Manipulating the position of DNA expression cassettes using location tags fused to dCas9 (Cas9-Lag) to improve metabolic pathway efficiency.

BACKGROUND: Deactivated Cas9 (dCas9) led to significant improvement of CRISPR/Cas9-based techniques because it can be fused with a variety of functional groups to form diverse molecular devices, which can manipulate or modify target DNA cassettes. One important metabolic engineering strategy is to localize the enzymes in proximity of their substrates for improved catalytic efficiency. In this work, we developed a novel molecular device to manipulate the cellular location of specific DNA cassettes either on plasmids or on the chromosome, by fusing location tags to dCas9 (Cas9-Lag), and applied the technique for synthetic biology applications. Carotenoids like ß-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane compartment. RESULTS: Carotenoids like ß-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane components. To improve the functional expression of membrane-bound enzymes and localize them in proximity to the substrates, Cas9-Lag was used to pull plasmids or chromosomal DNA expressing carotenoid enzymes onto the cell membrane. For this purpose, dCas9 was fused to the E. coli membrane docking tag GlpF, and gRNA was designed to direct this fusion protein to the DNA expression cassettes. With Cas9-Lag, the zeaxanthin and astaxanthin titer increased by 29.0% and 26.7% respectively. Due to experimental limitations, the electron microscopy images of cells expressing Cas9-Lag vaguely indicated that GlpF-Cas9 might have pulled the target DNA cassettes in close proximity to membrane. Similarly, protein mass spectrometry analysis of membrane proteins suggested an increased expression of carotenoid-converting enzymes in the membrane components. CONCLUSION: This work therefore provides a novel molecular device, Cas9-Lag, which was proved to increase zeaxanthin and astaxanthin production and might be used to manipulate DNA cassette location.

SMC complexes organize the bacterial chromosome by lengthwise compaction.

Structural maintenance of chromosomes (SMC) complexes are ancient and conserved molecular machines that organize chromosomes in all domains of life. We propose that the principles of chromosome folding needed to accommodate DNA inside a cell in an accessible form will follow similar principles in prokaryotes and eukaryotes. However, the exact contributions of SMC complexes to bacterial chromosome organization have been elusive. Recently, it was shown that the SMC homolog, MukBEF, organizes and individualizes the Escherichia coli chromosome by forming a filamentous axial core from which DNA loops emanate, similar to the action of condensin in mitotic chromosome formation. MukBEF action, along with its interaction with the partner protein, MatP, also facilitates chromosome individualization by directing opposite chromosome arms (replichores) to different cell halves. This contrasts with the situation in many other bacteria, where SMC complexes organise chromosomes in a way that the opposite replichores are aligned along the long axis of the cell. We highlight the similarities and differences of SMC complex contributions to chromosome organization in bacteria and eukaryotes, and summarize the current mechanistic understanding of the processes.

Growth Phase-Dependent Chromosome Condensation and Heat-Stable Nucleoid-Structuring Protein Redistribution in Escherichia coli under Osmotic Stress.

The heat-stable nucleoid-structuring (H-NS) protein is a global transcriptional regulator implicated in coordinating the expression of over 200 genes in Escherichia coli, including many involved in adaptation to osmotic stress. We have applied superresolved microscopy to quantify the intracellular and spatial reorganization of H-NS in response to a rapid osmotic shift. We found that H-NS showed growth phase-dependent relocalization in response to hyperosmotic shock. In stationary phase, H-NS detached from a tightly compacted bacterial chromosome and was excluded from the nucleoid volume over an extended period of time. This behavior was absent during rapid growth but was induced by exposing the osmotically stressed culture to a DNA gyrase inhibitor, coumermycin. This chromosomal compaction/H-NS exclusion phenomenon occurred in the presence of either potassium or sodium ions and was independent of the presence of stress-responsive sigma factor σS and of the H-NS paralog StpA.IMPORTANCE The heat-stable nucleoid-structuring (H-NS) protein coordinates the expression of over 200 genes in E. coli, with a large number involved in both bacterial virulence and drug resistance. We report on the novel observation of a dynamic compaction of the bacterial chromosome in response to exposure to high levels of salt. This stress response results in the detachment of H-NS proteins and their subsequent expulsion to the periphery of the cells. We found that this behavior is related to mechanical properties of the bacterial chromosome, in particular, to how tightly twisted and coiled is the chromosomal DNA. This behavior might act as a biomechanical response to stress that coordinates the expression of genes involved in adapting bacteria to a salty environment.

Construction of Fluorescent Pneumococci for In Vivo Imaging and Labeling of the Chromosome.

Advances in fluorescence imaging techniques and development and optimization of fluorescent proteins recent years have made major impacts on different fields of pneumococcal research. This chapter provides methodology for construction of fluorescent pneumococcal strains using fusions to DNA-binding proteins. By expressing fluorescent proteins fused to HlpA, a pneumococcal nucleoid binding protein, brightly fluorescent pneumococci are generated. HlpA fusions may be used both for in vivo imaging of pneumococci as well as for marking the nucleoid in cell biology studies. Furthermore, it also explains how to construct strains for imaging of specific chromosomal loci in pneumococci, using a heterologous ParBS system.

HflX protein protects Escherichia coli from manganese stress.

The ribosome-binding GTPase HflX is required for manganese homeostasis in E. coli. While under normal conditions ΔhflX cells behave like wild type E. coli with respect to growth pattern and morphology, deletion of hflX makes E. coli cells extremely sensitive to manganese, characterized by arrested cell growth and filamentation. Here we demonstrate that upon complementation by hflX, manganese stress is relieved. In phenotypic studies done in a manganese-rich environment, ΔhflX cells were highly sensitive to antibiotics that bind the penicillin binding protein 3 (PBP3), suggesting that the manganese stress led to impaired peptidoglycan biosynthesis. An irregular distribution of dark bands of constriction along filaments, delocalization of the dark bands from midcell towards poles and subpoles, lack of septum formation and arrested cell division were observed in ΔhflX cells under manganese stress. However, chromosome replication and segregation of nucleoids were unaffected under these conditions, as observed from confocal microscopy imaging and FACS studies. We conclude that absence of HflX leads to manganese accumulation in E. coli cells, affecting cell septum formation, probably by modulating the activity of the cell division protein PBP3 (FtsI), a major component of the divisome apparatus. We propose that HflX acts as a gatekeeper, regulating the influx of manganese into the cell.

Single molecule tracking reveals that the bacterial SMC complex moves slowly relative to the diffusion of the chromosome.

Structural Maintenance of Chromosomes (SMC) proteins and their complex partners (ScpA and ScpB in many bacteria) are involved in chromosome compaction and segregation in all kinds of organisms. We employed single molecule tracking (SMT), tracking of chromosomal loci, and single molecule counting in Bacillus subtilis to show that in slow growing cells, ∼30 Smc dimers move throughout the chromosome in a constrained mode, while ∼60 ScpA and ScpB molecules travel together in a complex, but independently of the nucleoid. Even an Smc truncation that lacks the ATP binding head domains still scans the chromosome, highlighting the importance of coiled coil arm domains. When forming a complex, 10-15 Smc/ScpAB complexes become essentially immobile, moving slower than chromosomal loci. Contrarily, SMC-like protein RecN, which forms assemblies at DNA double strand breaks, moves faster than chromosome sites. In the absence of Smc, chromosome sites investigated were less mobile than in wild type cells, indicating that Smc contributes to chromosome dynamics. Thus, our data show that Smc/ScpAB clusters occur at several sites on the chromosome and contribute to chromosome movement.

Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus.

We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in Escherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E's function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.

High-resolution 3D models of Caulobacter crescentus chromosome reveal genome structural variability and organization.

High-resolution three-dimensional models of Caulobacter crescentus nucleoid structures were generated via a multi-scale modeling protocol. Models were built as a plectonemically supercoiled circular DNA and by incorporating chromosome conformation capture based data to generate an ensemble of base pair resolution models consistent with the experimental data. Significant structural variability was found with different degrees of bending and twisting but with overall similar topologies and shapes that are consistent with C. crescentus cell dimensions. The models allowed a direct mapping of the genomic sequence onto the three-dimensional nucleoid structures. Distinct spatial distributions were found for several genomic elements such as AT-rich sequence elements where nucleoid associated proteins (NAPs) are likely to bind, promoter sites, and some genes with common cellular functions. These findings shed light on the correlation between the spatial organization of the genome and biological functions.

Mycobacterial nucleoid associated proteins: An added dimension in gene regulation.

Nucleoid associated proteins (NAPs) are known organisers of chromosomal structure and regulators of transcriptional expression. The number of proposed NAPs in mycobacteria are significantly lower than the number identified in other organisms. An interesting feature of mycobacterial NAPs is their low sequence similarity with those in other species, a property that has hindered their identification. In this review, we discuss the current evidence for the proposed classification of six mycobacterial proteins, Lsr2, EspR, mIHF, HupB, MDP2 and NapM, as NAPs in mycobacterial species with an emphasis on their roles in modulating chromosome structure and transcriptional regulation. In addition, we highlight the technical difficulties associated with investigating and providing evidence for the classification of proteins as NAPs in mycobacteria. We also address the role of mycobacterial NAPs as mediators of stress responses and highlight the recent developments aimed at targeting NAP-DNA interactions for the development of novel anti-TB drugs.

Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins.

As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to ∼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization.

A ring-polymer model shows how macromolecular crowding controls chromosome-arm organization in Escherichia coli.

Macromolecular crowding influences various cellular processes such as macromolecular association and transcription, and is a key determinant of chromosome organization in bacteria. The entropy of crowders favors compaction of long chain molecules such as chromosomes. To what extent is the circular bacterial chromosome, often viewed as consisting of "two arms", organized entropically by crowding? Using computer simulations, we examine how a ring polymer is organized in a crowded and cylindrically-confined space, as a coarse-grained bacterial chromosome. Our results suggest that in a wide parameter range of biological relevance crowding is essential for separating the two arms in the way observed with Escherichia coli chromosomes at fast-growth rates, in addition to maintaining the chromosome in an organized collapsed state. Under different conditions, however, the ring polymer is centrally condensed or adsorbed onto the cylindrical wall with the two arms laterally collapsed onto each other. We discuss the relevance of our results to chromosome-membrane interactions.

A novel nucleoid-associated protein coordinates chromosome replication and chromosome partition.

We searched for regulators of chromosome replication in the cell cycle model Caulobacter crescentus and found a novel DNA-binding protein (GapR) that selectively aids the initiation of chromosome replication and the initial steps of chromosome partitioning. The protein binds the chromosome origin of replication (Cori) and has higher-affinity binding to mutated Cori-DNA that increases Cori-plasmid replication in vivo. gapR gene expression is essential for normal rapid growth and sufficient GapR levels are required for the correct timing of chromosome replication. Whole genome ChIP-seq identified dynamic DNA-binding distributions for GapR, with the strongest associations at the partitioning (parABS) locus near Cori. Using molecular-genetic and fluorescence microscopy experiments, we showed that GapR also promotes the first steps of chromosome partitioning, the initial separation of the duplicated parS loci following replication from Cori. This separation occurs before the parABS-dependent partitioning phase. Therefore, this early separation, whose mechanisms is not known, coincides with the poorly defined mechanism(s) that establishes chromosome asymmetry: C. crescentus chromosomes are partitioned to distinct cell-poles which develop into replicating and non-replicating cell-types. We propose that GapR coordinates chromosome replication with asymmetry-establishing chromosome separation, noting that both roles are consistent with the phylogenetic restriction of GapR to asymmetrically dividing bacteria.

Features of genomic organization in a nucleotide-resolution molecular model of the Escherichia coli chromosome.

We describe structural models of the Escherichia coli chromosome in which the positions of all 4.6 million nucleotides of each DNA strand are resolved. Models consistent with two basic chromosomal orientations, differing in their positioning of the origin of replication, have been constructed. In both types of model, the chromosome is partitioned into plectoneme-abundant and plectoneme-free regions, with plectoneme lengths and branching patterns matching experimental distributions, and with spatial distributions of highly-transcribed chromosomal regions matching recent experimental measurements of the distribution of RNA polymerases. Physical analysis of the models indicates that the effective persistence length of the DNA and relative contributions of twist and writhe to the chromosome's negative supercoiling are in good correspondence with experimental estimates. The models exhibit characteristics similar to those of 'fractal globules,' and even the most genomically-distant parts of the chromosome can be physically connected, through paths combining linear diffusion and inter-segmental transfer, by an average of only ∼10 000 bp. Finally, macrodomain structures and the spatial distributions of co-expressed genes are analyzed: the latter are shown to depend strongly on the overall orientation of the chromosome. We anticipate that the models will prove useful in exploring other static and dynamic features of the bacterial chromosome.

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.

Protein depletion using the arabinose promoter in Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (X. citri) is a plant pathogen and the etiological agent of citrus canker, a severe disease that affects all the commercially important citrus varieties, and has worldwide distribution. Citrus canker cannot be healed, and the best method known to control the spread of X. citri in the orchards is the eradication of symptomatic and asymptomatic plants in the field. However, in the state of São Paulo, Brazil, the main orange producing area in the world, control is evolving to an integrated management system (IMS) in which growers have to use less susceptible plants, windshields to prevent bacterial spread out and sprays of cupric bactericidal formulations. Our group has recently proposed alternative methods to control citrus canker, which are based on the use of chemical compounds able to disrupt vital cellular processes of X. citri. An important step in this approach is the genetic and biochemical characterization of genes/proteins that are the possible targets to be perturbed, a task not always simple when the gene/protein under investigation is essential for the organism. Here, we describe vectors carrying the arabinose promoter that enable controllable protein expression in X. citri. These vectors were used as complementation tools for the clean deletion of parB in X. citri, a widespread and conserved gene involved in the essential process of bacterial chromosome segregation. Overexpression or depletion of ParB led to increased cell size, which is probably a resultant of delayed chromosome segregation with subsequent retard of cell division. However, ParB is not essential in X. citri, and in its absence the bacterium was fully competent to colonize the host citrus and cause disease. The arabinose expression vectors described here are valuable tools for protein expression, and especially, to assist in the deletion of essential genes in X. citri.

Interference-driven spacer acquisition is dominant over naive and primed adaptation in a native CRISPR-Cas system.

CRISPR-Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR-Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR-Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3'-5' translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition ('targeted acquisition') is a major contributor to adaptation in type I-F CRISPR-Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome.

Investigating Bacterial Chromosome Architecture.

How is the bacterial chromosome organized within the bacterial cell? Over the last 60 years, a variety of approaches have been used to investigate this question. More recently, the parallel development of epifluorescence microscopy and genetic tools has enabled the direct visualization of the intracellular positioning of DNA sequences in live cells and has consequently revolutionized our view of the architecture of the nucleoid in vivo. In this chapter I present a comprehensive methodology designed to characterize the architecture of the nucleoid DNA and the positioning of specific DNA sequences in live Escherichia coli cells. DNA localization systems, preparation of stable agarose-mounted microscopy slides, and basic image analysis tools are mentioned.

Structures of the nucleoid occlusion protein SlmA bound to DNA and the C-terminal domain of the cytoskeletal protein FtsZ.

Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called "nucleoid occlusion" (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator-CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA-DNA-FtsZ CTD regulatory interaction and provide insight into FtsZ-regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA-DNA-FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA-DNA-FtsZ interaction with implications for SlmA's NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins.

Control of Smc Coiled Coil Architecture by the ATPase Heads Facilitates Targeting to Chromosomal ParB/parS and Release onto Flanking DNA.

Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosome-presumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc.

A model for chromosome organization during the cell cycle in live E. coli.

Bacterial chromosomal DNA is a highly compact nucleoid. The organization of this nucleoid is poorly understood due to limitations in the methods used to monitor the complexities of DNA organization in live bacteria. Here, we report that circular plasmid DNA is auto-packaged into a uniform dual-toroidal-spool conformation in response to mechanical stress stemming from sharp bending and un-winding by atomic force microscopic analysis. The mechanism underlying this phenomenon was deduced with basic physical principles to explain the auto-packaging behaviour of circular DNA. Based on our observations and previous studies, we propose a dynamic model of how chromosomal DNA in E. coli may be organized during a cell division cycle. Next, we test the model by monitoring the development of HNS clusters in live E. coli during a cell cycle. The results were in close agreement with the model. Furthermore, the model accommodates a majority of the thus-far-discovered remarkable features of nucleoids in vivo.