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Nature

• Cooperation and conflict in quorum-sensing bacterial populations

Stephen P. Diggle1, Ashleigh S. Griffin2, Genevieve S. Campbell1 & Stuart A. West2

It has been suggested that bacterial cells communicate by releasing and sensing small diffusible signal molecules in a process commonly known as quorum sensing (QS)1–4. It is generally assumed that QS is used to coordinate cooperative behaviours at the population level3,5. However, evolutionary theory predicts that individuals who communicate and cooperate can be exploited6–10. Here we examine the social evolution of QS experimentally in the opportunistic pathogen Pseudomonas aeruginosa, and show that although QS can provide a benefit at the group level, exploitative individuals can avoid the cost of producing the QS signal or of performing the cooperative behaviour that is coordinated by QS, and can therefore spread. We also show that a solution to the problem of exploitation is kin selection, if interacting bacterial cells tend to be close relatives. These results show that the problem of exploitation, which has been the focus of considerable attention in animal communication, also arises in bacteria.

NATURE| Vol 450| 15 November 2007

• Coevolution with viruses drives the evolution of bacterial mutation rates

Csaba Pal1,2, Marı´a D. Macia´3, Antonio Oliver3, Ira Schachar1 & Angus Buckling1

Bacteria with greatly elevated mutation rates (mutators) are frequently found in natural1–3 and laboratory4,5 populations, and are often associated with clinical infections6,7. Although mutators may increase adaptability to novel environmental conditions, they are also prone to the accumulation of deleterious mutations. The longterm maintenance of high bacterial mutation rates is therefore likely to be driven by rapidly changing selection pressures8–14, in addition to the possible slow transition rate by point mutation from mutators to non-mutators15. One of the most likely causes of rapidly changing selection pressures is antagonistic coevolution with parasites16,17. Here we show whether coevolution with viral parasites could drive the evolution of bacterial mutation rates in laboratory populations of the bacterium Pseudomonas fluorescens 18. After fewer than 200 bacterial generations, 25% of the populations coevolving with phages had evolved 10- to 100-fold increases in mutation rates owing to mutations in mismatchrepair genes; no populations evolving in the absence of phages showed any significant change in mutation rate. Furthermore, mutator populations had a higher probability of driving their phage populations extinct, strongly suggesting that mutators have an advantage against phages in the coevolutionary arms race. Given their ubiquity, bacteriophages may play an important role in the evolution of bacterial mutation rates.

Vol 450| 13 December 2007| doi:10.1038/nature06350

• Isolation of rare circulating tumour cells in cancer patients by microchip technology

Sunitha Nagrath1*, Lecia V. Sequist2*, Shyamala Maheswaran2, Daphne W. Bell2{, Daniel Irimia1, Lindsey Ulkus2, Matthew R. Smith2, Eunice L. Kwak2, Subba Digumarthy2, Alona Muzikansky2, Paula Ryan2, Ulysses J. Balis1{, Ronald G. Tompkins1, Daniel A. Haber2 & Mehmet Toner1

Viable tumour-derived epithelial cells (circulating tumour cells or CTCs) have been identified in peripheral blood fromcancer patients and are probably the origin of intractable metastatic disease1–4. Although extremely rare, CTCs represent a potential alternative to invasive biopsies as a source of tumour tissue for the detection, characterization and monitoring of non-haematologic cancers5–8. The ability to identify, isolate, propagate and molecularly characterize CTC subpopulations could further the discovery of cancer stem cell biomarkers and expand the understanding of the biology of metastasis.Current strategies for isolatingCTCs are limited to complex analytic approaches that generate very low yield and purity9. Here we describe the development of a unique microfluidic platform (the ‘CTC-chip’) capable of efficient and selective separation of viable CTCs from peripheral whole blood samples, mediated by the interaction of target CTCs with antibody (EpCAM)-coated microposts under precisely controlled laminar flow conditions, and without requisite pre-labelling or processing of samples. The CTC-chip successfully identified CTCs in the peripheral blood of patients with metastatic lung, prostate, pancreatic, breast and colon cancer in 115 of 116 (99%) samples, with a range of 5–1,281CTCs per ml and approximately 50% purity. In addition, CTCs were isolated in 7/7 patients with early-stage prostate cancer. Given the high sensitivity and specificity of the CTC-chip, we tested its potential utility in monitoring response to anti-cancer therapy. In a small cohort of patients with metastatic cancer undergoing systemic treatment, temporal changes in CTC numbers correlated reasonably well with the clinical course of disease as measured by standard radiographic methods. Thus, the CTC-chip provides a new and effective tool for accurate identification and measurement of CTCs in patients with cancer. It has broad implications in advancing both cancer biology research and clinical cancer management, including the detection, diagnosis and monitoring of cancer10.

Vol 450|20/27 December 2007| doi:10.1038/nature06385

• CLOCK-mediated acetylation of BMAL1 controls circadian function

Jun Hirayama1, Saurabh Sahar1, Benedetto Grimaldi1, Teruya Tamaru2, Ken Takamatsu2, Yasukazu Nakahata1 & Paolo Sassone-Corsi1

Regulation of circadian physiology relies on the interplay of interconnected transcriptional–translational feedback loops1,2. The CLOCK–BMAL1 complex activates clock-controlled genes, including cryptochromes (Crys), the products of which act as repressors by interacting directly with CLOCK–BMAL13,4. We have demonstrated that CLOCK possesses intrinsic histone acetyltransferase activity and that this enzymatic function contributes to chromatin-remodelling events implicated in circadian control of gene expression5. Here we show that CLOCK also acetylates a non-histone substrate: its own partner, BMAL1, is specifically acetylated on a unique, highly conserved Lys 537 residue. BMAL1 undergoes rhythmic acetylation in mouse liver, with a timing that parallels the downregulation of circadian transcription of clock-controlled genes. BMAL1 acetylation facilitates recruitment of CRY1 to CLOCK–BMAL1, thereby promoting transcriptional repression. Importantly, ectopic expression of a K537R-mutated BMAL1 is not able to rescue circadian rhythmicity in a cellular model of peripheral clock. These findings reveal that the enzymatic interplay between two clock core components6,7 is crucial for the circadian machinery.

Vol 450| 13 December 2007| doi:10.1038/nature06394

• Stochastic gene expression out-of-steady-state in the cyanobacterial circadian clock

Jeffrey R. Chabot1{, Juan M. Pedraza1, Prashant Luitel1 & Alexander van Oudenaarden1

Recent advances in measuring gene expression at the single-cell level have highlighted the stochastic nature of messenger RNA and protein synthesis1–3. Stochastic gene expression creates a source of variability in the abundance of cellular components, even among isogenic cells exposed to an identical environment. Recent integrated experimental and modelling studies4–13 have shed light on the molecular sources of this variability. However, many of these studies focus on systems that have reached a steady state and therefore do not address a large class of dynamic phenomena including oscillatory gene expression. Here we develop a general protocol for analysing and predicting stochastic gene expression in systems that never reach steady states. Weuse this framework to analyse experimentally stochastic expression of genes driven by the Synechococcus elongatus circadian clock. We find that, although the average expression at two points in the circadian cycle separated by 12 hours is identical, the variability at these two time points can be different. We show that this is a general feature of out-of-steady-state systems. We demonstrate how intrinsic noise sources, owing to random births and deaths of mRNAs and proteins, or extrinsic noise sources, which introduce fluctuations in rate constants, affect the cell-to-cell variability. To distinguish experimentally between these sources, we measured how the correlation between expression fluctuations of two identical genes is modulated during the circadian cycle. This quantitative framework is generally applicable to any out-ofsteady- state system and will be necessary for understanding the fidelity of dynamic cellular systems.

Vol 450|20/27 December 2007| doi:10.1038/nature06395

• RNA-mediated epigenetic programming of a genome-rearrangement pathway

Mariusz Nowacki1, Vikram Vijayan2, Yi Zhou1, Klaas Schotanus1, Thomas G. Doak1 & Laura F. Landweber1

Genome-wideDNArearrangements occur in many eukaryotes but are most exaggerated in ciliates, making them ideal model systems for epigenetic phenomena. During development of the somatic macronucleus, Oxytricha trifallax destroys 95% of its germ line, severely fragmenting its chromosomes, and then unscrambles hundreds of thousands of remaining fragments by permutation or inversion. Here we demonstrate that DNA or RNA templates can orchestrate these genome rearrangements in Oxytricha, supporting an epigenetic model for sequence-dependent comparison between germline and somatic genomes. A complete RNA cache of the maternal somatic genome may be available at a specific stage during development to provide a template for correct and precise DNA rearrangement. We show the existence of maternal RNA templates that could guide DNA assembly, and that disruption of specific RNA molecules disables rearrangement of the corresponding gene. Injection of artificial templates reprogrammes the DNA rearrangement pathway, suggesting that RNA molecules guide genome rearrangement.

Vol 451| 10 January 2008| doi:10.1038/nature06452

• The coevolution of choosiness and cooperation

John M. McNamara1, Zoltan Barta2, Lutz Fromhage3 & Alasdair I. Houston3

Explaining the rise and maintenance of cooperation is central to our understanding of biological systems1,2 and human societies3,4. When an individual’s cooperativeness is used by other individuals as a choice criterion, there can be competition to be more generous than others, a situation called competitive altruism5. The evolution of cooperation between non-relatives can then be driven by a positive feedback between increasing levels of cooperativeness and choosiness6. Here we use evolutionary simulations to show that, in a situation where individuals have the opportunity to engage in repeated pairwise interactions, the equilibrium degree of cooperativeness depends critically on the amount of behavioural variation that is being maintained in the population by processes such as mutation. Because our model does not invoke complex mechanisms such as negotiation behaviour, it can be applied to a wide range of species. The results suggest an important role of lifespan in the evolution of cooperation.

Vol 451| 10 January 2008| doi:10.1038/nature06455

• Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA

Wenqiang Yu1, David Gius2, Patrick Onyango1, Kristi Muldoon-Jacobs2, Judith Karp3, Andrew P. Feinberg1* & Hengmi Cui1*

Tumour suppressor genes (TSGs) inhibiting normal cellular growth are frequently silenced epigenetically in cancer1. DNA methylation is commonly associated with TSG silencing1, yet mutations in the DNA methylation initiation and recognition machinery in carcinogenesis are unknown2. An intriguing possible mechanism for gene regulation involves widespread non-coding RNAs such as microRNA, Piwi-interacting RNA and antisense RNAs3–5. Widespread sense–antisense transcripts have been systematically identified in mammalian cells6, and global transcriptome analysis shows that up to 70% of transcripts have antisense partners and that perturbation of antisense RNA can alter the expression of the sense gene7. For example, it has been shown that an antisense transcript not naturally occurring but induced by genetic mutation leads to gene silencing and DNA methylation, causing thalassaemia in a patient8. Here we show that many TSGs have nearby antisense RNAs, and we focus on the role of one RNA in silencing p15, a cyclin-dependent kinase inhibitor implicated in leukaemia. We found an inverse relation between p15 antisense (p15AS) and p15 sense expression in leukaemia. A p15AS expression construct induced p15 silencing in cis and in trans through heterochromatin formation but not DNA methylation; the silencing persisted after p15AS was turned off, although methylation and heterochromatin inhibitors reversed this process. The p15ASinduced silencing was Dicer-independent. Expression of exogenous p15AS in mouse embryonic stem cells caused p15 silencing and increased growth, through heterochromatin formation, as well as DNA methylation after differentiation of the embryonic stem cells. Thus, natural antisense RNA may be a trigger for heterochromatin formation

Vol 451| 10 January 2008| doi:10.1038/nature06468

• The bacterial enzyme RppH triggers messenger RNA degradation by 59 pyrophosphate removal

Atilio Deana1, Helena Celesnik1 & Joel G. Belasco1

The long-standing assumption that messenger RNA (mRNA) degradation in Escherichia coli begins with endonucleolytic cleavage has been challenged by the recent discovery that RNA decay can be triggered by a prior non-nucleolytic event that marks transcripts for rapid turnover: the rate-determining conversion of the 59 terminus from a triphosphate to a monophosphate1. This modification creates better substrates for the endonuclease RNase E, whose cleavage activity at internal sites is greatly enhanced when the RNA 59 end is monophosphorylated2,3. Moreover, it suggests an explanation for the influence of 59 termini on the endonucleolytic cleavage of primary transcripts, which are triphosphorylated4– 8. However, no enzyme capable of removing pyrophosphate fromRNA59 ends has been identified in any bacterial species. Here we show that the E. coli protein RppH (formerly NudH/YgdP) is the RNA pyrophosphohydrolase that initiates mRNA decay by this 59-end-dependent pathway. In vitro, RppH efficiently removes pyrophosphate from the 59 end of triphosphorylated RNA, irrespective of the identity of the 59-terminal nucleotide. In vivo, it accelerates the degradation of hundreds of E. coli transcripts by converting their triphosphorylated 59 ends to a more labile monophosphorylated state that can stimulate subsequent ribonuclease cleavage. That the action of the pyrophosphohydrolase is impeded when the 59 end is structurally sequestered by a stem-loop helps to explain the stabilizing influence of 59-terminal base pairing on mRNA lifetimes. Together, these findings suggest a possible basis for the effect of RppH and its orthologues on the invasiveness of bacterial pathogens. Interestingly, this master regulator of 59-enddependent mRNA degradation in E. coli not only catalyses a process functionally reminiscent of eukaryotic mRNA decapping but also bears an evolutionary relationship to the eukaryotic decapping enzyme Dcp2.

Vol 451| 17 January 2008| doi:10.1038/nature06475

• Facultative cheater mutants reveal the genetic complexity of cooperation in social amoebae

Lorenzo A. Santorelli1, Christopher R. L. Thompson2{, Elizabeth Villegas2, Jessica Svetz2, Christopher Dinh3, Anup Parikh2,4, Richard Sucgang3, Adam Kuspa1,2,3, Joan E. Strassmann1, David C. Queller1 & Gad Shaulsky1,2,4

Cooperation is central to many major transitions in evolution, including the emergence of eukaryotic cells, multicellularity and eusociality1. Cooperation can be destroyed by the spread of cheater mutants that do not cooperate but gain the benefits of cooperation from others1,2. However, cooperation can be preserved if cheaters are facultative, cheating others but cooperating among themselves2. Several cheater mutants have been studied before, but no study has attempted a genome-scale investigation of the genetic opportunities for cheating. Here we describe such a screen in a social amoeba and show that cheating is multifaceted by revealing cheater mutations in well over 100 genes of diverse types. Many of these mutants cheat facultatively, producing more than their fair share of spores in chimaeras, but cooperating normally when clonal. These findings indicate that phenotypically stable cooperative systems may nevertheless harbour genetic conflicts. The opportunities for evolutionary moves and countermoves in such conflicts may select for the involvement of multiple pathways and numerous genes.

Vol 451|28 February 2008| doi:10.1038/nature06558

Nature Methods

• A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli

Chien-Sheng Chen1,2,4, Ekaterina Korobkova3,4, Hao Chen3, Jian Zhu1, Xing Jian3, Sheng-Ce Tao1, Chuan He3 & Heng Zhu1

Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.

NATURE METHODS | VOL.5 NO.1 | JANUARY 2008 | 69

• Highly inclined thin illumination enables clear single-molecule imaging in cells

Makio Tokunaga1–3, Naoko Imamoto4,5 & Kumiko Sakata-Sogawa2

We describe a simple illumination method of fluorescence microscopy for molecular imaging. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. A high ratio yielded clear single-molecule images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify molecular dynamics, interactions and kinetics in cells for molecular systems biology.

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 159

• An activity-independent selection system of thermostable protein variants

He´le`ne Chautard1, Emilio Blas-Galindo2, Thierry Menguy1, Laure Grand’Moursel1, Felipe Cava2, Jose´ Berenguer2 & Marc Delcourt1

We describe an activity-independent method for the selection of thermostable mutants of any protein. It is based on a fusion construct comprising the protein of interest and a thermostable antibiotic resistance reporter, in such a way that thermostable mutants provide increased resistance in a thermophile. We isolated thermostable mutants of three human interferons and of two enzymes to demonstrate the applicability of the system.

NATURE METHODS | VOL.4 NO.11 | NOVEMBER 2007 | 919

• In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit

Jose´ Luis Royo1,2,4, Pablo Daniel Becker2,4, Eva Marı´a Camacho1, Angel Cebolla3, Claudia Link2, Eduardo Santero1 & Carlos Alberto Guzma´n2

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20–150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine–converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.

NATURE METHODS | VOL.4 NO.11 | NOVEMBER 2007 | 937

• Gene synthesis by circular assembly amplification

Duhee Bang & George M Church

Here we report the development of a gene-synthesis technology, circular assembly amplification. In this approach, we first constructed exonuclease-resistant circular DNA via simultaneous ligation of oligonucleotides. Exonuclease- and subsequent mismatch cleaving endonuclease–mediated degradation of the resulting ligation mixture eliminated error-rich products, thereby substantially improving gene-synthesis quality. We used this method to construct genes encoding a small thermostable DNA polymerase, a highly repetitive DNA sequence and large (44 kb) constructs.

NATURE METHODS | VOL.5 NO.1 | JANUARY 2008 | 37

• Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies

Xiaodong Zhang1,7, Chunguang Guo2,7, Yueting Chen1,7, Hennady P Shulha3, Michael P Schnetz1, Thomas LaFramboise1, Cynthia F Bartels1, Sanford Markowitz4, Zhiping Weng3,5, Peter C Scacheri1 & Zhenghe Wang1,6

We developed a strategy to introduce epitope tag–encoding DNA into endogenous loci by homologous recombination–mediated ‘knock-in’. The tagging method is straightforward, can be applied to many loci and several human somatic cell lines, and can facilitate many functional analyses including western blot, immunoprecipitation, immunofluorescence and chromatin immunoprecipitation–microarray (ChIP-chip). The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available.

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 163

• Plasmid-chromosome shuffling for non-deletion alleles in yeast

Zhiwei Huang1,4, Richard S Sucgang1,4, Yu-yi Lin3, Xiaomin Shi1, Jef D Boeke3 & Xuewen Pan1,2

Here we describe a facile plasmid-chromosome shuffling technique for generating and analyzing non-deletion alleles in the yeast Saccharomyces cerevisiae. This technique takes advantage of an existing set of genome-wide haploid-convertible heterozygous diploid yeast knockout mutants. This simple method will facilitate characterization of essential gene functions and genome-wide investigation of protein structure-function relationships.

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 167

• High-density mapping of single-molecule trajectories with photoactivated localization microscopy

Suliana Manley1, Jennifer M Gillette1, George H Patterson1, Hari Shroff2, Harald F Hess2, Eric Betzig2 & Jennifer Lippincott-Schwartz1

We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 155

• Printing protein arrays from DNA arrays

Mingyue He1,4, Oda Stoevesandt1,3,4, Elizabeth A Palmer1,3, Farid Khan1,3, Olle Ericsson2 & Michael J Taussig1,3

We describe a method, DNA array to protein array (DAPA), which allows the ‘printing’ of replicate protein arrays directly from a DNA array template using cell-free protein synthesis. At least 20 copies of a protein array can be obtained from a single DNA array. DAPA eliminates the need for separate protein expression, purification and spotting, and also overcomes the problem of long-term functional storage of surface-bound proteins.

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 175

• A systematic library for comprehensive overexpression screens in Saccharomyces cerevisiae

Grace Marie Jones1, Jim Stalker2, Sean Humphray2, Anthony West2, Tony Cox2, Jane Rogers2, Ian Dunham2 & Gregory Prelich1

Modern genetic analysis requires the development of new resources to systematically explore gene function in vivo. Overexpression screens are a powerful method to investigate genetic pathways, but the goal of routine and comprehensive overexpression screens has been hampered by the lack of systematic libraries. Here we describe the construction of a systematic collection of the Saccharomyces cerevisiae genome in a high-copy vector and its validation in two overexpression screens.

NATURE METHODS | VOL.5 NO.3 | MARCH 2008 | 239

• A systematic library for comprehensive overexpression screens in Saccharomyces cerevisiaeHomogeneous reporter system enables

quantitative functional assessment of multiple transcription factors

Sergei Romanov1, Alexander Medvedev1, Maria Gambarian1, Natalia Poltoratskaya1, Matt Moeser1, Liubov Medvedeva1, Mikhail Gambarian2, Luda Diatchenko3 & Sergei Makarov1

We developed a high-content reporter system that allows quantitative assessment of activities of multiple transcription factors (TFs) in a eukaryotic cell. The system comprises a library of reporter constructs that are evaluated according to their transcription rates. All reporters produce essentially identical messages that are subjected to ‘processing’, which generates a spectrum of distinguishable fragments that are analyzed quantitatively. The homogeneity of the reporter library afforded inherently uniform detection conditions for all reporters and provided repeatability, accuracy and robustness of assessment. We showed that this technology can be used to identify pathways transmitting cell responses to inducers, and that the profile of TF activities generated using this system represents a stable and sustained cell signature that clearly distinguishes different cell types and pathological conditions. This technology provides a framework for functional characterization of signal transduction networks through profiling activities of multiple TFs.

NATURE METHODS | VOL.5 NO.3 | MARCH 2008 | 253

• MicroRNA quantitation from a single cell by PCR using SYBR® Green detection and LNA-based primers

We describe a new, highly sensitive and specific PCR approach for quantitation of microRNAs (miRNAs): the miRCURY™ LNA microRNA PCR system. The method, which allows detection of 10 copies of miRNA, is enabled by the use of Locked Nucleic Acids (LNA™). The LNA-conferred sensitivity facilitates accurate detection of miRNA expression in a single cell.

NATURE METHODS | FEBRUARY 2008 | iii ADVERTISING FEATURE/EXIQON