Biology subjects

Schaeper, S.

Publications and source records attributed to Schaeper, S..

5 recordsLinked to original sources

bioRxiv2026

Nucleoid-binding protein RicO anchors replication origins to the membrane to ensure correct chromosome segregation in Staphylococcus aureus

The mechanisms underlying chromosome segregation in bacteria, particularly in non-canonical models, remain incompletely understood. The coccoid bacterium Staphylococcus aureus encodes a limited set of conserved proteins involved in chromosome segregation, including the Structural Maintenance of Chromosomes (SMC) complex and an incomplete partition system. Here, we identified a protein of previously unknown function that interacts with the nucleoid and ensures accurate chromosome segregation in S. aureus. RicO (Regulator and Insulator of Chromosomal Origins) contains a conserved N-terminal DNA-binding domain that recognizes a specific sequence motif in the origin-proximal region of the chromosome, as well as a C-terminal amphipathic helix that interacts with the cell membrane, thereby bridging DNA and the membrane. RicO localizes in membrane-proximal foci at the tip of each hemisphere (or cell poles). Cells lacking RicO fail to correctly position replication origins at the cell poles and have weakened genomic interactions in the origin-proximal region, resulting in chromosome segregation defects. The combined absence of RicO and a functional SMC complex leads to nearly half of the population appearing as anucleate cells, indicating that these two systems are key contributors to chromosome segregation in S. aureus. Collectively, our data support a model in which RicO anchors chromosomal origins at the cell periphery to ensure genome maintenance in this spherical bacterial pathogen.

microbiology↗

bioRxiv2025

FtsW protein-protein interactions visualized in live Staphylococcus aureus cells by FLIM-FRET

The bacterial cell cycle relies on the coordinated and dynamic interactions between division proteins and those involved in peptidoglycan (PG) synthesis. However, visualizing these interactions in vivo remains technically challenging. Here, we established fluorescence-lifetime imaging microscopy combined with Forster resonance energy transfer (FLIM-FRET) as a robust, spatially resolved technique to visualize protein interactions in living Staphylococcus aureus using fluorescent proteins. After systematically optimizing growth conditions and the analysis pipeline, we validated the method with cytosolic and membrane-anchored control proteins, achieving FRET efficiencies of up to 40%. Using FLIM-FRET, we mapped the protein interactions of the critical glycosyltransferase FtsW within the septal PG synthesizing complex. We confirmed its interaction with the cognate transpeptidase PBP1 and the regulatory protein DivIB. Notably, we found that FtsW also self-interacts, an observation corroborated by an alternative FLIM-FRET method employing Halo-Tag labelled with Janelia Fluor dyes. These findings support the hypothesis that septal PG synthesis may be carried out by a complex of multimers, capable of simultaneously synthesizing more than one glycan strand. Inhibition of PG synthesis by directly targeting PBP1 with the beta-lactam antibiotic imipenem weakened the interaction between PBP1 and FtsW, whereas the FtsW self-interaction was enhanced in a dose-dependent manner. In contrast, inhibition of PG synthesis by targeting the lipid II flippase, therefore depleting the FtsW-PBP1 substrate from the outer surface of the cell membrane, had little effect on these interactions. This suggests that alterations in FtsW interactions result primarily from antibiotic-induced conformational changes or from uncoupling the activities of FtsW and PBP1, resulting in the presence of uncrosslinked glycans, rather than merely from a loss of PG synthesis activity.

microbiology↗

bioRxiv2025

Chromosome segregation dynamics during the cell cycle of Staphylococcus aureus

Research on chromosome organization and cell cycle progression in spherical bacteria, particularly Staphylococcus aureus, remains limited and fragmented. In this study, we established a working model to investigate chromosome dynamics in S. aureus using a Fluorescent Repressor-Operator System (FROS), which enabled precise localization of specific chromosomal loci. This approach revealed that the S. aureus cell cycle and chromosome replication cycle are not coupled, with cells exhibiting two segregated origins of replication at the start of the cell cycle. The chromosome has a specific origin-terminus-origin conformation, with origins localizing near the membrane, towards the tip of each hemisphere, or the "cell poles". We further used this system to assess the role of various proteins with a role in S. aureus chromosome biology, focusing on the ParB-parS and SMC-ScpAB systems. Our results demonstrate that ParB binds five parS chromosomal sequences and the resulting complexes influence chromosome conformation, but play a minor role in chromosome compaction and segregation. In contrast, the SMC-ScpAB complex plays a key role in S. aureus chromosome biology, contributing to chromosome compaction, segregation and spatial organization. Additionally, we systematically assessed and compared the impact of proteins linking chromosome segregation to cell division--Noc, FtsK, SpoIIIE and XerC--on origin and terminus number and positioning. This work provides a comprehensive study of the factors governing chromosome dynamics and organization in S. aureus, contributing to our knowledge on chromosome biology of spherical bacteria.

microbiology↗

bioRxiv2023

Processive movement of Staphylococcus aureus essential septal peptidoglycan synthases is independent of FtsZ treadmilling and drives cell constriction

Bacterial cell division is mediated by the tubulin-homolog FtsZ, which recruits peptidoglycan (PG) synthesis enzymes to the division site. Septal PG synthases promote inward growth of the division septum, but the mechanisms governing the spatiotemporal regulation of these enzymes are poorly understood. Recent studies on various organisms have proposed different models for the relationship between the movement and activity of septum-specific PG synthases and FtsZ treadmilling. Here, we studied the movement dynamics of conserved cell division proteins relative to the rates of septum constriction and FtsZ treadmilling in the Gram-positive pathogen Staphylococcus aureus. The septal PG synthesis enzyme complex FtsW/PBP1 and its putative activator protein, DivIB, moved processively, around the division site, with the same velocity. Impairing FtsZ treadmilling did not affect FtsW and DivIB velocities or septum constriction rates. Contrarily, inhibition of PG synthesis slowed down or completely stopped both septum constriction and the directional movement of FtsW/PBP1 and DivIB. Our findings support a model for S. aureus in which a single population of processively moving FtsW/PBP1 remains associated with DivIB to drive cell constriction independently of treadmilling FtsZ filaments.

microbiology↗

bioRxiv2022

A CRISPRi-based genetic resource to study essential Staphylococcus aureus genes

We have optimized a CRISPR interference system to facilitate gene knockdown in the gram-positive bacterial pathogen Staphylococcus aureus. Our approach used a CRISPRi system derived from Streptococcus pyogenes, which involves the co-expression of the dcas9 gene encoding a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). In our system, dcas9 is expressed from a single copy in the chromosome of methicillin resistant S. aureus (MRSA) strains COL or JE2, under the control of a tightly regulated promoter, inducible by anhydrotetracycline. The sgRNAs are expressed from a replicative plasmid under the control of a constitutively active promoter. This system enables efficient, inducible, knockdown of both essential and non-essential genes. Using this approach, we constructed the Lisbon CRISPRi Mutant Library (LCML) comprising 261 strains, in the JE2 background, containing sgRNAs targeting 200 essential genes/operons. This library facilitates the study of the function of essential S. aureus genes and is complementary to the Nebraska Transposon Mutant Library which consists of nearly 2000 strains, each carrying a transposon insertion within a non-essential gene. The availability of these two libraries will facilitate the study of S. aureus pathogenesis and biology. Abstract ImportanceStaphylococcus aureus is an important clinical pathogen that causes a high number of antibiotic resistant infections. The study of S. aureus biology, and particularly of the function of essential proteins, is of particular importance to develop new approaches to combat this pathogen. We have optimized a CRISPRi system that allows efficient targeting of essential S. aureus genes. Furthermore, we have used that system to construct a library of 261 strains which allow the depletion of essential proteins encoded in 200 genes/operons. This library, which we have named Lisbon CRISPRi Mutant Library (LCML), should facilitate the study of S. aureus pathogenesis and biology.

microbiology↗