Esteban Toro

A spindle-like apparatus guides bacterial chromosome segregation

Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. We show that the C. crescentus ATPase ParA forms linear polymers in vitro and assembles into a narrow linear structure in vivo. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. We propose that this ParB-stimulated ParA depolymerization activity moves the centromere to the opposite cell pole through a burnt bridge Brownian ratchet mechanism. Finally, we identify the pole-specific TipN protein as a new component of the Par system that is required to maintain the directionality of DNA transfer towards the new cell pole. Our results elucidate a bacterial chromosome segregation mechanism that features basic operating principles similar to eukaryotic mitotic machines, including a multivalent protein complex at the centromere that stimulates the dynamic disassembly of polymers to move chromosomes into daughter compartments.

Bacterial Chromosome Organization and Segregation

Bacterial chromosomes are generally approximately 1000 times longer than the cells in which they reside, and concurrent replication, segregation, and transcription/translation of this crowded mass of DNA poses a challenging organizational problem. Recent advances in cell-imaging technology with subdiffraction resolution have revealed that the bacterial nucleoid is reliably oriented and highly organized within the cell. Such organization is transmitted from one generation to the next by progressive segregation of daughter chromosomes and anchoring of DNA to the cell envelope. Active segregation by a mitotic machinery appears to be common; however, the mode of chromosome segregation varies significantly from species to species.

Caulobacter requires a dedicated mechanism to initiate chromosome segregation

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.

The Evolution of Jumping Performance in Caribbean Anolis Lizards: Solutions to Biomechanical Trade‐Offs

Adaptationist theory predicts that species will evolve functional specializations for occupying different ecological niches. However, whereas performance traits are often complex, most comparative functional studies examine only simple measures of performance (e.g., sprint speed). Here we examine multiple facets of jumping biomechanics in 12 species of Caribbean Anolis lizards. These 12 species represent six ecomorphs, which are distinct ecological and morphological entities that have independently evolved on different Caribbean islands. We first show that the optimal angles for jumping maximum horizontal distances range from 39° to 42°, but the average jump angle of the 12 species is about 36°. Interestingly, these “suboptimal” jumping angles result in only a small decrement in jump distance but substantial savings in flight duration and jump height. Further, our data show that the two key variables associated with increased jumping velocity (hindlimb length and takeoff acceleration) are independent of one another. Thus, there are two possible ways to achieve superior jumping capabilities: to evolve more muscular limbs—as stronger legs will produce more force and, hence, more acceleration—or evolve longer limbs. Our data show that anole species face trade‐offs that prevent them from simultaneously optimizing different aspects of jumping ability but that they appear to have evolved behaviors that partially overcome these trade‐offs.

A biomechanical analysis of intra- and interspecific scaling of jumping and morphology in Caribbean Anolis lizards

SUMMARY Scaling models predict how functional variables change as animals grow or increase in size evolutionarily. However, few experimental studies have found support for the predictions of these models. Here, we use a force plate to investigate the scaling of functional variables associated with jumping within (for three species) and across adults of 12 species of Anolis lizards. Both ontogenetically (with the exception of Anolis carolinensis) and across the 12 species examined, limb dimensions increased geometrically, making Anolis lizards an ideal study system to test the predictions of geometric scaling models. However, both the ontogenetic and interspecific scaling of functional variables deviated in several aspects from model predictions. Unexpectedly, the scaling of functional variables such as acceleration differed for different species. Whereas acceleration capacity increases with hindlimb length for A. carolinensis, no relationship was detected for the other two species. Interspecifically, the inclusion of two large species in our analysis appears to drive the absence of a correlation between acceleration capacity and hindlimb length across species. These data suggest that selection for enhanced jumping performance is relaxed in larger anoles and support the notion that no scaling model seems to be able to comprehensively predict changes in function with size across species; rather, natural selection seems to drive changes in the scaling relationships of some key variables such as force output or acceleration capacity.

Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement

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, 〈r〉, scale as n0.22, where n is the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n0.22 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 μm. 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.

Dynamic Chromosome Organization and Protein Localization Coordinate the Regulatory Circuitry that Drives the Bacterial Cell Cycle

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.

In vitro fertilization and artificial activation of eggs of the direct-developing anuran Eleutherodactylus coqui

Although much is known about the reproductive biology of pond-breeding frogs, there is comparatively little information about terrestrial-breeding anurans, a highly successful and diverse group. This study investigates the activation and in vitro fertilization of eggs of the Puerto Rican coqui frog obtained by hormonally induced ovulation. We report that spontaneous activation occurs in 34% of eggs, probably in response to mechanical stress during oviposition. Artificial activation, as evidenced by the slow block to polyspermy and the onset of zygote division, was elicited both by mechanical stimulation and calcium ionophore exposure in 64% and 83% of the cases, respectively. Finally, one in vitro fertilization protocol showed a 27% success rate, despite the fact that about one third of all unfertilized eggs obtained by hormone injection auto-activate. We expect these findings to aid in the conservation effort of Eleutherodactylus frogs, the largest vertebrate genus.

A versatile platform strain for high-fidelity multiplex genome editing

Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (1) a refactored λ-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (2) genetic modifications that boost recombination efficiency, and (3) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a seven-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context-neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.

Chapter 18 Multiplexed Quantitative Proteomics Using Mass Spectrometry

Publisher Summary This chapter discusses a proteomics approach that uses isobaric N-terminal peptide tagging with subsequent matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometry (MS) to perform multiplexed protein identification and quantitation. The protein quantitation approach makes use of isobaric tags to chemically modify peptide N-termini and lysine amine side chains. This technology consists of a set of four different reagents whose chemical structures and nominal molecular masses are the same. The reagent system uses an N-hydroxy succinimide ester (NHS ester) functional group to covalently attach to peptide amine groups in solution. There are several strengths of the isobaric tagging approach in the context of proteomics applications. Because there are four unique signature ions, up to four separate samples can be compared quantitatively in a single experiment. This is the highest degree of multiplexing currently available for widespread use in MS analysis of proteins. The fragmentation to produce low-mass signature ions appears to be efficient for a number of MS platforms utilizing both MALDI and electrospray ionization (ESI). This aspect of the approach lends itself to use in laboratories that already have access to MS instrumentation for protein and peptide analysis. Because all peptides in a mixture will be tagged, this technique is applicable to the analysis of posttranslational modifications. The chapter concludes by discussing two quantitative applications of multiplexed protein quantitation using isobaric tagging: bacterial cell cycle protein quantitation and characterization of a novel protease.