Biology subjects

Mazzuoli, M.-V.

Publications and source records attributed to Mazzuoli, M.-V..

4 recordsLinked to original sources

bioRxiv2024

Virulence regulates and boosts CRISPR-Cas9 immunity in Group B Streptococcus

CRISPR-Cas9 immune systems protect bacteria from foreign DNA. However, immune efficiency is constrained by Cas9 off-target cleavages and toxicity. How bacteria regulate Cas9 to maximize protection while preventing autoimmunity is not understood. Here, we show that the master regulator of virulence, CovR, regulates CRISPR-Cas9 immunity against mobile genetic elements in Streptococcus agalactiae, a pathobiont responsible for invasive neonatal infections. We show that CovR binds to and represses a distal promoter of the cas operon, integrating immunity within the virulence regulatory network. The CovR-regulated promoter provides a controlled increase in off-target cleavages to counteract mutations in the target DNA, restores the potency of old immune memory, and stimulates the acquisition of new memory in response to recent infections. Regulation of Cas9 by CovR is conserved at the species level, with lineage specificities suggesting different adaptive trajectories. Altogether, we describe the coordinated regulation of immunity and virulence that enhances the bacterial immune repertoire during host-pathogen interaction.

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↗

bioRxiv2024

Make-or-break prime editing for bacterial genome engineering

CRISPR-Cas9 has revolutionized genome engineering by allowing precise introductions of DNA double-strand breaks (DSBs). However, genome engineering in bacteria is still a complex, multi-step process requiring a donor DNA template for repair of DSBs. Prime editing circumvents this need as the repair template is indirectly provided within the prime editing guide RNA (pegRNA). Here, we developed make-or-break Prime Editing (mbPE) that allows for precise and effective genetic engineering in the opportunistic human pathogen Streptococcus pneumoniae. In contrast to traditional prime editing in which a nicking Cas9 is employed, mbPE harnesses wild type Cas9 in combination with a pegRNA that destroys the seed region or protospacer adjacent motif. Since most bacteria poorly perform template-independent end joining, correctly genome-edited clones are selectively enriched during mbPE. We show that mbPE is RecA-independent and can be used to introduce point mutations, deletions and targeted insertions, including protein tags such as a split luciferase, at selection efficiencies of over 93%. mbPE enables sequential genome editing, is scalable, and can be used to generate pools of mutants in a high-throughput manner. The mbPE system and pegRNA design guidelines described here will ameliorate future bacterial genome editing endeavors.

microbiology↗

bioRxiv2024

Signal-independent activation reveals two-component regulatory networks

Each bacterial species has specific regulatory systems to control physiology, adaptation, and host interactions. One challenge posed by this diversity is to define the evolving gene regulatory networks. This study aims to characterise two-component systems (TCS) in Streptococcus agalactiae, the main cause of neonatal meningitis. Here we demonstrate signal-independent activation of signalling pathways by systematically targeting the conserved mechanism of phosphatase activity of the 14 histidine kinases of the two main TCS families. Transcriptomic analysis resolves most pathways with high resolution, encompassing specialized, connected, and global regulatory systems. The activated network notably reveals the connection between CovRS and SaeRS signaling through the adhesin PbsP, linking the main regulators of host interactions to balance pathogenicity. Additionally, constitutive activation of the BceRS system reveals its role in cell envelope homeostasis beyond antimicrobial resistance. Overall, this study demonstrates the generalizability and versatility of TCS genetic activation to uncover regulatory logics and biological processes.

microbiology↗