig. S12).
8 MARCH 2013 VOL 339 SCIENCE has a low H+ permeability, preventing the acidi- symporters, and glycerol uptake facilitators (fig.
currently cannot distinguish between these alter- fication of the cell interior even at very high S23). Together, these metabolite transporters are native hypotheses owing to limited taxon repre- external H+ concentrations. G. sulphuraria shows likely to be essential for the exceptional ability of sentation in sequence databases.
an expansion of both, the intracellular chloride G. sulphuraria to grow heterotrophically on many G. sulphuraria can also survive saprophytically channel (CLIC) family and the chloride carrier/ different metabolites. In contrast, most other uni- by excreting catabolic enzymes that decompose channel (ClC) family (table S3), which do not cellular algae, including C. merolae, are strictly (or extracellular organic polymers into small metabo- conduct protons.
almost strictly) photoautotrophic.
lites for uptake by plasma membrane transporters.
G. sulphuraria copes with toxic metals typi- Again, phylogenetic analyses indicate that Proteomics (18) and/or bioinformatics analyses cally found in volcanic areas and acid mine drain- HGT contributed to G. sulphuraria's enormous (supplementary materials) indicate excretion of age by a variety of metal transporters (table S3).
metabolic flexibility. All acetate permeases (fig.
aspartyl proteases, which cleave peptide bonds at Different plasma membrane uptake systems for S21) appear to originate from bacteria, whereas acidic pH; b-galactosidases (fig. S26) and gluco- divalent metal cations permit the selective up- some of the amino acid–polyamine–organocation amylases, which degrade polysaccharides; and take of essential metals (such as iron or copper) transporters (fig. S22) seem to stem from thermo- acid phosphatases (fig. S27), which remove phos- at high concentrations of toxic metals (such as acidophilic archaea. G. sulphuraria can grow het- phate groups from organic molecules. These aluminum or cadmium). G. sulphuraria can also erotrophically on glycerol as sole carbon source excreted enzymes lack orthologs in other photo- neutralize biohazardous metals, making it po- (3) using a family of five glycerol uptake facil- synthetic eukaryotes, but show homology to ex- tentially useful in biotechnological applications.
itators and a family of three glycerol dehydro- creted enzymes encoded by fungi or bacteria. In For example, G. sulphuraria arsenite methyltrans- genases; both families apparently originate from particular, G. sulphuraria may have acquired acid ferases can biotransform arsenic, which is often HGT (figs. S23 and S24).
phosphatases (fig. S27) and some b-galactosidases found at very high concentrations in geothermal Some of G. sulphuraria's metabolic path- (fig. S26) from bacteria.
environments, into less toxic and possibly gaseous ways appear to be conserved from a hetero- Extensive gene transfer appears to have been methyl derivatives (17). In addition, two intron- trophic last common eukaryotic ancestor, but key to the genomic evolution of a metabolical- less G. sulphuraria genes encode the bacterial subsequently were lost from other photosynthetic ly versatile, extremophilic, red alga. Numerous arsenical membrane protein pump, ArsB. The se- eukaryotes. For example, animals (metazoa) use proteins acquired through HGT interact with quences most similar to G. sulphuraria ArsB are the methylmalonyl–coenzyme A pathway for G. sulphuraria's physico-chemical and meta- from thermoacidophilic bacteria (Fig. 3), again the degradation of odd-numbered chain fatty bolic environment. Protein families acquired hori- indicating a central role for HGT in extremophilic acids and leucine. Whereas green plants and zontally by G. sulphuraria are 3-fold enriched adaptation. Mercury is found at concentrations up C. merolae lack this pathway, it is present in in membrane transporters (10.5% for HGT fami- to 200 mg/g in soils from which G. sulphuraria G. sulphuraria, as well as in diatoms and brown lies, P = 0.010, Fisher's exact test) and 14-fold has been isolated. G. sulphuraria can reduce cy- algae. Furthermore, green plants, diatoms, and enriched in protein families also found in ex- totoxic Hg2+ into less toxic metallic mercury. The brown algae synthesize the essential nicotinamide tremophilic bacteria or archaea (86.8% for HGT enzyme responsible, mercuric reductase, was also adenine dinucleotide precursor quinolinate from families, P = 1.5 × 10−22). These findings for most likely acquired horizontally from Proteo- aspartate. In contrast, G. sulphuraria and C. merolae G. sulphuraria mirror the results of a previous bacteria (fig. S17).
produce quinolinate from tryptophan by way systematic study, which showed that proteo- In total, 5.2% of G. sulphuraria genes en- of kynurenine, a pathway common to animals bacterial adaptation relies on the horizontal code membrane transport proteins (mostly me- acquisition of genes that function at the bacte- tabolite transporters), which is more than has been G. sulphuraria contains a number of metab- ria's interface to the environment (19). Whereas discovered in most other eukaryotes (fig. S18).
olite transporter families that group with fungi the importance of HGT for evolution of Bacteria Gene family expansions (table S3) are found, and show less similarity to metabolite transporter and Archaea is well established, adaptation for example, in sugar porters (fig. S19), amino families from more closely related organisms (figs.
of a eukaryotic extremophile by gene transfer acid/auxin permeases (fig. S20), and putative ace- S19, S20, and S25). This unexpected phyloge- from Bacteria and Archaea is unexpected and tate transporters (fig. S21); these three protein fam- netic trace could be explained by the loss of genes shines a new light on the evolution of unicel- ilies are among the 20 largest in G. sulphuraria present in the common eukaryotic ancestor from lular eukaryotes.
(table S2). Further major expansions were found other clades, by HGT from unsequenced bacte- in amino acid–polyamine–organocation transporters ria or archaea into both eukaryotic lineages, or (fig. S22), glycoside-pentoside-hexuronide:cation by HGT between fungi and G. sulphuraria. We References and Notes 1. W. N. Doemel, T. D. Brock, J. Gen. Microbiol. 67, 17 Fig. 3. The phylogeny 2. L. J. Rothschild, R. L. Mancinelli, Nature 409, 1092 (Bayesian tree) of bac- 3. W. Gross, C. Schnarrenberger, Plant Cell Physiol. 36, terial arsenical resist- ance efflux pumps (ArsB) 4. V. Reeb, D. Bhattacharya, in Red Algae in the Genomic Age, indicates HGT into G.
J. Seckbach, D. J. Chapman, Eds. (Springer, Netherlands, sulphuraria (full tree 2010), pp. 409–426.
and details in fig. S16).
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The curly bracket marks (Springer, Dordrecht, Netherlands, 2007), vol. 11, G. sulphuraria and four pp. 487–502.
thermophilic and/or aci- 6. Materials and methods are available as supplementary dophilic bacteria (in ol- materials on Science Online.
ive), which live in the 7. M. Matsuzaki et al., Nature 428, 653 (2004).
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G. sulphuraria and from 9. T. J. Treangen, E. P. C. Rocha, PLoS Genet. 7, e1001284 which taxa the algal ArsB may have derived.
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We thank M. Hanikenne and E. Koonin for helpful 17, 155 (2012).
discussion, and A. Doust and P. Pelser for advice on 18. C. Oesterhelt, S. Vogelbein, R. P. Shrestha, phylogenetic analyses. We are grateful to B. Sears for Additional Data Table S4 M. Stanke, A. P. M. Weber, Planta 227, 353 (2008).
introduction to and assistance with CsCl purification 19. C. Pál, B. Papp, M. J. Lercher, Nat. Genet. 37, 1372 of bisbenzamide-treated nuclear DNA. Some of the computing for this project was performed at the OSU 20. D. D. Leipe, E. V. Koonin, L. Aravind, J. Mol. Biol.
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have been deposited at DNA Data Bank of Japan, EMBL, Cell Death from Antibiotics have known sequelae that ultimately lead to DNAdamage. Specifically, superoxide and hydrogenperoxide damage the iron-sulfur clusters of de- Without the Involvement of hydratases (11, 12), releasing iron atoms andelevating the pool of intracellular unincorporated Reactive Oxygen Species iron (13, 14). This iron can then react with hy-drogen peroxide in the Fenton reaction, generat-ing hydroxyl radicals that either directly damage Yuanyuan Liu and James A. Imlay* DNA (15) or indirectly oxidize the deoxynucleo-tide pool, which is subsequently incorporated into Recent observations have suggested that classic antibiotics kill bacteria by stimulating the DNA (16). This scenario could explain the ob- formation of reactive oxygen species (ROS). If true, this notion might guide new strategies to served oxidation of intracellular dyes, protection improve antibiotic efficacy. In this study, the model was directly tested. Contrary to the by scavengers and chelators, a requirement for hypothesis, antibiotic treatment did not accelerate the formation of hydrogen peroxide in respiration, and the sensitivity of DNA-repair Escherichia coli and did not elevate intracellular free iron, an essential reactant for the production of lethal damage. Lethality persisted in the absence of oxygen, and DNA repair mutants were This model is plausible, so we devised exper- not hypersensitive, undermining the idea that toxicity arose from oxidative DNA lesions. We iments to directly test the molecular events that conclude that these antibiotic exposures did not produce ROS and that lethality more likely underpin it. The bacterial strain (E. coli MG1655), resulted from the direct inhibition of cell-wall assembly, protein synthesis, and DNA replication.
growth medium (LB), and antibiotic doses werechosen to match those of previous studies (4).
In recent decades, the growing number of was postulated that interference with ribosome Kanamycinwasusedtotargettranslation,ampi- antibiotic-resistant pathogens has spurred ef- progression would release incomplete polypep- cillin to block cell-wall synthesis, and norfloxacin forts to further understand and improve the tides, some of which are translocated to the cell to disrupt DNA replication.
efficacy of the basic antibiotic classes. Most clin- membranes where they might trigger envelope Superoxide and hydrogen peroxide are gen- ically used antibiotics target cell-wall assembly, stress. The Arc regulatory system is perturbed, po- erated inside cells when flavoenzymes inadver- protein synthesis, or DNA replication. However, tentially accelerating respiration and thereby in- tently transfer a fraction of their electron flux recent reports have raised the possibility that creasing the flux of superoxide and hydrogen directly to molecular oxygen (15, 17). Thus, nei- although these antibiotics block growth by di- peroxide into the cell interior. These two oxidants ther of these ROS can be formed under anoxic rectly inhibiting the targets mentioned above,they may owe their lethal effects to the indirectcreation of reactive oxygen species (ROS) that Fig. 1. Antibiotic effica- 250 ng/ml Nor, WT 30 µg/ml Kan, WT then damage bacterial DNA (1–10).
cy does not require oxygen The evidence supporting this proposal includ- 2O2. (A to C) Wild-type ed the observation that cell-penetrating dyes were cells were treated with oxidized more quickly inside antibiotic-treated bacte- ampicillin (A), norfloxacin ria (3–8). Furthermore, iron chelators (3, 4, 7–9), (B), or kanamycin (C) in the which suppress hydroxyl-radical–generating Fenton presence (solid squares) Survivial (%) 0.01 or absence (open squares) chemistry, and thiourea (4, 6, 8–10), a potential of oxygen. In (C), anoxic kill- scavenger of hydroxyl radicals, lessened toxicity.
ing was also tested in the Mutations that diminish fluxes through the tri- presence of 40 mM KNO 250 ng/ml Nor, +O carboxylic acid cycle were protective (3–5), suggest- (gray squares). (D to F) Wild- ing a key role for respiration, and DNA-repair type cells (solid squares, mutants were somewhat sensitive (4, 8). Systems MG1655) and congenic analysis of aminoglycoside-treated Escherichia Hpx– cells (open diamonds, coli suggested a model that fits these data (5). It AL427) were treated with antibiotics ampicillin (D), Department of Microbiology, University of Illinois, Urbana, norfloxacin (E), or kanamy- IL 61801, USA.
cin (F). Results are repre- *To whom correspondence should be addressed. E-mail: sentative of at least three 8 MARCH 2013 VOL 339 SCIENCE


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Appendix e-3: AAN Parkinson's Disease Measurement Set Parkinson's Disease Physician Performance Measurement Set As of December 16, 2009 ©2009 American Academy of Neurology. All rights reserved. AAN BOD approved 12.21.09. Appendix e-3: AAN Parkinson's Disease Measurement Set Physician Performance Measures (measures) and related data specifications developed by the American Academy of

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