Brucella MucR acts as an H-NS-like protein to silence virulence genes and structure the nucleoid.

Histone-like nucleoid structuring (H-NS) and H-NS-like proteins serve as global gene silencers and work with antagonistic transcriptional activators (counter-silencers) to properly coordinate the expression of virulence genes in pathogenic bacteria. In Brucella, MucR has been proposed as a novel H-NS-like gene silencer, but direct experimental evidence is lacking. Here, we show that MucR serves as an H-NS-like silencer of the Brucella abortus genes encoding the polar autotransporter adhesins BtaE and BmaC, the c-di-GMP-specific phosphodiesterase BpdB, and the quorum-sensing regulator BabR. We also demonstrate that the MarR-type transcriptional activator MdrA can displace MucR from the btaE promoter, supporting the existence of MucR counter-silencers in Brucella. Moreover, our chromatin immunoprecipitation (ChIP)-seq analysis identified 546 MucR enrichment peaks along the genome, including in the promoters of the genes encoding the Type IV secretion machinery and effectors and the quorum-sensing regulator VjbR. Importantly, MucR ChIP-seq peaks overlap with the previously described binding sites for the transcriptional activators VjbR, BvrR, and CtrA suggesting that these regulators serve as MucR counter-silencers and work in concert with MucR to coordinate virulence gene expression in Brucella. In addition, using chromosome conformation capture (Hi-C), we show that like H-NS in Escherichia coli, MucR alters the global structure of the Brucella nucleoid. Finally, a copy of the E. coli hns rescues the distinctive growth defect and elevated btaE expression of a B. abortus mucR mutant. Together, these findings solidify the role of MucR as a novel type of H-NS-like protein and suggest that MucR's gene-silencing properties play a key role in virulence in Brucella. IMPORTANCE Histone-like nucleoid structuring (H-NS) and H-NS-like proteins coordinate host-associated behaviors in many pathogenic bacteria, often through forming silencer/counter-silencer pairs with signal-responsive transcriptional activators to tightly control gene expression. Brucella and related bacteria do not encode H-NS or homologs of known H-NS-like proteins, and it is unclear if they have other proteins that perform analogous functions during pathogenesis. In this work, we provide compelling evidence for the role of MucR as a novel H-NS-like protein in Brucella. We show that MucR possesses many of the known functions attributed to H-NS and H-NS-like proteins, including the formation of silencer/counter-silencer pairs to control virulence gene expression and global structuring of the nucleoid. These results uncover a new role for MucR as a nucleoid structuring protein and support the importance of temporal control of gene expression in Brucella and related bacteria.

A framework to validate fluorescently labeled DNA-binding proteins for single-molecule experiments.

Due to the enhanced labeling capability of maleimide-based fluorescent probes, lysine-cysteine-lysine (KCK) tags are frequently added to proteins for visualization. In this study, we employed an in vitro single-molecule DNA flow-stretching assay as a sensitive way to assess the impact of the KCK tag on the property of DNA-binding proteins. Using Bacillus subtilis ParB as an example, we show that, although no noticeable changes were detected by in vivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays, the KCK tag substantially altered ParB's DNA compaction rates and its response to nucleotide binding and to the presence of the specific sequence (parS) on the DNA. While it is typically assumed that short peptide tags minimally perturb protein function, our results urge researchers to carefully validate the use of tags for protein labeling. Our comprehensive analysis can be expanded and used as a guide to assess the impacts of other tags on DNA-binding proteins in single-molecule assays.

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.

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.

Using DNA flow-stretching assay as a tool to validate the tagging of DNA-binding proteins for single-molecule experiments.

Due to the enhanced labeling capability of maleimide-based fluorescent probes, lysine-cysteine-lysine (KCK) tags are frequently added to proteins for visualization. In this study, we employed in vitro single-molecule DNA flow-stretching assay as a sensitive way to assess the impact of the KCK-tag on the property of DNA-binding proteins. Using Bacillus subtilis ParB as an example, we show that, although no noticeable changes were detected by in vivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays, the KCK-tag substantially altered ParB's DNA compaction rates, its response to nucleotide binding and to the presence of the specific sequence (parS) on the DNA. While it is typically assumed that short peptide tags minimally perturb protein function, our results urge researchers to carefully validate the use of tags for protein labeling. Our comprehensive analysis can be expanded and used as a guide to assess the impacts of other tags on DNA-binding proteins in single-molecule assays.

Polyploidy, regular patterning of genome copies, and unusual control of DNA partitioning in the Lyme disease spirochete.

Borrelia burgdorferi, the tick-transmitted spirochete agent of Lyme disease, has a highly segmented genome with a linear chromosome and various linear or circular plasmids. Here, by imaging several chromosomal loci and 16 distinct plasmids, we show that B. burgdorferi is polyploid during growth in culture and that the number of genome copies decreases during stationary phase. B. burgdorferi is also polyploid inside fed ticks and chromosome copies are regularly spaced along the spirochete's length in both growing cultures and ticks. This patterning involves the conserved DNA partitioning protein ParA whose localization is controlled by a potentially phage-derived protein, ParZ, instead of its usual partner ParB. ParZ binds its own coding region and acts as a centromere-binding protein. While ParA works with ParZ, ParB controls the localization of the condensin, SMC. Together, the ParA/ParZ and ParB/SMC pairs ensure faithful chromosome inheritance. Our findings underscore the plasticity of cellular functions, even those as fundamental as chromosome segregation.

Roles of RodZ and class A PBP1b in the assembly and regulation of the peripheral peptidoglycan elongasome in ovoid-shaped cells of Streptococcus pneumoniae D39.

RodZ of rod-shaped bacteria functions to link MreB filaments to the Rod peptidoglycan (PG) synthase complex that moves circumferentially perpendicular to the long cell axis, creating hoop-like sidewall PG. Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus; Spn) that lack MreB, use a different modality for peripheral PG elongation that emanates from the midcell of dividing cells. Yet, S. pneumoniae encodes a RodZ homolog similar to RodZ in rod-shaped bacteria. We show here that the helix-turn-helix and transmembrane domains of RodZ(Spn) are essential for growth at 37°C. ΔrodZ mutations are suppressed by Δpbp1a, mpgA(Y488D), and ΔkhpA mutations that suppress ΔmreC, but not ΔcozE. Consistent with a role in PG elongation, RodZ(Spn) co-localizes with MreC and aPBP1a throughout the cell cycle and forms complexes and interacts with PG elongasome proteins and regulators. Depletion of RodZ(Spn) results in aberrantly shaped, non-growing cells and mislocalization of elongasome proteins MreC, PBP2b, and RodA. Moreover, Tn-seq reveals that RodZ(Spn), but not MreCD(Spn), displays a specific synthetic-viable genetic relationship with aPBP1b, whose function is unknown. We conclude that RodZ(Spn) acts as a scaffolding protein required for elongasome assembly and function and that aPBP1b, like aPBP1a, plays a role in elongasome regulation and possibly peripheral PG synthesis.

A dicentric bacterial chromosome requires XerC/D site-specific recombinases for resolution.

Unlike eukaryotes and archaea, which have multiple replication origins on their chromosomes, bacterial chromosomes usually contain a single replication origin.1 Here, we discovered a dicentric bacterial chromosome with two replication origins, which has resulted from the fusion of the circular and linear chromosomes in Agrobacterium tumefaciens. The fused chromosome is well tolerated, stably maintained, and retains similar subcellular organization and genome-wide DNA interactions found for the bipartite chromosomes. Strikingly, the two replication origins and their partitioning systems are both functional and necessary for cell survival. Finally, we discovered that the site-specific recombinases XerC and XerD2 are essential in cells harboring the fused chromosome but not in cells with bipartite chromosomes. Analysis of actively dividing cells suggests a model in which XerC/D are required to recombine the sister fusion chromosomes when the two centromeres on the same chromosome are segregated to opposite cell poles. Thus, faithful segregation of dicentric chromosomes in bacteria can occur because of site-specific recombination between the sister chromatids during chromosome partitioning. Our study provides a natural comparative platform to examine a bacterial chromosome with multiple origins and a possible explanation for the fundamental difference in bacterial genome architecture relative to eukaryotes and archaea.1.

Centromere Interactions Promote the Maintenance of the Multipartite Genome in Agrobacterium tumefaciens.

Many pathogens or symbionts of animals and plants contain multiple replicons, a configuration called a multipartite genome. Multipartite genomes enable those species to replicate their genomes faster and better adapt to new niches. Despite their prevalence, the mechanisms by which multipartite genomes are stably maintained are poorly understood. Agrobacterium tumefaciens is a plant pathogen that contains four replicons: a circular chromosome (Ch1), a linear chromosome (Ch2), and two large plasmids. Recent work indicates that their replication origins are clustered at the cell poles in a manner that depends on their ParB family centromeric proteins: ParB1 for Ch1 and individual RepB paralogs for Ch2 and the plasmids. However, understanding of these interactions and how they contribute to genome maintenance is limited. By combining genome-wide chromosome conformation capture (Hi-C) assays, chromatin-immunoprecipitation sequencing (ChIP-seq), and live cell fluorescence microscopy, we provide evidence here that centromeric clustering is mediated by interactions between these centromeric proteins. We further show that the disruption of centromere clustering results in the loss of replicons. Our data establish the role of centromeric clustering in multipartite genome stability. IMPORTANCE About 10% of sequenced bacteria have multiple replicons, also known as multipartite genomes. How these multipartite genomes are maintained is still poorly understood. Here, we use Agrobacterium tumefaciens as a model and show that the replication origins of the four replicons are clustered through direct interactions between the centromeric proteins; disruption of origin clustering leads to the loss of replicons. Thus, our study provided evidence that centromeric clustering is important for maintaining multipartite genomes.

Conformation and dynamic interactions of the multipartite genome in Agrobacterium tumefaciens.

Bacterial species from diverse phyla contain multiple replicons, yet how these multipartite genomes are organized and segregated during the cell cycle remains poorly understood. Agrobacterium tumefaciens has a 2.8-Mb circular chromosome (Ch1), a 2.1-Mb linear chromosome (Ch2), and two large plasmids (pAt and pTi). We used this alpha proteobacterium as a model to investigate the global organization and temporal segregation of a multipartite genome. Using chromosome conformation capture assays, we demonstrate that both the circular and the linear chromosomes, but neither of the plasmids, have their left and right arms juxtaposed from their origins to their termini, generating interarm interactions that require the broadly conserved structural maintenance of chromosomes complex. Moreover, our study revealed two types of interreplicon interactions: "ori-ori clustering" in which the replication origins of all four replicons interact, and "Ch1-Ch2 alignment" in which the arms of Ch1 and Ch2 interact linearly along their lengths. We show that the centromeric proteins (ParB1 for Ch1 and RepBCh2 for Ch2) are required for both types of interreplicon contacts. Finally, using fluorescence microscopy, we validated the clustering of the origins and observed their frequent colocalization during segregation. Altogether, our findings provide a high-resolution view of the conformation of a multipartite genome. We hypothesize that intercentromeric contacts promote the organization and maintenance of diverse replicons.

DNA-loop-extruding SMC complexes can traverse one another in vivo.

Chromosome organization mediated by structural maintenance of chromosomes (SMC) complexes is vital in many organisms. SMC complexes act as motors that extrude DNA loops, but it remains unclear what happens when multiple complexes encounter one another on the same DNA in living cells and how these interactions may help to organize an active genome. We therefore created a crash-course track system to study SMC complex encounters in vivo by engineering defined SMC loading sites in the Bacillus subtilis chromosome. Chromosome conformation capture (Hi-C) analyses of over 20 engineered strains show an amazing variety of chromosome folding patterns. Through three-dimensional polymer simulations and theory, we determine that these patterns require SMC complexes to bypass each other in vivo, as recently seen in an in vitro study. We posit that the bypassing activity enables SMC complexes to avoid traffic jams while spatially organizing the genome.

XerD unloads bacterial SMC complexes at the replication terminus.

Bacillus subtilis structural maintenance of chromosomes (SMC) complexes are topologically loaded at centromeric sites adjacent to the replication origin by the partitioning protein ParB. These ring-shaped ATPases then translocate down the left and right chromosome arms while tethering them together. Here, we show that the site-specific recombinase XerD, which resolves chromosome dimers, is required to unload SMC tethers when they reach the terminus. We identify XerD-specific binding sites in the terminus region and show that they dictate the site of unloading in a manner that depends on XerD but not its catalytic residue, its partner protein XerC, or the recombination site dif. Finally, we provide evidence that ParB and XerD homologs perform similar functions in Staphylococcus aureus. Thus, two broadly conserved factors that act at the origin and terminus have second functions in loading and unloading SMC complexes that travel between them.