Bacterial Chromosome Research Articles

Dynamics of chromosome organization in a minimal bacterial cell.

Computational models of cells cannot be considered complete unless they include the most fundamental process of life, the replication and inheritance of genetic material. By creating a computational framework to model systems of replicating bacterial chromosomes as polymers at 10bp resolution with Brownian dynamics, we investigate changes in chromosome organization during replication and extend the applicability of an existing whole-cell model (WCM) for a genetically minimal bacterium, JCVI-syn3A, to the entire cell-cycle. To achieve cell-scale chromosome structures that are realistic, we model the chromosome as a self-avoiding homopolymer with bending and torsional stiffnesses that capture the essential mechanical properties of dsDNA in Syn3A. In addition, the conformations of the circular DNA must avoid overlapping with ribosomes identitied in cryo-electron tomograms. While Syn3A lacks the complex regulatory systems known to orchestrate chromosome segregation in other bacteria, its minimized genome retains essential loop-extruding structural maintenance of chromosomes (SMC) protein complexes (SMC-scpAB) and topoisomerases. Through implementing the effects of these proteins in our simulations of replicating chromosomes, we find that they alone are sufficient for simultaneous chromosome segregation across all generations within nested theta structures. This supports previous studies suggesting loop-extrusion serves as a near-universal mechanism for chromosome organization within bacterial and eukaryotic cells. Furthermore, we analyze ribosome diffusion under the influence of the chromosome and calculate in silico chromosome contact maps that capture inter-daughter interactions. Finally, we present a methodology to map the polymer model of the chromosome to a Martini coarse-grained representation to prepare molecular dynamics models of entire Syn3A cells, which serves as an ultimate means of validation for cell states predicted by the WCM.

Robust ParB Binding to Half-parS Sites in Pseudomonas aeruginosa-A Mechanism for Retaining ParB on the Nucleoid?

Chromosome segregation in Pseudomonas aeruginosa is assisted by the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB) and its target parS sequence(s). ParB forms a nucleoprotein complex around four parSs (parS1-parS4) that overlaps oriC and facilitates relocation of newly synthesized ori domains inside the cells by ParA. Remarkably, ParB of P. aeruginosa also binds to numerous heptanucleotides (half-parSs) scattered in the genome. Here, using chromatin immunoprecipitation-sequencing (ChIP-seq), we analyzed patterns of ParB genome occupancy in cells growing under conditions of coupling or uncoupling between replication and cell division processes. Interestingly, a dissipation of ParB-parS complexes and a shift of ParB to half-parSs were observed during the transition from the exponential to stationary phase of growth on rich medium, suggesting the role of half-parSs in retaining ParB on the nucleoid within non-dividing P. aeruginosa cells. The ChIP-seq analysis of strains expressing ParB variants unable to dislocate from parSs showed that the ParB spreading ability is not required for ParB binding to half-parSs. Finally, a P. aeruginosa strain with mutated 25 half-parSs of the highest affinity towards ParB was constructed and analyzed. It showed altered ParB coverage of the oriC region and moderate changes in gene expression. Overall, this study characterizes a novel aspect of conserved bacterial chromosome segregation machinery.

Dual pathogenicity island transfer by piggybacking lateral transduction

Lateral transduction (LT) is the process by which temperate phages mobilize large sections of bacterial genomes. Despite its importance, LT has only been observed during prophage induction. Here, we report that superantigen-carrying staphylococcal pathogenicity islands (SaPIs) employ a related but more versatile and complex mechanism of gene transfer to drive chromosomal hypermobility while self-transferring with additional virulence genes from the host. We found that after phage infection or prophage induction, activated SaPIs form concatamers in the bacterial chromosome by switching between parallel genomic tracks in replication bubbles. This dynamic life cycle enables SaPIbov1 to piggyback its LT of staphylococcal pathogenicity island vSaα, which encodes an array of genes involved in host-pathogen interactions, allowing both islands to be mobilized intact and transferred in a single infective particle. Our findings highlight previously unknown roles of pathogenicity islands in bacterial virulence and show that their evolutionary impact extends beyond the genes they carry.

Physiological and biochemical and molecular genetic characteristics of the bacteria – the basis of the microbial preparation “Bioproductin”

Cultures of Bacillus amyloliquefaciens BIM B-1267 D, B. mojavensis BIM B-1268 D, Priestia megaterium BIM B-1269 D and B. amyloliquefaciens BIM B-1270 D bacteria, which form the basis of the bioproduct “Bioproductin”, have mutually complementary properties – pronounced antimicrobial, nitrogen-fixing, phosphate-mobilizing and phytostimulating activities, the ability to synthesize a number of hydrolytic enzymes (proteinase, endo-1.4-β-xylanase, endo-1.4-β-glucanase, α-amylase and phytase), indolylacetic acid, siderophores. In the genomes of the studied bacterial strains, the presence of genes encoding the synthesis of bacillen, bacillibactin, bacillomycin, difficidin, phengicin, macrolactin, surfactin, and iturin was established, which confirms their significant antimicrobial potential. It is also shown that the genes determining the synthesis of indolylacetic acid – the main hormone of plant growth – are present in the bacterial chromosome.

The gut virome and the relevance of temperate phages in human health.

Alterations in the gut virome impact human health. Bacteriophages, viruses that infect bacteria, dominate the gut virome and are mainly composed by virulent and temperate phages. While virulent phages exclusively replicate within and lyse their bacterial host's cell, temperate phages switch from an integrated state residing within their bacterial host's chromosome to an induced free virion state via an induction event. How often do these induction events occur and what are their implications on gut homeostasis? Here, we summarize the current knowledge of the gut virome based on metagenomics and present how the proportion of induced temperate phages varies amongst individuals, age, and disease states. Finally, we highlight the importance of building upon classical culture-dependent techniques and sequencing approaches to improve our understanding of temperate phages to enable their potential therapeutic use.

Mechanisms of Regulation of Cryptic Prophage-Encoded Gene Products in Escherichia coli.

The dicBF operon of Qin cryptic prophage in Escherichia coli K-12 encodes the small RNA (sRNA) DicF and small protein DicB, which regulate host cell division and are toxic when overexpressed. While new functions of DicB and DicF have been identified in recent years, the mechanisms controlling the expression of the dicBF operon have remained unclear. Transcription from dicBp, the major promoter of the dicBF operon, is repressed by DicA. In this study, we discovered that transcription of the dicBF operon and processing of the polycistronic mRNA is regulated by multiple mechanisms. DicF sRNA accumulates during stationary phase and is processed from the polycistronic dicBF mRNA by the action of both RNase III and RNase E. DicA-mediated transcriptional repression of dicBp can be relieved by an antirepressor protein, Rem, encoded on the Qin prophage. Ectopic production of Rem results in cell filamentation due to strong induction of the dicBF operon, and filamentation is mediated by DicF and DicB. Spontaneous derepression of dicBp occurs in a subpopulation of cells independent of the antirepressor. This phenomenon is reminiscent of the bistable switch of λ phage with DicA and DicC performing functions similar to those of CI and Cro, respectively. Additional experiments demonstrate stress-dependent induction of the dicBF operon. Collectively, our results illustrate that toxic genes carried on cryptic prophages are subject to layered mechanisms of control, some that are derived from the ancestral phage and some that are likely later adaptations. IMPORTANCE Cryptic or defective prophages have lost genes necessary to excise from the bacterial chromosome and produce phage progeny. In recent years, studies have found that cryptic prophage gene products influence diverse aspects of bacterial host cell physiology. However, to obtain a complete understanding of the relationship between cryptic prophages and the host bacterium, identification of the environmental, host, or prophage-encoded factors that induce the expression of cryptic prophage genes is crucial. In this study, we examined the regulation of a cryptic prophage operon in Escherichia coli encoding a small RNA and a small protein that are involved in inhibiting bacterial cell division, altering host metabolism, and protecting the host bacterium from phage infections.

Identification, phylogenetic analysis, and genome mining of the tetracycline-resistant Bacillus thuringiensis strain m401 reveal its potential for biotechnological and biocontrol applications

Bacillus thuringiensis is an entomopathogen belonging to the Bacillus cereus clade. We isolated a tetracycline-resistant strain called m401, recovered it from honey, and identified it as Bacillus thuringiensis sv. kumamotoensis based on the average nucleotide identity calculations (ANIb) comparison and the analysis of the gyrB gene sequences of different B. thuringiensis serovars. Sequences with homology to virulence factors [cytK, nheA, nheB, nheC, hblA, hblB, hblC, hblD, entFM, and inhA] and tetracycline resistance genes [tet(45), tet(V), and tet(M)/tet(W)/tet(O)/tet(S) family] were identified in the bacterial chromosome. The prediction of plasmid-coding regions revealed homolog sequences to the MarR and TetR/AcrR family of transcriptional regulators, toxins, and lantipeptides. The genome mining analysis revealed 12 regions of biosynthetic gene clusters responsible for synthesizing secondary metabolites. We identified biosynthetic gene clusters coding for bacteriocins, siderophores, ribosomally synthesized post-translationally modified peptide products, and non-ribosomal peptide synthetase clusters that provide evidence for the possible use of Bt m401 as a biocontrol agent. Furthermore, Bt m401 showed high inhibition against all Paenibacillus larvae genotypes tested in vitro. In conclusion, Bt m401 owns various genes involved in different biological processes, such as transductional regulators associated with antibiotic resistance, toxins, and antimicrobial peptides with potential biotechnological and biocontrol applications.

CRISPR gene technology—the next best thing in medicine

CRISPR gene technology—the next best thing in medicine

PlasBin-flow: a flow-based MILP algorithm for plasmid contigs binning.

The analysis of bacterial isolates to detect plasmids is important due to their role in the propagation of antimicrobial resistance. In short-read sequence assemblies, both plasmids and bacterial chromosomes are typically split into several contigs of various lengths, making identification of plasmids a challenging problem. In plasmid contig binning, the goal is to distinguish short-read assembly contigs based on their origin into plasmid and chromosomal contigs and subsequently sort plasmid contigs into bins, each bin corresponding to a single plasmid. Previous works on this problem consist of de novo approaches and reference-based approaches. De novo methods rely on contig features such as length, circularity, read coverage, or GC content. Reference-based approaches compare contigs to databases of known plasmids or plasmid markers from finished bacterial genomes. Recent developments suggest that leveraging information contained in the assembly graph improves the accuracy of plasmid binning. We present PlasBin-flow, a hybrid method that defines contig bins as subgraphs of the assembly graph. PlasBin-flow identifies such plasmid subgraphs through a mixed integer linear programming model that relies on the concept of network flow to account for sequencing coverage, while also accounting for the presence of plasmid genes and the GC content that often distinguishes plasmids from chromosomes. We demonstrate the performance of PlasBin-flow on a real dataset of bacterial samples. https://github.com/cchauve/PlasBin-flow.

Prophage identification and molecular analysis in the genomes of Pseudomonas aeruginosa strains isolated from critical care patients.

Prophages are bacteriophages integrated into the bacterial host's chromosome. This research aims to analyze and characterize the existing prophages within a collection of 53 Pseudomonas aeruginosa strains from intensive care units (ICUs) in Portugal and Spain. A total of 113 prophages were localized in the collection, with 18 of them being present in more than one strain simultaneously. After annotation, five of them were discarded as incomplete, and the 13 remaining prophages were characterized. Of 13, 10 belonged to the siphovirus tail morphology group, 2 to the podovirus tail morphology group, and 1 to the myovirus tail morphology group. All prophages had a length ranging from 20,199 to 63,401 bp and a GC% between 56.2% and 63.6%. The number of open reading frames (ORFs) oscillated between 32 and 88, and in 3/13 prophages, more than 50% of the ORFs had an unknown function. With our findings, we show that prophages are present in the majority of the P. aeruginosa strains isolated from Portuguese and Spanish critically ill patients, many of them found in more than one circulating strain at the same time and following a similar clonal distribution pattern. Although a great sum of ORFs had an unknown function, number of proteins in relation to viral defense (anti-CRISPR proteins, toxin/antitoxin modules, proteins against restriction-modification systems) as well as to prophage interference into their host's quorum sensing system and regulatory cascades were found. This supports the idea that prophages have an influence in bacterial pathogenesis and anti-phage defense. IMPORTANCE Despite being known for decades, prophages remain understudied when compared to the lytic phages employed in phage therapy. This research aims to shed some light into the nature, composition, and role of prophages found within a set of circulating strains of Pseudomas aeruginosa, with special attention to high-risk clones. Given the fact that prophages can effectively influence bacterial pathogenesis, prophage basic research constitutes a topic of growing interest. Furthermore, the abundance of viral defense and regulatory proteins within prophage genomes detected in this study evidences the importance of characterizing the most frequent prophages in circulating clinical strains and in high-risk clones if phage therapy is to be used.

Plassembler: an automated bacterial plasmid assembly tool.

With recent advances in sequencing technologies, it is now possible to obtain near-perfect complete bacterial chromosome assemblies cheaply and efficiently by combining a long-read-first assembly approach with short-read polishing. However, existing methods for assembling bacterial plasmids from long-read-first assemblies often misassemble or even miss bacterial plasmids entirely and accordingly require manual curation. Plassembler was developed to provide a tool that automatically assembles and outputs bacterial plasmids using a hybrid assembly approach. It achieves increased accuracy and computational efficiency compared to the existing gold standard tool Unicycler by removing chromosomal reads from the input read sets using a mapping approach. Plassembler is implemented in Python and is installable as a bioconda package using 'conda install -c bioconda plassembler'. The source code is available on GitHub at https://github.com/gbouras13/plassembler. The full benchmarking pipeline can be found at https://github.com/gbouras13/plassembler_simulation_benchmarking, while the benchmarking input FASTQ and output files can be found at https://doi.org/10.5281/zenodo.7996690.

Temperature-dependent carrier state mediated by H-NS promotes the long-term coexistence of Y. pestis and a phage in soil.

The study of carrier state phages challenged the canonical lytic-lysogenic binary, and carrier state appears to be ubiquitous and ecologically important. However, the mechanisms of the carrier state are not well elucidated due to the limited phage models. Herein, we reported phage HQ103, similar to Escherichia coli phage P2. In contrast to the temperate P2 phage, the HQ103 phage does not insert its genome into the bacterial chromosome and displays a dual behavior depending on the temperature. At 37°C, HQ103 lyses the host and forms clear plaques due to the truncation of repressor CI and mutation of promoter Pc. In contrast, HQ103 maintains a carrier state lifestyle with Y. pestis at an environmental temperature (21°C). Mechanistically, we found that the host-encoded histone-like nucleoid-structuring protein H-NS, which is highly expressed at 21°C to silence the Cox promoter Pe and inhibits the phage lytic cycle. Subsequently, the HQ103 carrier state Y. pestis could grow and co-exist with the phage in the soil at 21°C for one month. Thus, this study reveals a novel carrier state lifestyle of phage HQ103 due to the H-NS mediated xenogeneic silencing and demonstrates that the carrier state lifestyle could promote long-term phage-host coexist in nature.

Transconjugant range of PromA plasmids in microbial communities is predicted by sequence similarity with the bacterial host chromosome.

Nucleotide sequence similarity, including k-mer plasmid composition, has been used for prediction of plasmid evolutionary host range, representing the hosts in which a plasmid has replicated at some point during its evolutionary history. However, the relationships between the bacterial taxa of experimentally identified transconjugants and the predicted evolutionary host ranges are poorly understood. Here, four different PromA group plasmids showing different k-mer compositions were used as model plasmids. Filter mating assays were performed with a donor harbouring plasmids and recipients of bacterial communities extracted from environmental samples. A broad range of transconjugants was obtained with different bacterial taxa. A calculation of the dissimilarities in k-mer compositions as Mahalanobis distance between the plasmid and its sequenced transconjugant chromosomes revealed that each plasmid and transconjugant were significantly more similar than the plasmid and other non-transconjugant chromosomes. These results indicate that plasmids with different k-mer compositions clearly have different host ranges to which the plasmid will be transferred and replicated. The similarity of the nucleotide compositions could be used for predicting not only the plasmid evolutionary host range but also future host ranges.

Ensifer canadensis sp. nov. strain T173T isolated from Melilotus albus (sweet clover) in Canada possesses recombinant plasmid pT173b harbouring symbiosis and type IV secretion system genes apparently acquired from Ensifer medicae.

A bacterial strain, designated T173T, was previously isolated from a root-nodule of a Melilotus albus plant growing in Canada and identified as a novel Ensifer lineage that shared a clade with the non-symbiotic species, Ensifer adhaerens. Strain T173T was also previously found to harbour a symbiosis plasmid and to elicit root-nodules on Medicago and Melilotus species but not fix nitrogen. Here we present data for the genomic and taxonomic description of strain T173T. Phylogenetic analyses including the analysis of whole genome sequences and multiple locus sequence analysis (MLSA) of 53 concatenated ribosome protein subunit (rps) gene sequences confirmed placement of strain T173T in a highly supported lineage distinct from named Ensifer species with E. morelensis Lc04T as the closest relative. The highest digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of genome sequences of strain T173T compared with closest relatives (35.7 and 87.9%, respectively) are well below the respective threshold values of 70% and 95-96% for bacterial species circumscription. The genome of strain T173T has a size of 8,094,229 bp with a DNA G + C content of 61.0 mol%. Six replicons were detected: a chromosome (4,051,102 bp) and five plasmids harbouring plasmid replication and segregation (repABC) genes. These plasmids were also found to possess five apparent conjugation systems based on analysis of TraA (relaxase), TrbE/VirB4 (part of the Type IV secretion system (T4SS)) and TraG/VirD4 (coupling protein). Ribosomal RNA operons encoding 16S, 23S, and 5S rRNAs that are usually restricted to bacterial chromosomes were detected on plasmids pT173d and pT173e (946,878 and 1,913,930 bp, respectively) as well as on the chromosome of strain T173T. Moreover, plasmid pT173b (204,278 bp) was found to harbour T4SS and symbiosis genes, including nodulation (nod, noe, nol) and nitrogen fixation (nif, fix) genes that were apparently acquired from E. medicae by horizontal transfer. Data for morphological, physiological and symbiotic characteristics complement the sequence-based characterization of strain T173T. The data presented support the description of a new species for which the name Ensifer canadensis sp. nov. is proposed with strain T173T (= LMG 32374T = HAMBI 3766T) as the species type strain.

Fluoroquinolone resistance does not facilitate phage Φ13 integration or excision in Staphylococcus aureus.

Prophages of the ΦSa3int family are commonly found in human-associated strains of Staphylococcus aureus where they encode factors for evading the human innate immune system. In contrast, they are usually absent in livestock-associated methicillin-resistant S. aureus (LA-MRSA) strains where the phage attachment site is mutated compared to the human strains. However, ΦSa3int phages have been found in a subset of LA-MRSA strains belonging to clonal complex 398 (CC398), including a lineage that is widespread in pig farms in Northern Jutland, Denmark. This lineage contains amino acid changes in the DNA topoisomerase IV and the DNA gyrase encoded by grlA and gyrA, respectively, which have been associated with fluoroquinolone (FQ) resistance. As both of these enzymes are involved in DNA supercoiling, we speculated that the mutations might impact recombination between the ΦSa3int phage and the bacterial chromosome. To examine this, we introduced the FQ resistance mutations into S. aureus 8325-4attBLA that carry the mutated CC398-like bacterial attachment site for ΦSa3int phages. When monitoring phage integration and release of Φ13, a well-described representative of the ΦSa3int phage family, we did not observe any significant differences between the FQ-resistant mutant and the wild-type strain. Thus our results suggest that mutations in grlA and gyrA do not contribute to the presence of the ΦSa3int phages in LA-MRSA CC398.

Investigating the role of Bacillus subtilis type II toxin-antitoxin system in drought stress survival.

Toxin-antitoxin (TA) systems, present in plasmids and bacterial chromosomes, are widespread in bacteria such as Bacillus subtilis and are known to be involved in growth regulation, bacterial tolerance to environmental stress conditions as well as biofilm formation. The aim of the current study was to investigate the role of TA systems in drought condition stress in B. subtilis isolates. The presence of TA systems including mazF/mazE and yobQ/yobR in B. subtilis (strain 168) was investigated using the polymerase chain reaction (PCR) method. TA system expression at 438 and 548 g/L of ethylene glycol concentrations was evaluated using real-time PCR method and sigB gene was used as internal control. The expression rate (fold change) of mazF toxin gene treated with 438 and 548 g/L of ethylene glycol was 6 and 8.4, respectively. This indicates an increase in the expression of this toxin in drought stress condition. Also, the fold change of mazE antitoxin in the treatment with 438 and 548 g/L of ethylene glycol was 8.6 and 5, respectively. While yobQ/yobR showed a decrease in expression in 438 and 548 g/L of ethylene glycol concentrations. So that the highest expression reduction (8.3) was observed for yobQ gene at the concentration of 548 g/L of ethylene glycol. Results of this study revealed the significant role of B. subtilis TA systems in drought stress which can be considered as the resistance mechanism of this bacterium under stress conditions.

Optimized De Novo Eriodictyol Biosynthesis in Streptomyces albidoflavus Using an Expansion of the Golden Standard Toolkit for Its Use in Actinomycetes.

Eriodictyol is a hydroxylated flavonoid displaying multiple pharmaceutical activities, such as antitumoral, antiviral or neuroprotective. However, its industrial production is limited to extraction from plants due to its inherent limitations. Here, we present the generation of a Streptomyces albidoflavus bacterial factory edited at the genome level for an optimized de novo heterologous production of eriodictyol. For this purpose, an expansion of the Golden Standard toolkit (a Type IIS assembly method based on the Standard European Vector Architecture (SEVA)) has been created, encompassing a collection of synthetic biology modular vectors (adapted for their use in actinomycetes). These vectors have been designed for the assembly of transcriptional units and gene circuits in a plug-and-play manner, as well as for genome editing using CRISPR-Cas9-mediated genetic engineering. These vectors have been used for the optimization of the eriodictyol heterologous production levels in S. albidoflavus by enhancing the flavonoid-3'-hydroxylase (F3'H) activity (by means of a chimera design) and by replacing three native biosynthetic gene clusters in the bacterial chromosome with the plant genes matBC (involved in extracellular malonate uptake and its intracellular activation into malonyl-CoA), therefore allowing more malonyl-CoA to be devoted to the heterologous production of plant flavonoids in this bacterial factory. These experiments have allowed an increase in production of 1.8 times in the edited strain (where the three native biosynthetic gene clusters have been deleted) in comparison with the wild-type strain and a 13 times increase in eriodictyol overproduction in comparison with the non-chimaera version of the F3'H enzyme.

The membrane-binding bacterial toxin long direct repeat D inhibits protein translation

Bacterial plasmids and chromosomes widely contain toxin-antitoxin (TA) loci, which are implicated in stress response, growth regulation and even tolerance to antibiotics and environmental stress. Type I TA systems consist of a stable toxin-expressing mRNA, which is counteracted by an unstable RNA antitoxin. The Long Direct Repeat (LDR-) D locus, a type I TA system of Escherichia Coli (E. coli) K12, encodes a 35 amino acid toxic peptide, LdrD. Despite being characterized as a bacterial toxin, causing rapid killing and nucleoid condensation, little was known about its function and its mechanism of toxicity. Here, we show that LdrD specifically interacts with ribosomes which potentially blocks translation. Indeed, in vitro translation of LdrD-coding mRNA greatly reduces translation efficiency. The structure of LdrD in a hydrophobic environment, similar to the one found in the interior of ribosomes was determined by NMR spectroscopy in 100% trifluoroethanol solution. A single compact α-helix was found which would fit nicely into the ribosomal exit tunnel. Therefore, we conclude that rather than destroying bacterial membranes, LdrD exerts its toxic activity by inhibiting protein synthesis through binding to the ribosomes.

Kinetic principles of ParA2-ATP cycling guide dynamic subcellular localizations in Vibrio cholerae.

Dynamic protein gradients are exploited for the spatial organization and segregation of replicated chromosomes. However, mechanisms of protein gradient formation and how that spatially organizes chromosomes remain poorly understood. Here, we have determined the kinetic principles of subcellular localizations of ParA2 ATPase, an essential spatial regulator of chromosome 2 segregation in the multichromosome bacterium, Vibrio cholerae. We found that ParA2 gradients self-organize in V. cholerae cells into dynamic pole-to-pole oscillations. We examined the ParA2 ATPase cycle and ParA2 interactions with ParB2 and DNA. In vitro, ParA2-ATP dimers undergo a rate-limiting conformational switch, catalysed by DNA to achieve DNA-binding competence. This active ParA2 state loads onto DNA cooperatively as higher order oligomers. Our results indicate that the midcell localization of ParB2-parS2 complexes stimulate ATP hydrolysis and ParA2 release from the nucleoid, generating an asymmetric ParA2 gradient with maximal concentration toward the poles. This rapid dissociation coupled with slow nucleotide exchange and conformational switch provides for a temporal lag that allows the redistribution of ParA2 to the opposite pole for nucleoid reattachment. Based on our data, we propose a 'Tug-of-war' model that uses dynamic oscillations of ParA2 to spatially regulate symmetric segregation and positioning of bacterial chromosomes.

Recollections of a Helmstetter Disciple.

Nearly fifty years ago, it became possible to construct E. coli minichromosomes using recombinant DNA technology. These very small replicons, comprising the unique replication origin of the chromosome oriC coupled to a drug resistance marker, provided new opportunities to study the regulation of bacterial chromosome replication, were key to obtaining the nucleotide sequence information encoded into oriC and were essential for the development of a ground-breaking in vitro replication system. However, true authenticity of the minichromosome model system required that they replicate during the cell cycle with chromosome-like timing specificity. I was fortunate enough to have the opportunity to construct E. coli minichromosomes in the laboratory of Charles Helmstetter and, for the first time, measure minichromosome cell cycle regulation. In this review, I discuss the evolution of this project along with some additional studies from that time related to the DNA topology and segregation properties of minichromosomes. Despite the significant passage of time, it is clear that large gaps in our understanding of oriC regulation still remain. I discuss some specific topics that continue to be worthy of further study.