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This protocol describes the application of atomic force microscopy for structural analysis of prokaryotic and organellar nucleoids. It is based on a simple cell manipulation procedure that enables stepwise dissection of the nucleoid. The procedure includes (i) on-substrate lysis of cells and (ii) enzyme treatment, followed by atomic force microscopy. This type of dissection analysis permits analysis of nucleoid structure ranging from the fundamental units assembled on DNA to higher-order levels of organization. The combination with molecular-genetic and biochemical techniques further permits analysis of the functions of key nucleoid factors relevant to signal-induced structural reorganization or building up of basic structures, as seen for Dps in Escherichia coli and TrmBL2 in Thermococcus kodakarensis. These systems are described here as examples of the successful application of AFM for this purpose. Moreover, we describe the procedures needed for quantitative analysis of the data., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The Escherichia coli CrfC protein is an important regulator of nucleoid positioning and equipartition. Previously we revealed that CrfC homo-oligomers bind the clamp, a DNA-binding subunit of the DNA polymerase III holoenzyme, promoting colocalization of the sister replication forks, which ensures the nucleoid equipartition. In addition, CrfC localizes at the cell pole-proximal loci via an unknown mechanism. Here, we demonstrate that CrfC localizes to the distinct subnucleoid structures termed nucleoid poles (the cell pole-proximal nucleoid-edges) even in elongated cells as well as in wild-type cells. Systematic analysis of the nucleoid-associated proteins (NAPs) and related proteins revealed that HU, the most abundant NAP, and SlmA, the nucleoid occlusion factor regulating the localization of cell division apparatus, promote the specific localization of CrfC foci. When the replication initiator DnaA was inactivated, SlmA and HU were required for formation of CrfC foci. In contrast, when the replication initiation was inhibited with a specific mutant of the helicase-loader DnaC, CrfC foci were sustained independently of SlmA and HU. H-NS, which forms clusters on AT-rich DNA regions, promotes formation of CrfC foci as well as transcriptional regulation of crfC. In addition, MukB, the chromosomal structure mainetanice protein, and SeqA, a hemimethylated nascent DNA region-binding protein, moderately stimulated formation of CrfC foci. However, IHF, a structural homolog of HU, MatP, the replication terminus-binding protein, Dps, a stress-response factor, and FtsZ, an SlmA-interacting factor in cell division apparatus, little or only slightly affected CrfC foci formation and localization. Taken together, these findings suggest a novel and unique mechanism that CrfC localizes to the nucleoid poles in two steps, assembly and recruitment, dependent upon HU, MukB, SeqA, and SlmA, which is stimulated directly or indirectly by H-NS and DnaA. These factors might concordantly affect specific nucleoid substructures. Also, these nucleoid dynamics might be significant in the role for CrfC in chromosome partition.

The Escherichia coli CrfC protein is an important regulator of nucleoid positioning and equipartition. Previously we revealed that CrfC homo-oligomers bind the clamp, a DNA-binding subunit of the DNA polymerase III holoenzyme, promoting colocalization of the sister replication forks, which ensures the nucleoid equipartition. In addition, CrfC localizes at the cell pole-proximal loci via an unknown mechanism. Here, we demonstrate that CrfC localizes to the distinct subnucleoid structures termed nucleoid poles (the cell pole-proximal nucleoid-edges) even in elongated cells as well as in wild-type cells. Systematic analysis of the nucleoid-associated proteins (NAPs) and related proteins revealed that HU, the most abundant NAP, and SlmA, the nucleoid occlusion factor regulating the localization of cell division apparatus, promote the specific localization of CrfC foci. When the replication initiator DnaA was inactivated, SlmA and HU were required for formation of CrfC foci. In contrast, when the replication initiation was inhibited with a specific mutant of the helicase-loader DnaC, CrfC foci were sustained independently of SlmA and HU. H-NS, which forms clusters on AT-rich DNA regions, promotes formation of CrfC foci as well as transcriptional regulation of crfC. In addition, MukB, the chromosomal structure mainetanice protein, and SeqA, a hemimethylated nascent DNA region-binding protein, moderately stimulated formation of CrfC foci. However, IHF, a structural homolog of HU, MatP, the replication terminus-binding protein, Dps, a stress-response factor, and FtsZ, an SlmA-interacting factor in cell division apparatus, little or only slightly affected CrfC foci formation and localization. Taken together, these findings suggest a novel and unique mechanism that CrfC localizes to the nucleoid poles in two steps, assembly and recruitment, dependent upon HU, MukB, SeqA, and SlmA, which is stimulated directly or indirectly by H-NS and DnaA. These factors might concordantly affect specific nucleoid substructures. Also, these nucleoid dynamics might be significant in the role for CrfC in chromosome partition. [ABSTRACT FROM AUTHOR]

In the fast-growing Escherichia coli cells, RNA polymerase (RNAP) molecules are concentrated and form foci at clusters of ribosomal RNA (rRNA) operons resembling eukaryotic nucleolus. The bacterial nucleolus-like organization, spatially compartmentalized at the surface of the compact bacterial chromosome (nucleoid), serves as transcription factories for rRNA synthesis and ribosome biogenesis, which influences the organization of the nucleoid. Unlike wild type that has seven rRNA operons in the genome in a mutant that has six (Δ6rrn) rRNA operons deleted in the genome, there are no apparent transcription foci and the nucleoid becomes uncompacted, indicating that formation of RNAP foci requires multiple copies of rRNA operons clustered in space and is critical for nucleoid compaction. It has not been determined, however, whether a multicopy plasmid-borne rRNA operon (prrnB) could substitute the multiple chromosomal rRNA operons for the organization of the bacterial nucleolus-like structure in the mutants of Δ6rrn and Δ7rrn that has all seven rRNA operons deleted in the genome. We hypothesized that extrachromosomal nucleolus-like structures are similarly organized and functional in trans from prrnB in these mutants. In this report, using multicolor images of three-dimensional superresolution Structured Illumination Microscopy (3D-SIM), we determined the distributions of both RNAP and NusB that are a transcription factor involved in rRNA synthesis and ribosome biogenesis, prrnB clustering, and nucleoid structure in these two mutants in response to environmental cues. Our results found that the extrachromosomal nucleolus-like organization tends to be spatially located at the poles of the mutant cells. In addition, formation of RNAP foci at the extrachromosomal nucleolus-like structure condenses the nucleoid, supporting the idea that active transcription at the nucleolus-like organization is a driving force in nucleoid compaction.

In the fast-growing Escherichia coli cells, RNA polymerase (RNAP) molecules are concentrated and form foci at clusters of ribosomal RNA (rRNA) operons resembling eukaryotic nucleolus. The bacterial nucleolus-like organization, spatially compartmentalized at the surface of the compact bacterial chromosome (nucleoid), serves as transcription factories for rRNA synthesis and ribosome biogenesis, which influences the organization of the nucleoid. Unlike wild type that has seven rRNA operons in the genome in a mutant that has six (Δ6rrn) rRNA operons deleted in the genome, there are no apparent transcription foci and the nucleoid becomes uncompacted, indicating that formation of RNAP foci requires multiple copies of rRNA operons clustered in space and is critical for nucleoid compaction. It has not been determined, however, whether a multicopy plasmid-borne rRNA operon (prrnB) could substitute the multiple chromosomal rRNA operons for the organization of the bacterial nucleolus-like structure in the mutants of Δ6rrn and Δ7rrn that has all seven rRNA operons deleted in the genome. We hypothesized that extrachromosomal nucleoluslike structures are similarly organized and functional in trans from prrnB in these mutants. In this report, using multicolor images of three-dimensional superresolution Structured Illumination Microscopy (3D-SIM), we determined the distributions of both RNAP and NusB that are a transcription factor involved in rRNA synthesis and ribosome biogenesis, prrnB clustering, and nucleoid structure in these two mutants in response to environmental cues. Our results found that the extrachromosomal nucleolus-like organization tends to be spatially located at the poles of the mutant cells. In addition, formation of RNAP foci at the extrachromosomal nucleolus-like structure condenses the nucleoid, supporting the idea that active transcription at the nucleolus-like organization is a driving force in nucleoid compaction. [ABSTRACT FROM AUTHOR]

The hyperactive Tn5 transposase in the ATAC-seq method has been widely used to determine the open DNA regions and understand the overall epigenomic regulation in the chromatins of eukaryotic cells. Here, we describe POP-seq (Prokaryotic chromatin Openness Profiling sequencing), an adaptation of the ATAC-seq method, to interrogate changes in the openness of prokaryotic nucleoids., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Single-molecule fluorescence provides high resolution spatial distributions of ribosomes and RNA polymerase (RNAP) in live, rapidly growing E. coli. Ribosomes are more strongly segregated from the nucleoids (chromosomal DNA) than previous widefield fluorescence studies suggested. While most transcription may be co-translational, the evidence indicates that most translation occurs on free mRNA copies that have diffused from the nucleoids to a ribosome-rich region. Analysis of time-resolved images of the nucleoid spatial distribution after treatment with the transcription-halting drug rifampicin and the translation-halting drug chloramphenicol shows that both drugs cause nucleoid contraction on the 0-3 min timescale. This is consistent with the transertion hypothesis. We suggest that the longer-term (20-30 min) nucleoid expansion after Rif treatment arises from conversion of 70S-polysomes to 30S and 50S subunits, which readily penetrate the nucleoids. Monte Carlo simulations of a polymer bead model built to mimic the chromosomal DNA and ribosomes (either 70S-polysomes or 30S and 50S subunits) explain spatial segregation or mixing of ribosomes and nucleoids in terms of excluded volume and entropic effects alone. A comprehensive model of the transcription-translation-transertion system incorporates this new information about the spatial organization of the E. coli cytoplasm. We propose that transertion, which radially expands the nucleoids, is essential for recycling of 30S and 50S subunits from ribosome-rich regions back into the nucleoids. There they initiate co-transcriptional translation, which is an important mechanism for maintaining RNAP forward progress and protecting the nascent mRNA chain. Segregation of 70S-polysomes from the nucleoid may facilitate rapid growth by shortening the search time for ribosomes to find free mRNA concentrated outside the nucleoid and the search time for RNAP concentrated within the nucleoid to find transcription initiation sites.

Single-molecule fluorescence provides high resolution spatial distributions of ribosomes and RNA polymerase (RNAP) in live, rapidly growing Escherichia coli. Ribosomes are more strongly segregated from the nucleoids (chromosomal DNA) than previous widefield fluorescence studies suggested. While most transcription may be co-translational, the evidence indicates that most translation occurs on free mRNA copies that have diffused from the nucleoids to a ribosome-rich region. Analysis of time-resolved images of the nucleoid spatial distribution after treatment with the transcription-halting drug rifampicin and the translation-halting drug chloramphenicol shows that both drugs cause nucleoid contraction on the 0-3 min timescale. This is consistent with the transertion hypothesis. We suggest that the longer-term (20-30 min) nucleoid expansion after Rif treatment arises from conversion of 70S-polysomes to 30S and 50S subunits, which readily penetrate the nucleoids. Monte Carlo simulations of a polymer bead model built to mimic the chromosomal DNA and ribosomes (either 70S-polysomes or 30S and 50S subunits) explain spatial segregation or mixing of ribosomes and nucleoids in terms of excluded volume and entropic effects alone. A comprehensive model of the transcription-translation-transertion system incorporates this new information about the spatial organization of the E. coli cytoplasm. We propose that transertion, which radially expands the nucleoids, is essential for recycling of 30S and 50S subunits from ribosome-rich regions back into the nucleoids. There they initiate co-transcriptional translation, which is an important mechanism for maintaining RNAP forward progress and protecting the nascent mRNA chain. Segregation of 70Spolysomes from the nucleoid may facilitate rapid growth by shortening the search time for ribosomes to find free mRNA concentrated outside the nucleoid and the search time for RNAP concentrated within the nucleoid to find transcription initiation sites. [ABSTRACT FROM AUTHOR]

Despite an increasing number of studies over the last 15 years, aerobic anoxygenic photoheterotrophic (AAP) bacteria remain a puzzling functional group in terms of physiology, metabolism, and ecology. To contribute to a better knowledge of their environmental distribution, the present study aims at analyzing their diversity and structure at the boundary between the Norwegian, Greenland, and Barents Seas. The polymorphism of a marker gene encoding a sub-unit of the photosynthetic apparatus (pufM gene) was analyzed and attempted to be related to environmental parameters. The Atlantic or Arctic origin of water masses had a strong impact on the AAP bacterial community structure whose populations mostly belonged to the Alpha- and Gammaproteobacteria. A majority (>60%) of pufM sequences were affiliated to the Gammaproteobacteria reasserting that this class often represents the major component of the AAP bacterial community in oceanic regions. Two alphaproteobacterial groups dominate locally suggesting that they can constitute key players in this marine system transiently. We found that temperature is a major determinant of alpha diversity of AAP bacteria in this marine biome with specific clades emerging locally according to the partitioning of water masses. Whereas we expected specific AAP bacterial populations in this peculiar and newly explored ecosystem, most pufM sequences were highly related to sequences retrieved elsewhere. This observation highlights that the studied area does not favor AAP bacteria endemism but also opens new questions about the truthfulness of biogeographical patterns and on the extent of AAP bacterial diversity. [ABSTRACT FROM AUTHOR]

[Display omitted] • 3D whole cell modeling requires new bioinformatics and computational methods. • Information for generating 3D cell models is gathered and curated with Mesoscope. • A multi-step workflow generates structural models of an entire proteome. • Entire bacterial cells are interactively modeled and visualized with CellPACKgpu. • This work demonstrates the feasibility of building 3D models of an entire cell. Building structural models of entire cells has been a long-standing cross-discipline challenge for the research community, as it requires an unprecedented level of integration between multiple sources of biological data and enhanced methods for computational modeling and visualization. Here, we present the first 3D structural models of an entire Mycoplasma genitalium (MG) cell, built using the CellPACK suite of computational modeling tools. Our model recapitulates the data described in recent whole-cell system biology simulations and provides a structural representation for all MG proteins, DNA and RNA molecules, obtained by combining experimental and homology-modeled structures and lattice-based models of the genome. We establish a framework for gathering, curating and evaluating these structures, exposing current weaknesses of modeling methods and the boundaries of MG structural knowledge, and visualization methods to explore functional characteristics of the genome and proteome. We compare two approaches for data gathering, a manually-curated workflow and an automated workflow that uses homologous structures, both of which are appropriate for the analysis of mesoscale properties such as crowding and volume occupancy. Analysis of model quality provides estimates of the regularization that will be required when these models are used as starting points for atomic molecular dynamics simulations. [ABSTRACT FROM AUTHOR]

In enteric bacteria organization of the circular chromosomal DNA into a highly dynamic and toroidal-shaped nucleoid involves various factors, such as DNA supercoiling, nucleoid-associated proteins (NAPs), the structural maintenance of chromatin (SMC) complex, and macrodomain organizing proteins. Here, we show that ectopic expression of transcription regulators at high levels leads to nucleoid compaction. This serendipitous result was obtained by fluorescence microscopy upon ectopic expression of the transcription regulator and phosphodiesterase PdeL of Escherichia coli. Nucleoid compaction by PdeL depends on DNA-binding, but not on its enzymatic phosphodiesterase activity. Nucleoid compaction was also observed upon high-level ectopic expression of the transcription regulators LacI, RutR, RcsB, LeuO, and Cra, which range from single-target gene regulators to global regulators. In the case of LacI, its high-level expression in the presence of the gratuitous inducer IPTG (isopropyl-β-d-thiogalactopyranoside) also led to nucleoid compaction, indicating that compaction is caused by unspecific DNA-binding. In all cases nucleoid compaction correlated with misplacement of the FtsZ ring and loss of MukB foci, a subunit of the SMC complex. Thus, high levels of several transcription regulators cause nucleoid compaction with consequences for replication and cell division. IMPORTANCE The bacterial nucleoid is a highly organized and dynamic structure for simultaneous transcription, replication, and segregation of the bacterial genome. Compaction of the nucleoid and disturbance of DNA segregation and cell division by artificially high levels of transcription regulators, as described here, reveals that an excess of DNA-binding protein disturbs nucleoid structuring. The results suggest that ectopic expression levels of DNA-binding proteins for genetic studies of their function but also for their purification should be carefully controlled and adjusted.

Background: Periodic spacing of A-tracts (short runs of A or T) with the DNA helical period of ~10-11 bp is characteristic of intrinsically bent DNA. In eukaryotes, the DNA bending is related to chromatin structure and nucleosome positioning. However, the physiological role of strong sequence periodicity detected in many prokaryotic genomes is not clear. Results: We developed measures of intensity and persistency of DNA curvature-related sequence periodicity and applied them to prokaryotic chromosomes and phages. The results indicate that strong periodic signals present in chromosomes are generally absent in phage genomes. Moreover, chromosomes containing prophages are less likely to possess a persistent periodic signal than chromosomes with no prophages. Conclusions: Absence of DNA curvature-related sequence periodicity in phages could arise from constraints associated with DNA packaging in the viral capsid. Lack of prophages in chromosomes with persistent periodic signal suggests that the sequence periodicity and concomitant DNA curvature could play a role in protecting the chromosomes from integration of phage DNA. [ABSTRACT FROM AUTHOR]

The stochasticity of chromosome organization was investigated by fluorescently labeling genetic loci in live Escherichia coil cells. In spite of the common assumption that the chromosome is well modeled by an unstructured polymer, measurements of the locus distributions reveal that the E. coli chromosome is precisely organized into a nucleoid filament with a linear order. Loci in the body of the nucleoid show a precision of positioning within the cell of better than 10% of the cell length. The precision of interlocus distance genomically-proximate loci was better than 4% of the cell length. The measured dependence of the precision of interlocus distance on genomic distance singles out intranucleoid interactions as the mechanism responsible for chromosome organization. From the magnitude of the variance, we infer the existence of an as-yet- uncharacterized higher-order DNA organization in bacteria. We demonstrate that both the stochastic and average structure of the nucleoid is captured by a fluctuating elastic filament model. [ABSTRACT FROM AUTHOR]

The availability of complete genome sequences for a large variety of organisms is a major advance in understanding genome structure and function. One attribute of genome structure is chromosome organisation in terms of gene localisation and orientation. For example, bacterial operons, i.e. clusters of co-oriented genes that form transcription units, enable functionally related genes to be expressed simultaneously. The description of genome organisation was pioneered with the study of the distribution of genes of the Escherichia coli partial genetic map before the full genome sequence was known. Deploying powerful techniques from circular statistics and signal processing, we revisit the issue of gene localisation and orientation using 89 complete microbial chromosomes from the eubacterial and archaeal domains. We demonstrate that there is no characteristic size pertinent to the description of chromosome structure, e.g. there does not exist any single length appropriate to describe gene clustering. Our results show that, for all 89 chromosomes, gene positions and gene orientations share a common form of scale-invariant correlations known as “long-range correlations” that we can reveal for distances from the gene length, up to the chromosome size. This observation indicates that genes tend to assemble and to co-orient over any scale of observation greater than a few kilobases. This unexpected property of chromosome structure can be portrayed as an operon-like organisation at all scales and implies that a complete scale range extending over more than three orders of magnitudes of chromosome segment lengths is necessary to properly describe prokaryotic genome organisation. We propose that this pattern results from the effects of the superhelical context on gene expression coupled with the structure and dynamics of the nucleoid, possibly accommodating the diverse gene expression profiles needed during the different stages of cellular life. [Copyright &y& Elsevier]

This protocol describes the application of atomic force microscopy for structural analysis of the prokaryotic and organellar nucleoids. It is based on a simple cell manipulation procedure that enables step-wise dissection of the nucleoid. The procedure includes (1) on-substrate-lysis of cells, and (2) enzyme treatment, followed by atomic force microscopy. This type of dissection analysis permits analysis of nucleoid structure ranging from the fundamental units assembled on DNA to higher order levels of organization. The combination with molecular-genetic and biochemical techniques further permits analysis of the functions of key nucleoid factors relevant to signal-induced structural re-organization or building up of basic structures, as seen for Dps in Escherichia coli, and TrmBL2 in Thermococcus kodakarensis. These systems are described here as examples of the successful application of AFM for this purpose. Moreover, we describe the procedures needed for quantitative analysis of the data.