Biology subjects

Pinho, M. G.

Publications and source records attributed to Pinho, M. G..

16 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↗

bioRxiv2026

BAY 11-7082 potentiates select β-lactams to inhibit growth of methicillin-resistant Staphylococcus aureus.

{beta}-lactams are an important class of antibiotics that target penicillin-binding proteins (PBPs) essential to bacterial cell wall synthesis. They are used to treat serious bacterial infections, including those caused by the versatile pathogen Staphylococcus aureus. Some strains carry the resistance gene, mecA, encoding a {beta}-lactam-insensitive PBP2A that allows for peptidoglycan synthesis in the presence of {beta}-lactams like methicillin, limiting treatment options. We previously identified BAY 11-7082 as having antibacterial activity against methicillin-resistant S. aureus (MRSA) and showed that it re-sensitizes MRSA to {beta}-lactams like penicillin G, making BAY 11-7082 and its analogues promising candidates for the development of an antibiotic adjuvant. Although the direct antibacterial mechanism of BAY 11-7082 remains undefined, here we aimed to better understand how it potentiates {beta}-lactam activity. We tested BAY 11-7082 in combination with a variety of {beta}-lactams and identified a subset that were synergistic in MRSA but not methicillin-susceptible S. aureus, suggesting they may directly or indirectly impact the function of PBP2A. Electron and fluorescence microscopy studies revealed that unlike other {beta}-lactam adjuvants, such as those that target the biosynthesis of wall teichoic acids (WTAs), BAY 11-7082 failed to impact cell division, disrupt PBP2 localization, or activate a sensitive reporter of cell wall damage. However, the production of WTA was necessary for BAY 11-7082-{beta}-lactam synergy. Together these data suggest its mechanism of {beta}-lactam potentiation is distinct from known compounds, making BAY 11-7082 a useful tool to better understand the complexity of resistance in MRSA.

microbiology↗

bioRxiv2025

mAIcrobe: an open-source framework for high-throughput bacterial image analysis

Quantitative analysis in bacterial microscopy is often hindered by diverse cell morphologies, population heterogeneity, and the requirement for specialised computational expertise. To address these challenges, mAIcrobe is introduced as an open-source framework that broadens access to advanced bacterial image analysis by integrating a suite of deep learning models. mAIcrobe incorporates multiple segmentation algorithms, including StarDist, CellPose, and U-Net, alongside comprehensive morphological profiling and an adaptable neural network classifier, all within the napari ecosystem. This unified platform enables the analysis of a wide range of bacterial species, from spherical Staphylococcus aureus to rod-shaped Escherichia coli, across various microscopy modalities within a single environment. The biological utility of mAIcrobe is demonstrated through its application to antibiotic phenotyping in E. coli and the identification of cell cycle defects in S. aureus DnaA mutants. The modular design, supported by Jupyter notebooks, facilitates custom model development and extends AI-driven image analysis capabilities to the broader microbiology community. Building upon the foundation established by eHooke, mAIcrobe represents a substantial advancement in automated and reproducible bacterial microscopy.

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

ReScale4DL: Balancing Pixel and Contextual Information for Enhanced Bioimage Segmentation

Deep learning has established itself as the state-of-the-art approach for segmentation in bioimage analysis. However, these powerful algorithms present an intriguing paradox regarding image resolution: contrary to intuition, lower-resolution images can yield superior performance for specific image analysis carried out by deep learning. This phenomenon is particularly significant in microscopy imaging, where high-resolution acquisitions come with substantial costs in throughput, data storage requirements, and potential photodamage to specimens. Through systematic experimentation, we evaluate how varying image resolution impacts deep learning performance in cellular image segmentation tasks. We trained popular architectures on datasets downsampled to 6-50% of their original resolution, mimicking acquisitions at lower image magnification, and compared their performance against models trained on native-resolution images. Our results show that segmentation accuracy either improves (by up to 25% of mean Intersection over Union (IoU)) or experiences only minimal degradation (< 5% of mean IoU) when using images downsampled by up to a factor of 4 (25% of the original resolution). This downsampling proportionally increases information throughput while reducing data storage requirements and inference time. With these findings, we contribute systematic guidelines to deep learning practitioners in creating efficient experimental pipelines for image-driven discoveries. This approach improves the sustainability and cost-effectiveness of bioimaging studies by reducing data and computing needs while optimising microscopy techniques.

biophysics↗

bioRxiv2025

Physical mechanisms that enable bacteria to traverse channels half their width

Bacteria colonize surfaces in the environment and can also penetrate tissues and materials by entering micro- and nanoscale cracks and pores. Staphylococcus aureus has been observed within nanoscale channels in bone that are 2-3 times smaller than cell diameter. Once inside bone, bacteria are protected from host immunity and systemic antibiotics, potentially contributing to chronic and recurrent infections. The physical mechanisms that enable bacteria to enter channels smaller than cell width are unclear. It has been proposed that bacteria traverse narrow passages through division - such that daughter cells form within small channels and eventually create a chain of cells extending down the channel length. Here we use microfluidics to test the idea that S. aureus can traverse bone-like nanochannels through growth. We examined 322 individual cells trapped within tapered nanochannels ([~]1.5 to 0.4 mm in width). When cells were deformed below 606 nm (65% original width), growth and division were slowed or completely inhibited. When cell division did occur in nanochannels, daughter cells were more likely to travel towards the wider side of the channel. Hence, it is unlikely that cell division would preferentially enable transit of S. aureus into nanoscale channels. However, the magnitudes of fluid pressure needed to deform S. aureus to widths similar to that seen in bone (1 to 6.5 kPa), were small relative to fluid pressure in bone generated by physical forces in vivo (8-20 kPa). Thus, our findings suggest that colonization of nanoscale channels in bone or other tissues is more likely due to moderate fluid pressure rather than growth. ImportanceBacteria that colonize materials and tissues within the body can be difficult to remove, even with thorough cleaning and application of antibiotics. Recent studies show that bacteria not only colonize the surfaces of tissues in the body but can also squeeze into naturally occurring pores and channels and thereby gain protection from immune cells and antibiotics. Here we ask how physical forces and cell growth might enable bacteria to enter small pores within materials. We use microfluidic devices to study the growth and migration of the human pathogenic bacteria, S. aureus, which is the leading cause of chronic bone infections.

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

Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum β-lactam resistance in Staphylococcus aureus

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by Methicillin-Resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to Next Generation {beta}-lactams (NGB) such as Methicillin, Nafcillin, Oxacillin etc. Resistance to NGBs, which is alternatively known as broad-spectrum {beta}- lactam resistance is classically mediated by PBP2a, a Penicillin-Binding Protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as Methicillin-Resistant Lacking mec (MRLM) are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance towards NGBs at levels comparable to that of MRSAs. Our study, provides a fresh new perspective about alternative mechanisms of NGBs resistance, challenging our current overall understanding of high-level, broad-spectrum {beta}-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach towards diagnosis and treatment of {beta}-lactam resistant S. aureus infections.

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↗

bioRxiv2023

The role of GpsB in cell morphogenesis of Staphylococcus aureus

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.

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↗

bioRxiv2021

DeepBacs: Bacterial image analysis using open-source deep learning approaches

Deep Learning (DL) is rapidly changing the field of microscopy, allowing for efficient analysis of complex data while often out-performing classical algorithms. This revolution has led to a significant effort to create user-friendly tools allowing biomedical researchers with little background in computer sciences to use this technology effectively. Thus far, these approaches have mainly focused on analysing microscopy images from eukaryotic samples and are still underused in microbiology. In this work, we demonstrate how to use a range of state-of-the-art artificial neural-networks particularly suited for the analysis of bacterial microscopy images, using our recently developed ZeroCostDL4Mic platform. We showcase different DL approaches for segmenting bright field and fluorescence images of different bacterial species, use object detection to classify different growth stages in time-lapse imaging data, and carry out DL-assisted phenotypic profiling of antibiotic-treated cells. To also demonstrate the DL capacity to enhance low-phototoxicity live-cell microscopy, we showcase how image denoising can allow researchers to attain high-fidelity data in faster and longer imaging. Finally, artificial labelling of cell membranes and predictions of super-resolution images allow for accurate mapping of cell shape and intracellular targets. To aid in the training of novice users, we provide a purposefully-built database of training and testing data, enabling bacteriologists to quickly explore how to analyse their data through DL. We hope this lays a fertile ground for the efficient application of DL in microbiology and fosters the creation of novel tools for bacterial cell biology and antibiotic research.

microbiology↗

bioRxiv2019

The cell cycle in Staphylococcus aureus is regulated by an amidase that controls peptidoglycan synthesis

Bacteria are protected by a polymer of peptidoglycan that serves as an exoskeleton. In Staphylococcus aureus, the enzymes that assemble peptidoglycan move during the cell cycle from the periphery, where they are active during growth, to the division site where they build the partition between daughter cells. But how peptidoglycan synthesis is regulated throughout the cell cycle is not understood. Here we identify a membrane protein complex that spatially regulates S. aureus peptidoglycan synthesis. This complex consists of an amidase that removes peptide chains from uncrosslinked peptidoglycan and a partner protein that controls its activity. Typical amidases act after cell division to hydrolyze peptidoglycan between daughter cells so they can separate. However, we show that this amidase controls cell growth. In its absence, excess peptidoglycan synthesis occurs at the cell periphery, causing cells to grow so large that cell division is defective. We show that cell growth and division defects due to loss of this amidase can be mitigated by attenuating the polymerase activity of the major S. aureus peptidoglycan synthase. Our findings lead to a model wherein the amidase complex regulates the density of peptidoglycan assembly sites to control peptidoglycan synthase activity at a given cellular location. Removal of peptide chains from peptidoglycan at the cell periphery promotes synthase movement to midcell during cell division. This mechanism ensures that cell expansion is properly coordinated with cell division.

microbiology↗

bioRxiv2019

SEDS-bPBP pairs direct lateral and septal peptidoglycan synthesis in Staphylococcus aureus

Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely through the localisation of the late stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and the bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in cell elongation and cell division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 likely constitute cognate pairs of interacting proteins. Lack of RodA-PBP3 decreased cell eccentricity due to deficient pre-septal PGN synthesis, whereas the depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilising the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings/arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate lateral and septal PGN incorporation, respectively, and that the activity of these pairs must be balanced in order to maintain coccoid morphology.

microbiology↗

bioRxiv2018

The pentaglycine bridges of Staphylococcus aureus peptidoglycan are essential for cell integrity

Bacterial cells are surrounded by cell wall, whose main component is peptidoglycan (PG), a macromolecule that withstands the internal turgor of the cell. PG composition can vary considerably between species. The Gram-positive pathogen Staphylococcus aureus possesses highly crosslinked PG due to the presence of cross bridges containing five glycines, which are synthesised by the FemXAB protein family. FemX adds the first glycine of the cross bridge, while FemA and FemB add the second and the third, and the fourth and the fifth glycines, respectively. Of these, FemX was reported to be essential. To investigate the essentiality of FemAB, we constructed a conditional S. aureus mutant of the femAB operon. Depletion of femAB was lethal, with cells appearing as pseudomulticellular forms that eventually lyse due to extensive membrane rupture. This deleterious effect was mitigated by drastically increasing the osmolarity of the medium, indicating that pentaglycine crosslinks are required for S. aureus cells to withstand internal turgor. Despite the absence of canonical membrane targeting domains, FemA has been shown to localise at the membrane. To study its mechanism of localisation, we constructed mutants in key residues present in the putative transferase pocket and the 6 helix of FemA, possibly involved in tRNA binding. Mutations in the 6 helix led to a sharp decrease in protein activity in vivo and in vitro but did not impair correct membrane localisation, indicating that FemA activity is not required for localisation. Our data indicates that, contrarily to what was previously thought, S. aureus cells do not survive in the absence of a pentaglycine cross bridge.

microbiology↗

bioRxiv2018

The ClpX chaperone controls the Staphylococcus aureus cell cycle but can be bypassed by β-lactam antibiotics

The worldwide spread of Staphylococcus aureus strains resistant to almost all {beta}-lactam antibiotics is of major clinical concern. {beta}-lactams interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Here we show that sub-lethal doses of {beta}-lactams stimulate the growth of S. aureus mutants lacking the widely conserved chaperone ClpX. S. aureus clpX mutants have a severe growth defect at temperatures below 37{degrees}C, and we reasoned that a better understanding of this growth defect could provide novel insights into how {beta}-lactam antibiotics interfere with growth of S. aureus. We demonstrate that ClpX is important for coordinating the S. aureus cell cycle, and that S. aureus cells devoid of ClpX fail to divide, or lyze spontaneously, at high frequency unless {beta}-lactams are added to the growth medium. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30{degrees}C, but not at 37{degrees}C. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, {beta}-lactams restored septum synthesis and prevented premature autolytic splitting of clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with {beta}-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, underscoring a functional link between the PBP activity and WTA biosynthesis. The finding that {beta} -lactams can prevent lysis and restore septum synthesis of a mutant with dysregulated cell division lends support to the idea that PBPs function as coordinators of cell division and that {beta} -lactams do not kill S. aureus simply by weakening the cell wall.\n\nAuthor SummaryThe bacterium Staphylococcus aureus is a major cause of human disease, and the rapid spread of S. aureus strains that are resistant to almost all {beta}-lactam antibiotics has made treatment increasingly difficult. {beta}-lactams interfere with cross-linking of the bacterial cell wall but the killing mechanism of this important class of antibiotics is still not fully understood. Here we provide novel insight into this topic by examining a defined S. aureus mutant that has the unusual property of growing markedly better in the presence of {beta}-lactams. Without {beta}-lactams this mutant dies spontaneously at a high frequency due to premature separation of daughter cells during cell division. Cell death of the mutant can, however, be prevented either by exposure to {beta}-lactam antibiotics or by inhibiting synthesis of wall teichoic acid, a major component of the cell wall in Gram-positive bacteria with a conserved role in activation of autolytic splitting of daughter cells. The finding that the detrimental effect of {beta}-lactam antibiotics can be reversed by a mutation that affect the coordination of cell division emphasizes the idea that {beta}-lactams do not kill S. aureus simply by weakening the cell wall but rather by interference with the coordination of cell division.

microbiology↗