Bacterial Chromosomal DNA Research Articles

Creating a bacterium that forms eukaryotic nucleosome core particles

The nucleosome is one of the hallmarks of eukaryotes, a dynamic platform that supports many critical functions in eukaryotic cells. Here, we engineer the in vivo assembly of the nucleosome core in the model bacterium Escherichia coli. We show that bacterial chromosome DNA and eukaryotic histones can assemble in vivo to form nucleosome complexes with many features resembling those found in eukaryotes. The formation of nucleosomes in E. coli was visualized with atomic force microscopy and using tripartite split green fluorescent protein. Under a condition that moderate histones expression was induced at 1 µM IPTG, the nucleosome-forming bacterium is viable and has sustained growth for at least 110 divisions in longer-term growth experiments. It exhibits stable nucleosome formation, a consistent transcriptome across passages, and reduced growth fitness under stress conditions. In particular, the nucleosome arrays in E. coli genic regions have profiles resembling those in eukaryotic cells. The observed compatibility between the eukaryotic nucleosome and the bacterial chromosome machinery may reflect a prerequisite for bacteria-archaea union, providing insight into eukaryogenesis and the origin of the nucleosome.

Structure of the E. coli nucleoid-associated protein YejK reveals a novel DNA binding clamp.

Nucleoid-associated proteins (NAPs) play central roles in bacterial chromosome organization and DNA processes. The Escherichia coli YejK protein is a highly abundant, yet poorly understood NAP. YejK proteins are conserved among Gram-negative bacteria but show no homology to any previously characterized DNA-binding protein. Hence, how YejK binds DNA is unknown. To gain insight into YejK structure and its DNA binding mechanism we performed biochemical and structural analyses on the E. coli YejK protein. Biochemical assays demonstrate that, unlike many NAPs, YejK does not show a preference for AT-rich DNA and binds non-sequence specifically. A crystal structure revealed YejK adopts a novel fold comprised of two domains. Strikingly, each of the domains harbors an extended arm that mediates dimerization, creating an asymmetric clamp with a 30 Å diameter pore. The lining of the pore is electropositive and mutagenesis combined with fluorescence polarization assays support DNA binding within the pore. Finally, our biochemical analyses on truncated YejK proteins suggest a mechanism for YejK clamp loading. Thus, these data reveal YejK contains a newly described DNA-binding motif that functions as a novel clamp.

Leveraging Active Learning to Establish Efficient In Vitro Transcription and Translation from Bacterial Chromosomal DNA.

Gene expression is a fundamental aspect in the construction of a minimal synthetic cell, and the use of chromosomes will be crucial for the integration and regulation of complex modules. Expression from chromosomes in vitro transcription and translation (IVTT) systems presents limitations, as their large size and low concentration make them far less suitable for standard IVTT reactions. Here, we addressed these challenges by optimizing lysate-based IVTT systems at low template concentrations. We then applied an active learning tool to adapt IVTT to chromosomes as template DNA. Further insights into the dynamic data set led us to adjust the previous protocol for chromosome isolation and revealed unforeseen trends pointing at limiting transcription kinetics in our system. The resulting IVTT conditions allowed a high template DNA efficiency for the chromosomes. In conclusion, our system shows a protein-to-chromosome ratio that moves closer to in vivo biology and represents an advancement toward chromosome-based synthetic cells.

In Vitro Transcription Assay to Quantify Effects of H-NS Filaments on RNA Chain Elongation by RNA Polymerase.

Bacterial chromosomal DNA is structured and compacted by proteins known as bacterial chromatin proteins (i.e., nucleoid-associated proteins or NAPs). DNA-dependent RNA polymerase (RNAP) must frequently interact with bacterial chromatin proteins because they often bind DNA genome-wide. In some cases, RNAP must overcome barriers bacterial chromatin proteins impose on transcription. One key bacterial chromatin protein in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS. H-NS binds to DNA and forms nucleoprotein filaments. To investigate the effect of H-NS filaments on RNAP elongation, we developed an in vitro transcription assay to monitor RNAP progression on a DNA template bound by H-NS. In this method, initiation and elongation by RNAP are uncoupled by first initiating transcription in the presence of only three ribonucleoside triphosphates (rNTPs) to halt elongation just downstream of the promoter. Before elongation is restarted by addition of the fourth NTP, an H-NS filament is formed on the DNA so that transcript elongation occurs on an H-NS nucleoprotein filament template. Here, we provide detailed protocols for performing in vitro transcription through H-NS filaments, analysis of the transcription products, and visualization of H-NS filament formation on DNA by electrophoretic mobility shift assay (EMSA). These methods enable insight into how H-NS affects RNAP transcript elongation and provide a starting point to determine effects of other bacterial chromatin proteins on RNAP elongation.

RNA replication-independent, DNA linearization-dependent expression of reporter genes from a SARS-CoV-2 replicon-encoding DNA in human cells.

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.

Plasmid Profile and Enterobacterial Repetitive Intergenic Consensus-PCR Characterization of Salmonella Infantis Isolates Recovered From Poultry Sources

Background: Salmonella is known as one of the most important bacterial agents infecting both humans and animals. Salmonella Infantis has been reported as one of the 15 most prevalent serovars worldwide. Despite its clinical importance, there is little information on the molecular characteristics of S. Infantis in Iran. Objectives: This study was conducted to characterize S. Infantis isolates collected from poultry sources in the last decade. The isolates were typified by plasmid profile and enterobacterial repetitive intergenic consensus (ERIC-PCR). Methods: Forty S. Infantis isolates from poultry sources were subjected to plasmid profile and ERIC-PCR characterization. We used a commercial plasmid extraction kit to extract and purify plasmid DNA which then was separated by gel electrophoresis and viewed under a UV transilluminator. For ERIC-PCR, a commercial bacterial chromosomal DNA extraction kit was used. In this study, we chose ERIC2 primer for the ERIC-PCR test. Results: The plasmid profile revealed that 35% of isolates did not contain any plasmids, but the rest (65%) carried a variable number of plasmids with different molecular weights. Six plasmid profiles were found among 40 S. Infantis isolates. Using ERIC2 primer, 7 profiles were found among 40 S. Infantis isolates in ERIC-PCR. Bands with molecular weights ranging from 400 to 3000 bp were observed. Conclusion: This study provided some genetic data on S. Infantis isolates recovered from poultry sources, and these data can be used for a broader epidemiological study nationwide. These findings showed that although plasmid and ERIC profiles are valuable in epidemiological studies, they have some limitations, too.

Preparing Bacterial Genomic DNA.

We describe two alternative procedures for purifying bacterial chromosomal DNA. The first procedure incorporates the use of a commercial kit based on silica membrane technology. This approach relies on the selective binding of DNA to a silica-based column in the presence of chaotropic salts (guanidine salts). Polysaccharides and proteins do not bind well to the column and flow through. Their residual traces, along with the guanidine salts, are removed during alcohol-based wash steps. The DNA is then selectively eluted under low-salt conditions. This method is quick and easy and yields genomic DNA suitable for most downstream applications. The second procedure, implemented for many years in our laboratory before the appearance of commercial kits, is based on the ability of the cationic detergent cetyl trimethyl ammonium bromide (CTAB) to complex with polysaccharides and proteins, producing an emulsion that can be removed by chloroform-isoamyl alcohol extraction. This procedure is therefore especially suited to working with Gram-negative bacteria, which typically produce large amounts of polysaccharides. Its main advantage, besides cost-effectiveness, is the high yield of the DNA obtained; its main disadvantage is that the workflow is relatively cumbersome.

A fully automated high-throughput plasmid purification workstation for the generation of mammalian cell expression-quality DNA.

Early-stage antibody discovery and engineering typically require the cloning, expression, and screening of large numbers of proteins. Normally, DNA fragments encoding proteins of interest are cloned into extra-chromosomal plasmids that are amplified in Escherichia coli. Following purification from the bacteria, the plasmids are introduced into appropriate cells, and the expressed recombinant proteins screened for desired binding or function in a high-throughput manner. Even in a 96-well plate format, plasmid purification from E. coli is typically a labor intensive and time-consuming process. To further accelerate our existing biotherapeutic discovery workflows we designed, qualified, and enabled a fully integrated high-throughput plasmid purification and quantification workstation which we have termed AMPS (Automated Miniprep Plasmid Station). Using components from a commercially available kit, AMPS can purify plasmid preparations from twenty 96-deep-well plates of E. coli cultures, measure DNA absorbance at 260 nm, calculate plasmid concentrations, and prepare 96-deep-well plates for mammalian expression in an operator-independent manner. Plasmid yields and concentrations are equivalent to those obtained off-line. Furthermore, the quality of the DNA purified on the AMPS is equivalent to that obtained off-line in terms of DNA topology, and absence of contaminating bacterial chromosomal DNA and RNA. Most importantly, plasmids purified on the AMPS provide similar antibody titers following transfection in CHO cells as plasmids purified off-line. The AMPS bridges high-throughput E. coli colony picking capabilities typically available in an automation lab with downstream CHO expression needs and will facilitate screening of large numbers of biotherapeutics in binding and cell assay screens.

Evaluation of the Role of Bacterial Amyloid on Nucleoid Structure Using Cryo-Soft X-Ray Tomography.

Bacterial chromosomal DNA is packed within a non-membranous structure, the nucleoid, thanks to nucleoid associated proteins (NAPs). The role of bacterial amyloid has recently emerged among these NAPs, particularly with the nucleoid-associated protein Hfq that plays a direct role in DNA compaction. In this chapter, we present a 3D imaging technique, cryo-soft X-ray tomography (cryo-SXT) to obtain a detailed 3D visualization of subcellular bacterial structures, especially the nucleoid. Cryo-SXT imaging of native unlabeled cells enables observation of the nucleoid in 3D with a high resolution, allowing to evidence in vivo the role of amyloids on DNA compaction. The precise experimental methods to obtain 3D tomograms will be presented.

Characterization of a triad of genes in cyanophage S-2L sufficient to replace adenine by 2-aminoadenine in bacterial DNA

Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthetase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene (mazZ) located between purZ and datZ genes in S-2L and related phage genomes. We show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes – datZ, mazZ and purZ – was expressed in E. coli, resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA.

Virion Display: A High-Throughput Method to Express Functional Membrane Proteins.

Transmembrane proteins are responsible for many critical cellular functions and represent one of the largest families of drug targets. However, these proteins, especially multipass transmembrane proteins, are difficult to study because they must be embedded in a lipid bilayer to maintain their native conformations. The development of the virion display (VirD) technology enables transmembrane proteins to be integrated into the viral envelope of herpes simplex virus 1 (HSV-1). Combining high-throughput cloning, expression, and purification techniques, VirD technology has been applied to the largest set of human transmembrane proteins, namely G-protein-coupled receptors, and has allowed the identification of interactions that are both specific and functional. This article describes the procedures to integrate an open reading frame for any transmembrane protein into the HSV-1 genome and produce recombinant HSV-1 virus to ultimately generate pure VirD virions for biological and pharmaceutical studies. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Gateway cloning of transmembrane proteins Support Protocol 1: Ethanol precipitation of bacterial artificial chromosomal DNA Support Protocol 2: Preparation of competent cells Basic Protocol 2: Production of recombinant HSV-1 virions.

Bacterial Chromosome Replication and DNA Repair During the Stringent Response.

The stringent response regulates bacterial growth rate and is important for cell survival under changing environmental conditions. The effect of the stringent response is pleiotropic, affecting almost all biological processes in the cell including transcriptional downregulation of genes involved in stable RNA synthesis, DNA replication, and metabolic pathways, as well as the upregulation of stress-related genes. In this Review, we discuss how the stringent response affects chromosome replication and DNA repair activities in bacteria. Importantly, we address how accumulation of (p)ppGpp during the stringent response shuts down chromosome replication using highly different strategies in the evolutionary distant Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. Interestingly, (p)ppGpp-mediated replication inhibition occurs downstream of the origin in B. subtilis, whereas replication inhibition in E. coli takes place at the initiation level, suggesting that stringent cell cycle arrest acts at different phases of the replication cycle between E. coli and B. subtilis. Furthermore, we address the role of (p)ppGpp in facilitating DNA repair activities and cell survival during exposure to UV and other DNA damaging agents. In particular, (p)ppGpp seems to stimulate the efficiency of nucleotide excision repair (NER)-dependent repair of DNA lesions. Finally, we discuss whether (p)ppGpp-mediated cell survival during DNA damage is related to the ability of (p)ppGpp accumulation to inhibit chromosome replication.

Mammalian histones facilitate antimicrobial synergy by disrupting the bacterial proton gradient and chromosome organization

First proposed as antimicrobial agents, histones were later recognized for their role in condensing chromosomes. Histone antimicrobial activity has been reported in innate immune responses. However, how histones kill bacteria has remained elusive. The co-localization of histones with antimicrobial peptides (AMPs) in immune cells suggests that histones may be part of a larger antimicrobial mechanism in vivo. Here we report that histone H2A enters E. coli and S. aureus through membrane pores formed by the AMPs LL-37 and magainin-2. H2A enhances AMP-induced pores, depolarizes the bacterial membrane potential, and impairs membrane recovery. Inside the cytoplasm, H2A reorganizes bacterial chromosomal DNA and inhibits global transcription. Whereas bacteria recover from the pore-forming effects of LL-37, the concomitant effects of H2A and LL-37 are irrecoverable. Their combination constitutes a positive feedback loop that exponentially amplifies their antimicrobial activities, causing antimicrobial synergy. More generally, treatment with H2A and the pore-forming antibiotic polymyxin B completely eradicates bacterial growth.

Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Preparation of the A Homology Arm (A-Box) and B Homology Arm (B-Box).

The 700-bp A homology arm (A-box) and the 700-bp B homology arm (B-box) are amplified by polymerase chain reaction (PCR) using purified bacterial artificial chromosome (BAC) DNA as template for two-step BAC engineering. The resulting A-box PCR product contains an AscI site at its 5' end (the 5' primer incorporates an AscI site, and the 3' primer does not incorporate any restriction sites). The B-box PCR product contains an XmaI site at its 3' end (the 5' primer does not incorporate any restriction sites, and the 3' primer incorporates an XmaI site). The amplification products are then digested with the appropriate restriction endonucleases to render them suitable for cloning into the shuttle vector.

Molecular ring toss of circular BAC DNA using micropillar array for single-molecule studies.

This paper reports a method for trapping circular DNA molecules and imaging the dynamics with high spatial resolution using a micropillar-array device. We successfully trapped circular bacterial artificial chromosome DNA molecules at a micropillar-based "ring toss" in the laminar flow of a microchannel under a fluorescence microscope and demonstrated the imaging of their extension by flow and condensation process induced by spermine solution. DNA molecules were visualized in an extended loop conformation, allowing high spatial resolution, and the results showed that the dynamics is induced by the microfluidic control of the surrounding chemical environment. The method is expected to lead to the elucidation of the physical characteristics and the dynamics of circular DNA molecules.

Mutation and Recombination Rates Vary Across Bacterial Chromosome

Bacteria evolve as a result of mutations and acquisition of foreign DNA by recombination processes. A growing body of evidence suggests that mutation and recombination rates are not constant across the bacterial chromosome. Bacterial chromosomal DNA is organized into a compact nucleoid structure which is established by binding of the nucleoid-associated proteins (NAPs) and other proteins. This review gives an overview of recent findings indicating that the mutagenic and recombination processes in bacteria vary at different chromosomal positions. Involvement of NAPs and other possible mechanisms in these regional differences are discussed. Variations in mutation and recombination rates across the bacterial chromosome may have implications in the evolution of bacteria.

Examination of Bacterial Artificial Chromosome (BAC) DNA Quality and Quantity by Pulsed-Field Gel Electrophoresis.

Pulsed-field gel electrophoresis (PFGE) is the preferred method to measure the size of an insert in a high-capacity vector. After digestion with a restriction enzyme (e.g., PI-SceI or NotI), the DNA fragments are separated by PFGE through a 1% agarose gel.

Large-Scale Preparation and Linearization of Bacterial Artificial Chromosome (BAC) DNA.

A critical step for creating bacterial artificial chromosome (BAC) transgenic organisms is the production of high-quality BAC DNA. The method described here provides a source of DNA for further manipulation and modification, but can also be used for preparing modified BAC DNA, resulting from the one-step or two-step strategy, for use in other applications such as BAC transgenesis. An analysis of precautions to take when working with large DNA molecules is also provided.

Small-Scale Isolation of Bacterial Artificial Chromosome (BAC) DNA and Verification by Polymerase Chain Reaction (PCR).

In this protocol, small amounts of selected bacterial artificial chromosome (BAC) DNAs are prepared from 5-mL cultures of the Escherichia coli host transformed with the BAC clone. DNA is isolated by an adaptation of the alkaline lysis method. The yield is 0.1-0.4 µg, and the BAC DNA is suitable for analysis by polymerase chain reaction (PCR). Considerations for clone selection are also addressed.

Viral metagenomic analysis of the cheese surface: A comparative study of rapid procedures for extracting viral particles

The structure and functioning of microbial communities from fermented foods, including cheese, have been extensively studied during the past decade. However, there is still a lack of information about both the occurrence and the role of viruses in modulating the function of this type of spatially structured and solid ecosystems. Viral metagenomics was recently applied to a wide variety of environmental samples and standardized procedures for recovering viral particles from different type of materials has emerged. In this study, we adapted a procedure originally developed to extract viruses from fecal samples, in order to enable efficient virome analysis of cheese surface. We tested and validated the positive impact of both addition of a filtration step prior to virus concentration and substitution of purification by density gradient ultracentrifugation by a simple chloroform treatment to eliminate membrane vesicles. Viral DNA extracted from the several procedures, as well as a vesicle sample, were sequenced using Illumina paired-end MiSeq technology and the subsequent clusters assembled from the virome were analyzed to assess those belonging to putative phages, plasmid-derived DNA, or even from bacterial chromosomal DNA. The best procedure was then chosen, and used to describe the first cheese surface virome, using Epoisses cheese as example. This study provides the basis of future investigations regarding the ecological importance of viruses in cheese microbial ecosystems.