Picture of Jade Wang

Jade Wang

Associate Professor of Bacteriology

Address: 1550 Linden Drive, Room 6478
Phone: (608) 263-0307
Lab Phone: (608) 263-1072
Email: wang@bact.wisc.edu
Overview · Lab · Publications · Lab Website
J. Bacteriol., Mol. Microbiol., Mol. Cell, Cell, Phys Rev B Condens Matter., Nat. Rev. Microbiol., PLoS Genet., Curr. Opin. Microbiol., Proc. Natl. Acad. Sci. U.S.A., Nat Struct Biol. , Structure, Proc Natl Acad Sci U S A., Phys Rev Lett., MBio, Curr. Biol., Nature,

    Research Papers

  • Sankar TS, Wastuwidyaningtyas BD, Dong Y, Lewis SA, Wang JD (2016) The nature of mutations induced by replication–transcription collisions. Nature 535(7610):178-81 (PMC4945378) View Abstract · Pubmed Record
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    The DNA replication and transcription machineries share a common DNA template and thus can collide with each other co-directionally or head-on. Replication–transcription collisions can cause replication fork arrest, premature transcription termination, DNA breaks, and recombination intermediates threatening genome integrity. Collisions may also trigger mutations, which are major contributors to genetic disease and evolution. However, the nature and mechanisms of collision-induced mutagenesis remain poorly understood. Here we reveal the genetic consequences of replication–transcription collisions in actively dividing bacteria to be two classes of mutations: duplications/deletions and base substitutions in promoters. Both signatures are highly deleterious but are distinct from the previously well-characterized base substitutions in the coding sequence. Duplications/deletions are probably caused by replication stalling events that are triggered by collisions; their distribution patterns are consistent with where the fork first encounters a transcription complex upon entering a transcription unit. Promoter substitutions result mostly from head-on collisions and frequently occur at a nucleotide that is conserved in promoters recognized by the major σ factor in bacteria. This substitution is generated via adenine deamination on the template strand in the promoter open complex, as a consequence of head-on replication perturbing transcription initiation. We conclude that replication–transcription collisions induce distinct mutation signatures by antagonizing replication and transcription, not only in coding sequences but also in gene regulatory elements.

  • Li W, Bouveret E, Zhang Y, Liu K, Wang JD, Weisshaar JC (2016) Effects of amino acid starvation on RelA diffusive behavior in live Escherichia coli. Mol. Microbiol. 99(3):571-85 (PMC4962531) View Abstract · Pubmed Record
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    During amino acid starvation, bacterial cells rapidly synthesize the nucleotides (p)ppGpp, causing a massive re-programming of the transcriptional profile known as the stringent response. The (p)ppGpp synthase RelA is activated by ribosomes harboring an uncharged tRNA at the A site. It is unclear whether synthesis occurs while RelA is bound to the ribosome or free in the cytoplasm. We present a study of three Escherichia coli strains, each expressing a different RelA-fluorescent protein (RelA-FP) construct: RelA-YFP, RelA-mEos2 and RelA-Dendra2. Single-molecule localization and tracking studies were carried out under normal growth conditions and during amino acid starvation. Study of three labeling schemes enabled us to assess potential problems with FP labeling of RelA. The diffusive trajectories and axial spatial distributions indicate that amino acid starvation induces net binding of all three RelA-FP constructs to 70S ribosomes. The data are most consistent with a model in which RelA synthesizes (p)ppGpp while bound to the 70S ribosome. We suggest a 'short hopping time' model of RelA activity during starvation. Our results contradict an earlier study of RelA-Dendra2 diffusion that inferred off-ribosome synthesis of (p)ppGpp. The reasons for the discrepancy remain unclear.

  • Gaca AO, Kudrin P, Colomer-Winter C, Beljantseva J, Liu K, Anderson B, Wang JD, Rejman D, Potrykus K, Cashel M, Hauryliuk V, Lemos JA (2015) From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis. J. Bacteriol. 197(18):2908-19 (PMC4542164) View Abstract · Pubmed Record
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    The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQEf), we found that, in addition to (p)ppGpp, RelQEf is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

  • Liu K, Bittner AN, Wang JD (2015) Diversity in (p)ppGpp metabolism and effectors. Curr. Opin. Microbiol. 24:72-9 (PMC4380541) View Abstract · Pubmed Record
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    Bacteria produce guanosine tetraphosphate and pentaphosphate, collectively named (p)ppGpp, in response to a variety of environmental stimuli. These two remarkable molecules regulate many cellular processes, including the central dogma processes and metabolism, to ensure survival and adaptation. Work in Escherichia coli laid the foundation for understanding the molecular details of (p)ppGpp and its cellular functions. As recent studies expand to other species, it is apparent that there exists considerable variation, with respect to not only (p)ppGpp metabolism, but also to its mechanism of action. From an evolutionary standpoint, this diversification is an elegant example of how different species adapt a particular regulatory network to their diverse lifestyles.

  • Liu K, Myers AR, Pisithkul T, Claas KR, Satyshur KA, Amador-Noguez D, Keck JL, Wang JD (2015) Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp. Mol. Cell 57(4):735-49 (PMC4336630) View Abstract · Pubmed Record
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    The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. Here, we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK), revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A survey of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria, where (p)ppGpp regulates RNA polymerase (RNAP). We propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria.

  • Arjes HA, Kriel A, Sorto NA, Shaw JT, Wang JD, Levin PA (2014) Failsafe mechanisms couple division and DNA replication in bacteria. Curr. Biol. 24(18):2149-55 (PMC4175050) View Abstract · Pubmed Record
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    The past 20 years have seen tremendous advances in our understanding of the mechanisms underlying bacterial cytokinesis, particularly the composition of the division machinery and the factors controlling its assembly [1]. At the same time, we understand very little about the relationship between cell division and other cell-cycle events in bacteria. Here we report that inhibiting division in Bacillus subtilis and Staphylococcus aureus quickly leads to an arrest in the initiation of new rounds of DNA replication, followed by a complete arrest in cell growth. Arrested cells are metabolically active but are unable to initiate new rounds of either DNA replication or division when shifted to permissive conditions. Inhibiting DNA replication results in entry into a similar quiescent state in which cells are unable to resume growth or division when returned to permissive conditions. Our data suggest the presence of two failsafe mechanisms: one linking division to the initiation of DNA replication and another linking the initiation of DNA replication to division. These findings contradict the prevailing view of the bacterial cell cycle as a series of coordinated but uncoupled events. Importantly, the terminal nature of the cell-cycle arrest validates the bacterial cell-cycle machinery as an effective target for antimicrobial development.

  • Bittner AN, Kriel A, Wang JD (2014) Lowering GTP level increases survival of amino acid starvation but slows growth rate for Bacillus subtilis cells lacking (p)ppGpp. J. Bacteriol. 196(11):2067-76 (PMC4010990) View Abstract · Pubmed Record
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    Bacterial cells sense external nutrient availability to regulate macromolecular synthesis and consequently their growth. In the Gram-positive bacterium Bacillus subtilis, the starvation-inducible nucleotide (p)ppGpp negatively regulates GTP levels, both to resist nutritional stress and to maintain GTP homeostasis during growth. Here, we quantitatively investigated the relationship between GTP level, survival of amino acid starvation, and growth rate when GTP synthesis is uncoupled from its major homeostatic regulator, (p)ppGpp. We analyzed growth and nucleotide levels in cells that lack (p)ppGpp and found that their survival of treatment with a nonfunctional amino acid analog negatively correlates with both growth rate and GTP level. Manipulation of GTP levels modulates the exponential growth rate of these cells in a positive dose-dependent manner, such that increasing the GTP level increases growth rate. However, accumulation of GTP levels above a threshold inhibits growth, suggesting a toxic effect. Strikingly, adenine counteracts GTP stress by preventing GTP accumulation in cells lacking (p)ppGpp. Our results emphasize the importance of maintaining appropriate levels of GTP to maximize growth: cells can survive amino acid starvation by decreasing GTP level, which comes at a cost to growth, while (p)ppGpp enables rapid adjustment to nutritional stress by adjusting GTP level, thus maximizing fitness.

  • Zhang Y, Mooney RA, Grass JA, Sivaramakrishnan P, Herman C, Landick R, Wang JD (2014) DksA guards elongating RNA polymerase against ribosome-stalling-induced arrest. Mol. Cell 53(5):766-78 (PMC4023959) View Abstract · Pubmed Record
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    In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo.

  • Maduike NZ, Tehranchi AK, Wang JD, Kreuzer KN (2014) Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics. Mol. Microbiol. 91(1):39-56 (PMC3926323) View Abstract · Pubmed Record
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    DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons.

  • Kriel A, Brinsmade SR, Tse JL, Tehranchi AK, Bittner AN, Sonenshein AL, Wang JD (2014) GTP dysregulation in Bacillus subtilis cells lacking (p)ppGpp results in phenotypic amino acid auxotrophy and failure to adapt to nutrient downshift and regulate biosynthesis genes. J. Bacteriol. 196(1):189-201 (PMC3911124) View Abstract · Pubmed Record
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    The nucleotide (p)ppGpp inhibits GTP biosynthesis in the Gram-positive bacterium Bacillus subtilis. Here we examined how this regulation allows cells to grow in the absence of amino acids. We showed that B. subtilis cells lacking (p)ppGpp, due to either deletions or point mutations in all three (p)ppGpp synthetase genes, yjbM, ywaC, and relA, strongly require supplementation of leucine, isoleucine, valine, methionine, and threonine and modestly require three additional amino acids. This polyauxotrophy is rescued by reducing GTP levels. Reduction of GTP levels activates transcription of genes responsible for the biosynthesis of the five strongly required amino acids by inactivating the transcription factor CodY, which represses the ybgE, ilvD, ilvBHC-leuABCD, ilvA, ywaA, and hom-thrCB operons, and by a CodY-independent activation of transcription of the ilvA, ywaA, hom-thrCB, and metE operons. Interestingly, providing the eight required amino acids does not allow for colony formation of (p)ppGpp(0) cells when transitioning from amino acid-replete medium to amino acid-limiting medium, and we found that this is due to an additional role that (p)ppGpp plays in protecting cells during nutrient downshifts. We conclude that (p)ppGpp allows adaptation to amino acid limitation by a combined effect of preventing death during metabolic transitions and sustaining growth by activating amino acid biosynthesis. This ability of (p)ppGpp to integrate a general stress response with a targeted reprogramming of gene regulation allows appropriate adaptation and is likely conserved among diverse bacteria.

  • Gaca AO, Kajfasz JK, Miller JH, Liu K, Wang JD, Abranches J, Lemos JA (2013) Basal levels of (p)ppGpp in Enterococcus faecalis: the magic beyond the stringent response. MBio 4(5):e00646-13 (PMC3781836) View Abstract · Pubmed Record
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    The stringent response (SR), mediated by the alarmone (p)ppGpp, is a conserved bacterial adaptation system controlling broad metabolic alterations necessary for survival under adverse conditions. In Enterococcus faecalis, production of (p)ppGpp is controlled by the bifunctional protein RSH (for "Rel SpoT homologue"; also known as RelA) and by the monofunctional synthetase RelQ. Previous characterization of E. faecalis strains lacking rsh, relQ, or both revealed that RSH is responsible for activation of the SR and that alterations in (p)ppGpp production negatively impact bacterial stress survival and virulence. Despite its well-characterized role as the effector of the SR, the significance of (p)ppGpp during balanced growth remains poorly understood. Microarrays of E. faecalis strains producing different basal amounts of (p)ppGpp identified several genes and pathways regulated by modest changes in (p)ppGpp. Notably, expression of numerous genes involved in energy generation were induced in the rsh relQ [(p)ppGpp(0)] strain, suggesting that a lack of basal (p)ppGpp places the cell in a "transcriptionally relaxed" state. Alterations in the fermentation profile and increased production of H2O2 in the (p)ppGpp(0) strain substantiate the observed transcriptional changes. We confirm that, similar to what is seen in Bacillus subtilis, (p)ppGpp directly inhibits the activity of enzymes involved in GTP biosynthesis, and complete loss of (p)ppGpp leads to dysregulation of GTP homeostasis. Finally, we show that the association of (p)ppGpp with antibiotic survival does not relate to the SR but rather relates to basal (p)ppGpp pools. Collectively, this study highlights the critical but still underappreciated role of basal (p)ppGpp pools under balanced growth conditions. Drug-resistant bacterial infections continue to pose a significant public health threat by limiting therapeutic options available to care providers. The stringent response (SR), mediated by the accumulation of two modified guanine nucleotides collectively known as (p)ppGpp, is a highly conserved stress response that broadly remodels bacterial physiology to a survival state. Given the strong correlation of the SR with the ability of bacteria to survive antibiotic treatment and the direct association of (p)ppGpp production with bacterial infectivity, understanding how bacteria produce and utilize (p)ppGpp may reveal potential targets for the development of new antimicrobial therapies. Using the multidrug-resistant pathogen Enterococcus faecalis as a model, we show that small alterations to (p)ppGpp levels, well below concentrations needed to trigger the SR, severely affected bacterial metabolism and antibiotic survival. Our findings highlight the often-underappreciated contribution of basal (p)ppGpp levels to metabolic balance and stress tolerance in bacteria.

  • Denapoli J, Tehranchi AK, Wang JD (2013) Dose-dependent reduction of replication elongation rate by (p)ppGpp in Escherichia coli and Bacillus subtilis. Mol. Microbiol. 88(1):93-104 (PMC3640871) View Abstract · Pubmed Record
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    DNA replication is regulated in response to environmental constraints such as nutrient availability. While much is known about regulation of replication during initiation, little is known about regulation of replication during elongation. In the bacterium Bacillus subtilis, replication elongation is paused upon sudden amino acid starvation by the starvation-inducible nucleotide (p)ppGpp. However, in many bacteria including Escherichia coli, replication elongation is thought to be unregulated by nutritional availability. Here we reveal that the replication elongation rate in E. coli is modestly but significantly reduced upon strong amino acid starvation. This reduction requires (p)ppGpp and is exacerbated in a gppA mutant with increased pppGpp levels. Importantly, high levels of (p)ppGpp, independent of amino acid starvation, are sufficient to inhibit replication elongation even in the absence of transcription. Finally, in both E. coli and B. subtilis, (p)ppGpp inhibits replication elongation in a dose-dependent manner rather than via a switch-like mechanism, although this inhibition is much stronger in B. subtilis. This supports a model where replication elongation rates are regulated by (p)ppGpp to allow rapid and tunable response to multiple abrupt stresses in evolutionarily diverse bacteria.

  • Kriel A, Bittner AN, Kim SH, Liu K, Tehranchi AK, Zou WY, Rendon S, Chen R, Tu BP, Wang JD (2012) Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance. Mol. Cell 48(2):231-41 (PMC3483369) View Abstract · Pubmed Record
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    Cells constantly adjust their metabolism in response to environmental conditions, yet major mechanisms underlying survival remain poorly understood. We discover a posttranscriptional mechanism that integrates starvation response with GTP homeostasis to allow survival, enacted by the nucleotide (p)ppGpp, a key player in bacterial stress response and persistence. We reveal that (p)ppGpp activates global metabolic changes upon starvation, allowing survival by regulating GTP. Combining metabolomics with biochemical demonstrations, we find that (p)ppGpp directly inhibits the activities of multiple GTP biosynthesis enzymes. This inhibition results in robust and rapid GTP regulation in Bacillus subtilis, which we demonstrate is essential to maintaining GTP levels within a range that supports viability even in the absence of starvation. Correspondingly, without (p)ppGpp, gross GTP dysregulation occurs, revealing a vital housekeeping function of (p)ppGpp; in fact, loss of (p)ppGpp results in death from rising GTP, a severe and previously unknown consequence of GTP dysfunction.

  • Rymer RU, Solorio FA, Tehranchi AK, Chu C, Corn JE, Keck JL, Wang JD, Berger JM (2012) Binding mechanism of metal⋅NTP substrates and stringent-response alarmones to bacterial DnaG-type primases. Structure 20(9):1478-89 (PMC3438381) View Abstract · Pubmed Record
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    Primases are DNA-dependent RNA polymerases found in all cellular organisms. In bacteria, primer synthesis is carried out by DnaG, an essential enzyme that serves as a key component of DNA replication initiation, progression, and restart. How DnaG associates with nucleotide substrates and how certain naturally prevalent nucleotide analogs impair DnaG function are unknown. We have examined one of the earliest stages in primer synthesis and its control by solving crystal structures of the S. aureus DnaG catalytic core bound to metal ion cofactors and either individual nucleoside triphosphates or the nucleotidyl alarmones, pppGpp and ppGpp. These structures, together with both biochemical analyses and comparative studies of enzymes that use the same catalytic fold as DnaG, pinpoint the predominant nucleotide-binding site of DnaG and explain how the induction of the stringent response in bacteria interferes with primer synthesis.

  • Merrikh H, Zhang Y, Grossman AD, Wang JD (2012) Replication-transcription conflicts in bacteria. Nat. Rev. Microbiol. 10(7):449-58 (PMC3467967) View Abstract · Pubmed Record
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    DNA replication and transcription use the same template and occur concurrently in bacteria. The lack of temporal and spatial separation of these two processes leads to their conflict, and failure to deal with this conflict can result in genome alterations and reduced fitness. In recent years major advances have been made in understanding how cells avoid conflicts between replication and transcription and how such conflicts are resolved when they do occur. In this Review, we summarize these findings, which shed light on the significance of the problem and on how bacterial cells deal with unwanted encounters between the replication and transcription machineries.

  • Tehranchi AK, Blankschien MD, Zhang Y, Halliday JA, Srivatsan A, Peng J, Herman C, Wang JD (2010) The transcription factor DksA prevents conflicts between DNA replication and transcription machinery. Cell 141(4):595-605 (PMC2919171) View Abstract · Pubmed Record
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    Actively dividing cells perform robust and accurate DNA replication during fluctuating nutrient availability, yet factors that prevent disruption of replication remain largely unknown. Here we report that DksA, a nutrient-responsive transcription factor, ensures replication completion in Escherichia coli by removing transcription roadblocks. In the absence of DksA, replication is rapidly arrested upon amino acid starvation. This arrest requires active transcription and is alleviated by RNA polymerase mutants that compensate for DksA activity. This replication arrest occurs independently of exogenous DNA damage, yet it induces the DNA-damage response and recruits the main recombination protein RecA. This function of DksA is independent of its transcription initiation activity but requires its less-studied transcription elongation activity. Finally, GreA/B elongation factors also prevent replication arrest during nutrient stress. We conclude that transcription elongation factors alleviate fundamental conflicts between replication and transcription, thereby protecting replication fork progression and DNA integrity.

  • Srivatsan A, Tehranchi A, MacAlpine DM, Wang JD (2010) Co-orientation of replication and transcription preserves genome integrity. PLoS Genet. 6(1):e1000810 (PMC2797598) View Abstract · Pubmed Record
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    In many bacteria, there is a genome-wide bias towards co-orientation of replication and transcription, with essential and/or highly-expressed genes further enriched co-directionally. We previously found that reversing this bias in the bacterium Bacillus subtilis slows replication elongation, and we proposed that this effect contributes to the evolutionary pressure selecting the transcription-replication co-orientation bias. This selection might have been based purely on selection for speedy replication; alternatively, the slowed replication might actually represent an average of individual replication-disruption events, each of which is counter-selected independently because genome integrity is selected. To differentiate these possibilities and define the precise forces driving this aspect of genome organization, we generated new strains with inversions either over approximately 1/4 of the chromosome or at ribosomal RNA (rRNA) operons. Applying mathematical analysis to genomic microarray snapshots, we found that replication rates vary dramatically within the inverted genome. Replication is moderately impeded throughout the inverted region, which results in a small but significant competitive disadvantage in minimal medium. Importantly, replication is strongly obstructed at inverted rRNA loci in rich medium. This obstruction results in disruption of DNA replication, activation of DNA damage responses, loss of genome integrity, and cell death. Our results strongly suggest that preservation of genome integrity drives the evolution of co-orientation of replication and transcription, a conserved feature of genome organization.

  • Wang JD, Levin PA (2009) Metabolism, cell growth and the bacterial cell cycle. Nat. Rev. Microbiol. 7(11):822-7 (PMC2887316) View Abstract · Pubmed Record
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    Adaptation to fluctuations in nutrient availability is a fact of life for single-celled organisms in the 'wild'. A decade ago our understanding of how bacteria adjust cell cycle parameters to accommodate changes in nutrient availability stemmed almost entirely from elegant physiological studies completed in the 1960s. In this Opinion article we summarize recent groundbreaking work in this area and discuss potential mechanisms by which nutrient availability and metabolic status are coordinated with cell growth, chromosome replication and cell division.

  • Srivatsan A, Han Y, Peng J, Tehranchi AK, Gibbs R, Wang JD, Chen R (2008) High-precision, whole-genome sequencing of laboratory strains facilitates genetic studies. PLoS Genet. 4(8):e1000139 (PMC2474695) View Abstract · Pubmed Record
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    Whole-genome sequencing is a powerful technique for obtaining the reference sequence information of multiple organisms. Its use can be dramatically expanded to rapidly identify genomic variations, which can be linked with phenotypes to obtain biological insights. We explored these potential applications using the emerging next-generation sequencing platform Solexa Genome Analyzer, and the well-characterized model bacterium Bacillus subtilis. Combining sequencing with experimental verification, we first improved the accuracy of the published sequence of the B. subtilis reference strain 168, then obtained sequences of multiple related laboratory strains and different isolates of each strain. This provides a framework for comparing the divergence between different laboratory strains and between their individual isolates. We also demonstrated the power of Solexa sequencing by using its results to predict a defect in the citrate signal transduction pathway of a common laboratory strain, which we verified experimentally. Finally, we examined the molecular nature of spontaneously generated mutations that suppress the growth defect caused by deletion of the stringent response mediator relA. Using whole-genome sequencing, we rapidly mapped these suppressor mutations to two small homologs of relA. Interestingly, stable suppressor strains had mutations in both genes, with each mutation alone partially relieving the relA growth defect. This supports an intriguing three-locus interaction module that is not easily identifiable through traditional suppressor mapping. We conclude that whole-genome sequencing can drastically accelerate the identification of suppressor mutations and complex genetic interactions, and it can be applied as a standard tool to investigate the genetic traits of model organisms.

  • Srivatsan A, Wang JD (2008) Control of bacterial transcription, translation and replication by (p)ppGpp. Curr. Opin. Microbiol. 11(2):100-5 View Abstract · Pubmed Record
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    The small nucleotides pppGpp and ppGpp (or (p)ppGpp) are rapidly synthesized in response to nutritional stress. In Escherichia coli, the enzymes RelA and SpoT are triggered by different starvation signals to produce (p)ppGpp. In many Gram-positive bacteria this is carried out by RelA and two small homologs. (p)ppGpp, along with the transcription factor DksA, has profound effects on transcription initiation in E. coli. (p)ppGpp/DksA exert differential effects on promoters by playing upon their intrinsic kinetic parameters, and by facilitating the utilization of alternative sigma factors. (p)ppGpp also regulates replication and translation. These studies highlight (p)ppGpp as a key factor in bacterial physiology that responds rapidly to diverse stresses, by shutting down growth and priming cellular defensive and adaptive processes.

  • Wang JD, Sanders GM, Grossman AD (2007) Nutritional control of elongation of DNA replication by (p)ppGpp. Cell 128(5):865-75 (PMC1850998) View Abstract · Pubmed Record
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    DNA replication is highly regulated in most organisms. Although much research has focused on mechanisms that regulate initiation of replication, mechanisms that regulate elongation of replication are less well understood. We characterized a mechanism that regulates replication elongation in the bacterium Bacillus subtilis. Replication elongation was inhibited within minutes after amino acid starvation, regardless of where the replication forks were located on the chromosome. We found that small nucleotides ppGpp and pppGpp, which are induced upon starvation, appeared to inhibit replication directly by inhibiting primase, an essential component of the replication machinery. The replication forks arrested with (p)ppGpp did not recruit the recombination protein RecA, indicating that the forks are not disrupted. (p)ppGpp appear to be part of a surveillance mechanism that links nutrient availability to replication by rapidly inhibiting replication in starved cells, thereby preventing replication-fork disruption. This control may be important for cells to maintain genomic integrity.

  • Wang JD, Berkmen MB, Grossman AD (2007) Genome-wide coorientation of replication and transcription reduces adverse effects on replication in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 104(13):5608-13 (PMC1838449) View Abstract · Pubmed Record
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    In many bacteria, there is a strong bias for genes to be encoded on the leading strand of DNA, resulting in coorientation of replication and transcription. In Bacillus subtilis, transcription of the majority of genes (75%) is cooriented with replication. By using genome-wide profiling of replication with DNA microarrays, we found that this coorientation bias reduces adverse effects of transcription on replication. We found that in wild-type cells, transcription did not appear to affect the rate of replication elongation. However, in mutants with reversed transcription bias for an extended region of the chromosome, replication elongation was slower. This reduced replication rate depended on transcription and was limited to the region in which the directions of replication and transcription are opposed. These results support the hypothesis that the strong bias to coorient transcription and replication is due to selective pressure for processive, efficient, and accurate replication.

  • Goranov AI, Kuester-Schoeck E, Wang JD, Grossman AD (2006) Characterization of the global transcriptional responses to different types of DNA damage and disruption of replication in Bacillus subtilis. J. Bacteriol. 188(15):5595-605 (PMC1540033) View Abstract · Pubmed Record
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    DNA damage and perturbations in DNA replication can induce global transcriptional responses that can help organisms repair the damage and survive. RecA is known to mediate transcriptional responses to DNA damage in several bacterial species by inactivating the repressor LexA and phage repressors. To gain insight into how Bacillus subtilis responds to various types of DNA damage, we measured the effects of DNA damage and perturbations in replication on mRNA levels by using DNA microarrays. We perturbed replication either directly with p-hydroxyphenylazo-uracil (HPUra), an inhibitor of DNA polymerase, or indirectly with the DNA-damaging reagents mitomycin C (MMC) and UV irradiation. Our results indicate that the transcriptional responses to HPUra, MMC, and UV are only partially overlapping. recA is the major transcriptional regulator under all of the tested conditions, and LexA appears to directly repress the expression of 63 genes in 26 operons, including the 18 operons previously identified as LexA targets. MMC and HPUra treatments caused induction of an integrative and conjugative element (ICEBs1) and resident prophages (PBSX and SPbeta), which affected the expression of many host genes. Consistent with previous results, the induction of these mobile elements required recA. Induction of the phage appeared to require inactivation of LexA. Unrepaired UV damage and treatment with MMC also affected the expression of some of the genes that are controlled by DnaA. Furthermore, MMC treatment caused an increase in origin-proximal gene dosage. Our results indicate that different types of DNA damage have different effects on replication and on the global transcriptional profile.

  • Wang JD, Rokop ME, Barker MM, Hanson NR, Grossman AD (2004) Multicopy plasmids affect replisome positioning in Bacillus subtilis. J. Bacteriol. 186(21):7084-90 (PMC523195) View Abstract · Pubmed Record
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    The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.

  • Wang JD, Herman C, Tipton KA, Gross CA, Weissman JS (2002) Directed evolution of substrate-optimized GroEL/S chaperonins. Cell 111(7):1027-39 View Abstract · Pubmed Record
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    GroEL/S chaperonin ring complexes fold many unrelated proteins. To understand the basis and extent of the chaperonin substrate spectrum, we used rounds of selection and DNA shuffling to obtain GroEL/S variants that dramatically enhanced folding of a single substrate-green fluorescent protein (GFP). Changes in the substrate-optimized chaperonins increase the polarity of the folding cavity and alter the ATPase cycle. These findings reveal a surprising plasticity of GroEL/S, which can be exploited to aid folding of recombinant proteins. Our studies also reveal a conflict between specialization and generalization of chaperonins as increased GFP folding comes at the expense of the ability of GroEL/S to fold its natural substrates. This conflict and the nature of the ring structure may help explain the evolution of cellular chaperone systems.

  • Wang JD, Weissman JS (1999) Thinking outside the box: new insights into the mechanism of GroEL-mediated protein folding. Nat Struct Biol. 6(7):597-600 View Abstract · Pubmed Record
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    No abstract available.

  • Wang JD, Michelitsch MD, Weissman JS (1998) GroEL-GroES-mediated protein folding requires an intact central cavity. Proc Natl Acad Sci U S A. 95(21):12163-8 (PMC22802) View Abstract · Pubmed Record
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    No abstract available.

  • Johnson BL, Sachrajda AS, Kirczenow G, Feng Y, Taylor RP, Henning L, Wang J, Zawadzki P, Coleridge PT (1995) Quantum Hall effect and inter-edge-state tunneling within a barrier. Phys Rev B Condens Matter. 51(12):7650-7654 View Abstract · Pubmed Record
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    No abstract available.

  • Sachrajda AS, Feng Y, Taylor RP, Kirczenow G, Henning L, Wang J, Zawadzki P, Coleridge PT (1994) Magnetoconductance of a nanoscale antidot. Phys Rev B Condens Matter. 50(15):10856-10863 View Abstract · Pubmed Record
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    No abstract available.

  • Kirczenow G, Sachrajda AS, Feng Y, Taylor RP, Henning L, Wang J, Zawadzki P, Coleridge PT (1994) Artificial impurities in quantum wires: From classical to quantum behavior. Phys Rev Lett. 72(13):2069-2072 View Abstract · Pubmed Record
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    No abstract available.