Biology subjects

van Raaphorst, R.

Publications and source records attributed to van Raaphorst, R..

9 recordsLinked to original sources

bioRxiv2025

Dynamics and interplay of gene expression and chromosome organization across a predatory lifecycle

The obligate predatory bacterium Bdellovibrio bacteriovorus alternates between a motile attack phase and a growth phase inside another bacterium, during which it undergoes multiple rounds of DNA replication followed by non-binary division and progeny release. How its chromosome is dynamically reorganized across these contrasting states remains unclear. Here, we used synchronized predatory infections combined with time-resolved RNA-seq, Hi-C, ChIP-seq, and quantitative microscopy to map transcriptional activity and chromosome architecture across seven key stages of the predatory lifecycle. We uncover a dramatic shift in nucleoid organization - from an ultra-compact, transcriptionally restricted state in attack-phase cells to a progressively decompacted, ori-centered conformation during intracellular replication. Notably, distinct transcriptional and chromosome folding states not only mark the canonical attack and growth phases, but also critical transitions such as prey invasion, replication onset, and predator division. Temporal gene expression profiles reflect the coordinated regulation of core cellular functions across the predatory cycle. Furthermore, spatial localization of RNA polymerase, ribosomes, DNA-binding proteins, and freely diffusing proteins highlight the tight, cell cycle-dependent coupling between nucleoid compaction, chromosome accessibility, and transcriptional activity. Altogether, our findings reveal the intricate dynamics and coordination of genome folding and gene expression in a complex bacterial cell cycle driven by prey-predator interactions.

microbiology↗

bioRxiv2024

HU promotes higher-order chromosome organisation and influences DNA replication rates in Streptococcus pneumoniae

Nucleoid-associated proteins (NAPs) are crucial for maintaining chromosomal compaction and architecture and are actively involved in DNA replication, recombination, repair, and gene regulation. In the opportunistic pathogen Streptococcus pneumoniae, HU is the only identified NAP, and its role in chromosome conformation and other essential processes has not yet been investigated. Here, we use a multi-scale approach to explore the role of HU in chromosome conformation and segregation dynamics. By combining superresolution microscopy and whole-genome binding analysis, we describe the nucleoid as a dynamic structure where HU binds transiently across the entire nucleoid, with a preference for the origin of replication over the terminus. Reducing cellular HU levels impacts nucleoid maintenance and disrupts robust nucleoid scaling with cell size. This effect is similar to the distortion caused by fluoroquinolone-antibiotics, supporting earlier observations that HU is essential for maintaining DNA supercoiling. Furthermore, in cells lacking HU, the replication machinery is misplaced, and cells are unable to initiate and proceed with on-going replication. Chromosome conformation capture (Hi-C) experiments revealed that HU is required to maintain cohesion between the two chromosomal arms, in a similar way to the structural maintenance of the chromosome complex SMC. Together, we show that by promoting long-range chromosome interactions and supporting the architecture of the domain encompassing the origin, HU is fundamental for chromosome integrity and the intimately related processes of chromosome replication and segregation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/615122v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@10cfa9eorg.highwire.dtl.DTLVardef@119ae12org.highwire.dtl.DTLVardef@f35cd4org.highwire.dtl.DTLVardef@1537e7b_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Rewiring capsule production by CRISPRi-based genetic oscillators demonstrates a functional role of phenotypic variation in pneumococcal-host interactions

Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence1-8, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes9-14, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation15-20. In this study, we used synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference together with live cell microscopy and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.

microbiology↗

bioRxiv2022

Modulation of prey size reveals adaptability and robustness in the cell cycle of an intracellular predator

Despite the remarkable diversity of bacterial lifestyles, the sophisticated regulatory networks underlying bacterial replication have only been investigated in a limited number of model species so far. In bacteria that do not rely on canonical binary division for proliferation, the coordination of major cellular processes is still mysterious. Moreover, bacterial growth and division remain largely unexplored within spatially confined niches where nutrients are limited. This includes the lifecycle of the model endobiotic predatory bacterium Bdellovibrio bacteriovorus, which grows by filamentation within its host or prey cells and produces a variable number of daughter cells. Here, we examined how the size of the micro-compartment in which predators replicate (i.e., the prey bacterium) impacts their cell cycle progression at the single-cell level. Using Escherichia coli with genetically encoded size differences, we show that the duration of the predator cell cycle scales with prey size. As a result, the size of the prey determines the number of predator offspring, through a relationship that is maintained across prey species. Strikingly, the nutritional quality of the prey cell determined the specific growth rate of predators regardless of prey size, reminiscent of the effect of medium composition on the growth of other bacteria. Tuning the predatory cell cycle by modulating prey dimensions also allowed us to reveal invariable temporal connections between key cellular processes. Altogether, our data uncover adaptability and robustness shaping the enclosed replication of B. bacteriovorus. Consequently, predators optimally exploit the finite prey resources and space while ensuring a strict cell cycle progression. This study opens the way to explore diverse cell cycle control strategies by extending their characterization to intracellular lifestyles, beyond canonical models and growth conditions.

microbiology↗

bioRxiv2020

Chromosome dynamics and spatial organization during the non-binary cell cycle of a predatory bacterium

In bacteria, the dynamics of chromosome replication and segregation are tightly coordinated with cell cycle progression, and largely rely on specific spatiotemporal arrangement of the chromosome. Whereas these key processes are mostly investigated in species that divide by binary fission, they remain mysterious in bacteria producing larger number of descendants. Here, we establish the predatory bacterium Bdellovibrio bacteriovorus as a model to investigate the non-binary processing of a circular chromosome. Our data reveal its extreme compaction in a dense polarized nucleoid. We also show that a first binary-like cycle of replication and asymmetric segregation is followed by multiple asynchronous rounds of replication and progressive ParABS-dependent partitioning, uncoupled from cell division. Surprisingly, ParB localization at the centromere is cell-cycle regulated. Altogether, our findings support a model of complex chromosome choreography, leading to the generation of variable numbers of offspring, highlighting the adaptation of conserved mechanisms to achieve non-binary reproduction in bacteria.

microbiology↗

bioRxiv2020

Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

The rapid spread of antimicrobial resistance and vaccine escape in the opportunistic human pathogen Streptococcus pneumoniae can be largely attributed to competence-induced transformation. To better understand the dynamics of competence-induced transformation, we studied this process at the single-cell level. We show that within isogenic populations, all cells become naturally competent and bind exogenous DNA. In addition, we find that transformation is highly efficient and that the chromosomal location of the integration site or whether the transformed gene is encoded on the leading or lagging strand has limited influence on recombination efficiency. Indeed, we have observed multiple recombination events in single recipients in real-time. However, because of saturation of the DNA uptake and integration machinery and because a single stranded donor DNA replaces the original allele, we find that transformation efficiency has an upper threshold of approximately 50% of the population. Counterintuitively, in the presence of multiple transforming DNAs, the fraction of untransformed cells increases to more than 50%. The fixed mechanism of transformation results in a fail-safe strategy for the population as half of the population generally keeps an intact copy of the original genome. Together, this work advances our understanding of pneumococcal genome plasticity.

microbiology↗

bioRxiv2019

Spatio-temporal control of DNA replication by the pneumococcal cell cycle regulator CcrZ

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance is poorly understood. Here, we identified a conserved and essential protein in pneumococci and related Firmicutes named CcrZ (for Cell Cycle Regulator protein interacting with FtsZ) that couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae directly interacts with the cytoskeleton protein FtsZ to place it in the middle of the newborn cell where the DnaA-bound origin is positioned. Together, this work uncovers a new mechanism for the control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.

microbiology↗

bioRxiv2019

BactMAP: an R package for integrating, analyzing and visualizing bacterial microscopy data

High-throughput analyses of single-cell microscopy data is a critical tool within the field of bacterial cell biology. Several programs have been developed to specifically segment bacterial cells from phase-contrast images. Together with spot and object detection algorithms, these programs offer powerful approaches to quantify observations from microscopy data, ranging from cell-to-cell genealogy to localization and movement of proteins. Most segmentation programs contain specific post-processing and plotting options, but these options vary between programs and possibilities to optimize or alter the outputs are often limited. Therefore, we developed BactMAP (Bacterial toolbox for Microscopy Analysis & Plotting), a software package that allows researchers to transform cell segmentation and spot detection data generated by different programs automatically into various plots. Furthermore, BactMAP makes it possible to perform custom analyses and change the layout of the output. Because BactMAP works independently of segmentation and detection programs, inputs from different sources can be compared within the same analysis pipeline. BactMAP runs in R, which enables the use of advanced statistical analysis tools as well as easily adjustable plot graphics in every operating system. Using BactMAP we visualize key cell cycle parameters in Bacillus subtilis and Staphylococcus aureus, and demonstrate that the DNA replication forks in Streptococcus pneumoniae dissociate and associate before splitting of the cell, after the Z-ring is formed at the new quarter positions. BactMAP is available from https://veeninglab.com/bactmap.

microbiology↗

bioRxiv2016

Chromosome segregation drives division site selection in Streptococcus pneumoniae

Accurate spatial and temporal positioning of the tubulin-like protein FtsZ is key for proper bacterial cell division. Streptococcus pneumoniae (pneumococcus) is an oval-shaped, symmetrically dividing human pathogen lacking the canonical systems for division site control (nucleoid occlusion and the Min-system). Recently, the early division protein MapZ was identified and implicated in pneumococcal division site selection. We show that MapZ is important for proper division plane selection; thus the question remains what drives pneumococcal division site selection. By mapping the cell cycle in detail, we show that directly after replication both chromosomal origin regions localize to the future cell division sites, prior to FtsZ. Perturbing the longitudinal chromosomal organization by mutating the condensin SMC, by CRISPR/Cas9-mediated chromosome cutting or by poisoning DNA decatenation resulted in mistiming of MapZ and FtsZ positioning and subsequent cell elongation. Together, we demonstrate an intimate relationship between DNA replication, chromosome segregation and division site selection in the pneumococcus, providing a simple way to ensure equally sized daughter cells without the necessity for additional protein factors.

microbiology↗