Bacterial Genome Organization Research Articles

Principles of bacterial genome organization, a conformational point of view.

Bacterial chromosomes are large molecules that need to be highly compacted to fit inside the cells. Chromosome compaction must facilitate and maintain key biological processes such as gene expression and DNA transactions (replication, recombination, repair, and segregation). Chromosome and chromatin 3D-organization in bacteria has been a puzzle for decades. Chromosome conformation capture coupled to deep sequencing (Hi-C) in combination with other "omics" approaches has allowed dissection of the structural layers that shape bacterial chromosome organization, from DNA topology to global chromosome architecture. Here we review the latest findings using Hi-C and discuss the main features of bacterial genome folding.

Origin, evolution, and maintenance of gene-strand bias in bacteria.

Gene-strand bias is a characteristic feature of bacterial genome organization wherein genes are preferentially encoded on the leading strand of replication, promoting co-orientation of replication and transcription. This co-orientation bias has evolved to protect gene essentiality, expression, and genomic stability from the harmful effects of head-on replication-transcription collisions. However, the origin, variation, and maintenance of gene-strand bias remain elusive. Here, we reveal that the frequency of inversions that alter gene orientation exhibits large variation across bacterial populations and negatively correlates with gene-strand bias. The density, distance, and distribution of inverted repeats show a similar negative relationship with gene-strand bias explaining the heterogeneity in inversions. Importantly, these observations are broadly evident across the entire bacterial kingdom uncovering inversions and inverted repeats as primary factors underlying the variation in gene-strand bias and its maintenance. The distinct catalytic subunits of replicative DNA polymerase have co-evolved with gene-strand bias, suggesting a close link between replication and the origin of gene-strand bias. Congruently, inversion frequencies and inverted repeats vary among bacteria with different DNA polymerases. In summary, we propose that the nature of replication determines the fitness cost of replication-transcription collisions, establishing a selection gradient on gene-strand bias by fine-tuning DNA sequence repeats and, thereby, gene inversions.

Organization and replicon interactions within the highly segmented genome of Borrelia burgdorferi.

Borrelia burgdorferi, a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/Smc. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that Smc and the Smc-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC. Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.

A chromosomal loop anchor mediates bacterial genome organization.

Nucleoprotein complexes play an integral role in genome organization of both eukaryotes and prokaryotes. Apart from their role in locally structuring and compacting DNA, several complexes are known to influence global organization by mediating long-range anchored chromosomal loop formation leading to spatial segregation of large sections of DNA. Such megabase-range interactions are ubiquitous in eukaryotes, but have not been demonstrated in prokaryotes. Here, using a genome-wide sedimentation-based approach, we found that a transcription factor, Rok, forms large nucleoprotein complexes in the bacterium Bacillus subtilis. Using chromosome conformation capture and live-imaging of DNA loci, we show that these complexes robustly interact with each other over large distances. Importantly, these Rok-dependent long-range interactions lead to anchored chromosomal loop formation, thereby spatially isolating large sections of DNA, as previously observed for insulator proteins in eukaryotes.

Generating High-Resolution Hi-C Contact Maps of Bacteria.

During the past decade, Chromosome Conformation Capture (3C/Hi-C)-based methods have been used to probe the 3D structure and organization of bacterial genomes, revealing fundamental aspects of chromosome dynamics. However, the current protocols are expensive, inefficient, and limited in their resolution. Here we present a simple, cost-effective Hi-C approach that is readily applicable to a range of Gram-positive and Gram-negative bacteria.

Serodiagnosis and Bacterial Genome of Helicobacter pylori Infection.

The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori.

The Exploration of Novel Regulatory Relationships Drives Haloarchaeal Operon-Like Structural Dynamics over Short Evolutionary Distances.

Operons are a dominant feature of bacterial and archaeal genome organization. Numerous investigations have related aspects of operon structure to operon function, making operons exemplars for studies aimed at deciphering Nature’s design principles for genomic organization at a local scale. We consider this understanding to be both fundamentally important and ultimately useful in the de novo design of increasingly complex synthetic circuits. Here we analyze the evolution of the genomic context of operon-like structures in a set of 76 sequenced and annotated species of halophilic archaea. The phylogenetic depth and breadth of this dataset allows insight into changes in operon-like structures over shorter evolutionary time scales than have been studied in previous cross-species analysis of operon evolution. Our analysis, implemented in the updated software package JContextExplorer finds that operon-like context as measured by changes in structure frequently differs from a sequence divergence model of whole-species phylogeny and that changes seem to be dominated by the exploration of novel regulatory relationships.

The functional determinants in the organization of bacterial genomes.

Bacterial genomes are now recognized as interacting intimately with cellular processes. Uncovering organizational mechanisms of bacterial genomes has been a primary focus of researchers to reveal the potential cellular activities. The advances in both experimental techniques and computational models provide a tremendous opportunity for understanding these mechanisms, and various studies have been proposed to explore the organization rules of bacterial genomes associated with functions recently. This review focuses mainly on the principles that shape the organization of bacterial genomes, both locally and globally. We first illustrate local structures as operons/transcription units for facilitating co-transcription and horizontal transfer of genes. We then clarify the constraints that globally shape bacterial genomes, such as metabolism, transcription and replication. Finally, we highlight challenges and opportunities to advance bacterial genomic studies and provide application perspectives of genome organization, including pathway hole assignment and genome assembly and understanding disease mechanisms.

The Ingenuity of Bacterial Genomes.

The genomes of bacteria contain fewer genes and substantially less noncoding DNA than those of eukaryotes, and as a result, they have much less raw material to invent new traits. Yet, bacteria are vastly more taxonomically diverse, numerically abundant, and globally successful in colonizing new habitats compared to eukaryotes. Although bacterial genomes are generally considered to be optimized for efficient growth and rapid adaptation, nonadaptive processes have played a major role in shaping the size, contents, and compact organization of bacterial genomes and have allowed the establishment of deleterious traits that serve as the raw materials for genetic innovation.

The Impact of Global Transcriptional Regulation on Bacterial Gene Order.

SummaryBacterial gene expression depends on the allocation of limited transcriptional resources provided a particular growth rate and growth condition. Early studies in a few genes suggested this global regulation to generate a unifying hyperbolic expression pattern. Here, we developed a large-scale method that generalizes these experiments to quantify the response to growth of over 700 genes that a priori do not exhibit any specific control. We distinguish a core subset following a promoter-specific hyperbolic response. Within this group, we sort genes with regard to their responsiveness to the global regulatory program to show that those with a particularly sensitive linear response are located near the origin of replication. We then find evidence that this genomic architecture is biologically significant by examining position conservation of E. coli genes in 100 bacteria. The response to the transcriptional resources of the cell results in an additional feature contributing to bacterial genome organization.

Direct imaging of the circular chromosome in a live bacterium

Although the physical properties of chromosomes, including their morphology, mechanics, and dynamics are crucial for their biological function, many basic questions remain unresolved. Here we directly image the circular chromosome in live E. coli with a broadened cell shape. We find that it exhibits a torus topology with, on average, a lower-density origin of replication and an ultrathin flexible string of DNA at the terminus of replication. At the single-cell level, the torus is strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. Our data show a domain organization underlying the chromosome structure of E. coli, where MatP proteins induce site-specific persistent domain boundaries at Ori/Ter, while transcription regulators HU and Fis induce weaker transient domain boundaries throughout the genome. These findings provide an architectural basis for the understanding of the dynamic spatial organization of bacterial genomes in live cells.

Defined chromosome structure in the genome-reduced bacterium Mycoplasma pneumoniae

DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems.

Identification of Novel Raft Marker Protein, FlotP in Bacillus anthracis.

Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated lipid molecule on membrane of BA. In addition, treatment with ZA decreased secretion of anthrax toxins and FlotP expression, suggesting potential role of raft in pathogenesis and physiology of BA. Thus, the present study not only suggest the existence and role of raft like entity in pathophysiology of BA but also its possible use for the development of novel drugs or vaccines against anthrax.

Complete Genome Sequence of the African Strain AXO1947 of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae is the etiological agent of bacterial rice blight. Three distinct clades of X. oryzae pv. oryzae are known. We present the complete annotated genome of the African clade strain AXO194 using long-read single-molecule PacBio sequencing technology. The genome comprises a single chromosome of 4,674,975 bp and encodes for nine transcriptional activator-like (TAL) effectors. The approach and data presented in this announcement provide information for complex bacterial genome organization and the discovery of new virulence effectors, and they facilitate target characterization of TAL effectors.

A biophysical model of supercoiling dependent transcription predicts a structural aspect to gene regulation.

BackgroundTranscription in Escherichia coli generates positive supercoiling in the DNA, which is relieved by the enzymatic activity of gyrase. Recently published experimental evidence suggests that transcription initiation and elongation are inhibited by the buildup of positive supercoiling. It has therefore been proposed that intermittent binding of gyrase plays a role in transcriptional bursting. Considering that transcription is one of the most fundamental cellular processes, it is desirable to be able to account for the buildup and release of positive supercoiling in models of transcription.ResultsHere we present a detailed biophysical model of gene expression that incorporates the effects of supercoiling due to transcription. By directly linking the amount of positive supercoiling to the rate of transcription, the model predicts that highly transcribed genes’ mRNA distributions should substantially deviate from Poisson distributions, with enhanced density at low mRNA copy numbers. Additionally, the model predicts a high degree of correlation between expression levels of genes inside the same supercoiling domain.ConclusionsOur model, incorporating the supercoiling state of the gene, makes specific predictions that differ from previous models of gene expression. Genes in the same supercoiling domain influence the expression level of neighboring genes. Such structurally dependent regulation predicts correlations between genes in the same supercoiling domain. The topology of the chromosome therefore creates a higher level of gene regulation, which has broad implications for understanding the evolution and organization of bacterial genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13628-016-0027-0) contains supplementary material, which is available to authorized users.

Gene regulation by H-NS as a function of growth conditions depends on chromosomal position in Escherichia coli.

Cellular adaptation to changing environmental conditions requires the coordinated regulation of expression of large sets of genes by global regulatory factors such as nucleoid associated proteins. Although in eukaryotic cells genomic position is known to play an important role in regulation of gene expression, it remains to be established whether in bacterial cells there is an influence of chromosomal position on the efficiency of these global regulators. Here we show for the first time that genome position can affect transcription activity of a promoter regulated by the histone-like nucleoid-structuring protein (H-NS), a global regulator of bacterial transcription and genome organization. We have used as a local reporter of H-NS activity the level of expression of a fluorescent reporter protein under control of an H-NS−regulated promoter (Phns) at different sites along the genome. Our results show that the activity of the Phns promoter depends on whether it is placed within the AT-rich regions of the genome that are known to be bound preferentially by H-NS. This modulation of gene expression moreover depends on the growth phase and the growth rate of the cells, reflecting the changes taking place in the relative abundance of different nucleoid proteins and the inherent heterogeneous organization of the nucleoid. Genomic position can thus play a significant role in the adaptation of the cells to environmental changes, providing a fitness advantage that can explain the selection of a gene’s position during evolution.

The Ghost in the Machine: Is the Bacterial Chromosome a Phantom Chain?

In the classic picture of bacterial genome organization, the bacterial chromosome is considered to be a disordered polymer, like spaghetti confined in a bag. Progress in high-resolution microscopy radically advanced our understanding of chromosomal organization and completely toppled this classic picture. Many experiments in recent years have revealed intriguing order in chromosomal structure at different length- and timescales (1,2). The questions that arise from these observations include: What is the origin of multiscale order? And what does it mean?

Insights into structural variations and genome rearrangements in prokaryotic genomes.

Structural variations (SVs) are genomic rearrangements that affect fairly large fragments of DNA. Most of the SVs such as inversions, deletions and translocations have been largely studied in context of genetic diseases in eukaryotes. However, recent studies demonstrate that genome rearrangements can also have profound impact on prokaryotic genomes, leading to altered cell phenotype. In contrast to single-nucleotide variations, SVs provide a much deeper insight into organization of bacterial genomes at a much better resolution. SVs can confer change in gene copy number, creation of new genes, altered gene expression and many other functional consequences. High-throughput technologies have now made it possible to explore SVs at a much refined resolution in bacterial genomes. Through this review, we aim to highlight the importance of the less explored field of SVs in prokaryotic genomes and their impact. We also discuss its potential applicability in the emerging fields of synthetic biology and genome engineering where targeted SVs could serve to create sophisticated and accurate genome editing.

A Tight Link between Orthologs and Bidirectional Best Hits in Bacterial and Archaeal Genomes

Orthologous relationships between genes are routinely inferred from bidirectional best hits (BBH) in pairwise genome comparisons. However, to our knowledge, it has never been quantitatively demonstrated that orthologs form BBH. To test this “BBH-orthology conjecture,” we take advantage of the operon organization of bacterial and archaeal genomes and assume that, when two genes in compared genomes are flanked by two BBH show statistically significant sequence similarity to one another, these genes are bona fide orthologs. Under this assumption, we tested whether middle genes in “syntenic orthologous gene triplets” form BBH. We found that this was the case in more than 95% of the syntenic gene triplets in all genome comparisons. A detailed examination of the exceptions to this pattern, including maximum likelihood phylogenetic tree analysis, showed that some of these deviations involved artifacts of genome annotation, whereas very small fractions represented random assignment of the best hit to one of closely related in-paralogs, paralogous displacement in situ, or even less frequent genuine violations of the BBH–orthology conjecture caused by acceleration of evolution in one of the orthologs. We conclude that, at least in prokaryotes, genes for which independent evidence of orthology is available typically form BBH and, conversely, BBH can serve as a strong indication of gene orthology.

Multidisciplinary perspectives on bacterial genome organization and dynamics

Bacterial genomes are organized by a plethora of chromatin proteins and physical mechanisms. This organization appears to be hierarchical with DNA folding events at the nm scale influencing higher levels of chromosome organization. Besides acting in shaping the genome these factors also play important regulatory roles in numerous DNA transactions. While DNA folding mechanisms operating at the nm scale are fairly well understood, it has been hard to translate this knowledge into accurate models that describe the complete dynamics of the genome. In recent years new techniques have evolved that are key to filling the current gaps in understanding. Particularly insightful in this light appear techniques that probe architectural properties of chromatin proteins on single molecules, techniques that map the binding of protein components and spatial structure on a genome-wide basis and improved imaging techniques that provide resolutions capable of resolving substructures/heterogeneities in the nucleoid. Moreover, bioinformatic and polymer physics approaches are starting to provide novel insights. In our opinion, an important aim in the field is to generate an accurate and complete description of the nucleoid and its dynamics at all scales. A first step towards this aim has now been set by bringing together people from diverse disciplinary backgrounds at the Lorentz centre workshop 'Biology and Physics of Bacterial Genome Organization' in Leiden, the Netherlands from 18 to 22 June 2012.