Bacteriophage Lambda Research Articles

Histones direct site-specific CRISPR spacer acquisition in model archaeon.

CRISPR-Cas systems provide heritable immunity against viruses and other mobile genetic elements by incorporating fragments of invader DNA into the host CRISPR array as spacers. Integration of new spacers is localized to the 5' end of the array, and in certain Gram-negative Bacteria this polarized localization is accomplished by the integration host factor. For most other Bacteria and Archaea, the mechanism for 5' end localization is unknown. Here we show that archaeal histones play a key role in directing integration of CRISPR spacers. In Pyrococcus furiosus, deletion of either histone A or B impairs integration. In vitro, purified histones are sufficient to direct integration to the 5' end of the CRISPR array. Archaeal histone tetramers and bacterial integration host factor induce similar U-turn bends in bound DNA. These findings indicate a co-evolution of CRISPR arrays with chromosomal DNA binding proteins and a widespread role for binding and bending of DNA to facilitate accurate spacer integration.

Rapid Fluorescence-Free Detection of DNA Loop-Mediated Isothermal Amplification on Bare ITO Surface Under EG-FET Scheme

An extended gate field effect transistor (EG-FET)-based chip with a precision temperature control system is developed for real-time and fluorescence-free loop-mediated isothermal amplification (LAMP) detection. A label-free, non-optical instant method was used to detect the hydrogen ions released during nucleic acid amplification, rather than indirect measurements. The system comprises an EG-FET sensor, a temperature sensor, a heating block, and a signal processing system. Industrial grade bare indium tin oxide (ITO) is firstly demonstrated as the sensing material for nucleic acid amplification detection, such that stable and high hydrogen ion detection performance of -80.1±0.03 mV/pH with R <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> of 0.998 is achieved with the developed sensor. Moreover, a printed circuit board (PCB) with high thermal conductivity was used as the sensor base to provide a uniform temperature distribution during the reaction. The results show that the system maintains a stable LAMP assay temperature of 63°C with a variation of just 0.25°C. Moreover, in a pH-sensitive LAMP assay, the developed sensor achieves a limit-of-detection down to 10 genomic copies per reaction of lambda phage DNA within 17 minutes. The novel and performance iso-thermal EG-FET system is capable of real-time detecting the LAMP DNA amplification, and thus has significant potential for future biomedical applications.

Bacterial Swarm-Mediated Phage Transportation Disrupts a Biofilm Inherently Protected from Phage Penetration.

Physical forces that arise due to bacterial motility and growth play a significant role in shaping the biogeography of the human oral microbiota. Bacteria of the genus Capnocytophaga are abundant in the human oral microbiota and yet very little is known about their physiology. The human oral isolate Capnocytophaga gingivalis exhibits robust gilding motility that is driven by the rotary type 9 secretion system (T9SS), and cells of C. gingivalis transport nonmotile oral microbes as cargo. Phages, i.e., viruses that infect bacteria, are found in abundance within the microbiota. By tracking fluorescently labeled lambda phages that do not infect C. gingivalis, we report active phage transportation by C. gingivalis swarms. Lambda phage-carrying C. gingivalis swarms were propagated near an Escherichia coli colony. The rate of disruption of the E. coli colony increased 10 times compared with a control where phages simply diffused to the E. coli colony. This finding suggests a mechanism where fluid flows produced by motile bacteria increase the rate of transport of phages to their host bacterium. Additionally, C. gingivalis swarms formed tunnel-like structures within a curli fiber-containing E. coli biofilm that increased the efficiency of phage penetration. Our data suggest that invasion by a C. gingivalis swarm changes the spatial structure of the prey biofilm and further increases the penetration of phages. IMPORTANCE Dysbiosis of the human oral microbiota is associated with several diseases, but the factors that shape the biogeography of the oral microbiota are mostly opaque. Biofilms that form in the human supragingival and subgingival regions have a diverse microbial community where some microbes form well-defined polymicrobial structures. C. gingivalis, a bacterium abundant in human gingival regions, has robust gliding motility that is powered by the type 9 secretion system. We demonstrate that swarms of C. gingivalis can transport phages through a complex biofilm which increases the death rate of the prey biofilm. These findings suggest that C. gingivalis could be used as a vehicle for the transportation of antimicrobials and that active phage transportation could shape the spatial structure of a microbial community.

Identification, Characterization, and Genome Analysis of Two Novel Temperate Pseudomonas protegens Phages PseuP_222 and PseuP_224.

Two novel P. protegens bacteriophages PseuP_222 and Pseu_224 and their host P. protegens CEMTC 4060 were isolated from the same sample (Inya river, Siberia). Both phages have siphovirus morphology and belong to lambdoid phages. Comparative genome analysis revealed a low nucleotide and amino acid sequence similarity of PseuP_222 and PseuP_224 between themselves, and between them and other lambdoid phages. Bioinformatics analysis indicated that PseuP_222 and PseuP_224 are members of a genetically diverse group of phages of environmental Pseudomonas spp.; this group is distant from a large group of P. aeruginosa phages. In phylogenetic trees, the positioning of the terminase large subunits, major capsid proteins, tail tape measure proteins, and CI-like repressors of PseuP_222 and PseuP_224 were remote and changed relative to those of the Escherichia lambda phage and lambdoid phages of Pseudomonas spp. However, the nucleoid-associated protein NdpA/YejK and P5-like structural protein from both phages showed high similarity and were not found in lambda phage and other lambdoid phages of Pseudomonas spp. Substantial divergences of the PseuP_222 and PseuP_224 genomes and proteomes indicated that the evolutionary history of these phages was mostly independent and they probably began to use one host only recently.

A Microstrip Transmission Line Biosensor to Measure the Interaction between Microliter Aqueous Solutions and 1.0-17.0 GHz Radio Frequencies.

Radio frequency (RF) biosensors are an expanding field of interest because of the ability to design noninvasive, label-free, low-production-cost sensing devices. Previous works identified the need for smaller experimental devices, requiring nanoliter to milliliter sampling volumes and increased capability of repeatable and sensitive measurement capability. The following work aims to verify a millimeter-sized, microstrip transmission line biosensor design with a microliter well operating on a broadband radio frequency range of 1.0-17.0 GHz. Three successive experiments were performed to provide evidence for (1) repeatability of measurements after loading/unloading the well, (2) sensitivity of measurement sets, and (3) methodology verification. Materials under test (MUTs) loaded into the well included deionized water, Tris-EDTA buffer, and lambda DNA. S-parameters were measured to determine interaction levels between the radio frequencies and MUTs during the broadband sweep. MUTs increasing in concentration were repeatably detected and demonstrated high measurement sensitivity, with the highest error value observed being 0.36%. Comparing Tris-EDTA buffer versus lambda DNA suspended in Tris-EDTA buffer suggests that introducing lambda DNA into the Tris-EDTA buffer repeatably alters S-parameters. The innovative aspect of this biosensor is that it can measure interactions of electromagnetic energy and MUTs in microliter quantities with high repeatability and sensitivity.

The ecological consequences and evolution of retron-mediated suicide as a way to protect Escherichia coli from being killed by phage.

Retrons were described in 1984 as DNA sequences that code for a reverse transcriptase and a unique single-stranded DNA/RNA hybrid called multicopy single-stranded DNA (msDNA). It would not be until 2020 that a function was shown for retrons, when compelling evidence was presented that retrons activate an abortive infection pathway in response to bacteriophage (phage) infection. When infected with the virulent mutant of the phage lambda, λVIR, and to a lesser extent, other phages, a retron designated Ec48 is activated, the Escherichia coli bearing this retron element dies, and the infecting phage is lost. With the aid of a mathematical model, we explore the a priori conditions under which retrons will protect bacterial populations from predation by phage and the conditions under which retron-bearing bacteria will evolve in populations without this element. Using isogenic E. coli with and without Ec48 and λVIR, we estimated the parameters of our model and tested the hypotheses generated from our analysis of its properties. Our models and experiments demonstrate that cells expressing a retron-mediated abortive infection system can protect bacterial populations. Our results demonstrate that retron bearing bacteria only have a competitive advantage under a limited set of conditions.

Development of a disposable and easy-to-fabricate microfluidic PCR device for DNA amplification

The production of microfluidic PCR devices within lab settings in a straightforward process and large numbers is limited due to complex methods of fabrication and high cost of development. In this study, we developed a unique disposable, low-cost, and easy-to-fabricate microfluidic PCR chip, comprised of a passive micromixer, reaction well, microheater, and a dynamic temperature control system. In addition to being compact, this device has the advantages of microfluidic PCR systems, such as low power and sample consumption, as well as being disposable and easy to fabricate. The disposable part of the chip is easy to fabricate and eliminates the run-to-run carryover contamination while its integration with a reusable heating system reduces the cost of producing a micro PCR device. The geometry of the micromixer and the pattern of microheater electrodes are analyzed through experiments to achieve comparable amplification to a conventional thermocycler. The temperature control system is designed to monitor the temperature within the reaction well with ±1 °C precision. Finally, the reliability and repeatability of the microfluidic device were verified by amplifying lambda (λ) DNA. The proposed device demonstrated high-quality amplification comparable to the conventional PCR and is an adaptable and low-cost on-chip PCR device for reliable molecular diagnostics.

Novel molecular requirements for CRISPR RNA-guided transposition.

CRISPR-associated transposases(CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system(VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CAST systems.

Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara.

Bacteria colonize specific niches in the animal gut. However, the genetic basis of these associations is often unclear. The proteobacterium Frischella perrara is a widely distributed gut symbiont of honey bees. It colonizes a specific niche in the hindgut and causes a characteristic melanization response. Genetic determinants required for the establishment of this association, or its relevance for the host, are unknown. Here, we independently isolated three point mutations in genes encoding the DNA-binding protein integration host factor (IHF) in F. perrara. These mutants abolished the production of an aryl polyene metabolite causing the yellow colony morphotype of F. perrara. Inoculation of microbiota-free bees with one of the mutants drastically decreased gut colonization of F. perrara. Using RNAseq, we found that IHF affects the expression of potential colonization factors, including genes for adhesion (type 4 pili), interbacterial competition (type 6 secretion systems), and secondary metabolite production (colibactin and aryl polyene biosynthesis). Gene deletions of these components revealed different colonization defects depending on the presence of other bee gut bacteria. Interestingly, one of the T6SS mutants did not induce the scab phenotype anymore despite colonizing at high levels, suggesting an unexpected role in bacteria-host interaction. IHF is conserved across many bacteria and may also regulate host colonization in other animal symbionts.

CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes.

CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution.

Extremely early onset of nonlinear viscoelasticity in dynamic shear of ideally monodisperse DNA

AbstractThis work reports that the rheology of ideally monodisperse lambda () phage DNA solutions is extremely sensitive to the applied strain in dynamic oscillatory experiments. DNA exhibits nonlinearity at strains far smaller than what is observed in conventional synthetic polymers. However, it is found that polydisperse calf thymus DNA does not exhibit the extreme strain sensitivity. By mixing samples of monodisperse DNA with the polydisperse calf thymus DNA, we correlate the molecular weight distribution (or polydispersity) to the onset of nonlinear viscoelasticity. We demonstrate that the strain sensitivity weakens as the polydispersity index increases. This is the first work correlating the inception of nonlinear viscoelasticity in entangled polymer system such as lambda DNA solution to the polydispersity of the samples. The experimental data suggests that ideally monodisperse polymer systems are very sensitive to the applied strain and have a very early transition to the nonlinear regime. We conclude that nominally monodisperse synthetic polymers are not sufficiently monodispersed to exhibit the early onset.

Helper-embedded satellites from an integrase clade that repeatedly targets prophage late genes.

Satellites such as phage-induced chromosomal islands (PICIs) are mobile genetic elements relying on helper phages for their mobilization, through trans-regulatory interactions. We discovered a PICI with a more intimate cis-regulatory configuration, integrated within a late gene of its helper prophage. This helper-embedded PICI (HE-PICI) configuration delays expression of the interrupted helper late gene until the satellite excises and provides passive helper-driven components to both HE-PICI replication and late transcription. Upon induction of a helper-satellite composite, precise excision of the entire composite was observed, followed by composite replication, then satellite excision. We mapped 491 additional HE-PICIs to one of 14 sites in cognates of phage lambda late genes. Associated integrases form a single phylogenetic clade with subclades respecting the 14 site groups, exhibiting repeated tropism for prophage late genes as new integration sites evolve. Four ordered zones in a general gram-negative PICI genome organization are: an integration zone encoding integrase and AlpA, a dynamic zone encoding members of the Bro-N network of domain-swapping DNA-interactive proteins and immunity repressor RNAs, a replication zone, and a dynamic late zone in which clusters as large as 17 consecutive helper prophage late genes have been captured. Helper-embedded satellites present new dimensions in satellite/helper relationships.

Isolation of an Ecotropic Porcine Endogenous Retrovirus PERV-C from a Yucatan SLAD/D Inbred Miniature Swine.

Xenotransplantation may compensate the limited number of human allografts for transplantation using pigs as organ donors. Porcine endogenous retroviruses inherit infectious potential if pig cells, tissues, or organs were transplanted to immunosuppressed human recipients. Particularly, ecotropic PERV-C that could recombine with PERV-A to highly replication-competent human-tropic PERV-A/C should be excluded from pig breeds designed for xenotransplantation. Because of their low proviral background, SLAD/D (SLA, swine leukocyte antigen) haplotype pigs are potential candidates as organ donors as they do not bear replication-competent PERV-A and -B, even if they carry PERV-C. In this work, we characterized their PERV-C background isolating a full-length PERV-C proviral clone number 561 from a SLAD/D haplotype pig genome displayed in a bacteriophage lambda library. The provirus truncated in env due to cloning in lambda was complemented by PCR, and the recombinants were functionally characterized, confirming an increased infectivity in vitro compared to other PERV-C. Recombinant clone PERV-C(561) was chromosomally mapped by its 5'-proviral flanking sequences. Full-length PCR using 5'-and 3'-flanking primers specific to the PERV-C(561) locus verified that this specific SLAD/D haplotype pig harbors at least one full-length PERV-C provirus. The chromosomal location is different from that of the previously described PERV-C(1312) provirus, which was derived from the porcine cell-line MAX-T. The sequence data presented here provide further knowledge about PERV-C infectivity and contribute to targeted knockout in order to generate PERV-C-free founder animals. IMPORTANCE Yucatan SLAD/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro, the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.

Mycobacterial IHF is a highly dynamic nucleoid-associated protein that assists HupB in organizing chromatin.

Nucleoid-associated proteins (NAPs) crucially contribute to organizing bacterial chromatin and regulating gene expression. Among the most highly expressed NAPs are the HU and integration host factor (IHF) proteins, whose functional homologues, HupB and mycobacterial integration host factor (mIHF), are found in mycobacteria. Despite their importance for the pathogenicity and/or survival of tubercle bacilli, the role of these proteins in mycobacterial chromosome organization remains unknown. Here, we used various approaches, including super-resolution microscopy, to perform a comprehensive analysis of the roles of HupB and mIHF in chromosome organization. We report that HupB is a structural agent that maintains chromosome integrity on a local scale, and that the lack of this protein alters chromosome morphology. In contrast, mIHF is a highly dynamic protein that binds DNA only transiently, exhibits susceptibility to the chromosomal DNA topology changes and whose depletion leads to the growth arrest of tubercle bacilli. Additionally, we have shown that depletion of Mycobacterium smegmatis integration host factor (msIHF) leads to chromosome shrinkage and replication inhibition.

The book of Lambda does not tell us that naturally occurring lysogens of Escherichia coli are likely to be resistant as well as immune

The most significant difference between bacteriophages functionally and ecologically is whether they are purely lytic (virulent) or temperate. Virulent phages can only be transmitted horizontally by infection, most commonly with the death of their hosts. Temperate phages can also be transmitted horizontally, but upon infection of susceptible bacteria, their genomes can be incorporated into that of their host's as a prophage and be transmitted vertically in the course of cell division by their lysogenic hosts. From what we know from studies with the temperate phage Lambda and other temperate phages, in laboratory culture, lysogenic bacteria are protected from killing by the phage coded for by their prophage by immunity; where upon infecting lysogens, the free temperate phage coded by their prophage is lost. Why are lysogens not only resistant but also immune to the phage coded by their prophage since immunity does not confer protection against virulent phages? To address this question, we used a mathematical model and performed experiments with temperate and virulent mutants of the phage Lambda in laboratory culture. Our models predict and experiments confirm that selection would favor the evolution of resistant and immune lysogens, particularly if the environment includes virulent phage that shares the same receptors as the temperate. To explore the validity and generality of this prediction, we examined 10 lysogenic Escherichia coli from natural populations. All 10 were capable of forming immune lysogens, but their original hosts were resistant to the phage coded by their prophage.

Early antitermination in the atypical coliphage mEp021 mediated by the Gp17 protein

The coliphage mEp021 belongs to a phage group with a unique immunity repressor, and its life cycle requires the host factor Nus. mEp021 has been classified as non-lambdoid based on its specific characteristics. The mEp021 genome carries a gene encoding an Nλ-like antiterminator protein, termed Gp17, and three nut sites (nutL, nutR1, and nutR2). Analysis of plasmid constructs containing these nut sites, a transcription terminator, and a GFP reporter gene showed high levels of fluorescence when Gp17 was expressed, but not in its absence. Like lambdoid N proteins, Gp17 has an arginine-rich motif (ARM), and mutations in its arginine codons inhibit its function. In infection assays using the mutant phage mEp021ΔGp17::Kan (where gp17 has been deleted), gene transcripts located downstream of transcription terminators were obtainedonly when Gp17 was expressed. In contrast to phage lambda, mEp021 virus particle production was partially restored (>1/3 relative to wild type) when nus mutants (nusA1, nusB5, nusC60, and nusE71) were infected with mEp021 and Gp17 was overexpressed. Our results suggest that RNA polymerase reads through the third nut site (nutR2), which is more than 7.9 kbp downstream of nutR1.

Laboratory strains of Escherichia coli K-12: things are seldom what they seem.

Escherichia coli K-12 was originally isolated 100 years ago and since then it has become an invaluable model organism and a cornerstone of molecular biology research. However, despite its pedigree, since its initial isolation E. coli K-12 has been repeatedly cultured, passaged and mutagenized, resulting in an organism that carries many genetic changes. To understand more about this important model organism, we have sequenced the genomes of two ancestral K-12 strains, WG1 and EMG2, considered to be the progenitors of many key laboratory strains. Our analysis confirms that these strains still carry genetic elements such as bacteriophage lambda (λ) and the F plasmid, but also indicates that they have undergone extensive laboratory-based evolution. Thus, scrutinizing the genomes of ancestral E. coli K-12 strains leads us to examine whether E. coli K-12 is a sufficiently robust model organism for 21st century microbiology.

Fluidification of Entanglements by a DNA Bending Protein

In spite of the nanoscale and single-molecule insights into nucleoid associated proteins (NAPs), their role in modulating the mesoscale viscoelasticity of entangled DNA has been overlooked so far. By combining microrheology and molecular dynamics simulation, we find that the abundant NAP "integration host factor" (IHF) lowers the viscosity of entangled λDNA 20-fold at physiological concentrations and stoichiometries. Our results suggest that IHF may play a previously unappreciated role in resolving DNA entanglements and in turn may be acting as a "genomic fluidizer" for bacterial genomes.

Regulation of the phage lambda viral packaging motor's grip and DNA end-clamp mechanism

Many viruses utilize ATP-powered motors to package DNA into viral capsids. Here we use single DNA manipulation with optical tweezers and rapid solution exchange to interrogate the dynamics of DNA gripping by the phage lambda motor. Binding of poorly hydrolyzed ATP analogs results in persistent gripping and when slips occasionally occur, the release velocity is very slow, indicating significant friction between the motor and DNA. ADP induces more frequent transitions between gripping and faster slipping. Independent of nucleotides, an “end clamp” mechanism arrests slipping when the end of the DNA is about to exit the capsid. These features are qualitatively similar to those exhibited by the phage T4 motor, although there are some notable differences. Specifically, there are quantitative differences in the DNA grip/release kinetics and their dependence on nucleotides and applied force. But the most striking difference is that there are frequent long pauses during DNA exit from the capsid even in the absence of nucleotides. This pausing occurs at low capsid filling and does not increase with length of DNA packaged, indicating that it is not caused by jamming/knotting of the packaged DNA, but is attributable to fluctuation of apo-state motor subunits into a DNA gripping conformation. This finding supports our previous hypothesis that pauses which are frequently observed during packaging are caused by fluctuation of motor subunits into such a conformation. Overall, compared with T4, the lambda motor exhibits stronger interactions with DNA, which may be attributable to the small terminase subunit in the lambda packaging motor complex.

Role of DNA sliding friction and nonequilibrium dynamics in viral DNA ejection and packaging.

In many viruses DNA is packaged to high densities, resulting in high “internal force” inside the capsid shell. This helps drive DNA ejection through the portal channel when viruses infect cells. Imaging of the in vitro ejection of fluorescent-labeled DNA from phages T5 and lambda found that it was initially slow despite the internal force being highest when the capsid is full. This was attributed to hydrodynamic drag on the tightly-packed DNA, but this hypothesis has not been tested. Here we introduce a new method for probing the DNA mobility by using optical tweezers to pull it out from phage phi29 capsids. This allows us to measure the dynamics with higher precision, avoid using DNA-binding dyes that alter the dynamics, and apply variable forces. We measure even slower DNA exit than reported previously, a steeper, exponential velocity increase as the first 20% of the genome length exits, and find that exit velocity is not proportional to driving force as expected for hydrodynamic friction. Rather, our findings are consistent with theoretical predictions for DNA-DNA sliding friction. We also observe large heterogeneity in the exit dynamics and stochastic pausing, indicating that ejection is a nonequilibrium process, in qualitative agreement with simulations. Features of the pausing suggest it is related to the phenomenon of “clogging” in soft-matter physics, which occurs in the flow of disordered materials through constrictions. Our results indicate that intramolecular friction and nonequilibrium pausing control the kinetics of ejection but that this friction does not significantly affect DNA packaging.