Bio

Academic Appointments


Honors & Awards


  • John Scott Award, John Scott Trust, administrated by City of Philadelphia (2009)
  • Fellow, American Academy of Microbiologists (2006)

Professional Education


  • PhD, Rice University, Physics (1967)
  • MA, Rice University, Physics (1965)
  • MS, U. Illinois (Urbana), Physics (1962)
  • BS, Texas A&M, Physics (1960)

Research & Scholarship

Current Research and Scholarly Interests


We are focused on understanding the nature of bacterial genetic regulation. We use a variety of tools: gene expression microarrays, advanced techniques of fluorescence and electron microscopy, and a broad range of computational tools for bioinformatics and modeling.

The aquatic bacterium, Caulobacter crescentus, is our primary model organism. The members of the McAdams group include Electrical Engineers, Physicists, and Physical Chemists who are interested in biologically focused research. We work closely with geneticists and biochemists who are graduate students and postdoctoral fellows in Lucy Shapiro's group to bring a multi-disciplinary approach to the analysis of bacterial regulation and control.

Our interests range far beyond "conventional" approaches to genetic regulation to include the effects of dynamic spatial and temporal positioning of regulatory proteins and the regulatory effects working through the dynamic three-dimensional organization of the chromosome in the dividing cell. We are exploring how regulatory molecules are dynamically localized to particular cell positions and how this localization is integrated into the control mechanisms of the cell.

Teaching

2013-14 Courses


Graduate and Fellowship Programs


Publications

Journal Articles


  • Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle. Proceedings of the National Academy of Sciences of the United States of America Kozdon, J. B., Melfi, M. D., Luong, K., Clark, T. A., Boitano, M., Wang, S., Zhou, B., Gonzalez, D., Collier, J., Turner, S. W., Korlach, J., Shapiro, L., McAdams, H. H. 2013; 110 (48): E4658-67

    Abstract

    The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N(6)-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.

    View details for DOI 10.1073/pnas.1319315110

    View details for PubMedID 24218615

  • Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hong, S., Toro, E., Mortensen, K. I., de la Rosa, M. A., Doniach, S., Shapiro, L., Spakowitz, A. J., McAdams, H. H. 2013; 110 (5): 1674-1679

    Abstract

    We measured the distance between fluorescent-labeled DNA loci of various interloci contour lengths in Caulobacter crescentus swarmer cells to determine the in vivo configuration of the chromosome. For DNA segments less than about 300 kb, the mean interloci distances, , scale as n(0.22), where n is the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n(0.22) with broad cell-to-cell variability. For DNA segments greater than about 300 kb, the mean interloci distances scale as n, in agreement with previous observations. The 0.22 value of the scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. Predictions from Brownian dynamics simulations of the packing of supercoiled DNA polymers in an elongated cell-like confinement are also consistent with a branched DNA structure, and simulated interloci distance distributions predict that confinement leads to "freezing" of the supercoiled configuration. Lateral positions of labeled loci at comparable positions along the length of the cell are strongly correlated when the longitudinal locus positions differ by <0.16 ?m. We conclude that the chromosome structure is supercoiled locally and elongated at large length scales and that substantial cell-to-cell variability in the interloci distances indicates that in vivo crowding prevents the chromosome from reaching an equilibrium arrangement. We suggest that the force causing rapid transport of loci remote from the parS centromere to the distal cell pole may arise from the release at the polar region of potential energy within the supercoiled DNA.

    View details for DOI 10.1073/pnas.1220824110

    View details for Web of Science ID 000314558100027

    View details for PubMedID 23319648

  • Compaction and transport properties of newly replicated Caulobacter crescentus DNA MOLECULAR MICROBIOLOGY Hong, S., McAdams, H. H. 2011; 82 (6): 1349-1358

    Abstract

    Upon initiating replication of the Caulobacter chromosome, one copy of the parS centromere remains at the stalked pole; the other moves to the distal pole. We identified the segregation dynamics and compaction characteristics of newly replicated Caulobacter DNA during transport (highly variable from cell to cell) using time-lapse fluorescence microscopy. The parS centromere and a length (also highly variable) of parS proximal DNA on each arm of the chromosome are segregated with the same relatively slow transport pattern as the parS locus. Newly replicated DNA further than about 100 kb from parS segregates with a different and faster pattern, while loci at 48 kb from parS segregate with the slow pattern in some cells and the fast pattern in others. The observed parS-proximal DNA compaction characteristics have scaling properties that suggest the DNA is branched. HU2-deletion strains exhibited a reduced compaction phenotype except near the parS site where only the ?HU1?HU2 double mutant had a compaction phenotype. The chromosome shows speed-dependent extension during translocation suggesting the DNA polymer is under tension. While DNA segregation is highly reliable and succeeds in virtually all wild-type cells, the high degree of cell to cell variation in the segregation process is noteworthy.

    View details for DOI 10.1111/j.1365-2958.2011.07899.x

    View details for Web of Science ID 000298087300006

    View details for PubMedID 22085253

  • Direct inference of protein-DNA interactions using compressed sensing methods PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA AlQuraishi, M., McAdams, H. H. 2011; 108 (36): 14819-14824

    Abstract

    Compressed sensing has revolutionized signal acquisition, by enabling complex signals to be measured with remarkable fidelity using a small number of so-called incoherent sensors. We show that molecular interactions, e.g., protein-DNA interactions, can be analyzed in a directly analogous manner and with similarly remarkable results. Specifically, mesoscopic molecular interactions act as incoherent sensors that measure the energies of microscopic interactions between atoms. We combine concepts from compressed sensing and statistical mechanics to determine the interatomic interaction energies of a molecular system exclusively from experimental measurements, resulting in a "de novo" energy potential. In contrast, conventional methods for estimating energy potentials are based on theoretical models premised on a priori assumptions and extensive domain knowledge. We determine the de novo energy potential for pairwise interactions between protein and DNA atoms from (i) experimental measurements of the binding affinity of protein-DNA complexes and (ii) crystal structures of the complexes. We show that the de novo energy potential can be used to predict the binding specificity of proteins to DNA with approximately 90% accuracy, compared to approximately 60% for the best performing alternative computational methods applied to this fundamental problem. This de novo potential method is directly extendable to other biomolecule interaction domains (enzymes and signaling molecule interactions) and to other classes of molecular interactions.

    View details for DOI 10.1073/pnas.1106460108

    View details for Web of Science ID 000294543400027

    View details for PubMedID 21825146

  • The essential genome of a bacterium MOLECULAR SYSTEMS BIOLOGY Christen, B., Abeliuk, E., Collier, J. M., Kalogeraki, V. S., Passarelli, B., Coller, J. A., Fero, M. J., McAdams, H. H., Shapiro, L. 2011; 7

    Abstract

    Caulobacter crescentus is a model organism for the integrated circuitry that runs a bacterial cell cycle. Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of a bacterial cell. Using hyper-saturated transposon mutagenesis coupled with high-throughput sequencing, we determined the essential Caulobacter genome at 8 bp resolution, including 1012 essential genome features: 480 ORFs, 402 regulatory sequences and 130 non-coding elements, including 90 intergenic segments of unknown function. The essential transcriptional circuitry for growth on rich media includes 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor. We identified all essential promoter elements for the cell cycle-regulated genes. The essential elements are preferentially positioned near the origin and terminus of the chromosome. The high-resolution strategy used here is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species.

    View details for DOI 10.1038/msb.2011.58

    View details for Web of Science ID 000294537800007

    View details for PubMedID 21878915

  • The Architecture and Conservation Pattern of Whole-Cell Control Circuitry JOURNAL OF MOLECULAR BIOLOGY McAdams, H. H., Shapiro, L. 2011; 409 (1): 28-35

    Abstract

    The control circuitry that directs and paces Caulobacter cell cycle progression involves the entire cell operating as an integrated system. This control circuitry monitors the environment and the internal state of the cell, including the cell topology, as it orchestrates orderly activation of cell cycle subsystems and Caulobacter's asymmetric cell division. The proteins of the Caulobacter cell cycle control system and its internal organization are co-conserved across many alphaproteobacteria species, but there are great differences in the regulatory apparatus' functionality and peripheral connectivity to other cellular subsystems from species to species. This pattern is similar to that observed for the "kernels" of the regulatory networks that regulate development of metazoan body plans. The Caulobacter cell cycle control system has been exquisitely optimized as a total system for robust operation in the face of internal stochastic noise and environmental uncertainty. When sufficient details accumulate, as for Caulobacter cell cycle regulation, the system design has been found to be eminently rational and indeed consistent with good design practices for human-designed asynchronous control systems.

    View details for DOI 10.1016/j.jmb.2011.02.041

    View details for Web of Science ID 000290779500004

    View details for PubMedID 21371478

  • An essential transcription factor, SciP, enhances robustness of Caulobacter cell cycle regulation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tan, M. H., Kozdon, J. B., Shen, X., Shapiro, L., McAdams, H. H. 2010; 107 (44): 18985-18990

    Abstract

    A cyclical control circuit composed of four master regulators drives the Caulobacter cell cycle. We report that SciP, a helix-turn-helix transcription factor, is an essential component of this circuit. SciP is cell cycle-controlled and co-conserved with the global cell cycle regulator CtrA in the ?-proteobacteria. SciP is expressed late in the cell cycle and accumulates preferentially in the daughter swarmer cell. At least 58 genes, including many flagellar and chemotaxis genes, are regulated by a type 1 incoherent feedforward motif in which CtrA activates sciP, followed by SciP repression of ctrA and CtrA target genes. We demonstrate that SciP binds to DNA at a motif distinct from the CtrA binding motif that is present in the promoters of genes co-regulated by SciP and CtrA. SciP overexpression disrupts the balance between activation and repression of the CtrA-SciP coregulated genes yielding filamentous cells and loss of viability. The type 1 incoherent feedforward circuit motif enhances the pulse-like expression of the downstream genes, and the negative feedback to ctrA expression reduces peak CtrA accumulation. The presence of SciP in the control network enhances the robustness of the cell cycle to varying growth rates.

    View details for DOI 10.1073/pnas.1014395107

    View details for Web of Science ID 000283749000046

    View details for PubMedID 20956288

  • System-level design of bacterial cell cycle control FEBS LETTERS McAdams, H. H., Shapiro, L. 2009; 583 (24): 3984-3991

    Abstract

    Understanding of the cell cycle control logic in Caulobacter has progressed to the point where we now have an integrated view of the operation of an entire bacterial cell cycle system functioning as a state machine. Oscillating levels of a few temporally-controlled master regulator proteins in a cyclical circuit drive cell cycle progression. To a striking degree, the cell cycle regulation is a whole cell phenomenon. Phospho-signaling proteins and proteases dynamically deployed to specific locations on the cell wall are vital. An essential phospho-signaling system integral to the cell cycle circuitry is central to accomplishing asymmetric cell division.

    View details for DOI 10.1016/j.febslet.2009.09.030

    View details for Web of Science ID 000273208000016

    View details for PubMedID 19766635

  • Three enhancements to the inference of statistical protein-DNA potentials PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS AlQuraishi, M., McAdams, H. H. 2013; 81 (3): 426-442

    Abstract

    The energetics of protein-DNA interactions are often modeled using so-called statistical potentials, that is, energy models derived from the atomic structures of protein-DNA complexes. Many statistical protein-DNA potentials based on differing theoretical assumptions have been investigated, but little attention has been paid to the types of data and the parameter estimation process used in deriving the statistical potentials. We describe three enhancements to statistical potential inference that significantly improve the accuracy of predicted protein-DNA interactions: (i) incorporation of binding energy data of protein-DNA complexes, in conjunction with their X-ray crystal structures, (ii) use of spatially-aware parameter fitting, and (iii) use of ensemble-based parameter fitting. We apply these enhancements to three widely-used statistical potentials and use the resulting enhanced potentials in a structure-based prediction of the DNA binding sites of proteins. These enhancements are directly applicable to all statistical potentials used in protein-DNA modeling, and we show that they can improve the accuracy of predicted DNA binding sites by up to 21%.

    View details for DOI 10.1002/prot.24201

    View details for Web of Science ID 000314179600007

    View details for PubMedID 23042633

  • The Three-Dimensional Architecture of a Bacterial Genome and Its Alteration by Genetic Perturbation MOLECULAR CELL Umbarger, M. A., Toro, E., Wright, M. A., Porreca, G. J., Bau, D., Hong, S., Fero, M. J., Zhu, L. J., Marti-Renom, M. A., McAdams, H. H., Shapiro, L., Dekker, J., Church, G. M. 2011; 44 (2): 252-264

    Abstract

    We have determined the three-dimensional (3D) architecture of the Caulobacter crescentus genome by combining genome-wide chromatin interaction detection, live-cell imaging, and computational modeling. Using chromosome conformation capture carbon copy (5C), we derive ~13 kb resolution 3D models of the Caulobacter genome. The resulting models illustrate that the genome is ellipsoidal with periodically arranged arms. The parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Repositioning these elements resulted in rotations of the chromosome that changed the subcellular positions of most genes. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Collectively, our data suggest that genome folding is globally dictated by the parS sites and chromosome segregation.

    View details for DOI 10.1016/j.molcel.2011.09.010

    View details for Web of Science ID 000296212100011

    View details for PubMedID 22017872

  • Assembly of the Caulobacter cell division machine MOLECULAR MICROBIOLOGY Goley, E. D., Yeh, Y., Hong, S., Fero, M. J., Abeliuk, E., McAdams, H. H., Shapiro, L. 2011; 80 (6): 1680-1698

    Abstract

    Cytokinesis in Gram-negative bacteria is mediated by a multiprotein machine (the divisome) that invaginates and remodels the inner membrane, peptidoglycan and outer membrane. Understanding the order of divisome assembly would inform models of the interactions among its components and their respective functions. We leveraged the ability to isolate synchronous populations of Caulobacter crescentus cells to investigate assembly of the divisome and place the arrival of each component into functional context. Additionally, we investigated the genetic dependence of localization among divisome proteins and the cell cycle regulation of their transcript and protein levels to gain insight into the control mechanisms underlying their assembly. Our results revealed a picture of divisome assembly with unprecedented temporal resolution. Specifically, we observed (i) initial establishment of the division site, (ii) recruitment of early FtsZ-binding proteins, (iii) arrival of proteins involved in peptidoglycan remodelling, (iv) arrival of FtsA, (v) assembly of core divisome components, (vi) initiation of envelope invagination, (vii) recruitment of polar markers and cytoplasmic compartmentalization and (viii) cell separation. Our analysis revealed differences in divisome assembly among Caulobacter and other bacteria that establish a framework for identifying aspects of bacterial cytokinesis that are widely conserved from those that are more variable.

    View details for DOI 10.1111/j.1365-2958.2011.07677.x

    View details for Web of Science ID 000292106100020

    View details for PubMedID 21542856

  • Regulatory Response to Carbon Starvation in Caulobacter crescentus PLOS ONE Britos, L., Abeliuk, E., Taverner, T., Lipton, M., McAdams, H., Shapiro, L. 2011; 6 (4)

    Abstract

    Bacteria adapt to shifts from rapid to slow growth, and have developed strategies for long-term survival during prolonged starvation and stress conditions. We report the regulatory response of C. crescentus to carbon starvation, based on combined high-throughput proteome and transcriptome analyses. Our results identify cell cycle changes in gene expression in response to carbon starvation that involve the prominent role of the FixK FNR/CAP family transcription factor and the CtrA cell cycle regulator. Notably, the SigT ECF sigma factor mediates the carbon starvation-induced degradation of CtrA, while activating a core set of general starvation-stress genes that respond to carbon starvation, osmotic stress, and exposure to heavy metals. Comparison of the response of swarmer cells and stalked cells to carbon starvation revealed four groups of genes that exhibit different expression profiles. Also, cell pole morphogenesis and initiation of chromosome replication normally occurring at the swarmer-to-stalked cell transition are uncoupled in carbon-starved cells.

    View details for DOI 10.1371/journal.pone.0018179

    View details for Web of Science ID 000289354100006

    View details for PubMedID 21494595

  • The Caulobacter Tol-Pal Complex Is Essential for Outer Membrane Integrity and the Positioning of a Polar Localization Factor JOURNAL OF BACTERIOLOGY Yeh, Y., Comolli, L. R., Downing, K. H., Shapiro, L., McAdams, H. H. 2010; 192 (19): 4847-4858

    Abstract

    Cell division in Caulobacter crescentus involves constriction and fission of the inner membrane (IM) followed about 20 min later by fission of the outer membrane (OM) and daughter cell separation. In contrast to Escherichia coli, the Caulobacter Tol-Pal complex is essential. Cryo-electron microscopy images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. In wild-type cells, components of the Tol-Pal complex localize to the division plane in early predivisional cells and remain predominantly at the new pole of swarmer and stalked progeny upon completion of division. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane-associated polar proteins tested. Coimmunoprecipitation experiments show that both TolA and Pal interact directly or indirectly with TipN. We propose that disruption of the trans-envelope Tol-Pal complex releases TipN from its subcellular position. The Caulobacter Tol-Pal complex is thus a key component of cell envelope structure and function, mediating OM constriction at the final step of cell division as well as the positioning of a protein localization factor.

    View details for DOI 10.1128/JB.00607-10

    View details for Web of Science ID 000281866900006

    View details for PubMedID 20693330

  • Caulobacter PopZ forms a polar subdomain dictating sequential changes in pole composition and function MOLECULAR MICROBIOLOGY Bowman, G. R., Comolli, L. R., Gaietta, G. M., Fero, M., Hong, S., Jones, Y., Lee, J. H., Downing, K. H., Ellisman, M. H., McAdams, H. H., Shapiro, L. 2010; 76 (1): 173-189

    Abstract

    The bacterium Caulobacter crescentus has morphologically and functionally distinct cell poles that undergo sequential changes during the cell cycle. We show that the PopZ oligomeric network forms polar ribosome exclusion zones that change function during cell cycle progression. The parS/ParB chromosomal centromere is tethered to PopZ at one pole prior to the initiation of DNA replication. During polar maturation, the PopZ-centromere tether is broken, and the PopZ zone at that pole then switches function to act as a recruitment factor for the ordered addition of multiple proteins that promote the transformation of the flagellated pole into a stalked pole. Stalked pole assembly, in turn, triggers the initiation of chromosome replication, which signals the formation of a new PopZ zone at the opposite cell pole, where it functions to anchor the newly duplicated centromere that has traversed the long axis of the cell. We propose that pole-specific control of PopZ function co-ordinates polar development and cell cycle progression by enabling independent assembly and tethering activities at the two cell poles.

    View details for DOI 10.1111/j.1365-2958.2010.07088.x

    View details for Web of Science ID 000276036000013

    View details for PubMedID 20149103

  • High-throughput identification of protein localization dependency networks PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Christen, B., Fero, M. J., Hillson, N. J., Bowman, G., Hong, S., Shapiro, L., McAdams, H. H. 2010; 107 (10): 4681-4686

    Abstract

    Bacterial cells are highly organized with many protein complexes and DNA loci dynamically positioned to distinct subcellular sites over the course of a cell cycle. Such dynamic protein localization is essential for polar organelle development, establishment of asymmetry, and chromosome replication during the Caulobacter crescentus cell cycle. We used a fluorescence microscopy screen optimized for high-throughput to find strains with anomalous temporal or spatial protein localization patterns in transposon-generated mutant libraries. Automated image acquisition and analysis allowed us to identify genes that affect the localization of two polar cell cycle histidine kinases, PleC and DivJ, and the pole-specific pili protein CpaE, each tagged with a different fluorescent marker in a single strain. Four metrics characterizing the observed localization patterns of each of the three labeled proteins were extracted for hundreds of cell images from each of 854 mapped mutant strains. Using cluster analysis of the resulting set of 12-element vectors for each of these strains, we identified 52 strains with mutations that affected the localization pattern of the three tagged proteins. This information, combined with quantitative localization data from epitasis experiments, also identified all previously known proteins affecting such localization. These studies provide insights into factors affecting the PleC/DivJ localization network and into regulatory links between the localization of the pili assembly protein CpaE and the kinase localization pathway. Our high-throughput screening methodology can be adapted readily to any sequenced bacterial species, opening the potential for databases of localization regulatory networks across species, and investigation of localization network phylogenies.

    View details for DOI 10.1073/pnas.1000846107

    View details for Web of Science ID 000275368400036

    View details for PubMedID 20176934

  • Why and How Bacteria Localize Proteins SCIENCE Shapiro, L., McAdams, H. H., Losick, R. 2009; 326 (5957): 1225-1228

    Abstract

    Despite their small size, bacteria have a remarkably intricate internal organization. Bacteria deploy proteins and protein complexes to particular locations and do so in a dynamic manner in lockstep with the organized deployment of their chromosome. The dynamic subcellular localization of protein complexes is an integral feature of regulatory processes of bacterial cells.

    View details for DOI 10.1126/science.1175685

    View details for Web of Science ID 000272117900037

    View details for PubMedID 19965466

  • Dynamic chromosome organization and protein localization coordinate the regulatory circuitry that drives the bacterial cell cycle. Cold Spring Harbor symposia on quantitative biology Goley, E. D., Toro, E., McAdams, H. H., Shapiro, L. 2009; 74: 55-64

    Abstract

    The bacterial cell has less internal structure and genetic complexity than cells of eukaryotic organisms, yet it is a highly organized system that uses both temporal and spatial cues to drive its cell cycle. Key insights into bacterial regulatory programs that orchestrate cell cycle progression have come from studies of Caulobacter crescentus, a bacterium that divides asymmetrically. Three global regulatory proteins cycle out of phase with one another and drive cell cycle progression by directly controlling the expression of 200 cell-cycle-regulated genes. Exploration of this system provided insights into the evolution of regulatory circuits and the plasticity of circuit structure. The temporal expression of the modular subsystems that implement the cell cycle and asymmetric cell division is also coordinated by differential DNA methylation, regulated proteolysis, and phosphorylation signaling cascades. This control system structure has parallels to eukaryotic cell cycle control architecture. Remarkably, the transcriptional circuitry is dependent on three-dimensional dynamic deployment of key regulatory and signaling proteins. In addition, dynamically localized DNA-binding proteins ensure that DNA segregation is coupled to the timing and cellular position of the cytokinetic ring. Comparison to other organisms reveals conservation of cell cycle regulatory logic, even if regulatory proteins, themselves, are not conserved.

    View details for DOI 10.1101/sqb.2009.74.005

    View details for PubMedID 19687139

  • Caulobacter requires a dedicated mechanism to initiate chromosome segregation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Toro, E., Hong, S., McAdams, H. H., Shapiro, L. 2008; 105 (40): 15435-15440

    Abstract

    Chromosome segregation in bacteria is rapid and directed, but the mechanisms responsible for this movement are still unclear. We show that Caulobacter crescentus makes use of and requires a dedicated mechanism to initiate chromosome segregation. Caulobacter has a single circular chromosome whose origin of replication is positioned at one cell pole. Upon initiation of replication, an 8-kb region of the chromosome containing both the origin and parS moves rapidly to the opposite pole. This movement requires the highly conserved ParABS locus that is essential in Caulobacter. We use chromosomal inversions and in vivo time-lapse imaging to show that parS is the Caulobacter site of force exertion, independent of its position in the chromosome. When parS is moved farther from the origin, the cell waits for parS to be replicated before segregation can begin. Also, a mutation in the ATPase domain of ParA halts segregation without affecting replication initiation. Chromosome segregation in Caulobacter cannot occur unless a dedicated parS guiding mechanism initiates movement.

    View details for DOI 10.1073/pnas.0807448105

    View details for Web of Science ID 000260360500041

    View details for PubMedID 18824683

  • Architecture and inherent robustness of a bacterial cell-cycle control system PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shen, X., Collier, J., Dill, D., Shapiro, L., Horowitz, M., McAdams, H. H. 2008; 105 (32): 11340-11345

    Abstract

    A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. The hybrid cell-cycle simulation implementation is inherently extensible and provides a promising approach for development of whole-cell behavioral models that can replicate the observed functionality of the cell and its responses to changing environmental conditions.

    View details for DOI 10.1073/pnas.0805258105

    View details for Web of Science ID 000258560700056

    View details for PubMedID 18685108

  • Small non-coding RNAs in Caulobacter crescentus MOLECULAR MICROBIOLOGY Landt, S. G., Abeliuk, E., McGrath, P. T., Lesley, J. A., McAdams, H. H., Shapiro, L. 2008; 68 (3): 600-614

    Abstract

    Small non-coding RNAs (sRNAs) are active in many bacterial cell functions, including regulation of the cell's response to environmental challenges. We describe the identification of 27 novel Caulobacter crescentus sRNAs by analysis of RNA expression levels assayed using a tiled Caulobacter microarray and a protocol optimized for detection of sRNAs. The principal analysis method involved identification of sets of adjacent probes with unusually high correlation between the individual intergenic probes within the set, suggesting presence of a sRNA. Among the validated sRNAs, two are candidate transposase gene antisense RNAs. The expression of 10 of the sRNAs is regulated by either entry into stationary phase, carbon starvation, or rich versus minimal media. The expression of four of the novel sRNAs changes as the cell cycle progresses. One of these shares a promoter motif with several genes expressed at the swarmer-to-stalked cell transition; while another appears to be controlled by the CtrA global transcriptional regulator. The probe correlation analysis approach reported here is of general use for large-scale sRNA identification for any sequenced microbial genome.

    View details for DOI 10.1111/j.1365-2958.2008.06172.x

    View details for Web of Science ID 000254641600007

    View details for PubMedID 18373523

  • A DNA methylation ratchet governs progression through a bacterial cell cycle PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Collier, J., McAdams, H. H., Shapiro, L. 2007; 104 (43): 17111-17116

    Abstract

    The Caulobacter cell cycle is driven by a cascade of transient regulators, starting with the expression of DnaA in G(1) and ending with the expression of the essential CcrM DNA methyltransferase at the completion of DNA replication. The timing of DnaA accumulation was found to be regulated by the methylation state of the dnaA promoter, which in turn depends on the chromosomal position of dnaA near the origin of replication and restriction of CcrM synthesis to the end of the cell cycle. The dnaA gene is preferentially transcribed from a fully methylated promoter. DnaA initiates DNA replication and activates the transcription of the next cell-cycle regulator, GcrA. With the passage of the replication fork, the dnaA promoter becomes hemimethylated, and DnaA accumulation drops. GcrA then activates the transcription of the next cell-cycle regulator, CtrA, once the replication fork passes through the ctrA P1 promoter, generating two hemimethylated copies of ctrA. The ctrA gene is preferentially transcribed from a hemimethylated promoter. CtrA then activates the transcription of ccrM, to bring the newly replicated chromosome to the fully methylated state, promoting dnaA transcription and the start of a new cell cycle. We show that the cell-cycle timing of CcrM is critical for Caulobacter fitness. The sequential changes in the chromosomal methylation state serve to couple the progression of DNA replication to cell-cycle events regulated by the master transcriptional regulatory cascade, thus providing a ratchet mechanism for robust cell-cycle control.

    View details for Web of Science ID 000250487600069

    View details for PubMedID 17942674

  • High-throughput identification of transcription start sites, conserved promoter motifs and predicted regulons NATURE BIOTECHNOLOGY McGrath, P. T., Lee, H., Zhang, L., Iniesta, A. A., Hottes, A. K., Tan, M. H., Hillson, N. J., Hu, P., Shapiro, L., McAdams, H. H. 2007; 25 (5): 584-592

    Abstract

    Using 62 probe-level datasets obtained with a custom-designed Caulobacter crescentus microarray chip, we identify transcriptional start sites of 769 genes, 53 of which are transcribed from multiple start sites. Transcriptional start sites are identified by analyzing probe signal cross-correlation matrices created from probe pairs tiled every 5 bp upstream of the genes. Signals from probes binding the same message are correlated. The contribution of each promoter for genes transcribed from multiple promoters is identified. Knowing the transcription start site enables targeted searching for regulatory-protein binding motifs in the promoter regions of genes with similar expression patterns. We identified 27 motifs, 17 of which share no similarity to the characterized motifs of other C. crescentus transcriptional regulators. Using these motifs, we predict coregulated genes. We verified novel promoter motifs that regulate stress-response genes, including those responding to uranium challenge, a stress-response sigma factor and a stress-response noncoding RNA.

    View details for DOI 10.1038/nbt1294

    View details for Web of Science ID 000246369400027

    View details for PubMedID 17401361

  • Systems biology of Caulobacter ANNUAL REVIEW OF GENETICS Laub, M. T., Shapiro, L., McAdams, H. H. 2007; 41: 429-441

    Abstract

    The dynamic range of a bacterial species' natural environment is reflected in the complexity of its systems that control cell cycle progression and its range of adaptive responses. We discuss the genetic network and integrated three-dimensional sensor/response systems that regulate the cell cycle and asymmetric cell division in the bacterium Caulobacter crescentus. The cell cycle control circuitry is tied closely to chromosome replication and morphogenesis by multiple feedback pathways from the modular functions that implement the cell cycle. The sophistication of the genetic regulatory circuits and the elegant integration of temporally controlled transcription and protein synthesis with spatially dynamic phosphosignaling and proteolysis pathways, and epigenetic regulatory mechanisms, form a remarkably robust living system.

    View details for DOI 10.1146/annurev.genet.41.110306.130346

    View details for Web of Science ID 000252359500018

    View details for PubMedID 18076330

  • Graemlin: General and robust alignment of multiple large interaction networks GENOME RESEARCH Flannick, J., Novak, A., Srinivasan, B. S., McAdams, H. H., Batzoglou, S. 2006; 16 (9): 1169-1181

    Abstract

    The recent proliferation of protein interaction networks has motivated research into network alignment: the cross-species comparison of conserved functional modules. Previous studies have laid the foundations for such comparisons and demonstrated their power on a select set of sparse interaction networks. Recently, however, new computational techniques have produced hundreds of predicted interaction networks with interconnection densities that push existing alignment algorithms to their limits. To find conserved functional modules in these new networks, we have developed Graemlin, the first algorithm capable of scalable multiple network alignment. Graemlin's explicit model of functional evolution allows both the generalization of existing alignment scoring schemes and the location of conserved network topologies other than protein complexes and metabolic pathways. To assess Graemlin's performance, we have developed the first quantitative benchmarks for network alignment, which allow comparisons of algorithms in terms of their ability to recapitulate the KEGG database of conserved functional modules. We find that Graemlin achieves substantial scalability gains over previous methods while improving sensitivity.

    View details for DOI 10.1101/gr.5235706

    View details for Web of Science ID 000240238600011

    View details for PubMedID 16899655

  • Bacterial stalks are nutrient-scavenging antennas PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA McAdams, H. H. 2006; 103 (31): 11435-11436

    View details for DOI 10.1073/pnas.0605027103

    View details for Web of Science ID 000239616400003

    View details for PubMedID 16868078

  • A phospho-signaling pathway controls the localization and activity of a protease complex critical for bacterial cell cycle progression PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Iniesta, A. A., McGrath, P. T., Reisenauer, A., McAdams, H. H., Shapiro, L. 2006; 103 (29): 10935-10940

    Abstract

    Temporally and spatially controlled master regulators drive the Caulobacter cell cycle by regulating the expression of >200 genes. Rapid clearance of the master regulator, CtrA, by the ClpXP protease is a critical event that enables the initiation of chromosome replication at specific times in the cell cycle. We show here that a previously unidentified single domain-response regulator, CpdR, when in the unphosphorylated state, binds to ClpXP and, thereby, causes its localization to the cell pole. We further show that ClpXP localization is required for CtrA proteolysis. When CpdR is phosphorylated, ClpXP is delocalized, and CtrA is not degraded. Both CtrA and CpdR are phosphorylated via the same CckA histidine kinase phospho-signaling pathway, providing a reinforcing mechanism that simultaneously activates CtrA and prevents its degradation by delocalizing the CpdR/ClpXP complex. In swarmer cells, CpdR is in the phosphorylated state, thus preventing ClpXP localization and CtrA degradation. As swarmer cells differentiate into stalked cells (G1/S transition), unphosphorylated CpdR accumulates and is localized to the stalked cell pole, where it enables ClpXP localization and CtrA proteolysis, allowing the initiation of DNA replication. Dynamic protease localization mediated by a phospho-signaling pathway is a novel mechanism to integrate spatial and temporal control of bacterial cell cycle progression.

    View details for DOI 10.1073/pnas.0604554103

    View details for Web of Science ID 000239327200022

    View details for PubMedID 16829582

  • A dynamically localized protease complex and a polar specificity factor control a cell cycle master regulator CELL McGrath, P. T., Iniesta, A. A., Ryan, K. R., Shapiro, L., McAdams, H. H. 2006; 124 (3): 535-547

    Abstract

    Regulated proteolysis is essential for cell cycle progression in both prokaryotes and eukaryotes. We show here that the ClpXP protease, responsible for the degradation of multiple bacterial proteins, is dynamically localized to specific cellular positions in Caulobacter where it degrades colocalized proteins. The CtrA cell cycle master regulator, that must be cleared from the Caulobacter cell to allow the initiation of chromosome replication, interacts with the ClpXP protease at the cell pole where it is degraded. We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. RcdA is required for CtrA polar localization and degradation by ClpXP. The localization pattern of RcdA is coincident with and dependent upon ClpX localization. Thus, a dynamically localized ClpXP proteolysis complex in concert with a cytoplasmic factor provides temporal and spatial specificity to protein degradation during a bacterial cell cycle.

    View details for DOI 10.1016/j.cell.2005.12.033

    View details for Web of Science ID 000235464000016

    View details for PubMedID 16469700

  • Cytokinesis signals truncation of the PodJ polarity factor by a cell cycle-regulated protease EMBO JOURNAL Chen, J. C., Hottes, A. K., McAdams, H. H., McGrath, P. T., Viollier, P. H., Shapiro, L. 2006; 25 (2): 377-386

    Abstract

    We demonstrate that successive cleavage events involving regulated intramembrane proteolysis (Rip) occur as a function of time during the Caulobacter cell cycle. The proteolytic substrate PodJ(L) is a polar factor that recruits proteins required for polar organelle biogenesis to the correct cell pole at a defined time in the cell cycle. We have identified a periplasmic protease (PerP) that initiates the proteolytic sequence by truncating PodJ(L) to a form with altered activity (PodJ(S)). Expression of perP is regulated by a signal transduction system that activates cell type-specific transcription programs and conversion of PodJ(L) to PodJ(S) in response to the completion of cytokinesis. PodJ(S), sequestered to the progeny swarmer cell, is subsequently released from the polar membrane by the membrane metalloprotease MmpA for degradation during the swarmer-to-stalked cell transition. This sequence of proteolytic events contributes to the asymmetric localization of PodJ isoforms to the appropriate cell pole. Thus, temporal activation of the PerP protease and spatial restriction of the polar PodJ(L) substrate cooperatively control the cell cycle-dependent onset of Rip.

    View details for DOI 10.1038/sj.emboj.7600935

    View details for Web of Science ID 000234952500011

    View details for PubMedID 16395329

  • Integrated Protein Interaction Networks for 11 Microbes Proceedings of the Tenth Annual International Conference on Computational Molecular Biology, (RECOMB 2006) Srinivasan, B., Novak A, Flannick J, Batzoglou S, McAdams H. 2006: 1-14
  • Whole-genome transcriptional analysis of heavy metal stresses in Caulobacter crescentus JOURNAL OF BACTERIOLOGY Hu, P., Brodie, E. L., Suzuki, Y., McAdams, H. H., Andersen, G. L. 2005; 187 (24): 8437-8449

    Abstract

    The bacterium Caulobacter crescentus and related stalk bacterial species are known for their distinctive ability to live in low-nutrient environments, a characteristic of most heavy metal-contaminated sites. Caulobacter crescentus is a model organism for studying cell cycle regulation with well-developed genetics. We have identified the pathways responding to heavy-metal toxicity in C. crescentus to provide insights for the possible application of Caulobacter to environmental restoration. We exposed C. crescentus cells to four heavy metals (chromium, cadmium, selenium, and uranium) and analyzed genome-wide transcriptional activities postexposure using an Affymetrix GeneChip microarray. C. crescentus showed surprisingly high tolerance to uranium, a possible mechanism for which may be the formation of extracellular calcium-uranium-phosphate precipitates. The principal response to these metals was protection against oxidative stress (up-regulation of manganese-dependent superoxide dismutase sodA). Glutathione S-transferase, thioredoxin, glutaredoxins, and DNA repair enzymes responded most strongly to cadmium and chromate. The cadmium and chromium stress response also focused on reducing the intracellular metal concentration, with multiple efflux pumps employed to remove cadmium, while a sulfate transporter was down-regulated to reduce nonspecific uptake of chromium. Membrane proteins were also up-regulated in response to most of the metals tested. A two-component signal transduction system involved in the uranium response was identified. Several differentially regulated transcripts from regions previously not known to encode proteins were identified, demonstrating the advantage of evaluating the transcriptome by using whole-genome microarrays.

    View details for DOI 10.1128/JB.187.24.8437-8449.2005

    View details for Web of Science ID 000233880000026

    View details for PubMedID 16321948

  • DnaA coordinates replication initiation and cell cycle transcription in Caulobacter crescentus MOLECULAR MICROBIOLOGY Hottes, A. K., Shapiro, L., McAdams, H. H. 2005; 58 (5): 1340-1353

    Abstract

    The level of DnaA, a key bacterial DNA replication initiation factor, increases during the Caulobacter swarmer-to-stalked transition just before the G1/S transition. We show that DnaA coordinates DNA replication initiation with cell cycle progression by acting as a global transcription factor. Using DnaA depletion and induction in synchronized cell populations, we have analysed global transcription patterns to identify the differential regulation of normally co-expressed genes. The DnaA regulon includes genes encoding several replisome components, the GcrA global cell cycle regulator, the PodJ polar localization protein, the FtsZ cell division protein, and nucleotide biosynthesis enzymes. In cells depleted of DnaA, the G1/S transition is temporally separated from the swarmer-to-stalked cell differentiation, which is normally coincident. In the absence of DnaA, the CtrA master regulator is cleared by proteolysis during the swarmer-to-stalked cell transition as usual, but DNA replication initiation is blocked. In this case, expression of gcrA, which is directly repressed by CtrA, does not increase in conjunction with the disappearance of CtrA until DnaA is subsequently induced, showing that gcrA expression requires DnaA. DnaA boxes are present upstream of many genes whose expression requires DnaA, and His6-DnaA binds to the promoters of gcrA, ftsZ and podJ in vitro. This redundant control of gcrA transcription by DnaA (activation) and CtrA (repression) forms a robust switch controlling the decision to proceed through the cell cycle or to remain in the G1 stage.

    View details for DOI 10.1111/j.1365-2958.2005.04912.x

    View details for Web of Science ID 000233170700012

    View details for PubMedID 16313620

  • Distinct constrictive processes, separated in time and space, divide Caulobacter inner and outer membranes JOURNAL OF BACTERIOLOGY Judd, E. M., Comolli, L. R., Chen, J. C., Downing, K. H., Moerner, W. E., McAdams, H. H. 2005; 187 (20): 6874-6882

    Abstract

    Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed pre-fission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.

    View details for DOI 10.1128/JB.187.20.6874-6882.2005

    View details for Web of Science ID 000232509500003

    View details for PubMedID 16199556

  • Conserved modular design of an oxygen sensory/signaling network with species-specific output PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Crosson, S., McGrath, P. T., Stephens, C., McAdams, H. H., Shapiro, L. 2005; 102 (22): 8018-8023

    Abstract

    Principles of modular design are evident in signaling networks that detect and integrate a given signal and, depending on the organism in which the network module is present, transduce this signal to affect different metabolic or developmental pathways. Here we report a global transcriptional analysis of an oxygen sensory/signaling network in Caulobacter crescentus consisting of the sensor histidine kinase FixL, its cognate response regulator FixJ, the transcriptional regulator FixK, and the kinase inhibitor FixT. It is known that in rhizobial bacteria these proteins form a network that regulates transcription of genes required for symbiotic nitrogen fixation, anaerobic and microaerobic respiration, and hydrogen metabolism under hypoxic conditions. We have identified a positive feedback loop in this network and present evidence that the negative feedback regulator, FixT, acts to inhibit FixL by mimicking a response regulator. Overall, the core circuit topology of the Fix network is conserved between the rhizobia and C. crescentus, a free-living aerobe that cannot fix nitrogen, respire anaerobically, or metabolize hydrogen. In C. crescentus, the Fix network is required for normal cellular growth during hypoxia and controls expression of genes encoding four distinct aerobic respiratory terminal oxidases and multiple carbon and nitrogen metabolic enzymes. Thus, the Fix network is a conserved sensory/signaling module whose transcriptional output has been adapted to the unique physiologies of C. crescentus and the nitrogen-fixing rhizobia.

    View details for DOI 10.1073/pnas.0503022102

    View details for Web of Science ID 000229531000043

    View details for PubMedID 15911751

  • Global approaches to the bacterial cell as an integrated system in The Bacterial Chrosome, edited by N. Patrick Higgins, ASM Press M. T. Laub, Lucy Shapiro, Harley McAdams 2005
  • Integrated Protein Interaction Networks for 230 Microbes BCATS Srinivasan, B., A. F. Novak, J. A. Flannick, S. Batzoglou, H. H. McAdams 2005
  • Visualization of the movement of single histidine kinase molecules in live Caulobacter cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Deich, J., Judd, E. M., McAdams, H. H., Moerner, W. E. 2004; 101 (45): 15921-15926

    Abstract

    The bacterium Caulobacter crescentus divides asymmetrically as part of its normal life cycle. This asymmetry is regulated in part by the membrane-bound histidine kinase PleC, which localizes to one pole of the cell at specific times in the cell cycle. Here, we track single copies of PleC labeled with enhanced yellow fluorescent protein (EYFP) in the membrane of live Caulobacter cells over a time scale of seconds. In addition to the expected molecules immobilized at one cell pole, we observed molecules moving throughout the cell membrane. By tracking the positions of these molecules for several seconds, we determined a diffusion coefficient (D) of 12 +/- 2 x 10(-3) microm(2)/s for the mobile copies of PleC not bound at the cell pole. This D value is maintained across all cell cycle stages. We observe a reduced D at poles containing localized PleC-EYFP; otherwise D is independent of the position of the diffusing molecule within the bacterium. We did not detect any directional bias in the motion of the PleC-EYFP molecules, implying that the molecules are not being actively transported.

    View details for DOI 10.1073/pnas.0404200101

    View details for Web of Science ID 000225196800020

    View details for PubMedID 15522969

  • A genetic oscillator and the regulation of cell cycle progression in Caulobacter crescentus CELL CYCLE Crosson, S., McAdams, H., Shapiro, L. 2004; 3 (10): 1252-1254

    Abstract

    Analyses of cell polarity, division, and differentiation in prokaryotes have identified several regulatory proteins that exhibit dramatic changes in expression and spatial localization over the course of a cell cycle. The dynamic behavior of these proteins is often intrinsically linked to their function as polarity determinants.(1-3) In the alpha-proteobacterium, Caulobacter crescentus, the CtrA global transcriptional regulator exhibits a spatially and temporally dynamic expression pattern across the cell cycle. CtrA plays key roles in asymmetric cell division and in the timing of chromosome replication.(3,4) An additional global regulator, GcrA, has recently been discovered that both regulates and is regulated by CtrA.(5) Together, these regulatory proteins create a genetic circuit in which the cellular concentrations of CtrA and GcrA oscillate spatially and temporally to control daughter cell differentiation and cell cycle progression.

    View details for Web of Science ID 000225590800012

    View details for PubMedID 15467452

  • Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Viollier, P. H., Thanbichler, M., McGrath, P. T., West, L., Meewan, M., McAdams, H. H., Shapiro, L. 2004; 101 (25): 9257-9262

    Abstract

    The chromosomal origin and terminus of replication are precisely localized in bacterial cells. We examined the cellular position of 112 individual loci that are dispersed over the circular Caulobacter crescentus chromosome and found that in living cells each locus has a specific subcellular address and that these loci are arrayed in linear order along the long axis of the cell. Time-lapse microscopy of the location of the chromosomal origin and 10 selected loci in the origin-proximal half of the chromosome showed that during DNA replication, as the replisome sequentially copies each locus, the newly replicated DNA segments are moved in chronological order to their final subcellular destination in the nascent half of the predivisional cell. Thus, the remarkable organization of the chromosome is being established while DNA replication is still in progress. The fact that the movement of these 10 loci is, like that of the origin, directed and rapid, and occurs at a similar rate, suggests that the same molecular machinery serves to partition and place many, if not most, chromosomal loci at defined subcellular sites.

    View details for DOI 10.1073/pnas.0402606101

    View details for Web of Science ID 000222278600018

    View details for PubMedID 15178755

  • Oscillating global regulators control the genetic circuit driving a bacterial cell cycle SCIENCE Holtzendorff, J., Hung, D., Brende, P., Reisenauer, A., Viollier, P. H., McAdams, H. H., Shapiro, L. 2004; 304 (5673): 983-987

    Abstract

    A newly identified cell-cycle master regulator protein, GcrA, together with the CtrA master regulator, are key components of a genetic circuit that drives cell-cycle progression and asymmetric polar morphogenesis in Caulobacter crescentus. The circuit drives out-of-phase temporal and spatial oscillation of GcrA and CtrA concentrations, producing time- and space-dependent transcriptional regulation of modular functions that implement cell-cycle processes. The CtrA/GcrA regulatory circuit controls expression of polar differentiation factors and the timing of DNA replication. CtrA functions as a silencer of the replication origin and GcrA as an activator of components of the replisome and the segregation machinery.

    View details for DOI 10.1126/science.1095191

    View details for Web of Science ID 000221383300040

    View details for PubMedID 15087506

  • Setting the pace: mechanisms tying Caulobacter cell-cycle progression to macroscopic cellular events CURRENT OPINION IN MICROBIOLOGY McGrath, P. T., Viollier, P., McAdams, H. H. 2004; 7 (2): 192-197

    Abstract

    The bacterium Caulobacter crescentus divides asymmetrically, producing daughter cells with differing polar structures, different cell fates and asymmetric regulation of the initiation of chromosome replication. Complex intracellular signaling is required to keep the organelle developmental processes at the cell poles synchronized with other cell cycle events. Two recently characterized switch mechanisms controlling cell cycle progress are triggered by relatively large-scale developmental events in the cell: the progress of the DNA replication fork and the physical compartmentalization of the cell that occurs well before division. These mechanisms invoke rapid, precisely timed and even spatially differentiated regulatory responses at important points in the cell cycle.

    View details for DOI 10.1016/j.mib.2004.02.012

    View details for Web of Science ID 000221065800016

    View details for PubMedID 15063858

  • Codon usage between genomes is constrained by genome-wide mutational processes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, S. L., Lee, W., Hottes, A. K., Shapiro, L., McAdams, H. H. 2004; 101 (10): 3480-3485

    Abstract

    Analysis of genome-wide codon bias shows that only two parameters effectively differentiate the genome-wide codon bias of 100 eubacterial and archaeal organisms. The first parameter correlates with genome GC content, and the second parameter correlates with context-dependent nucleotide bias. Both of these parameters may be calculated from intergenic sequences. Therefore, genome-wide codon bias in eubacteria and archaea may be predicted from intergenic sequences that are not translated. When these two parameters are calculated for genes from nonmammalian eukaryotic organisms, genes from the same organism again have similar values, and genome-wide codon bias may also be predicted from intergenic sequences. In mammals, genes from the same organism are similar only in the second parameter, because GC content varies widely among isochores. Our results suggest that, in general, genome-wide codon bias is determined primarily by mutational processes that act throughout the genome, and only secondarily by selective forces acting on translated sequences.

    View details for DOI 10.1073/pnas.0307827100

    View details for Web of Science ID 000220163800029

    View details for PubMedID 14990797

  • The evolution of genetic regulatory systems in bacteria NATURE REVIEWS GENETICS McAdams, H. H., Srinivasan, B., Arkin, A. P. 2004; 5 (3): 169-178

    View details for DOI 10.1038/nrg1292

    View details for Web of Science ID 000189334500012

    View details for PubMedID 14970819

  • Transcriptional profiling of Caulobacter crescentus during growth on complex and minimal media JOURNAL OF BACTERIOLOGY Hottes, A. K., Meewan, M., Yang, D., Arana, N., Romero, P., McAdams, H. H., Stephens, C. 2004; 186 (5): 1448-1461

    Abstract

    Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation.

    View details for DOI 10.1128/JB.186.5.1448-1461.2004

    View details for Web of Science ID 000189117900027

    View details for PubMedID 14973021

  • Genomics-Based Analysis of the Bacterial Cell Cycle Microbial Genomes, Ed. by Fraser, C. M., Read, T., Nelson, K. E. Laub, M.T., McAdams, H. H., Shapiro L. 2004
  • A bacterial cell-cycle regulatory network operating in time and space SCIENCE McAdams, H. H., Shapiro, L. 2003; 301 (5641): 1874-1877

    Abstract

    Transcriptional regulatory circuits provide only a fraction of the signaling pathways and regulatory mechanisms that control the bacterial cell cycle. The CtrA regulatory network, important in control of the Caulobacter cell cycle, illustrates the critical role of nontranscriptional pathways and temporally and spatially localized regulatory proteins. The system architecture of Caulobacter cell-cycle control involves top-down control of modular functions by a small number of master regulatory proteins with cross-module signaling coordinating the overall process. Modeling the cell cycle probably requires a top-down modeling approach and a hybrid control system modeling paradigm to treat its combined discrete and continuous characteristics.

    View details for Web of Science ID 000185536700040

    View details for PubMedID 14512618

  • Fluorescence bleaching reveals asymmetric compartment formation prior to cell division in Caulobacter PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Judd, E. M., Ryan, K. R., Moerner, W. E., Shapiro, L., McAdams, H. H. 2003; 100 (14): 8235-8240

    Abstract

    Asymmetric cell division in Caulobacter crescentus yields daughter cells that have different cell fates. Compartmentalization of the predivisional cell is a critical event in the establishment of the differential distribution of regulatory factors that specify cell fate. To determine when during the cell cycle the cytoplasm is compartmentalized so that cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments, we designed a fluorescence loss in photobleaching assay. Individual cells containing enhanced GFP were exposed to a bleaching laser pulse tightly focused at one cell pole. In compartmentalized cells, fluorescence disappears only in the compartment receiving the bleaching beam; in noncompartmentalized cells, fluorescence disappears from the entire cell. In a 135-min cell cycle, the cells were compartmentalized 18 +/- 5 min before the progeny cells separated. Clearance of the 22000 CtrA master transcriptional regulator molecules from the stalked portion of the predivisional cell is a controlling element of Caulobacter asymmetry. Monitoring of a fluorescent marker for CtrA showed that the differential degradation of CtrA in the nascent stalk cell compartment occurs only after the cytoplasm is compartmentalized.

    View details for DOI 10.1073/pnas.1433105100

    View details for Web of Science ID 000184222500037

    View details for PubMedID 12824468

  • Generating and exploiting polarity in bacteria SCIENCE Shapiro, L., McAdams, H. H., Losick, R. 2002; 298 (5600): 1942-1946

    Abstract

    Bacteria are often highly polarized, exhibiting specialized structures at or near the ends of the cell. Among such structures are actin-organizing centers, which mediate the movement of certain pathogenic bacteria within the cytoplasm of an animal host cell; organized arrays of membrane receptors, which govern chemosensory behavior in swimming bacteria; and asymmetrically positioned septa, which generate specialized progeny in differentiating bacteria. This polarization is orchestrated by complex and dynamic changes in the subcellular localization of signal transduction and cytoskeleton proteins as well as of specific regions of the chromosome. Recent work has provided information on how dynamic subcellular localization occurs and how it is exploited by the bacterial cell. The main task of a bacterial cell is to survive and duplicate itself. The bacterium must replicate its genetic material and divide at the correct site in the cell and at the correct time in the cell cycle with high precision. Each kind of bacterium also executes its own strategy to find nutrients in its habitat and to cope with conditions of stress from its environment. This involves moving toward food, adapting to environmental extremes, and, in many cases, entering and exploiting a eukaryotic host. These activities often involve processes that take place at or near the poles of the cell. Here we explore some of the schemes bacteria use to orchestrate dynamic changes at their poles and how these polar events execute cellular functions. In spite of their small size, bacteria have a remarkably complex internal organization and external architecture. Bacterial cells are inherently asymmetric, some more obviously so than others. The most easily recognized asymmetries involve surface structures, e.g., flagella, pili, and stalks that are preferentially assembled at one pole by many bacteria. "New" poles generated at the cell division plane differ from old poles from the previous round of cell division. Even in Escherichia coli, which is generally thought to be symmetrical, old poles are more static than new poles with respect to cell wall assembly (1), and they differ in the deposition of phospholipid domains (2). There are many instances of differential polar functions; among these is the preferential use of old poles when attaching to host cells as in the interaction of Bradyrhizobium with plant root hairs (3) or the polar pili-mediated attachment of the Pseudomonas aeruginosa pathogen to tracheal epithelia (4). An unusual polar organelle that mediates directed motility on solid surfaces is found in the nonpathogenic bacterium Myxococcus xanthus. The gliding motility of this bacterium is propelled by a nozzle-like structure that squirts a polysaccharide-containing slime from the pole of the cell (5). Interestingly, M. xanthus, which has nozzles at both poles, can reverse direction by closing one nozzle and opening the other in response to end-to-end interactions between cells.

    View details for Web of Science ID 000179629200032

    View details for PubMedID 12471245

  • Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Laub, M. T., Chen, S. L., Shapiro, L., McAdams, H. H. 2002; 99 (7): 4632-4637

    Abstract

    Studies of the genetic network that controls the Caulobacter cell cycle have identified a response regulator, CtrA, that controls, directly or indirectly, one-quarter of the 553 cell cycle-regulated genes. We have performed in vivo genomic binding site analysis of the CtrA protein to identify which of these genes have regulatory regions bound directly by CtrA. By combining these data with previous global analysis of cell cycle transcription patterns and gene expression profiles of mutant ctrA strains, we have determined that CtrA directly regulates at least 95 genes. The total group of CtrA-regulated genes includes those involved in polar morphogenesis, DNA replication initiation, DNA methylation, cell division, and cell wall metabolism. Also among the genes in this notably large regulon are 14 that encode regulatory proteins, including 10 two-component signal transduction regulatory proteins. Identification of additional regulatory genes activated by CtrA will serve to directly connect new regulatory modules to the network controlling cell cycle progression.

    View details for DOI 10.1073/pnas.062065699

    View details for Web of Science ID 000174856000089

    View details for PubMedID 11930012

  • Global analysis of the genetic network controlling a bacterial cell cycle SCIENCE Laub, M. T., McAdams, T. H., Feldblyum, T., Fraser, C. M., Shapiro, L. 2000; 290 (5499): 2144-2148

    Abstract

    This report presents full-genome evidence that bacterial cells use discrete transcription patterns to control cell cycle progression. Global transcription analysis of synchronized Caulobacter crescentus cells was used to identify 553 genes (19% of the genome) whose messenger RNA levels varied as a function of the cell cycle. We conclude that in bacteria, as in yeast, (i) genes involved in a given cell function are activated at the time of execution of that function, (ii) genes encoding proteins that function in complexes are coexpressed, and (iii) temporal cascades of gene expression control multiprotein structure biogenesis. A single regulatory factor, the CtrA member of the two-component signal transduction family, is directly or indirectly involved in the control of 26% of the cell cycle-regulated genes.

    View details for Web of Science ID 000165870600056

    View details for PubMedID 11118148

  • Toggles and oscillators: new genetic circuit designs BIOESSAYS Judd, E. M., Laub, M. T., McAdams, H. H. 2000; 22 (6): 507-509

    Abstract

    Two recent papers report the de novo design of a functioning biological circuit using well-characterized genetic elements.(1,2) Gardner et al. designed and constructed a genetic toggle switch while Elowitz and Leibler built an oscillating genetic circuit. Both circuits were designed with the aid of mathematical models. These papers demonstrate progress towards the unification of theory and experiment in the study of genetic circuits. Comparison of the predicted and observed behavior of the circuits, however, shows that the models explain only some of the circuits' properties. Further study of the observed behaviors not predicted by the model would lead to new insight into the properties of genetic networks. BioEssays 22:507-509, 2000.

    View details for Web of Science ID 000087379200003

    View details for PubMedID 10842304

  • Towards a circuit engineering discipline. Current biology McAdams, H. H., Arkin, A. 2000; 10 (8): R318-20

    Abstract

    Genetic circuits can now be engineered that perform moderately complicated switching functions or exhibit predictable dynamical behavior. These 'forward engineering' techniques may have to be combined with directed evolution techniques to produce robustness comparable with naturally occurring circuits.

    View details for PubMedID 10801411

  • Gene regulation: Towards a circuit engineering discipline CURRENT BIOLOGY McAdams, H. H., Arkin, A. 2000; 10 (8): R318-R320
  • It's a noisy business! Genetic regulation at the nanomolar scale TRENDS IN GENETICS McAdams, H. H., Arkin, A. 1999; 15 (2): 65-69

    Abstract

    Many molecules that control genetic regulatory circuits act at extremely low intracellular concentrations. Resultant fluctuations (noise) in reaction rates cause large random variation in rates of development, morphology and the instantaneous concentration of each molecular species in each cell. To achieve regulatory reliability in spite of this noise, cells use redundancy in genes as well as redundancy and extensive feedback in regulatory pathways. However, some regulatory mechanisms exploit this noise to randomize outcomes where variability is advantageous.

    View details for Web of Science ID 000079419400005

    View details for PubMedID 10098409

  • Stochastic kinetic analysis of developmental pathway bifurcation in phage lambda-infected Escherichia coli cells GENETICS Arkin, A., Ross, J., McAdams, H. H. 1998; 149 (4): 1633-1648

    Abstract

    Fluctuations in rates of gene expression can produce highly erratic time patterns of protein production in individual cells and wide diversity in instantaneous protein concentrations across cell populations. When two independently produced regulatory proteins acting at low cellular concentrations competitively control a switch point in a pathway, stochastic variations in their concentrations can produce probabilistic pathway selection, so that an initially homogeneous cell population partitions into distinct phenotypic subpopulations. Many pathogenic organisms, for example, use this mechanism to randomly switch surface features to evade host responses. This coupling between molecular-level fluctuations and macroscopic phenotype selection is analyzed using the phage lambda lysis-lysogeny decision circuit as a model system. The fraction of infected cells selecting the lysogenic pathway at different phage:cell ratios, predicted using a molecular-level stochastic kinetic model of the genetic regulatory circuit, is consistent with experimental observations. The kinetic model of the decision circuit uses the stochastic formulation of chemical kinetics, stochastic mechanisms of gene expression, and a statistical-thermodynamic model of promoter regulation. Conventional deterministic kinetics cannot be used to predict statistics of regulatory systems that produce probabilistic outcomes. Rather, a stochastic kinetic analysis must be used to predict statistics of regulatory outcomes for such stochastically regulated systems.

    View details for Web of Science ID 000075232100002

    View details for PubMedID 9691025

  • Simulation of prokaryotic genetic circuits ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE McAdams, H. H., Arkin, A. 1998; 27: 199-224

    Abstract

    Biochemical and genetic approaches have identified the molecular mechanisms of many genetic reactions, particularly in bacteria. Now a comparably detailed understanding is needed of how groupings of genes and related protein reactions interact to orchestrate cellular functions over the cell cycle, to implement preprogrammed cellular development, or to dynamically change a cell's processes and structures in response to environmental signals. Simulations using realistic, molecular-level models of genetic mechanisms and of signal transduction networks are needed to analyze dynamic behavior of multigene systems, to predict behavior of mutant circuits, and to identify the design principles applicable to design of genetic regulatory circuits. When the underlying design rules for regulatory circuits are understood, it will be far easier to recognize common circuit motifs, to identify functions of individual proteins in regulation, and to redesign circuits for altered functions.

    View details for Web of Science ID 000074324000009

    View details for PubMedID 9646867

  • Stochastic mechanisms in gene expression PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA McAdams, H. H., Arkin, A. 1997; 94 (3): 814-819

    Abstract

    In cellular regulatory networks, genetic activity is controlled by molecular signals that determine when and how often a given gene is transcribed. In genetically controlled pathways, the protein product encoded by one gene often regulates expression of other genes. The time delay, after activation of the first promoter, to reach an effective level to control the next promoter depends on the rate of protein accumulation. We have analyzed the chemical reactions controlling transcript initiation and translation termination in a single such "genetically coupled" link as a precursor to modeling networks constructed from many such links. Simulation of the processes of gene expression shows that proteins are produced from an activated promoter in short bursts of variable numbers of proteins that occur at random time intervals. As a result, there can be large differences in the time between successive events in regulatory cascades across a cell population. In addition, the random pattern of expression of competitive effectors can produce probabilistic outcomes in switching mechanisms that select between alternative regulatory paths. The result can be a partitioning of the cell population into different phenotypes as the cells follow different paths. There are numerous unexplained examples of phenotypic variations in isogenic populations of both prokaryotic and eukaryotic cells that may be the result of these stochastic gene expression mechanisms.

    View details for Web of Science ID A1997WG23400009

    View details for PubMedID 9023339

  • CIRCUIT SIMULATION OF GENETIC NETWORKS SCIENCE McAdams, H. H., Shapiro, L. 1995; 269 (5224): 650-656

    Abstract

    Genetic networks with tens to hundreds of genes are difficult to analyze with currently available techniques. Because of the many parallels in the function of these biochemically based genetic circuits and electrical circuits, a hybrid modeling approach is proposed that integrates conventional biochemical kinetic modeling within the framework of a circuit simulation. The circuit diagram of the bacteriophage lambda lysislysogeny decision circuit represents connectivity in signal paths of the biochemical components. A key feature of the lambda genetic circuit is that operons function as active integrated logic components and introduce signal time delays essential for the in vivo behavior of phage lambda.

    View details for Web of Science ID A1995RM70200022

    View details for PubMedID 7624793

  • Dynamic nuclear polarization of liquid helium-three by optical pumping Phys. Rev. McAdams, H. H., Walters, G. K. 1968; 170 (1): 276
  • New Liquid 4HE Experimental cryostat Rev. Sci. Instr. McAdams, H. H. 1968; 39 (1)
  • Dynamic polarization of liquid helium three Physical Review Letters McAdams, H. H., Walters, G. K. 1967; 18 (12): 436

Conference Proceedings


  • Assembly of a bacterial cell division machine. Goley, E. D., Yeh, Y., McAdams, H., Shapiro, L. AMER SOC CELL BIOLOGY. 2011
  • Integrated protein interaction networks for 11 microbes Srinivasan, B. S., Novak, A. F., Flannick, J. A., Batzoglou, S., McAdams, H. H. SPRINGER-VERLAG BERLIN. 2006: 1-14

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