António D. Brito

Universidade Nova de Lisboa

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Bruno M. Saraiva

Instituto Gulbenkian de Ciência

Ricardo Henriques

MRC Laboratory for Molecular Cell Biology

Simon Schäper

Universidade Nova de Lisboa

Robert Haase

TU Dresden

Guillaume Jacquemet

Turku Centre for Computer Science

Pedro M. Pereira

Universidade Nova de Lisboa

Mariana G. Pinho

Universidade Nova de Lisboa

Inês Cunha

Instituto Gulbenkian de Ciência

Gautier Follain

University of Turku

Raquel Portela

Universidade Nova de Lisboa

Cooperative Institutions

Universidade Nova de Lisboa

Universidade de São Paulo

Instituto de Tecnología Química

Harvard University

Instituto Gulbenkian de Ciência

University of Lisbon

Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo

Universidade Federal do Rio de Janeiro

Instituto de Medicina Tropical

Center for Systems Biology

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Chromosome segregation dynamics during the cell cycle of Staphylococcus aureus

bioRxiv (Cold Spring Harbor Laboratory) (2025)

Adrian Izquierdo-Martinez Simon Schäper António D. Brito Qin Liao Coralie Tesseur

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.

Dynamics

10.1101/2025.02.18.638847

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Processive movement ofStaphylococcus aureusessential septal peptidoglycan synthases is independent of FtsZ treadmilling and drives cell constriction

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Simon Schäper António D. Brito Bruno M. Saraiva Georgia R. Squyres Matthew J. Holmes

Abstract 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.

FtsZ

Treadmilling

10.1101/2023.06.29.547026

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NanoPyx: super-fast bioimage analysis powered by adaptive machine learning

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Bruno M. Saraiva Inês Cunha António D. Brito Gautier Follain Raquel Portela

To overcome the challenges posed by large and complex microscopy datasets, we have developed NanoPyx, an adaptive bioimage analysis framework designed for high-speed processing. At the core of NanoPyx is the Liquid Engine, an agent-based machine-learning system that predicts acceleration strategies for image analysis tasks. Unlike traditional single-algorithm methods, the Liquid Engine generates multiple CPU and GPU code variations using a meta-programming system, creating a competitive environment where different algorithms are benchmarked against each other to achieve optimal performance under the user”s computational environment. In initial experiments focusing on super-resolution analysis methods, the Liquid Engine demonstrated an over 10-fold computational speed improvement by accurately predicting the ideal scenarios to switch between algorithmic implementations. NanoPyx is accessible to users through a Python library, code-free Jupyter notebooks, and a napari plugin, making it suitable for individuals regardless of their coding proficiency. Furthermore, the optimisation principles embodied by the Liquid Engine have broader implications, extending their applicability to various high-performance computing fields.

Python

Plug-in

Implementation

10.1101/2023.08.13.553080

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The role of GpsB in cell morphogenesis ofStaphylococcus aureus

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Sara F. Costa Bruno M. Saraiva Helena Veiga Leonor B. Marques Simon Schäper

Abstract For decades, cells of the gram-positive bacterial pathogen Staphylococcus aureus were thought to lack a dedicated elongation machinery. However, S. aureus cells were recently shown to elongate before division, in a process that requires a SEDS (Shape Elongation Division and Sporulation) / PBP (Penicillin Binding Protein) pair for peptidoglycan synthesis, consisting of the glycosyltransferase RodA and the transpeptidase PBP3. In ovococci and rod-shaped bacteria the elongation machinery, known as elongasome, is composed of various proteins besides a dedicated SEDS/PBP pair. To identify proteins involved in the elongation of S. aureus , we screened the Nebraska Transposon Mutant Library, which contains transposon mutants in virtually all non-essential staphylococcal genes, for mutants with modified cell shape. We confirmed the roles of RodA/PBP3 in S. aureus elongation and identified GpsB, SsaA, and RodZ as additional proteins involved in this process. The gpsB mutant showed the strongest phenotype, mediated by the partial delocalization from the division septum of PBP2, the only bifunctional PBP in S. aureus , with both glycosyltransferase and transpeptidase activity, and of the PBP4 transpeptidase. Increased levels of these PBPs at the cell periphery result in higher levels of peptidoglycan insertion throughout the entire cell, overriding the RodA/PBP3-mediated peptidoglycan synthesis at the outer edge of the septum, which leads to cell elongation. As a consequence, in the absence of GpsB, S. aureus cells become more spherical.

Penicillin binding proteins

10.1101/2023.06.16.545294

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Efficiently accelerated bioimage analysis with NanoPyx, a Liquid Engine-powered Python framework

Nature Methods (2025)

Bruno M. Saraiva Inês Cunha António D. Brito Gautier Follain Raquel Portela

Abstract The expanding scale and complexity of microscopy image datasets require accelerated analytical workflows. NanoPyx meets this need through an adaptive framework enhanced for high-speed analysis. At the core of NanoPyx, the Liquid Engine dynamically generates optimized central processing unit and graphics processing unit code variations, learning and predicting the fastest based on input data and hardware. This data-driven optimization achieves considerably faster processing, becoming broadly relevant to reactive microscopy and computing fields requiring efficiency.

Python

Graphics processing unit

10.1038/s41592-024-02562-6

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Cell constriction requires processive septal peptidoglycan synthase movement independent of FtsZ treadmilling in Staphylococcus aureus

Nature Microbiology (2024)

Simon Schäper António D. Brito Bruno M. Saraiva Georgia R. Squyres Matthew J. Holmes

Abstract Bacterial cell division requires recruitment of peptidoglycan (PG) synthases to the division site by the tubulin homologue, FtsZ. Septal PG synthases promote septum growth. FtsZ treadmilling is proposed to drive the processive movement of septal PG synthases and septal constriction in some bacteria; however, the precise mechanisms spatio-temporally regulating PG synthase movement and activity and FtsZ treadmilling are poorly understood. Here using single-molecule imaging of division proteins in the Gram-positive pathogen Staphylococcus aureus , we showed that the septal PG synthase complex FtsW/PBP1 and its putative activator protein, DivIB, move with similar velocity around the division site. Impairing FtsZ treadmilling did not affect FtsW or DivIB velocities or septum constriction rates. Contrarily, PG synthesis inhibition decelerated or stopped directional movement of FtsW and DivIB, and septum constriction. Our findings suggest that a single population of processively moving FtsW/PBP1 associated with DivIB drives cell constriction independently of FtsZ treadmilling in S. aureus .

FtsZ

Treadmilling

10.1038/s41564-024-01629-6

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The role of GpsB in Staphylococcus aureus cell morphogenesis

mBio (2024)

Sara F. Costa Bruno M. Saraiva Helena Veiga Leonor B. Marques Simon Schäper

For decades, cells of the Gram-positive bacterial pathogen

10.1128/mbio.03235-23

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Citations (8)

NanoPyx - Figures' Data

Zenodo (CERN European Organization for Nuclear Research) (2023)

Bruno M. Saraiva Inês Cunha António D. Brito Gautier Follain Raquel Portela

a549_mt.tif Data used in Figure "Microscopy image processing workflow using NanoPyx methods". Fixed A549 cells immunolabelled against ß-tubulin with Alexa Fluor™ 647. Acquired using a Nanoimager microscope (Oxford Nanoimaging; ONI) equipped with a 100 × oil-immersion objective (Olympus 100x NA 1.45) Image size 10000x283x283 (pixel size 117 nm) .tif 16-bit huvec_nuclei.tif Data used in Figure "Comparative run times of multiple implementations of an algorithm, ran on a consumer-grade laptop and a professional workstation". Human Umbilical Vein Endothelial Cells (HUVEC) stained with DAPI Acquired in a Marianas spinning-disk confocal microscope. The objective used was a 63x oil (NA 1.4 oil, Plan-Apochromat, M27). Image size 100x512x512 .tif 16-bit

10.5281/zenodo.8318395

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Techniques used to identify the Brazilian variant of HIV-1 subtype B

Brazilian Journal of Medical and Biological Research (2007)

Shirley Vasconcelos Komninakis Ligia M.I. Fukumori Rosana Alcalde María E. Cortina Ludmila Abdala

The purpose of the present study was to compare the sensitivity and specificity of V3 enzyme immunoassay (solid phase EIA and EIA inhibition) and restriction fragment length polymorphism (RFLP) with the DNA sequencing "gold standard" to identify the Brazilian HIV-1 variants of subtype B and B"-GWGR. Peripheral blood mononuclear cells were collected from 61 HIV-1-infected individuals attending a clinic in São Paulo. Proviral DNA was amplified and sequentially cleaved with the Fok I restriction enzyme. Plasma samples were submitted to a V3-loop biotinylated synthetic peptide EIA. Direct partial DNA sequencing of the env gene was performed on all samples. Based on EIA results, the sensitivity for detecting B-GPGR was 70%, compared to 64% for the Brazilian variant B"-GWGR while, the specificity of B-GPGR detection was 85%, compared to 88% for GWGR. The assessment of RFLP revealed 68% sensitivity and 94% specificity for the B-GPGR strain compared to 84 and 90% for the B"-GWGR variant. Moreover, direct DNA sequencing was able to detect different base sequences corresponding to amino acid sequences at the tip of the V3 loop in 22 patients. These results show a similar performance of V3 serology and RLFP in identifying the Brazilian variant GWGR. However, V3 peptide serology may give indeterminate results. Therefore, we suggest that V3 serology be used instead of DNA sequencing where resources are limited. Samples giving indeterminate results by V3 peptide serology should be analyzed by direct DNA sequencing to distinguish between B-GPGR and the Brazilian variant B"-GWGR.

V3 loop

10.1590/s0100-879x2007000300003

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NanoPyx - Figures' Data

Zenodo (CERN European Organization for Nuclear Research) (2023)

Bruno M. Saraiva Inês Cunha António D. Brito Gautier Follain Raquel Portela

10.5281/zenodo.8318394

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Citations (1)