Cell splitting in Staphylococcus aureus is controlled by an adaptor protein facilitating degradation of a peptidoglycan hydrolase

Maria Disen Barbuti; Eivind Frøyland Skjennum; Viktor Hundtofte Mebus; Danae Morales Angeles; Matilde Hustad; Marita Torrissen Mårli; Dorte Frees; Morten Kjos

doi:10.1371/journal.pgen.1011841

. 2025 Sep 5;21(9):e1011841. doi: 10.1371/journal.pgen.1011841

Cell splitting in Staphylococcus aureus is controlled by an adaptor protein facilitating degradation of a peptidoglycan hydrolase

Maria Disen Barbuti ^1,^#, Eivind Frøyland Skjennum ^1,^#, Viktor Hundtofte Mebus ², Danae Morales Angeles ¹, Matilde Hustad ¹, Marita Torrissen Mårli ¹, Dorte Frees ², Morten Kjos ^1,^*

Editor: Shumin Tan³

PMCID: PMC12443321 PMID: 40911637

Abstract

Regulated protein degradation by Clp proteases is a highly conserved post-translational control mechanism in bacteria. In Staphylococcus aureus, the ClpXP complex targets the peptidoglycan hydrolase Sle1, maintaining a tightly regulated balance between peptidoglycan biosynthesis and hydrolysis, which is required to ensure proper cell splitting without compromising cell integrity. β-lactams antibiotics disturb this balance, leading to their bactericidal effects. The mechanism underlying the specific targeting of Sle1 by the conserved ClpXP complex remains unknown. From a genome-wide screen for determinants of penicillin G susceptibility in S. aureus, we here identify the uncharacterized protein CxaR (for ClpXP-associated autolytic regulator). Growth defects, premature cell splitting, and increased cell lysis were observed in the absence of CxaR. Interestingly, these defects were mitigated by sublethal concentrations of β-lactams. Through sequencing cxaR suppressor mutants, followed by immunoblotting, we show that the cxaR phenotypes are caused by excessive Sle1 accumulation. Indeed, exposure to β-lactams reduces Sle1 levels, thereby rescuing the cells lacking CxaR. Furthermore, in vivo protein-protein interaction assays demonstrated that CxaR directly interacts with both ClpXP and Sle1, whereas no direct interaction was detected between Sle1 and ClpX. In line with this, CxaR was found to co-localize with ClpX adjacent to the septum. Taken together, these findings reveal that CxaR is a new regulatory factor controlling staphylococcal cell splitting by acting as an adaptor protein for controlled ClpXP-mediated degradation of Sle1.

Author summary

A delicate balance between peptidoglycan biosynthesis and hydrolysis is required to ensure proper cell division in the important pathogen Staphylococcus aureus. β-lactam antibiotics are thought to disturb this balance, leading to their bactericidal effects. To maintain the biosynthesis-hydrolysis balance, the peptidoglycan hydrolase Sle1 undergoes controlled degradation by the proteolytic complex ClpXP. However, the mechanism regulating this degradation remains unknown. Here, we uncover that CxaR, a previously uncharacterized protein, facilitates the ClpXP-mediated degradation of Sle1. This work thus unravels a novel, critical regulatory factor of staphylococcal cell division. As such, CxaR represents a potential target for future therapeutics. Additionally, this study provides new insight into the mechanism of action of widely used antibiotic classes, including β-lactams.

Introduction

Bacterial proliferation depends on the precise coordination of cell division, a complex process that culminates in the separation of daughter cells. In the Gram-positive coccoid pathogen Staphylococcus aureus, division initiates with the synthesis of a septal cross-wall at midcell, generating two hemispherical daughter cells that remain transiently connected via a peripheral wall ring [1–4]. Only after the septum is fully formed does this septum-adjacent peripheral cell wall undergo rapid resolution (“popping”) [1,2]. This step, occurring in less than two milliseconds, is highly regulated to prevent premature splitting of the septum, which would compromise cell integrity and viability, ultimately leading to cell lysis [5,6]. Septal splitting is facilitated by peptidoglycan hydrolases (PGHs) that create small perforations in the peripheral peptidoglycan ring, causing mechanical crack propagation [1,7]. S. aureus encodes at least 20 PGHs with different physiological roles during bacterial growth and division [5,8]. Among these, the amidase Sle1, which cleaves the amide bond linking the stem peptide to the glycan chain, plays a particularly important role in cell splitting, as its absence results in cell clustering, delays in cell cycle progression, and the formation of tetrad cells [2,7,9,10]. Sle1 is translocated to the extracellular milieu by active secretion, facilitated by its N-terminal signal peptide. Three cell wall-binding LysM domains ensure localization to the division septum, where the catalytic CHAP domain facilitates peptidoglycan cleavage [11,12].

As with all PGHs, Sle1 activity must be tightly regulated both spatially and temporally to prevent unintended breaches in the cell wall [5,6]. Targeted recruitment of these enzymes to specific regions of the cell serves as a key regulatory mechanism in many bacteria, including S. aureus [11,13,14]. The localization of Sle1 is dependent on the anionic glycopolymers wall teichoic acids (WTAs). The reduced levels of WTA at the septum promote Sle1 accumulation and peptidoglycan cleavage in this region [11,15]. The two-component systems WalKR and GraSR, facilitate transcriptional regulation of Sle1, ensuring proper coordination of cell wall remodeling and division [16–19]. Furthermore, recent studies have identified the DNA translocase and cell division protein FtsK [10] as well as a non-coding RNA named Rbc1 [20] as positive regulators of Sle1. Finally, and importantly, Sle1 levels are regulated by the ClpXP protease through targeted degradation [7].

Regulated protein degradation by Clp proteases affects all biological pathways, playing a crucial role in both general protein quality control and the targeted degradation of specific proteins in bacterial cells [21–23]. The ClpXP complex consist of the ATP-dependent hexameric ClpX ring, which recognizes, unfolds, and translocates specific protein substrates into the ClpP proteolytic chamber for degradation [24]. While ClpXP can directly target various substrates, efficient recognition of certain proteins requires separately encoded adaptor proteins that recognize specific substrates and assist in their delivery to ClpXP for degradation [21–23]. The vital roles of ClpXP adaptor proteins have been characterized in different bacteria, including Escherichia coli with SspB [25–27], UmuD [28,29], and RssB [30,31]. The only known adaptor protein for ClpXP in S. aureus is YjbH, which targets the stress-associated transcriptional regulator Spx for degradation [32–34], similar to its role in Bacillus subtilis [35,36].

Only a few ClpXP substrates have been verified in S. aureus, including Spx and Sle1 [37–39]. Unlike Spx, the mechanism by which ClpXP recognizes Sle1 is unknown. Nonetheless, the targeted degradation of Sle1 has been shown to be crucial for the survival of staphylococcal cells [7,39–41]. Deletion of clpX results in abnormal septum formation and premature splitting of daughter cells [40–42]. This phenotype is mainly attributed to the impaired proteolytic regulation and subsequent accumulation of PGHs, particularly Sle1 [7,38–40,42,43]. Beyond proteolysis, ClpX also appears to contribute to cell division independently of ClpP, as only clpX mutants exhibit heat stability and cold sensitivity [39,44], a temperature dependent growth defect that can be alleviated by a mutation in the essential cell division protein FtsA [43]. ClpX has additionally been found to impact transcription of genes involved in peptidoglycan synthesis and cell division independently of ClpP [45]. Interestingly, addition of β-lactams, inhibition of lipoteichoic acid (LTA) and wall teichoic acid (WTA) biosynthesis, or inactivation of Sle1 can rescue growth of clpX mutants [41,42], further evidencing a tight interplay between ClpXP, Sle1 and cell wall biogenesis in S. aureus.

The wide range of regulatory mechanisms invested into governing PGHs, and particularly Sle1, underlines the importance of maintaining control of these enzymes. Indeed, the bactericidal mechanism of β-lactam antibiotics appears to involve the dysregulation of PGH activity [46]. β-lactams inhibit the peptidoglycan cross-linking capacity of penicillin-binding proteins (PBPs) by covalently binding their active site [47–49]. This is believed to disturb the delicate balance between polymerization and autolytic degradation of peptidoglycan, which ultimately result in cell lysis [46,50–52]. The relationship between PBPs and PGHs appears to be highly complex. On one hand, low-dose treatment with β-lactams result in thickened septal walls, delay cell separation, and significantly reduce the levels of specific PGHs, including Sle1 [7,41,53–56]. These findings suggest that β-lactams may exert their bactericidal effects in part by lowering the activity of specific PGHs, resulting in aberrant septum formation that does not permit further cell growth and division [49,50,57]. On the other hand, β-lactam treatment has frequently been associated with activation of PGHs, leading to excessive cell wall degradation and subsequent cell lysis [50–52,57]. Studies in various bacteria, including Streptococcus pneumoniae and B. subtilis, have shown that when autolytic activity is suppressed, exposure to penicillin primarily results in growth inhibition rather than lysis [49,50,57]. The contrasting observations underscore that the exact mode of action of β-lactams is more complex than initially assumed and may depend on species-specific factors.

In the current study, we show that SAOUHSC_00659, a hitherto uncharacterized protein renamed CxaR (for ClpXP-associated autolytic regulator), controls cellular levels of Sle1 by mediating its targeted degradation by the ClpXP complex. Deletion or knockdown of the gene encoding CxaR resulted in accumulation of Sle1, which in turn contributed to growth deficiencies, accelerated daughter cell splitting, and increased cell lysis. Co-localization analyses combined with protein-protein interaction assays revealed that CxaR depends on ClpX for its septal-adjacent localization. While both proteins directly interact with each other, our results suggest that only CxaR interacts with Sle1. Together, these findings identify and elucidate the mechanism of a new adaptor protein required for spatiotemporal regulation of bacterial cell splitting.

Results

Sublethal concentrations of cell wall targeting antibiotics stimulate growth of cells lacking cxaR

A genome-wide, pooled CRISPR interference sequencing (CRISPRi-seq) screen [58–60] was conducted to identify genetic factors modulating susceptibility to β-lactams in S. aureus NCTC8325–4 (see S1 Table and S1 Fig). By knocking down the expression of transcriptional units, both in the presence and absence of inhibitory concentrations of penicillin G, we could quantify the associated growth fitness cost for each unit. The most prominent hit from this screen was a gene of unknown function, SAOUHSC_00659 (hereafter referred to as cxaR), whose knockdown led to reduced fitness, an effect that appeared to be counteracted by the presence of penicillin G (S1 Fig).

To validate this result, a cxaR CRISPRi knockdown strain was created in S. aureus NCTC8325–4. CxaR depletion resulted in a distinct growth profile in TSB at 37°C, characterized by reduced initial growth rate, followed by a transient decline in optical density (OD), and a subsequent gradual recovery of growth (Fig 1A). The growth defect of cxaR knockdown was similar, although even more pronounced, in the MRSA strain JE2 (Fig 1A). A cxaR deletion mutant was also constructed in NCTC8325–4 by allelic exchange with a spectinomycin resistance cassette. The resulting strain (NCTC8325–4 ΔcxaR::spc) exhibited the same biphasic growth (Fig 1B), confirming that the growth phenotype was not caused by the CRISPRi-system. Finally, to exclude the possibility that the phenotype observed upon knockdown or deletion of cxaR was caused by polar effect on neighboring genes, we designed sgRNAs for CRISPRi knockdown of five adjacent genetic loci, located both upstream and downstream of cxaR (S2A Fig). Growth analyses revealed that none of these strains exhibited growth patterns similar to the cxaR mutant (S2B–S2C Fig), confirming that the observed effects are specifically attributable to cxaR.

Fig 1 — (A) Growth curves of the *cxaR* knockdown mutants (*cxaR*_down) and CRISPRi control strains in TSB at 37°C for both NCTC8325-4 and JE2. Gene knockdown was induced with addition of 500 μM IPTG, and growth of the *cxaR*_down mutants was restored with addition of penicillin G (1 ng/mL for NCTC8325-4 and 5 ng/mL for JE2). The graphs represent averages with standard deviation from triplicate measurements. (B) Growth curves of the NCTC8325-4 *cxaR* deletion mutant and wild-type in TSB at 37°C. Growth of the Δ*cxaR* mutant was restored with addition of penicillin G (1 ng/mL). The graphs represent averages with standard deviation from triplicate measurements. (C) Growth of the NCTC8325-4 *cxaR* deletion mutant and wild-type on TSA with or without penicillin G (1 ng/mL) after 10 hours of incubation at 37°C. The bacterial cultures were sampled after 10 hours of incubation in TSB (± penicillin G) and 10-fold dilution series were spotted onto TSA plates (± penicillin G). (D) Relative growth of the JE2 *cxaR*_down mutant compared to the CRISPRi control after 8 hours of incubation at 37°C in the presence or absence of MIC₁ concentrations of various antibiotics. Data represent the average growth ratio ± standard deviation from the mean of two biological replicates for the antibiotic-treated samples, and from 30 biological replicates for the untreated control. A ratio of 1 indicates that the two strains have reached the exact same OD₆₀₀ after 8 hours of incubation. In the absence of antibiotics, the ratio was approximately 0.16. Notably, several of the antibiotics stimulated growth of the *cxaR*_down mutant, leading to a significant increase in the growth ratio. For the individual growth of the *cxaR*_down and CRISPRi control strain with increasing concentrations of the various antibiotics, see S3 Fig.(E) The AlphaFold 3-predicted structure of CxaR, with its three proposed anti-parallel sets of TPR-like helices colored red, blue, and purple. These helices form a slightly concave surface groove, as indicated in the figure.

Strikingly, and in line with the results from the CRISPRi-seq screen, subinhibitory concentrations of penicillin G rescued the growth defects of the cxaR knockdown and deletion mutants. When treated with penicillin G, the mutants grew similarly to the controls, both in liquid and on solid medium (Figs 1A–1C and S3A). The minimal inhibitory concentration (MIC) of the cxaR mutants, however, remained unaltered (S3A Fig).

To determine whether the penicillin G-mediated growth stimulation was a general response to growth inhibition or specific to certain antibiotics, the JE2 cxaR knockdown strain was tested against a panel of antibiotics, targeting different cellular processes (Figs 1D and S3A–S3O). Subinhibitory concentrations of all tested β-lactams (penicillin G, oxacillin, and cefoxitin) fully rescued growth of CxaR depleted cells. Similarly, other antibiotics targeting the cell wall, such as moenomycin (inhibiting transglycosylation activity) and tunicamycin (inhibiting early stages in wall teichoic acid and peptidoglycan biosynthesis), also fully restored growth. Partial growth rescue was observed for bacitracin (inhibiting lipid carrier recycling) and teicoplanin (glycopeptide inhibiting cell wall synthesis by binding to the D-Ala-D-Ala terminus of peptidoglycan precursors), while another glycopeptide with the same mechanism of action, vancomycin, did not seem to stimulate growth. Furthermore, translation inhibitors binding to the 30S ribosomal subunit, including kanamycin and streptomycin, as well as the DNA gyrase inhibitor novobiocin, partially restored growth of the CxaR depleted strain. On the other hand, the tested antibiotics targeting topoisomerase IV (ciprofloxacin), folate biosynthesis (trimethoprim), transcription (rifampicin), and the 50S ribosomal subunit (erythromycin) did not stimulate growth at any concentration.

CxaR is conserved among Staphylococcaceae and has structural resemblance to TPR motifs

cxaR is part of the S. aureus core genome, and phylogenetic analysis of CxaR homologues from 55 different species within the Staphylococcaceae family demonstrated a high degree of conservation among the homologs from the closely related genera Staphylococcus, Macrococcus, and Mammaliicoccus, with 44 out of 165 amino acids being completely invariable (S4A–S4B Fig). cxaR does not encode any signal peptide nor any transmembrane helices. The high-confidence AlphaFold-predicted structure of CxaR consists of two short N-terminal α-helices, six similarly sized antiparallel α-helices forming a slightly concave groove, and finally a longer C-terminal α-helix protruding from the rest of the structure (Fig 1E). When mapping residue conservation onto the predicted structure of CxaR, we found that highly conserved residues were primarily located at positions mediating hydrophobic interactions between the antiparallel α-helices and within the groove (Figs 1E and S4). HHpred [61] and HMMER [62] were used to identify potential protein domains of CxaR. Interestingly, both tools revealed a substantial number of significant hits with tetratricopeptide repeat (TPR) motifs. TPR motifs consist of 34-residue antiparallel helix pairs, typically repeated 3–16 times to form grooved, amphipathic binding surfaces that facilitate protein-protein interactions [63–65]. Although the sequences of TPR motifs are highly variable and therefore at times difficult to detect, they are involved in a wide range of cellular processes [63,66]. The most probable TPR helix pairs of CxaR, along with the resulting binding groove, are labelled in Fig 1E.

CxaR affects splitting of daughter cells

To better understand the growth phenotypes of the cxaR mutants, phase contrast and epifluorescence microscopy were performed on samples taken at different stages of growth (indicated in Fig 2A). Cells lacking CxaR clearly displayed increased lytic activity. Even before the drop in OD, a noticeably higher proportion of mutant cells appeared lysed compared to wild-type cells (hour 6 and 8, Figs 2B and S5). While the morphology of the live mutant cells did not significantly diverge from the wild-type, including comparable cell size (Fig 2C), we often observed cxaR mutant cells with a nick at septum displaying DNA dispersal, as visualized by DAPI-staining (Fig 2B and 2D). After the temporal decline in OD, few intact cells remained, and high levels of cellular debris were observed (hour 10, Figs 2B and S5). During the subsequent growth recovery phase, less lysis was observed. Strikingly, addition of penicillin G to cells lacking CxaR significantly reduced lysis at all recorded time points, with a particularly notable reduction at the 10-hour mark, when the majority of CxaR-deficient cells would otherwise have lysed (S5 Fig).

CxaR overexpression was also analyzed (Fig 2E–2F). Elevated levels of CxaR did not result in any growth deficiency. In fact, these cells reach a slightly higher final OD compared to those with normal CxaR levels (Fig 2F). Morphologically, cells with excess CxaR clustered together, frequently forming tetrads, where a second round of division is initiated before the previous pair of daughter cells have separated (Fig 2E). Notably, very few of the CxaR overexpressing cells were observed without a septum, indicating that excess CxaR does not impair septum synthesis but rather disrupts the separation of daughter cells after septum completion.

The lysis phenotype was further investigated by transmission electron microscopy (TEM). As expected, TEM analysis of CxaR depleted cells also revealed a significantly higher proportion of lysed cells compared to the control strain (Fig 3A–3C). Consistent with earlier observations, penicillin treatment resulted in a drastic reduction in lysis of CxaR depleted cells compared to untreated samples (Fig 3A–3C). Interestingly, in contrast to control cells (Fig 3D), the cxaR mutant cells frequently displayed premature splitting of daughter cells, where the septum appeared to be degraded before it was completed (marked with red asterisks in Fig 3A and 3E–3G). Variations of this phenotype were observed, with some cells exhibiting a nick prior to or at early stages of septal growth (Fig 3E), while others underwent splitting just before septum completion (Fig 3F). Some cells also underwent splitting at both ends of the septum simultaneously (Fig 3G). Furthermore, while most live cxaR cells resembled the control cells (Fig 3H), a subset exhibited a significantly thicker cell wall (Fig 3I), potentially a strategy adapted by some mutant cells to evade lysis. Taken together, these findings indicate that the lysis observed in cells lacking CxaR is due to premature septum splitting, and that penicillin G mitigates this by preventing spontaneous lysis in the mutant.

Fig 3 — (A) TEM images of the NCTC8325-4 *cxaR* knockdown mutant (*cxaR*_down) and CRISPRi control strain grown in TSB for 11 hours at 37°C, in the absence or presence of sublethal concentrations of penicillin G (1 ng/mL). Red asterisks mark cells that have undergone cell splitting prior to septum completion. The scale bars are 2 µm.(B) Growth curves of NCTC8325-4 *cxaR*_down and CRISPRi control, with (dashed line) or without (solid line) sublethal concentrations of penicillin G (1 ng/mL), showing the sampling timepoint for TEM imaging. The graphs represent averages from triplicate measurements.(C) Box plots showing the proportion of lysed cells, as observed by TEM, that retained minimal structural integrity. Significant differences between the strains are indicated with asterisks (***, P-value < 0.0005, Tukey’s Honest Significant Difference test). The number of cells analyzed, from left to right, is 231, 303, 241, and 250.(D-I) Representative TEM images of CRISPRi control and *cxaR*_down cells, highlighting their characteristic morphologies. (D) Control cells displaying normal coccoid morphology. *cxaR* knockdown cells displaying: (E) a septal nick prior to or at early stages of septal growth, (F) premature daughter cell splitting occurring just before septum completion, (G) simultaneous splitting at both ends of the forming septum, (H) wild-type-like morphology, and (I) significantly thicker cell wall. Black asterisks mark cells in which hydrolysis of the peripheral septal wall occurred after septum completion, while red asterisks mark cells initiating daughter cell separation, despite an incomplete septum. The scale bars are 200 nm.

Reduced Sle1 activity can compensate for the lack of CxaR

Since cell splitting in S. aureus is known to be temperature-responsive [20], we examined the growth of cxaR mutant cells across a range of temperatures. The mutants exhibited a clear temperature-dependent growth phenotype (Figs 4A, S6A and S6B); the growth defect was exaggerated at 30°C, while it was recovered at higher temperatures (42–45°C). Notably, when plated at 30°C, the cxaR deletion mutant displayed heterogeneous colony size, with both small and large-sized colonies, in contrast to the uniform, large colony size of the wild-type (Fig 4B). The colony size variation also appeared at 37°C, although to a lesser extent (Figs 4A and S6B). We hypothesized that the larger sized colonies at 30°C had obtained suppressor mutations. Five large ΔcxaR colonies were picked and shown to exhibit wild-type-like growth patterns (Fig 4B–4C). Whole-genome re-sequencing of these mutants revealed that each had independently acquired additional mutations associated with the PGH sle1 (S2 Table). Two of the suppressor mutants had substitutions in the catalytically active CHAP-domain, responsible for the hydrolytic activity of Sle1 (M292I and G315C), two resulted in truncated versions of Sle1 (Q197* and V5*), while the last one acquired an alteration in the Shine-Dalgarno sequence of sle1 (G > A in the RBS). The mutations thus likely resulted in inactivation and/or reduced activity of Sle1 (see below).

Fig 4 — (A) Spotting assays comparing growth of the NCTC8325-4 *cxaR* knockdown mutant (*cxaR*_down) and CRISPRi control at 30°C, 37°C, and 42°C. The bacterial cultures were grown at 37°C to an OD₆₀₀ of 0.5, 10-fold serially diluted, and 5 µL of each dilution was spotted onto TSA plates that were incubated at the indicated temperatures. Compared to the control, the *cxaR*_down mutant was cold-sensitive and heat-stable.(B) At 30°C, the NCTC8325-4 Δ*cxaR* mutant formed both very small colonies and larger suppressor colonies, unlike the homogeneous colony size observed for wild-type. When overnight cultures of Δ*cxaR* grown at 37°C were plated on TSA and incubated at 30°C, some larger colonies appeared among the very small ones (day 1). When single colonies from these plates were grown overnight at 30°C and replated on TSA at 30°C, the plates exhibited predominantly large suppressor colonies (day 2).(C) Growth of five independently isolated large Δ*cxaR* colonies, all found to have different *sle1*-associated mutations, in TSB at 37°C. All five suppressor mutants displayed growth patterns more closely resembling the wild-type than the original Δ*cxaR* strain. The graphs represent averages from triplicate measurements.(D) Western blot analysis of Sle1 levels in the extracellular and cell wall-associated protein fractions of the Δ*cxaR* suppressors with *sle1*-associated mutations. The Sle1 levels of the suppressor mutants were compared to those of wild-type, *cxaR* deletion, and *cxaR* overexpressing cells.

To further confirm that the growth defects caused by a lack of CxaR can be rescued by the absence of sle1, sle1 was knocked down in the ΔcxaR strain (S7 Fig). As expected, depletion of Sle1 in the cxaR mutant resulted in loss of the biphasic growth and thus a reversion to a wild-type-like growth phenotype (S7 Fig).

Sle1 levels are highly elevated in the absence of CxaR and reduced upon CxaR overexpression

To investigate the apparent functional link between CxaR and Sle1 levels, we performed Western blot analyses using an antibody that recognizes Sle1 (35.8 kDa) [20]. Strikingly, a clear accumulation of Sle1 was detected in cells lacking CxaR (ΔcxaR and cxaR_down in Figs 5 and S8). Sle1 accumulation was observed both in the cell wall fraction, the extracellular fraction, and the whole cell lysate. In contrast, when CxaR is overexpressed, Sle1 completely disappears in all fractions (Figs 5 and S8). Thus, the cellular levels of Sle1 are controlled by CxaR, as both its presence and absence directly correlate with Sle1 protein levels.

Fig 5 — Western blot analysis of Sle1 levels in extracellular protein fractions, cell wall-associated protein fractions, and whole-cell lysates using an anti-Sle1 antibody. The blots show that Sle1 levels were elevated in NCTC8325-4 cells lacking CxaR (Δ*cxaR* and *cxaR*_down) or ClpX (Δ*clpX*), while Sle1 was depleted in NCTC8325-4 cells overexpressing CxaR (Δ*cxaR*, pLOW-*cxaR*), compared to the wild-type and CRISPRi control cells.

We then performed immunoblot assays with cell wall-associated and extracellular protein extracts from the cxaR suppressor mutants (Fig 4D). This revealed reduced levels of cell wall-associated Sle1 in cells with the sle1 RBS mutation as well as in those encoding truncated Sle1 versions, compared to the wild-type. The mutants with single CHAP-domain substitutions also exhibited slightly reduced Sle1 levels, although not restored to wild-type levels. This further supports the notion that reducing Sle1 levels or activity can compensate for the loss of CxaR.

Given that reduced levels of Sle1 seem to rescue growth of cells lacking CxaR, we investigated whether the same mechanism was at play when growth was restored at high temperatures and/or with the addition of penicillin G. Indeed, both incubation at 42°C and treatment with penicillin G reduced Sle1 levels, both in wild-type cells and cells without CxaR (S9A–S9B Fig). We also tested whether the other antibiotics that stimulated growth of the cxaR_down mutant had a similar effect on Sle1 levels (Fig 1D). Indeed, a reduction in cell wall-associated Sle1 was observed, although the extent of reduction varied. Oxacillin, bacitracin, moenomycin, tunicamycin, and kanamycin all appeared to decrease Sle1 levels in the ΔcxaR strain to some degree. However, for teicoplanin and novobiocin, the effect on Sle1 levels was less evident (S9C Fig), consistent with their comparable limited ability to rescue growth of the cxaR_down mutant. Together, this suggests that the stimulatory effects of antibiotics observed for the ΔcxaR strain at least partially can be explained by reduced levels of Sle1.

CxaR regulates Sle1 levels in a ClpXP-dependent manner

Previous studies have shown that the cellular levels of Sle1 are regulated by the ClpXP protease complex [38,39] (Fig 6A), and similarly, we observed a strong accumulation of Sle1 in ΔclpX cells and cells devoid of ClpXP complex formation (ClpX_I265E) (Figs 5 and S10). In fact, cxaR mutant cells phenocopy clpX mutants to a large degree, including the observed temperature-dependent growth defect and growth rescue by cell wall targeting antibiotics [39,41,44]. These parallels suggest that CxaR plays a role in the ClpXP-mediated Sle1 degradation. Notably, the level of Sle1 accumulation in cells lacking either ClpXP activity or CxaR was comparable (Figs 5 and S10). If CxaR was primarily involved in regulating sle1 expression, one would expect the combined loss of CxaR and ClpXP to produce a significantly stronger Sle1 accumulation compared to either single mutant. However, ClpX_I265E cells with CxaR depletion showed no greater Sle1 accumulation than ClpX_I265E cells (S10 Fig). In addition, there was no reduction in cell wall-associated Sle1 when CxaR was overexpressed in the ΔclpX mutant, indicating that the mechanism in which CxaR controls Sle1 levels is not active when clpX is deleted (S10 Fig). Furthermore, a cxaR clpX double knockdown strain did not exacerbate the phenotypes of the two single knockdowns (S11A–S11C Fig). Together, this suggests that CxaR and ClpXP likely operate within a shared pathway rather than through independent mechanisms.

CxaR directly interacts with ClpXP and Sle1

Seeing that structural modeling of CxaR suggests it may contain a TPR-like domain (Fig 1E) commonly associated with protein-protein interactions [63,64,65], we investigated the potential role of CxaR in mediating interactions between Sle1 and ClpXP. We employed the split luciferase system [67], where the proteins of interest are conjugated to the complementary small (SmBit) and large (LgBit) subunits of luciferase, which produce a quantifiable luminescence signal if they come into close proximity when expressed in S. aureus.

Initially, CxaR self-interaction was tested, as TPR-containing proteins commonly exhibit self-oligomerization when involved in regulatory roles [68,69]. Indeed, CxaR appears to oligomerize, as the self-interaction displayed a significant 10-fold higher value than the control (Fig 6B). Next, interactions between CxaR, ClpX, and ClpP were tested. Luminescence readings between all three were highly significant (Fig 6B–6C). While it is well established that ClpX and ClpP form a proteolytic complex (Fig 6A and 6C), CxaR also appears to interact with both proteins, displaying a 25-fold and 13-fold increase in signal compared to the control, respectively (Fig 6B). However, the interaction between CxaR and ClpX appears to be lost when ClpX contains the I265E mutation (Fig 6C), suggesting that CxaR only interacts with the ClpXP complex. Notably, the interaction of CxaR with ClpX generated a stronger signal than the interaction with ClpP.

Moreover, CxaR also displayed a significant interaction with the precursor of Sle1, with a 4-fold increase in luminescence signal compared to the control (Fig 6D–6E). Interestingly, however, this interaction was not observed when Sle1 was expressed without the signal peptide (Fig 6D–6E). Indeed, in the AlphaFold3 predicted interaction model, supported by a high interaction score, the first LysM domain of Sle1 (located in the N-terminal part close to the signal sequence) interacts with the predicted binding groove of CxaR (Figs 1E and S12). Neither the precursor nor the mature form of Sle1 showed any detectable interaction with ClpX (Fig 6F). Together this strongly suggests that CxaR functions as an adaptor or intermediary to mediate the interaction between Sle1 and the ClpXP complex, facilitating Sle1 degradation. Since the Sle1 precursor is transported across the cytoplasmic membrane via its N-terminal signal peptide (Fig 6D), it is expected that only the precursor of Sle1 is accessible for degradation by the cytoplasmic ClpXP complex.

CxaR localization is ClpX-dependent

ClpX has been shown to localize in single foci near the membrane, positioned close to the ingrowing septum, in S. aureus cells undergoing septation [45]. By expressing an inducible ClpX-mCherry fusion, a similar localization was observed at the higher inducer concentrations (S13A Fig). Furthermore, a C-terminal CxaR-GFP fusion appeared to localize at midcell in dividing cells (Figs 7A, 7B, S13B and S13C). Indeed, when co-expressing ClpX-mCherry and CxaR-GFP, a clear co-localization of the two proteins was observed, with their fluorescent signals appearing at the exact same positions in approximately 92% of cells (n = 635) (Fig 7C–7D). To determine whether CxaR depends on ClpX for its localization, a plasmid encoding CxaR-GFP was transformed into the ΔclpX mutant. In these cells, the distinct signal pattern was lost (Fig 7E), suggesting that CxaR requires ClpX for its spatiotemporal localization. However, when ClpX-mCherry was introduced into ΔcxaR cells, the localization remained unchanged (Fig 7F), indicating that ClpX localization is independent of CxaR. It should be noted that while the CxaR-GFP fusion seems to be functional in terms of growth, cells expressing CxaR-GFP, either from its native promoter on the chromosome or from a plasmid, exhibited cell separation defects, including cell clumping (Figs 7A, 7B, S13B and S13C).

Fig 7 — (A) Fluorescence microscopy of the NCTC8325-4 strain expressing chromosomally integrated CxaR-GFP under its native promoter, showing that CxaR-GFP localizes at midcell in dividing cells. The scale bar represents 2 µm.(B) Growth curves of NCTC8325-4 wild-type and a strain in which *cxaR* is replaced with a *cxaR-gfp* fusion, demonstrating that GFP tagging of CxaR does not impair growth. Cells were grown in TSB at 37°C. The graphs represent averages from triplicate measurements.(C) Fluorescent microscopy of NCTC8325-4 cells co-expressing CxaR-GFP (from the chromosome) and ClpX-mCherry (from a plasmid induced with 80 μM IPTG), showing clear co-localization of the two proteins. The scale bar represents 2 µm.(D) Quantification of CxaR-GFP and ClpX-mCherry co-localization in NCTC8325-4 cells expressing both fusion proteins. Co-localization was analyzed by manual examination of the fluorescence signals in 300–350 randomly selected cells from two biological replicates (n = 635). CxaR-GFP and ClpX-mCherry were co-localized in 92.4 ± 2.4% of cells, 6.9 ± 1.9% of cells displayed only CxaR-GFP signal, and 0.6 ± 0.5% showed only ClpX-mCherry, confirming spatiotemporal co-localization of the two proteins during cell division.(E-F) Fluorescence microscopy of (E) Δ*clpX* cells expressing CxaR-GFP from a plasmid and (F) Δ*cxaR* cells expressing ClpX-mCherry from a plasmid. Expression of the plasmids were induced with the addition of 10 μM and 160 uM IPTG, respectively. The scale bar represents 2 µm. In the absence of ClpX, CxaR-GFP loses septal-adjacent localization, indicating that CxaR requires ClpX for its spatiotemporal localization. In contrast, ClpX-mCherry retained its focal localization pattern in Δ*cxaR* cells, demonstrating that ClpX localization does not depend on the presence of CxaR.

Discussion

In this work, we have discovered a novel factor critical for regulating cell splitting in S. aureus. Our results demonstrate that the hitherto unstudied protein CxaR acts as an adaptor protein, controlling the levels of Sle1, a PGH crucial for staphylococcal cell separation. Specifically, our model proposes that CxaR binds the Sle1 precursor prior to its export and translocates it to the ClpXP protease for degradation (Fig 8).

Fig 8 — Under normal conditions, CxaR regulates Sle1 levels by delivering its precursor to ClpXP for degradation, ensuring that cell splitting is triggered only after septum completion. In the absence of CxaR, ClpX and ClpP still form a proteolytic complex capable of degrading various proteins, but not Sle1, as its mode of delivery is missing. Consequently, Sle1 accumulates to high levels, causing hydrolysis of the incomplete septum and subsequent cell lysis.

This model is supported by several experimental observations. First, we observed that cxaR mutants phenocopy clpX mutants in a number of aspects, including biphasic growth profiles, temperature-dependent growth phenotypes with exacerbation of the growth defect at suboptimal and improved growth at supraoptimal temperatures [44], high lysis frequency due to premature cell splitting [41], and accumulation of Sle1 [38]. These defects were found to be alleviated by certain antibiotics, including β-lactams and tunicamycin, as well as inactivation of Sle1, similar to what has previously been observed for cells lacking ClpX [41]. Consistently, overexpression of CxaR led to depletion of cellular Sle1 due to enhanced degradation, causing severely impaired cell splitting. Second, split luciferase assays, used to assess protein-protein interactions in vivo, showed that CxaR interacts directly with the ClpXP complex, as well as Sle1. However, no direct interaction between Sle1 and ClpX was detected in these assays, suggesting that an intermediate protein facilitates the delivery of Sle1 to ClpXP for targeted degradation. Third, subcellular localization analysis showed that CxaR co-localizes with ClpX near the septum in dividing S. aureus cells. Additionally, the localization of CxaR was dependent on the presence of ClpX, indicating that its function is closely linked to ClpX and likely cell division. Moreover, it should also be noted that CxaR exhibited the highest score among identified staphylococcal ClpXP substrates when a proteolytically inactive ClpP variant (ClpP^trap) was expressed in vivo in S. aureus [38]. However, CxaR-GFP does not appear to accumulate in S. aureus cells lacking ClpXP activity (S13D Fig), suggesting that its retention in the ClpP^trap is likely due to other interactions than direct degradation. Finally, structural analyses revealed that CxaR resembles proteins containing TPR motifs, which are typically involved in facilitating protein-protein interactions [63,64,65]. This structural similarity suggests that CxaR may bind specific substrates to mediate their interactions or delivery. Thus, dysregulation of CxaR levels have detrimental effects on cell splitting and growth; Sle1 accumulates in the absence of CxaR, resulting in cell lysis due to premature splitting, while high levels of CxaR, on the other hand, significantly delay cell splitting, leading to cell clumping and cells with multiple septa, by reducing Sle1 abundances.

cxaR was uncharacterized prior to this study, but its transcription has been shown to increase under various stress conditions, including treatment with vancomycin [70], oxacillin [71], mersacidin [72], brilacidin [73], and under heat shock [74]. This pattern of transcriptional upregulation in response to envelope damage is consistent with the finding that expression of cxaR likely is regulated by the two-component system VraSR (vancomycin-resistance associated sensor/regulator). VraSR is activated by cell wall targeting antimicrobials, such as β-lactams and glycopeptides, and controls transcriptional induction of several cell wall-associated genes [71,75]. While Sle1 and other PGHs are not directly regulated by the VraSR system, it can thus be speculated that VraSR-mediated upregulation of CxaR serves as a regulatory mechanism to reduce Sle1 levels under these conditions, thereby delaying cell splitting in response to cell wall stress. This delay could provide the cells with enough time to repair damaged peptidoglycan and reinforce cell wall integrity, ultimately enhancing bacterial survival.

While our results show that CxaR regulates Sle1 levels via ClpXP-mediated degradation, it may have additional roles in the cell that remain to be elucidated. For example, when performing localization microscopy with fluorescently tagged versions of CxaR, we surprisingly observed that overexpression of CxaR-GFP in ΔclpX appeared to improve growth of the deletion strain. This was also confirmed with a non-tagged CxaR in the clpX deletion mutant (S14 Fig). Western blot assays did not indicate any decrease in cell wall-associated Sle1 during CxaR overexpression in ΔclpX cells (S10 Fig), which suggests that CxaR may have an additional role which helps the cells mitigate the impact of elevated Sle1 levels, potentially by rendering Sle1 non-functional. Additionally, the CxaR overexpression phenotype appears to be more severe than the expected defects from sle1 deletion alone [2,7,9,10], more closely resembling mutants with deletions of several PGHs [9,76]. This could indicate an additional role of CxaR in cell division, or that CxaR facilitates ClpXP-mediated degradation of other proteins in addition to Sle1.

Sublethal concentrations of different antibiotics could rescue the growth defects observed in cxaR mutants, a phenomenon previously reported in clpX mutants [41]. For some antibiotics, the growth rescue can be directly attributed to reduced levels of Sle1 in the treated cells (S9B–S9C Fig). This is in line with previous observations that expression of certain PGHs, including sle1, is positively correlated with PBP transpeptidase activity [55,56]. The reduction in Sle1 levels upon β-lactam exposure is independent of mecA, as the same occurs in both MRSA (JE2) and non-MRSA (NCTC8325–4) strains [7]. Sle1 levels are also highly temperature dependent, increasing at 30°C and decreasing at temperatures exceeding 40°C [20]. The temperature-dependent growth phenotype of cells lacking CxaR is likely attributed to these fluctuating Sle1 levels. The exact mechanism by which temperature influences the levels of Sle1 is unknown, but the observed upregulation of CxaR under heat [74] and envelope stress [71,75] suggest that elevated temperatures may enhance CxaR abundances, leading to increased Sle1 degradation.

Beyond the direct impact antibiotics have on hydrolase activity, additional mechanisms are likely involved in the antibiotic-mediated growth rescue of cxaR mutants. The growth stimulation mediated by sublethal concentrations of tunicamycin may be attributed to the role of teichoic acids in directing septal hydrolysis, in addition to its effect on Sle1 levels. A decrease in wall teichoic acid abundance has been shown to reduce septum-directed localization of PGHs and thus delay septal resolution [11,15,77,78]. The same growth-rescue by tunicamycin has also been observed in a ΔclpX mutant [41], which additionally acquired spontaneous, growth-rescuing loss-of-function mutations in the gene encoding the lipoteichoic acid synthase, ltaS [42]. Furthermore, sublethal concentrations of β-lactams have been observed to prolong the duration of peptidoglycan biosynthesis at the septal wall, leading to cells with thickened, unresolved septa, due to the retention of septal PBPs [41,53,54]. A thicker septum could potentially mitigate the effects of increased autolytic activity caused by Sle1 accumulation in cxaR mutants.

S. aureus employs a complex network of regulatory mechanisms to effectively control the autolytic activity driving cell splitting. Given the important role of Sle1 in septum resolution, our findings establish CxaR as a key regulatory factor in maintaining the balance between peptidoglycan synthesis and hydrolysis – a balance which is critical to the function of antibiotics. As CxaR represents a previously unrecognized component in this process, further studies investigating its potential additional roles, as well as functional analogs in other species, may provide new insight into the mode of action of widely used antibiotic classes, including β-lactams.

Materials and methods

Bacterial strains and growth conditions

Strains used in this work are listed in S3 Table. All S. aureus strains were grown in tryptic soy broth (TSB) and E. coli strains were grown in lysogeny broth (LB) aerobically at 37°C, unless stated otherwise. Growth in liquid media was carried out in an orbital shaker at 180 rpm, while for growth on solid media 1.5% (w/v) agar was added to the respective medium base. When appropriate, the following antibiotics were added for selection: ampicillin (Amp, 100 µg/mL), chloramphenicol (Cam, 10 or 25 µg/mL), spectinomycin (Spc, 100 or 1,000 µg/mL for NCTC8325–4 and JE2, respectively), and erythromycin (Ery, 5 µg/mL). In addition, isopropyl β-D-1-thiogalactopyranoside (IPTG) or anhydrotetracycline (aTc) was added for induction of gene expression when needed.

For E. coli transformation, chemically competent IM08B cells were prepared using calcium chloride treatment, followed by heat shock transformation according to standard protocols. Plasmids isolated from E. coli IM08B were transformed into electrocompetent S. aureus by electroporation, as previously described [79].

Strain and plasmid construction

See S3-S5 Tables for strains, plasmids, and oligonucleotides used in this work. Every construct was verified by PCR and sequencing.

Construction of CRISPRi knockdown strains.

For gene knockdown, we used the two-plasmid CRISPR interference (CRISPRi) system described previously [59,80,81]. In this system, dCas9 is expressed from an IPTG-inducible promoter on the plasmid pLOW-dcas9, while the gene-specific single guide RNA (sgRNA) is constitutively expressed from a second plasmid (pCG248-sgRNA(x) or pVL2336-sgRNA(x), where x denotes the targeted gene). Upon co-expression, the dCas9-sgRNA complex binds the gene of interest thereby blocking transcription by the RNA polymerase. The sgRNA plasmids were constructed using inverse PCR for pCG248 [80] or Golden Gate cloning for pVL2336 [59], with the oligonucleotide primers listed in S5 Table.

Construction of the cxaR deletion mutant.

cxaR was replaced by a spectinomycin resistance cassette in its native loci in S. aureus NCTC8325–4 using the temperature-sensitive pMAD system. First, the pMAD-cxaR::spc plasmid was constructed by assembling a fragment containing the cxaR up- and downstream regions (amplified using primer pairs mk591/mk592 and mk593/mk594, respectively) flanking a spectinomycin resistance cassette (amplified from pCN55 with primers mk188 and mk189) using overlap extension PCR. The fragment was ligated into pMAD using restriction cloning with NcoI and BamHI, and the resulting plasmid was transformed into E. coli IM08B. Construction of ΔcxaR::spc in strain NCTC8325–4 was then performed using a standard pMAD protocol with temperature shifts, as described before [82].

Construction of suppressor mutants.

When the NCTC8325–4 cxaR deletion mutant was plated at 30°C, larger colonies consistently appeared among the very small colonies observed at 37°C. To determine whether these larger colonies had restored growth due to acquisition of spontaneous suppressor mutations, we performed a genetic suppressor screen similar to what has been described previously [42]. The ΔcxaR mutant was streaked for single colonies on TSA plates and incubated at 37°C overnight. Single small colonies were picked, resuspended in TSB, and grown overnight at 37°C with shaking. The following day, each independent overnight culture was plated on TSA and incubated overnight at 30°C. On some plates, visibly larger colonies had appeared. In those cases, a single large colony from each independent culture was selected, grown overnight in TSB at 30°C with shaking, and stored. For cultures without initially detectable larger colonies, a random colony was picked, grown overnight in TSB at 30°C, and then replated on TSA at 30°C. By the next day, all plates had a majority of large colonies. One large colony per plate was picked, grown overnight in TSB at 30°C, and stored. The growth of the stored cultures was analyzed, and five were selected for whole-genome re-sequencing.

Construction of plasmids for complementation and overexpression.

cxaR and clpX were amplified from S. aureus genomic DNA using the primer pairs mhu28/mhu27 and mdb98/mdb99, respectively. The resulting fragments were digested with SalI and EcoRI and subsequently ligated into the corresponding restriction sites of plasmid pLOW-m(sf)gfp-SA1477 to produce pLOW-cxaR and pLOW-clpX, which enable IPTG-inducible expression of the respective genes.

Construction of plasmids for localization analysis of CxaR and ClpX.

To study the subcellular localization of C-terminally tagged CxaR, cxaR was amplified from S. aureus genomic DNA using primers mhu28 and mhu29, digested with SalI and BamHI, and ligated into the respective sites of pLOW-SA0948-m(sf)gfp. For N-terminally tagged CxaR, cxaR was amplified from S. aureus genomic DNA using primers mhu26 and mhu27, digested with BamHI and EcoRI, and ligated into the respective sites of pLOW-m(sf)gfp-SA1477. To study the subcellular localization of C-terminally tagged ClpX, clpX was amplified from S. aureus genomic DNA using primers mdb98 and mdb102, digested with SalI and BamHI, and ligated into the respective sites of pLOW-lacA-mCherry.

Construction of a chromosomally integrated cxaR-m(sf)gfp fusion.

The temperature-sensitive pMAD system was used to GFP-tag cxaR in its native loci in S. aureus NCTC8325–4. To construct pMAD-cxaR-m(sf)gfp_spc the following four fragments were amplified: (1) the upstream region of cxaR with primers mdb58 and mdb59 using gDNA from S. aureus NCTC8325–4 as a template, (2) the cxaR-m(sf)gfp fusion from plasmid pLOW-cxaR-m(sf)gfp using primers mdb60 and mdb61, (3) the spectinomycin resistance cassette using primers mk503 and mk504 from plasmid pCN55, and (4) the downstream region of cxaR from S. aureus NCTC8325–4 gDNA using mdb62 and mdb63. These fragments were subsequently fused by overlap extension PCR and ligated into pMAD using the NcoI and SalI restriction sites introduced by the outer primers. Finally, chromosomal integration of the fusion construct was performed following a standard pMAD protocol [82].

Construction of split luciferase plasmids.

To C-terminally fuse CxaR, ClpX, and ClpX_I265E to the small luciferase subunit (SmBit), cxaR, clpX, and clpX_I265E were amplified from S. aureus NCTC8325–4 WT or clpX_I265E genomic DNA using the primer pairs efs7/efs8 for cxaR and mdb93/mdb94 for clpX and clpX_I265E. The amplified fragments and vectors pAF256-P_tet-hupA-smbit/lgbit and pAP118-P_tet-hupA-smbit/hupA-lgbit were then digested with SacI and XhoI. The digested fragments were ligated into the vectors and subsequently transformed into E. coli IM08B. The pAF256 vectors, encoding CxaR, ClpX, or ClpX_I265E fused to SmBit and LgBit not part of a fusion protein, served as negative controls in the split luciferase assays. The pAP118 vectors were further modified to C-terminally fuse CxaR, ClpX, ClpP, Sle1, or Sle1 (without signal peptide) to the large luciferase subunit (LgBit). All fragments were amplified using NCTC8325–4 WT genomic DNA as template and the following primer pairs: (1) efs20/efs21 for cxaR, (2) efs5/efs6 for clpX, (3) efs9/efs10 for clpP, (4) mdb88/mdb89 for sle1, and (5) mdb90/mdb89 for sle1 (without signal peptide). The amplified fragments and the corresponding pAP118 vectors were then digested with PvuI and NotI, ligated, and subsequently transformed into E. coli.

Genome re-sequencing and analysis

Whole-genome sequencing of ΔcxaR (MK2047), the strain expressing chromosomally integrated CxaR-GFP (MDB376), and the cxaR suppressor mutants (EFS104, EFS105, MDB390, MDB401, and MDB404) was performed. Genomic DNA was isolated from the respective strains using The Wizard Genomic DNA Purification Kit (Promega) and sent to Novogene for Illumina sequencing. Sequence assembly to the reference genomes and SNP detection were done using Geneious Prime [83].

CRISPRi-sequencing screen

The explorative CRISPRi-seq screen for penicillin G susceptibility determinants was performed as described before, using an already constructed genome-wide CRISPRi library in the S. aureus strain NCTC8325–4 [59,60]. Briefly, the library (pre-grown in BHI to OD₆₀₀ = 0.8) was diluted 1:1,000 in 100 mL ultra-high temperature (UHT) treated milk (Tine SA, Norway) and incubated at 37 °C with shaking for 7 hours. After, it was reinoculated 1:1,000 in fresh UHT milk and grown for another 6 hours. Chloramphenicol (10 µg/mL) was added for selection, and when appropriate, 30 ng/mL anhydrotetracycline was added for induction of the CRISPRi-system. Furthermore, to identify fitness determinants affected by β-lactam treatment, the library was grown in the presence and absence of 0.008 µg/mL penicillin G (corresponding to the MIC for NCTC8325–4 in milk). Four parallels were included per condition. After growth, cells were harvested by centrifugation, and plasmids were isolated as previously described [60]. Illumina amplicon libraries were prepared, and sequencing was performed on a NovaSeq platform using 150 bp paired-end reads (performed by Novogene, UK) following established protocols [60]. CRISPRi-seq differential enrichment analyses was done as described by Bakker et al. [58] and Mårli et al. [60]. Notably, due to the inhibitory concentration of penicillin G used in this explorative screen, the number of generations for growth of the penicillin G treated library was significantly lower than for the untreated library.

Growth assays in liquid media

To assess bacterial growth in a liquid medium, overnight cultures of the strains to be monitored were diluted 1:1,000 in TSB medium supplemented with the appropriate antibiotics and inducers. For MIC analysis, the following panel of antibiotics were tested against the JE2 cxaR knockdown mutant and CRISPRi control strain in two-fold dilution series: penicillin G (PEN), oxacillin (OXA), cefoxitin (CXI), vancomycin (VAN), teicoplanin (TEC), bacitracin (BAC), moenomycin (MOE), tunicamycin (TUN), ciprofloxacin (CIP), novobiocin (NOV), trimethoprim (TMP), rifampicin (RIF), erythromycin (ERY), kanamycin (KAN), and streptomycin (STR). Bacterial dilutions were added to a 96-well microtiter plate and incubated in a plate reader at 37°C for 20 hours, unless otherwise stated. OD₆₀₀ measurements were taken every 10 minutes, with a brief shaking of the plate for 5 seconds prior to each measurement. All growth curves in this work are the mean of three replicate measurements, with the exception of the MIC panel (S3 Fig), growth at different temperatures (S6A Fig), and CxaR/ClpX overexpression (S14 Fig) which represent the mean value of two replicates, and they are all representative of at least two independent experiments.

Spotting assays

Overnight cultures diluted 1:1,000 in TSB with the appropriate antibiotics were grown at 37°C until they reached an OD₆₀₀ of 0.5 or the indicated time point. The cultures were diluted in a ten-fold dilution series from 10^-1 to 10^-8 and 5 µL from each dilution was spotted on TSA. The TSA, in addition to the appropriate selective antibiotics, contained 500 µM IPTG for CRISPRi knockdown strains and 1 ng/mL penicillin G or 6 ng/mL oxacillin when assessing growth rescue by β-lactams. The plates were incubated for 24 hours at their indicated temperature. All presented spotting assays are representative of three technical replicates from at least three independent experiments.

Western blot analyses of Sle1 levels

Cell harvesting.

Overnight cultures were diluted 1:1,000 in TSB medium with the appropriate antibiotics and inducers and incubated at either 37°C or 42°C with shaking until an OD₆₀₀ of approximately 0.4 was reached.

Preparation of extracellular and cell wall-associated protein extracts.

Culture volumes of 50 mL were harvested by centrifugation at 6,000 x g for five minutes at 4°C. To extract extracellular proteins, 30 mL of the supernatants were mixed with 30 mL 96% ethanol and incubated overnight at 4°C. To extract cell wall-associated proteins, the pellets were washed with 10 mL cold 0.9% (w/v) NaCl and centrifuged at 6,000 x g at 4°C for five minutes. The supernatants were discarded, and cell wall-associated proteins were eluted with 10 mL 4% (w/v) SDS at 25°C for 45 minutes with shaking. The samples were then centrifuged at 6,000 x g for five minutes at 4°C, and the resulting supernatants were mixed with 10 mL 96% ethanol and left to precipitate at 4°C overnight. The following day, the protein extracts were pelleted by centrifugation at 10,000 x g for 30 minutes at 4°C, dried in a fume hood, and normalized with 50 mM Tris-HCl (pH 7.4). The normalized extracts were mixed 1:1 with 2x SDS loading buffer and heated at 95°C for five minutes prior to SDS-PAGE.

Preparation of whole cell lysate.

Culture volumes of 50 mL, normalized to an OD₆₀₀ of 0.4, were harvested by centrifugation at 4,000 x g for five minutes at 4°C. The pellets were resuspended in 100 µL TBS buffer and mechanically lysed using the Fast Prep method with ≤106 µm glass beads in three cycles of 20 seconds at 6 m/s, with one minute incubation on ice between cycles. Insoluble material was removed by centrifugation at 20,000 × g for 2 minutes. Next, the supernatants were mixed with equal volumes 2x SDS loading buffer and heated at 95°C for 5 minutes prior to SDS-PAGE.

SDS-PAGE, Western blotting, and detection.

The protein samples were separated by SDS-PAGE on a polyacrylamide gel, which consisted of a 12% separation gel with a 4% stacking gel layered on top. The proteins were then electroblotted onto a PVDF membrane using the Trans-Blot Turbo System (Bio-Rad). Afterward, the membrane was trimmed to retain proteins below 60 kDa and blocked in TBST, containing 5% (w/v) bovine serum albumin (BSA) and 1 μg/mL human IgG, for 1 hour at room temperature, followed by overnight incubation at 4°C. After washing the membrane with TBST, the following day, it was incubated with an anti-Sle1 primary antibody [20] (diluted 1:10,000 in TBST with 2% (w/v) BSA and 1 μg/mL human IgG) for 1 hour. Next, unbound antibodies were removed by three TBST washes, and the membrane was then incubated for another hour with an anti-mouse IgG HRP-conjugate secondary antibody (Promega) (diluted 1:10,000 in TBST with 1 μg/mL human IgG). After another series of washes, the membrane was finally developed using the SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific), and blot images were captured with an Azure Imager c400 (Azure Biosystems).

Western blot analysis of GFP-tagged CxaR

Overnight cultures of MDB376 (NCTC8325–4 with a chromosomally integrated cxaR-m(sf)gfp fusion), MDB430 (NCTC8325–4 carrying pLOW-cxaR-m(sf)gfp), and MDB395 (NCTC8325–4 clpX_I265E carrying pLOW-cxaR-m(sf)gfp) were diluted 1:1,000 in TSB medium supplemented with 100 µg/mL spectinomycin (for MDB376) or 5 µg/mL erythromycin and 10 µM IPTG (for MDB430 and MDB395). The bacterial cultures were incubated at 37°C until they reached an OD₆₀₀ of approximately 0.5 and 1. They were then normalized to an OD₆₀₀ of 0.5, and harvested by centrifugation at 4,000 × g for 5 minutes at 4°C. The pellets were resuspended in 200 µL TBS buffer and lysed mechanically using the Fast Prep method with ≤106 µm glass beads at 6 m/s for three cycles of 20 seconds each. Insoluble material was removed by centrifugation at 20,000 × g for 2 minutes. Next, the supernatants were mixed with equal volumes 2x SDS loading buffer and heated at 95°C for 5 minutes.

The subsequent SDS-PAGE, blotting, and GFP detection were performed as described above for Sle1 immunoblotting, with the exception of antibody selection. GFP-tagged CxaR was detected using an anti-GFP primary antibody (Invitrogen) (diluted 1:4,000 in TBST) and an anti-rabbit IgG HRP-conjugated secondary antibody (Promega) (diluted 1:5,000 in TBST).

Phase contrast and epifluorescence microscopy

Microscopy.

Overnight cultures were diluted 1:1,000 in TSB medium with the appropriate antibiotics and inducers. These cultures were incubated at 37°C with shaking until they reached the desired optical density (OD₆₀₀ ~ 0.4) or time point, followed by cooling on ice. In some cases, cells were then stained with fluorescent vancomycin (VanFL, 0.8 µg/mL [Invitrogen]) to visualize the cell wall and/or 4′,6-diamidino-2-phenylindole (DAPI, 7.5 µg/mL [Invitrogen]) to visualize the nucleoid. Bacterial cells were immobilized on 1.2% (w/v) agarose pads before imaging on either a Zeiss Axio Observer microscope or a Leica DMi8 microscope. The bacteria were visualized with a 100 × phase contrast objective, and images were captured using an ORCA-Flash4.0 V3 Digital CMOS (for the Zeiss microscope) or a K8 Scientific CMOS (for the Leica microscope) camera.

Image analysis.

Cell size distribution among different S. aureus strains was determined using MicrobeJ [84]. Bacterial cells were automatically detected using phase contrast image stacks, and every image was subsequently manually corrected by adding unidentified cells or removing incorrectly identified cells. Additionally, co-localization of CxaR-GFP and ClpX-mCherry was analyzed manually by examining both signals in 300–350 randomly selected cells from two biological replicates (n = 635).

Transmission electron microscopy

Overnight cultures of the NCTC8325–4 cxaR CRISPRi knockdown and control strains were diluted 1:1,000 in 10 mL TSB with the appropriate selective antibiotics and inducers, with and without 1 ng/mL penicillin G. The cultures were grown at 37°C for 11 hours, corresponding to the transient minimum of the cxaR knockdown strain. The cultures were added to fixative to a final concentration of 1.25% (v/v) glutaraldehyde, 2% (v/v) paraformaldehyde, and 100 mM cacodylate (CaCo) buffer. The cells were fixed for one hour at room temperature and overnight at 4°C, before being washed in 100 mM CaCo buffer three times. The final pellets were embedded in 3% (w/v) low-melt agarose and stored in 100 mM CaCo buffer. Post-fixation with 1% osmium tetroxide was performed for one hour. Excess osmium tetroxide was removed by washing with 100 mM CaCo buffer and the pellets were dehydrated using gradually increasing concentrations of cold acetone. The dehydrated pellets were embedded in EPON epoxy resin and polymerized at 60°C. The samples were sectioned 60 nm thick and stained with uranyl acetate and lead citrate. A Jeol JEM-2100Plus was used for imaging.

Split luciferase assays

Pairwise protein-protein interactions were assessed in vivo in S. aures NCTC8325–4 using the split luciferase system developed by Paiva et al. [67] for C. difficile, following the same approach as described before [85].

Structural and phylogenetic analysis

The presence of general structural features, including transmembrane helices and signal peptides, were predicted using SignalP-6.0 and DeepTMHMM [86,87]. Homologs of CxaR within Staphylococcaceae were identified through BLASTp, using the NCTC8325–4 CxaR peptide sequence as the query [88]. Sequence representatives from 55 species within Staphylococcus, Mammaliicoccus, and Macrococcus were aligned by Clustal Omega, and the multiple sequence alignment (MSA) was visualized using Jalview [89,90]. Phylogenetic trees from the homologs were generated using MEGA11 [91]. HMMER and HHpred were applied for sensitive hidden Markov model-based homolog searches to detect structural motifs [61,62]. TPRpred from the HH-suite was used to detect TPR-like motifs in CxaR homologs [92].

In silico protein interaction modelling

AlphaFold 3 [93] was used for the modelling of every protein structure and protein interactions in this work. The top ranked predictions were selected and visualized using either ChimeraX [94] or PyMOL [95].

Supporting information

S1 Fig. CRISPRi-seq identified genetic factors modulating susceptibility to β-lactams in S. aureus.

The growth fitness impact of knocking down each transcriptional unit was assessed under conditions with (x-axis) and without (y-axis) penicillin G (0.008 µg/mL). sgRNAs found to be significantly reduced upon penicillin G treatment, indicating that knockdown of these transcriptional units increases sensitivity to penicillin G, are highlighted in blue (log₂FC < -1, P_adj < 0.05). In contrast, sgRNAs that were significantly enriched, indicating that their knockdown reduces sensitivity to penicillin G, are highlighted in red (log₂FC > 1, P_adj < 0.05). Among the significantly enriched sgRNA, SAOUHSC_00659 (cxaR) was the top hit.

(TIF)

pgen.1011841.s001.tif^{(13.1MB, tif)}

S2 Fig. The phenotype observed upon knockdown or deletion of cxaR is not caused by polar effect on neighboring genes.

(A) Schematic overview of the genes surrounding cxaR in its operon. Red lines indicate the approximate location of sgRNA targets used for CRISPRi knockdown. (B) Growth curves of an NCTC8325–4 cxaR_down strain in TSB at 37°C with (dashed line) and without (solid line) 1 ng/mL of penicillin G. Knockdown was induced by 30 ng/mL aTc. (C) Growth curves showing the effects of CRISPRi knockdown of genes in NCTC8325–4 located upstream (SAOUHSC_00655, SAOUHSC_00656 and SAOUHSC_00658) and downstream (S245 and SAOUHSC_00660) of cxaR, with (dashed line) and without (solid line) 1 ng/mL of penicillin G. The strains were grown at 37°C in TSB with 30 ng/mL aTc to induce knockdown.

(TIF)

pgen.1011841.s002.tif^{(15MB, tif)}

S3 Fig. Sublethal concentrations of various antibiotics stimulate growth of cells lacking cxaR.

The JE2 cxaR_down strain (left panels) and the CRISPRi control (middle panels) subjected to various concentrations of different antibiotics during growth in TSB at 37°C. Knockdown was induced by 500 µM IPTG. The antibiotics tested included the β-lactams (A) penicillin G, (B) oxacillin, and (C) cefoxitin, the glycopeptides (D) vancomycin and (E) teicoplanin, the polypeptide antibiotic (F) bacitracin, the phosphoglycolipid antibiotic (G) moenomycin, the WTA-inhibiting nucleoside antibiotic (H) tunicamycin, the fluoroquinolone (I) ciprofloxacin, the aminocoumarin (J) novobiocin, the folate biosynthesis inhibitor (K) trimethoprim, the ansamycin (L) rifampicin, the macrolide (M) erythromycin, and the aminoglycosides (N) kanamycin and (O) streptomycin. The right panels show the OD₆₀₀ for each strain after 8 hours of growth for each concentration of the antibiotics tested.

(PDF)

pgen.1011841.s003.pdf^{(1.3MB, pdf)}

S4 Fig. CxaR is conserved among Staphylococcaceae.

(A) Phylogenetic tree of CxaR homologues from 55 species within the Staphylococcaceae family. The phylogenetic relatedness of the homologues generally clustered by genera, with the exception of Staphylococcus schleiferi which appeared to utilize a CxaR homolog more closely resembling those found in Mammaliicoccus. (B) AlphaFold 3-predicted structure of CxaR with residue conservation from the phylogenetic analysis in A mapped on to it. The colors represent conservation scores ranging from 0 (least conserved) to 11 (most conserved), accounting for both conservation of specific amino acids and their stereochemical properties. (C) Coulombic electrostatic potential of the predicted CxaR structure, with red for negative potential through white to blue for positive potential. (D) Hydrophobicity (molecular lipophilicity potential) of the predicted CxaR structure, with dark cyan for most hydrophilic through white to dark goldenrod for most hydrophobic.

(TIF)

pgen.1011841.s004.tif^{(21.3MB, tif)}

S5 Fig. Sublethal concentrations penicillin G significantly reduce lysis of cell lacking cxaR.

Phase contrast and fluorescence microscopy of the JE2 cxaR_down strain after 6, 8, 10, and 12 hours of incubation in TSB at 37°C, with and without 5 ng/mL penicillin G. CRISPRi knockdown of cxaR expression was induced by adding IPTG to a final concentration of 500 µM. The cells were stained with VanFL (cell wall) and DAPI (nucleoid). White arrows indicate abnormal and recently lysed cells. Scale bars represent 2 µm.

(TIF)

pgen.1011841.s005.tif^{(33.7MB, tif)}

S6 Fig. cxaR mutants exhibit heat stability and cold sensitivity.

(A) The NCTC8325–4 cxaR_down and CRISPRi control strains grown in TSB at 30°C, 37°C, and 45°C with different concentrations of penicillin G. Gene knockdown was induced with the addition of 500 μM IPTG. The graphs represent averages from duplicate measurements. (B) Spotting of the NCTC8325–4 cxaR_down and CRISPRi control strains at different temperatures with or without addition on oxacillin. The strains were grown until an OD₆₀₀ of 0.5 was reached, serially diluted in 10-fold dilution series, and 5 µL of each dilution was spotted on TSA containing 500 μM IPTG, with or without 6 ng/mL oxacillin. The plates were incubated at 30°C, 37°C, or 42°C for 24 hours before imaging.

(TIF)

pgen.1011841.s006.tif^{(31.4MB, tif)}

S7 Fig. Reduced Sle1 activity can compensate for the lack of CxaR.

Growth of an NCTC8325–4 ΔcxaR mutant with (green) and without (grey) knockdown of sle1 in TSB at 37°C. Gene knockdown was induced with the addition of 500 μM IPTG. The graphs represent averages from triplicate measurements.

(TIF)

pgen.1011841.s007.tif^{(8.4MB, tif)}

S8 Fig. Sle1 levels are elevated in the absence of CxaR and reduced upon CxaR overexpression.

Western blot analysis of Sle1 levels in (A) cell wall-associated protein fractions and (B) whole-cell lysates from NCTC8325–4 wild-type, cxaR deletion, and cxaR overexpressing cells. Band intensities were quantified using ImageJ and normalized to the wild-type signal. (A) In cell wall-associated fractions, Sle1 levels were increased in ΔcxaR cells (4.681 ± 1.270) and reduced in CxaR overexpressing cells (0.007 ± 0.003), relative to the wild-type (1.000 ± 0.220). (B) Similarly, in whole-cell lysates, Sle1 levels were highly elevated in ΔcxaR cells (147.847 ± 45.768) and depleted in CxaR overexpressing cells (0.015 ± 0.043), compared to the wild-type (1.000 ± 1.095). Statistical significance was assessed using one-way ANOVA followed by Tukey’s Honest Significant Difference (HSD) test (***, P-value < 0.001). Column height represents the mean, and error bars the standard deviation from four biological replicates. Representative Sle1 blots are shown below the corresponding graphs.

(TIF)

pgen.1011841.s008.tif^{(11.9MB, tif)}

S9 Fig. Supraoptimal temperatures and various antibiotics reduce the level of cell wall-associated Sle1.

(A) Western blots displaying the difference in cell wall-associated and extracellular Sle1 levels in the NCTC8325–4 cxaR_down strain and CRISPRi control incubated at 37°C and 42°C. IPTG (500 μM) was added for gene knockdown. Band intensities were quantified using ImageJ. Values for cell wall-associated Sle1 were normalized to the control band at 37 °C, while extracellular Sle1 values were normalized to the cxaR_down band at 37 °C. The intensities, shown below the corresponding bands, represent the mean of two biological replicates, with standard deviation. (B) The effect of sublethal concentrations of penicillin G (1 ng/mL) on the cell wall-associated and extracellular levels of Sle1 in the NCTC8325–4 wild-type and ΔcxaR mutant, shown by Western blotting. Band intensities were quantified using ImageJ. Values for cell wall-associated Sle1 were normalized to the wild-type band without penicillin G, while extracellular Sle1 values were normalized to the ΔcxaR band without penicillin G. The intensities, shown below the corresponding bands, represent the mean of two biological replicates, with standard deviation. (C) Western blotting of cell wall-associated Sle1 in the NCTC8325–4 ΔcxaR mutant in response to sublethal concentrations of various antibiotics. From left to right; - (without antibiotics), OXA (7 ng/mL oxacillin), TEC (62.5 ng/mL teicoplanin), BAC (1 μg/mL bacitracin), MOE (7.5 ng/mL moenomycin), TUN (200 ng/mL tunicamycin), NOV (15 ng/mL novobiocin), KAN (300 ng/mL kanamycin). Band intensities were quantified using ImageJ. Values for cell wall-associated Sle1 were normalized to the ΔcxaR band without antibiotics. The intensities, shown below the corresponding bands, represent the mean of two biological replicates, with standard deviation.

(TIF)

pgen.1011841.s009.tif^{(17.4MB, tif)}

S10 Fig. CxaR regulates Sle1 levels in a ClpXP-dependent manner.

(A) Western blots of extracellular, cell wall-associated, and whole cell Sle1 levels in the NCTC8325–4 wildtype, ΔcxaR mutant, cxaR overexpressing strain, ΔclpX mutant, ΔclpX mutant overexpressing cxaR, clpX_I265E mutant, clpX_I265E mutant with knockdown of cxaR, and cxaR knockdown strain. Gene knockdown and overexpression were induced with the addition of 500 μM IPTG. (B) Growth curves displaying the growth phenotypes of the strains Western blotted in A at 37°C in TSB. Gene knockdown and overexpression were induced with the addition of 500 μM IPTG. The graphs represent averages from triplicate measurements.

(TIF)

pgen.1011841.s010.tif^{(20.1MB, tif)}

S11 Fig. cxaR mutant cells resemble clpX mutants, exhibiting a comparable temperature-dependent growth defect.

(A) The effect of concurrent CRISPRi knockdown of clpX and cxaR (purple) in NCTC8325–4 compared to the CRISPRi control (grey) and single knockdown of clpX (green) or cxaR (red) during growth in TSB at 37°C. Gene knockdown was induced with addition of 500 μM IPTG. The graphs represent averages from triplicate measurements. (B) Growth of an NCTC8325–4 ΔclpX mutant in TSB at 37°C with (red) and without (grey) knockdown of cxaR. Gene knockdown was induced with addition of 500 μM IPTG. The graphs represent averages from triplicate measurements. (C) Spotting assay of cells with single knockdown of either clpX (clpX_down) or cxaR (cxaR_down), and double knockdown of both clpX and cxaR (clpX_down cxaR_down) compared to the CRISPRi control at 30°C, 37°C, and 42°C. The strains were grown to mid-exponential phase (OD₆₀₀ of 0.5), 10-fold serially diluted, and spotted in volumes of 5 µL on TSA with 500 μM IPTG.

(TIF)

pgen.1011841.s011.tif^{(25MB, tif)}

S12 Fig. The AlphaFold 3-predicted interaction between CxaR and Sle1.

The domains of Sle1 are colored in accordance with the structure in Fig 6D, with the signal peptide in purple, LysM domains in light green, and CHAP domain in dark green. The predicted complex has a high confidence interaction score (ipTM) of 0.86.

(TIF)

pgen.1011841.s012.tif^{(12.4MB, tif)}

S13 Fig. Fluorescent fusions of ClpX and CxaR.

(A) Phase contrast and fluorescence microscopy of a plasmid-borne C-terminal ClpX-mCherry fusion in NCTC8325–4, with inducer concentrations between 5 µM and 320 µM IPTG. Scale bar represents 2 µm. (B) Phase contrast and fluorescence microscopy of NCTC8325–4 carrying plasmids encoding an N-terminal (left) and C-terminal (right) CxaR-GFP fusion, with and without induction of expression by 50 µM IPTG. Scale bar represents 2 µm. (C) Various induction levels of the plasmid-borne C-terminal CxaR-GFP fusion in NCTC8325–4. The concentrations of the IPTG inducer ranged from 0.5 µM to 100 µM, and localization was assessed with phase contrast and fluorescence microscopy. Scale bar represents 2 µm. (D) Western blot of the C-terminal CxaR-GFP fusion from whole cell lysate expressed from the native cxaR chromosomal locus in NCTC8325–4 (MDB376), from the pLOW plasmid in the NCTC8325–4 wild-type and from the pLOW plasmid in the NCTC8325–4 clpX_I265E mutant. Expression of the fusion from pLOW was induced by 10 µM IPTG. Cultures were harvested at OD₆₀₀ 0.5 and 1.0.

(TIF)

pgen.1011841.s013.tif^{(32.7MB, tif)}

S14 Fig. Overexpression of CxaR can compensate for the absence of ClpX, whereas ClpX overexpression further exacerbates the growth defect of ΔcxaR.

Overexpression of cxaR and clpX in the NCTC8325–4 wild-type (green), ΔclpX mutant (blue) and ΔcxaR mutant (red) from pLOW-cxaR and pLOW-clpX, respectively. Concentrations of IPTG ranged from 0 to 500 µM.

(TIF)

pgen.1011841.s014.tif^{(10.4MB, tif)}

S1 Table. CRISPRi-seq identified genetic factors modulating susceptibility to β-lactams in S. aureus.

The CRISPRi library, targeting over 97% of the genomic features of S. aureus NCTC8325–4 [59], was transformed into a NCTC8325–4 strain where dCas9 expression is controlled by a tetracycline-inducible promoter on the chromosome [60]. The induced library was grown in the presence and absence of 0.008 µg/mL penicillin G for ~20 generations at 37ºC in milk. Plasmids were subsequently isolated, and the sgRNA abundances were determined by Illumina sequencing, as previously described [58]. Log₂ fold-changes (Log₂FC) between penicillin G treated (PenG) and non-treated (minPenG) samples are presented for each sgRNA, along with the corresponding p-values (P_adj). Explanation of columns headings: sgRNA; sgRNA identification number, Locus tag; locus tag of the gene in S. aureus NCTC8325–4 targeted by the sgRNA, name; gene name, minPenG essentiality; whether the targeted gene is classified as essential, neutral or costly for growth without penicillin G added, PenG essentiality; whether the targeted gene is classified as essential, neutral or costly for growth with 0.008 µg/mL penicillin G added, Log₂FC; log₂ fold-change in sgRNA abundance between conditions with or without penicillin G; P_adj; adjusted P-value, Interactions; whether (yes or no) there were significant (P_adj < 0.05) difference in log₂FC between conditions with or without penicillin G. Transcriptional units (sgRNAs) whose knockdown were found to significantly alter sensitivity to penicillin are labelled with colors, where blue indicates increased sensitivity and red indicates reduced sensitivity. SAOUHSC_00659 (cxaR) is shown in bold.

(XLSX)

pgen.1011841.s015.xlsx^{(135.3KB, xlsx)}

S2 Table. The spontaneous mutations acquired when the cxaR deletion mutant was grown at a 30°C.

List of mutations, with over 90% variant frequency, identified when re-sequencing five independently isolated, visibly larger NCTC8325–4 ΔcxaR colonies formed at 30°C.

(PDF)

pgen.1011841.s016.pdf^{(144.4KB, pdf)}

S3 Table. Strains and mutants used in this work.

(PDF)

pgen.1011841.s017.pdf^{(240.2KB, pdf)}

S4 Table. Plasmids used in this work.

(PDF)

pgen.1011841.s018.pdf^{(188.2KB, pdf)}

S5 Table. Oligonucleotide primers used in this work.

(PDF)

pgen.1011841.s019.pdf^{(183.1KB, pdf)}

Acknowledgments

We would like to thank the NMBU Imaging Center for help with TEM, and Zhian Salehian and Anne Ohren Nordraak at NMBU for laboratory assistance.

Data Availability

Sequencing data generated in this study is available on SRA (accession number PRJNA1263195).

Funding Statement

M.K. was supported by a JPI-AMR grant from the Research Council of Norway (Grant No. 296906). D.F. was supported by the Independent Research Fund Denmark Technology and Production grant 0136-00200. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. CRISPRi-seq identified genetic factors modulating susceptibility to β-lactams in S. aureus.

(TIF)

pgen.1011841.s001.tif^{(13.1MB, tif)}

S2 Fig. The phenotype observed upon knockdown or deletion of cxaR is not caused by polar effect on neighboring genes.

(TIF)

pgen.1011841.s002.tif^{(15MB, tif)}

S3 Fig. Sublethal concentrations of various antibiotics stimulate growth of cells lacking cxaR.

(PDF)

pgen.1011841.s003.pdf^{(1.3MB, pdf)}

S4 Fig. CxaR is conserved among Staphylococcaceae.

(TIF)

pgen.1011841.s004.tif^{(21.3MB, tif)}

S5 Fig. Sublethal concentrations penicillin G significantly reduce lysis of cell lacking cxaR.

(TIF)

pgen.1011841.s005.tif^{(33.7MB, tif)}

S6 Fig. cxaR mutants exhibit heat stability and cold sensitivity.

(TIF)

pgen.1011841.s006.tif^{(31.4MB, tif)}

S7 Fig. Reduced Sle1 activity can compensate for the lack of CxaR.

(TIF)

pgen.1011841.s007.tif^{(8.4MB, tif)}

S8 Fig. Sle1 levels are elevated in the absence of CxaR and reduced upon CxaR overexpression.

(TIF)

pgen.1011841.s008.tif^{(11.9MB, tif)}

S9 Fig. Supraoptimal temperatures and various antibiotics reduce the level of cell wall-associated Sle1.

(TIF)

pgen.1011841.s009.tif^{(17.4MB, tif)}

S10 Fig. CxaR regulates Sle1 levels in a ClpXP-dependent manner.

(TIF)

pgen.1011841.s010.tif^{(20.1MB, tif)}

S11 Fig. cxaR mutant cells resemble clpX mutants, exhibiting a comparable temperature-dependent growth defect.

(TIF)

pgen.1011841.s011.tif^{(25MB, tif)}

S12 Fig. The AlphaFold 3-predicted interaction between CxaR and Sle1.

(TIF)

pgen.1011841.s012.tif^{(12.4MB, tif)}

S13 Fig. Fluorescent fusions of ClpX and CxaR.

(TIF)

pgen.1011841.s013.tif^{(32.7MB, tif)}

S14 Fig. Overexpression of CxaR can compensate for the absence of ClpX, whereas ClpX overexpression further exacerbates the growth defect of ΔcxaR.

(TIF)

pgen.1011841.s014.tif^{(10.4MB, tif)}

S1 Table. CRISPRi-seq identified genetic factors modulating susceptibility to β-lactams in S. aureus.

(XLSX)

pgen.1011841.s015.xlsx^{(135.3KB, xlsx)}

S2 Table. The spontaneous mutations acquired when the cxaR deletion mutant was grown at a 30°C.

List of mutations, with over 90% variant frequency, identified when re-sequencing five independently isolated, visibly larger NCTC8325–4 ΔcxaR colonies formed at 30°C.

(PDF)

pgen.1011841.s016.pdf^{(144.4KB, pdf)}

S3 Table. Strains and mutants used in this work.

(PDF)

pgen.1011841.s017.pdf^{(240.2KB, pdf)}

S4 Table. Plasmids used in this work.

(PDF)

pgen.1011841.s018.pdf^{(188.2KB, pdf)}

S5 Table. Oligonucleotide primers used in this work.

(PDF)

pgen.1011841.s019.pdf^{(183.1KB, pdf)}

Data Availability Statement

Sequencing data generated in this study is available on SRA (accession number PRJNA1263195).

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PERMALINK

Cell splitting in Staphylococcus aureus is controlled by an adaptor protein facilitating degradation of a peptidoglycan hydrolase

Maria Disen Barbuti

Eivind Frøyland Skjennum

Viktor Hundtofte Mebus

Danae Morales Angeles

Matilde Hustad

Marita Torrissen Mårli

Dorte Frees

Morten Kjos

Roles

Abstract

Author summary

Introduction

Results

Sublethal concentrations of cell wall targeting antibiotics stimulate growth of cells lacking cxaR

Fig 1. Lack of CxaR leads to a biphasic growth defect that can be rescued by sublethal concentrations of various antibiotics.

CxaR is conserved among Staphylococcaceae and has structural resemblance to TPR motifs

CxaR affects splitting of daughter cells

Fig 2. cxaR deletion increases lytic activity, while overexpression impairs cell division.

Fig 3. The increased lysis observed in the absence of CxaR is due to premature splitting of daughter cells, a phenomenon significantly mitigated by penicillin G treatment.

Reduced Sle1 activity can compensate for the lack of CxaR

Fig 4. Spontaneous sle1-associated mutations frequently rescued the defects of the cxaR deletion mutant at 30°C.

Sle1 levels are highly elevated in the absence of CxaR and reduced upon CxaR overexpression

Fig 5. Sle1 accumulates in the absence of CxaR, while overexpression of CxaR causes Sle1 depletion below detectable levels.

CxaR regulates Sle1 levels in a ClpXP-dependent manner

Fig 6. CxaR interacts with ClpX and Sle1.

CxaR directly interacts with ClpXP and Sle1

CxaR localization is ClpX-dependent

Fig 7. CxaR localizes adjacent to the septum in a ClpX-dependent manner.

Discussion

Fig 8. CxaR facilitates ClpXP-mediated degradation of Sle1, ensuring proper separation of staphylococcal daughter cells.

Materials and methods

Bacterial strains and growth conditions

Strain and plasmid construction

Construction of CRISPRi knockdown strains.

Construction of the cxaR deletion mutant.

Construction of suppressor mutants.

Construction of plasmids for complementation and overexpression.

Construction of plasmids for localization analysis of CxaR and ClpX.

Construction of a chromosomally integrated cxaR-m(sf)gfp fusion.

Construction of split luciferase plasmids.

Genome re-sequencing and analysis

CRISPRi-sequencing screen

Growth assays in liquid media

Spotting assays

Western blot analyses of Sle1 levels

Cell harvesting.

Preparation of extracellular and cell wall-associated protein extracts.

Preparation of whole cell lysate.

SDS-PAGE, Western blotting, and detection.

Western blot analysis of GFP-tagged CxaR

Phase contrast and epifluorescence microscopy

Microscopy.

Image analysis.

Transmission electron microscopy

Split luciferase assays

Structural and phylogenetic analysis

In silico protein interaction modelling

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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