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ABSTRACT Bacterial two-component systems (TCSs) are signaling modules that control physiology, adaptation, and host interactions. A typical TCS consists of a histidine kinase (HK) that

activates a response regulator via phosphorylation in response to environmental signals. Here, we systematically test the effect of inactivating the conserved phosphatase activity of HKs to

activate TCS signaling pathways. Transcriptome analyses of 14 HK mutants in _Streptococcus agalactiae_, the leading cause of neonatal meningitis, validate the conserved HK phosphatase

mechanism and its role in the inhibition of TCS activity in vivo. Constitutive TCS activation, independent of environmental signals, enables high-resolution mapping of the regulons for

several TCSs (e.g., SaeRS, BceRS, VncRS, DltRS, HK11030, HK02290) and reveals the functional diversity of TCS signaling pathways, ranging from highly specialized to interconnected global

regulatory networks. Targeted analysis shows that the SaeRS-regulated PbsP adhesin acts as a signaling molecule to activate CovRS signaling, thereby linking the major regulators of

host-pathogen interactions. Furthermore, constitutive BceRS activation reveals drug-independent activity, suggesting a role in cell envelope homeostasis beyond antimicrobial resistance. This

study highlights the versatility of constitutive TCS activation, via phosphatase-deficient HKs, to uncover regulatory networks and biological processes. SIMILAR CONTENT BEING VIEWED BY

OTHERS AN _E. COLI_ DISPLAY METHOD FOR CHARACTERIZATION OF PEPTIDE–SENSOR KINASE INTERACTIONS Article 08 December 2022 HIGHLY-CONSERVED REGULATORY ACTIVITY OF THE ANR FAMILY IN THE VIRULENCE

OF DIARRHEAGENIC BACTERIA THROUGH INTERACTION WITH MASTER AND GLOBAL REGULATORS Article Open access 29 April 2023 IMPACT OF _ABAI_ MUTATION ON VIRULENCE, BIOFILM DEVELOPMENT, AND ANTIBIOTIC

SUSCEPTIBILITY IN _ACINETOBACTER BAUMANNII_ Article Open access 14 September 2024 INTRODUCTION Two-component systems (TCSs) are one of the main bacterial signalling mechanisms. In their

simplest form, an environmental signal activates a histidine kinase (HK), which phosphorylates a cognate response regulator (RR), leading to the transcription of specific genes that mediate

the cellular response to the stimuli1. Actually, TCSs are sophisticated molecular machinery with buffering and insulating mechanisms that dynamically control specific or global cellular

responses2,3,4. Considerable effort has been made to define TCS regulatory networks in both model and pathogenic species, including by comprehensive analysis5,6. Although knowledge gained in

one species can provide information about homologous systems, TCS are characterised by their diversity, plasticity and evolvability7,8. This prevents global inferences even between closely

related species9,10. This evolution of regulatory networks is sustained by several mechanisms, including mutations, horizontal gene transfer, duplication followed by neofunctionalization,

and rewiring that shapes adaptation and speciation11,12,13. Functional, evolutionary, and system analyses require characterising individual signalling pathways and integrating them into the

cellular regulatory network. Traditionally, regulons are characterised using inactivated TCS mutants. One common pitfall is that TCSs are not active until the specific, but usually unknown,

stimulus is provided. Current approaches to overcome signal requirements are based on phosphomimetic mutation of the RR14,15 and profiling of protein-DNA interaction16,17,18. An alternative

approach exploits the distinct HK enzymatic activities. The HK cytoplasmic core called the transmitter module, is composed of the DHp (Dimerisation and Histidine phosphotransfer) and CA

(catalytic and ATP-binding) domains19,20,21. The two domains are dynamically structured in specific conformations that catalyse three distinct reactions: autophosphorylation of a conserved

histidine residue in the DHp domain, phosphotransfer to a conserved aspartate on the RR, and RR dephosphorylation. Pioneering studies have identified mutations abolishing the HK phosphatase

activity leading to increased RR phosphorylation and signalling pathway activation22,23,24. The importance of HK phosphatase activity in vivo has been initially debated, especially when

considering the lability of RR phosphorylation and spontaneous dephosphorylation rate25. Nowadays, phosphatase activity is recognised as essential for the dynamics of the response and to

ensure that the RR is activated by the cognate HK only26,27. Co-evolving residues and HKs conformational rearrangements ensure the specificity and directionality of enzymatic

reactions28,29,30. While the activation mechanism involving the conserved histidine residue is fundamentally conserved among HKs, the phosphatase mechanism has remained more elusive due to

variations in the DHp domain31. Then, a seminal study proposed a conserved phosphatase mechanism for the two main HisKA and HisKA_3 families, identifying conserved motifs and specific

catalytic residues needed for the correct positioning of nucleophilic attack31,32. Substitution of the catalytic residues abolishes the phosphatase activity without impacting the autokinase

and phosphotransfer activities, resulting in increased RR phosphorylation and pathway activation for the individual HKs reported to date32,33,34,35,36,37. This study aims to systematically

test the proposed conserved mechanism of phosphatase activity, the in vivo effect of phosphatase-deficient HK, and the activation of the regulatory network in all HisKA and HisKA_3 systems

in a bacterium. We focused on _Streptococcus agalactiae_ (Group B _Streptococcus_, GBS), a pathobiont that is commensal in adults but pathogenic during pregnancy and in neonates, for whom it

is the leading cause of invasive infections38,39. We report that targeting HK phosphatase activity provides high-resolution views of signalling pathways for most TCSs independently from

environmental signals. In addition, regulatory network activation resolves the connectivity between TCSs involved in host-pathogen interactions and reveals the physiological function of a

TCS involved in antimicrobial resistance. This systematic analysis argues for the widespread adoption of this gain-of-function approach to decipher TCSs signalling in genetically manipulable

species. RESULTS THE HK+ COLLECTION TARGETS THE PHOSPHATASE ACTIVITY OF HISTIDINE KINASE We undertook a genetic approach to systematically test the hypothesis of a conserved

dephosphorylation mechanism in the two major HK families32. The genome of the BM110 strain belonging to the hypervirulent clonal complex 17 (CC–17) encodes 20 HKs40, among which 12 and 2

have a HisKA and HisKA_3 DHp domain, respectively (Supplementary Data 1). Their H-box motif always contains the conserved phospho-acceptor histidine, immediately followed by the predicted

phosphatase motif (Fig. 1A). Eleven of the twelve HisKA proteins have the E/DxxT/N motif with a putative threonine catalytic residue, while the remaining HisKA protein (BceS) has a divergent

sequence composition (QMKV) with a valine at the predicted catalytic position (Fig. 1A). The two HisKA_3 proteins have the DxxxQ/H motif with the predicted glutamine or histidine catalytic

residue (Fig. 1A). The 14 HKs encoding genes are organised in operon with their cognate response regulator (RR) belonging to the OmpR (with HisKA) or LuxR (with HisKA_3) family, but one

system is not functional (HK10655-RR10650fs) due to a pseudogenization of the RR in the CC-17 hypervirulent GBS lineage (Supplementary Data 1). We generated 14 strains, called the HK+

collection, with an alanine substitution of the predicted phosphatase catalytic residue (Fig. 1A). Whole-genome sequencing confirmed the chromosomal substitution of targeted base pairs and

the absence of secondary mutations in 11 out of the 14 HK+ strains. In the three remaining HK+ (CovST282A, VicKT221A, and RelST208A), we sequenced independent mutants and selected one with a

single secondary mutation (Supplementary Data 2). Notably, the selected VicKT221A mutant possesses a non-synonymous polymorphism in the glutamine transporter GlnPQ, which we cannot dismiss

as a potential compensatory mutation. Four independently constructed VicKT221A mutants exhibit putative compensatory mutations (Supplementary Data 2), a phenomenon commonly observed in

mutants within the homologous WalRK system, which is essential for cell wall remodelling during growth and division41,42,43. Individual growth curves show a significant effect ( | F | > 

0.1, Mann Whitney test _p_ < 10−4) of the HK+ mutation for four mutants (Fig. 1B). The CovST282A and CiaHT228A have a reproducible fitness advantage compared to the WT strain, while the

VicKT221A and SaeST133A have fitness defect. Notably, the slow-growing VicKT221A mutant is unstable and gives rise to faster-growing cultures, likely due to additional mutations, while the

SaeST133A mutant exhibits a density-dependent phenotype characterised by a decreasing growth rate in the exponential phase and a lower final OD (Supplementary Fig. 1). In addition, two

mutants have increased antibiotic susceptibilities: the VicKT221A mutant against beta-lactams, in agreement with a conserved function in cell wall metabolism, and the RelS mutant against

fosfomycin (Supplementary Data 3). HK+ ACTIVATE POSITIVE FEEDBACK LOOPS To test TCS activation, we first relied on positive feedback loops. This autoregulation is often observed through

direct transcriptional activation of the TCS operon by the activated RR2. We therefore analysed the transcription of all HKs and RRs encoding genes (_n_ = 41, including non-HisKA and HisKA_3

TCSs and an orphan RR) in each HK+ mutant by RNA-sequencing from cultures grown in a standardised condition (THY: Todd Hewitt supplemented with 1% Yeast extract, pH 7.4, 37 °C, exponential

growth phase OD600 = 0.5). A positive feedback loop, indicated by a significant fold change greater than two relative to the WT strain (log2 FC > 1; p-adj < 10−4) for the HK and RR

genes, is observed in seven HK+ mutants (Fig. 1C). Furthermore, two TCSs are significantly regulated in an unrelated HK+ mutant: the HK11050-RR11055 system, which does not contain a HisKA

and HisKA_3 domain, in the VicKT221A mutant and the RelRS system, which is not positively auto-regulated, in the CiaHT228A mutant (Fig. 1C). As an independent approach to test TCS

activation, we introduced in each mutant a vector expressing an epitope-tagged copy of the cognate regulator. For two mutants (VicKT221A and RelST208A), an increased level of phosphorylation

of the ectopically expressed regulator is detected in the HK+ mutant compared to the WT strain after Phos-Tag electrophoresis and western analysis with anti-FLAG antibodies (Fig. 1D).

However, due to competition between WT and epitope-tagged regulators and variability in the stability of phosphorylated aspartate, no conclusions could be drawn for most regulators. This

highlights the need to quantify the level of phosphorylation using specific antibodies directed against each native RR. Overall, by considering epitope-tagged RR activation by

phosphorylation and positive feedback loops, the majority (8/14) of HK+ mutations appear to activate the corresponding TCS signalling pathway. THE ACTIVATED GENE REGULATORY NETWORK To

characterise the activated pathways, we analysed the RNA-seq profiles of each HK+ mutant grown under standardised conditions, independent of specific environmental cues (i.e., exponential

phase in THY pH 7.4 at 37 °C). Statistical analysis of differentially expressed genes (DEGs: Supplementary Data 4) grouped the HK+ mutants into three main categories based on the adjusted

_p_-values. Six HK+ mutants (HK11030T245A, VncST245A, SaeST133A, BceSV124A, HK02290H188A, and DltST184A) show DEGs associated with striking statistical significance (p-adj < 10−250),

revealing the activated regulons with high resolution (Fig. 2A and Supplementary Fig. 2). Four additional mutants (RelST208A, CiaHT228A, VicKT221A, and CovST282A) show DEGs with lower

statistical significance (p-adj > 10−150), suggesting complex regulons or intermediate TCS activation (Supplementary Fig. 2). The remaining four mutants (HssST150A, LiaSQ149A, PhoST345A,

and the HK10655T267A with a frameshifted RR) gave no or low significant signals (p-adj > 10−10) (Supplementary Fig. 2). Since most RRs are transcriptional activators, we focused the

analysis on activated genes. By applying strict thresholds (FC > 3, p-adj < 10−4) for normalisation between samples and excluding genes with very low read counts in all samples and

genes localised in mobile genetic elements, 219 genes (11.9% of the 1838 genes analysed) are transcriptionally activated in at least one HK+ mutant (Supplementary Data 4E). Transcriptional

activation can be up to 8000-fold, with an average fold change of 61.6-fold and an uneven distribution between HK+ mutants (Fig. 2B). The number of activated genes ranges from 3

(HK11030T245A) to 139 (VicKT221A) (Fig. 2B and Supplementary Data 4F). Five regulatory systems activate a specific genetic programme, four of them (HK11030T245A, HK02290H188A, VncST245A,

DltST184A) positively regulating a single functional genetic module composed of their own operon and at least one additional gene involved in the cellular response localised into, or

adjacent to, the TCS operon (Fig. 2C), and one system (CiaHT228A) coordinating the activation of at least six independent loci (Supplementary Fig. 3). Four additional TCSs activate specific

genes but share 1 to 3 activated genes with the VicKT221A mutant (Fig. 2D). One of these connected systems (HssST150A) is specialised in haem detoxification via the transcriptional

activation of the _hrtBA_ genes encoding a specific ABC transporter44, which is similarly activated in the VicKT221A mutant. The three additional connected systems activate several loci

involved in host-pathogen interaction (SaeST133A: adhesins and secreted proteins), drug resistance (BceSV124A: transporters and peptidase), or nucleotide metabolism (RelST208A: de novo

purine synthesis and ectonucleotidases), with (SaeST133A, BceSV124A) or without (RelST208A) a positive feedback loop (Fig. 2D and Supplementary Fig. 3). POSITIVE AND NEGATIVE INTERACTION

BETWEEN TCS SYSTEMS Overall, the HK+ mutation activates the signalling pathway for 10 out of 12 TCSs (Fig. 2D), excluding the CovST282A repressing system analysed separately and the negative

control HK10655T267A with a frameshifted RR. Notably, each HK+ mutant is associated with the activation of specific genes, except the global regulator VicRK (Fig. 2D and Supplementary Data 

4E). As expected, the VicKT221A regulon included several operons involved in cell wall metabolism (Supplementary Fig. 3). However, constitutive activation of VicK probably leads to the

activation of related stress and cell-wall signalling pathways. To identify relationships between TCS pathways involved in related processes, we analysed the 219 genes activated in at least

one HK+ mutant for their expression in the whole RNA-seq dataset. This analysis confirmed the partial activation of SaeRS signalling in the VicKT221A mutant (a shared CovR connection, in

fact, see specific section below) sustained by genes with 1 < FC < 3 and significant but higher p-adj value compared to the SaeST133A activated system (Supplementary Data 4H).

Similarly, by considering significantly regulated genes with lower thresholds (1 < | FC | < 3, p-adj< 0.05), significant positive or antagonistic interactions were detected between

signalling pathways (e.g., DltS activating CiaH and VicK, CiaH antagonising RelS, HK02290 antagonising HK11030). Finally, relaxing the thresholds also reveals the first five genes of the

_phoRS_ operon as the most and only significantly up-regulated genes (1,5 < FC < 1,75; 7.10−3 < p-adj< 10−5) in the PhoST345A mutant (Supplementary Data 4D), suggesting a

conserved mechanism of phosphatase activity but an inefficient activation of the PhoR regulator in the corresponding HK+ mutant. ACTIVATION OF THE GLOBAL REPRESSOR OF VIRULENCE COVRS The

CovRS system is the major regulator of virulence in GBS and, in contrast to canonical TCS, acts as a global repressor of gene transcription45. Analysis of RNA-seq to identify negative

regulation using similar thresholds (− 3 > FC, p-adj < 10−4) revealed the repression of 32 genes in the whole dataset (Supplementary Data 4G). Almost all repressed genes are regulated

by the two global regulators, VicK (17 genes) and CovS (14 genes). Notably, the most highly repressed gene (22-fold in VicKT221A) encodes a D-L endopeptidase, highlighting the conservation

of negative regulation of cell wall hydrolases between WalRK homologous systems41. On the other hand, a comparative analysis of repressed genes in CovST282A with the known CovR regulon shows

the limitations of the HK+ approach for characterising the CovRS system. Indeed, only 5 out of 14 repressed genes in the CovST282A mutant belong to the CovR regulon of 153 genes previously

determined with loss of function mutants45. This difference can be attributed to another specific feature of the CovRS system, which is active in the absence of an environmental signal45.

The CovST282A transcriptome, therefore, suggests that CovR over-activation does not translate into increased repression of targeted genes. To further analyse the CovRS system, we compared

the transcriptome of the activated CovST282A mutant with that of the inactivated CovRD53A mutant, which cannot be phosphorylated by CovS, and also included our genome-wide CovR binding

analysis done by ChIP-seq45. Side-by-side transcriptome comparison showed that the inactivation of CovR activates the signalling pathway repressed by the active CovRS system, without a

general inverse relationship in the CovST282A mutant (Fig. 3A). Nevertheless, a significant inverse correlation between the CovST282A and CovRD53A transcriptomes is observed for genes that

do not belong to the direct CovR regulon (Fig. 3B), suggesting that overactivation of CovR primarily increases binding to co-regulated promoters and low-affinity binding sites45. Since the

inactivation of the CovRS repressor system is more informative than its over-activation, we included the CovRD53A transcriptome in the HK+ dataset. The global gene network reveals

connections between CovR-repressed genes and SaeST133A, VicKT221A, or BceSV124A activated genes (Fig. 2D). Notably, SaeST133A is highly connected with the direct CovR-repressed genes, while

BceSV124A activates only three CovR indirectly regulated genes. Functional assays using pigmented beta-haemolysin/cytolysin (ß-h/c) production as a natural reporter of CovR activity first

confirmed the non-pigmented and non-haemolytic phenotypes of the CovST282A mutant (Fig. 3C), in agreement with CovR directly repressing the _cyl_ operon encoding the ß-h/c synthesis and

export machineries45,46. The phenotype of six additional HK+ mutants are different from the WT strain on selective media, either increasing (SaeST133A, VicKT221A, and RelST208A) or

decreasing (BceSV124A, CiaHT228A, and LiaSQ149A) pigmentation and/or haemolytic activity (Fig. 3C). However, the absence of correlation between the transcription of the _cyl_ operon and the

pigmentation/haemolytic phenotypes in several HK+ mutants (Fig. 3C) suggests that ß-h/c activity depends on post-transcriptional regulatory mechanisms in addition to CovR regulation of the

_cyl_ operon. This is potentially the case in mutants with altered cell surface composition, where the toxin interaction occurs47,48. THE PBSP ADHESIN CONNECTS SAERS AND COVRS SIGNALLING We

sought to decipher the connection between the CovRS and SaeRS systems, two main regulators of host-pathogen interactions. Published transcriptomes with _saeRS_ deletion mutants define a

large regulon of 400–600 genes depending on growth conditions49. In contrast, analysis of the SaeST133A transcriptome reveals the highly significant (60 < FC < 8000-fold, p-adj < 

10−250) activation of four genes only, along with a partial activation of the CovR regulon (Fig. 4A). We confirmed the stratification of the SaeST133A differentially regulated genes by

RT-qPCR, validating 3 groups: the _pbsP_ and _bvaP_ genes, the _saeRS_ operon, and the CovR-regulated genes represented by the directly repressed genes _cylE_ and _hvgA_ (Fig. 4B). Intrigued

by the almost 50-fold difference between _pbsP_ and _saeRS_ up-regulation, we analysed the genomic locus in detail. The 112 bp _pbsP_-_saeRS_ intergenic region contains a P_saeR_ promoter

but no canonical transcriptional terminator. The integration of such a terminator precisely after the _pbsP_ stop codon in the SaeST133A mutant abolishes _saeRS_ overexpression while having

no impact on other activated genes (Fig. 4B). Quantification of promoter activities using _ß-_galactosidase reporters confirms a similar activity of P_saeR_ in the WT and in SaeST133A

mutant, and the strong activation of P_pbsP_ upon activation of the SaeRS system (Fig. 4C). This shows an indirect positive feedback loop of the _saeRS_ operon, which is transcribed by its

constitutive promoter and regulated by _pbsP_ termination readthrough. Interestingly, the basal level of _saeRS_ transcription in SaeST133A with the _pbsP_ terminator is sufficient to fully

activate _pbsP_ and _bvaP_ (Fig. 4B), implying that the indirect feedback loop may be physiologically relevant only for controlling the kinetics of the signal-dependent response, but not its

amplitude. We next analysed the connection between SaeRS and CovRS signalling. The activation of CovR-regulated genes in the SaeST133A mutant is intermediate when compared to the CovRD53A

mutant (Figs. 3A, 4A). One hypothesis could be a competitive binding between SaeR and CovR, but it is unlikely that all binding sites will allow both SaeR-activation and CovR-repression. As

an alternative, we hypothesised that the two genes specifically regulated in the SaeST133A mutant, encoding the PbsP cell-wall anchored adhesin50,51 and the BvaP secreted protein52, could be

involved in the activation of the CovR regulon. Indeed, the deletion of _pbsP_, but not of _bvaP_, in the SaeST133A mutant, restores a WT haemolytic activity (Fig. 4D). In agreement with

the phenotypes, the deletion of _pbsP_ in the SaeST133A mutant restores a WT level of the CovR-regulated genes _cylE_ and _hvgA_, while the _saeRS_ and _bvaP_ genes are still similarly

up-regulated (Fig. 4E). After cleavage by the enzyme sortase A and anchoring to the cell wall, the remaining carboxy-terminal domain of an LPxTG adhesin can act as a signalling molecule by

interacting with the transmembrane domain of a specific HK, as demonstrated in _Streptococcus gordonii_53. We, therefore, considered this C-peptide mechanism and constructed mutants

expressing truncated PbsP variants in the SaeST133A mutant. In-frame deletion of the PbsP C-peptide (e.g., 108 bp deletion including the LPxTG cell-wall anchoring motif until the penultimate

codon) has no effect on the induction of the CovR-regulated haemolytic activity (Fig. 4D). In contrast, in-frame deletion of the PbsP adhesin domain (1239 bp deletion leaving the signal

peptide and the LPxTG cell wall anchoring motif intact) restores the haemolytic activity of the SaeST133A mutant to WT level (Fig. 4D). Furthermore, the growth defect of the SaeST133A

mutant, which is similar to the growth defect of the CovRD53A mutant, is suppressed by deletion of _pbsP_ or of the adhesin part of _pbsP_ (Supplementary Fig. 4). Thus, the PbsP adhesin

domain triggers CovR signalling either by interacting with CovS or co-regulatory proteins54 or by inducing surface perturbations specifically sensed by the CovRS system (Fig. 4F).

DRUG-INDEPENDENT ACTIVITY OF THE BCERS THREE-COMPONENT SYSTEM We next sought to decipher the function of the BceRS system, which, upon activation, shows hypo-pigmented and haemolytic

phenotypes that are independent of CovRS regulation of the _cyl_ operon (Fig. 3C). The BceRS system belongs to a conserved TCS family that relies on a transporter to sense and transmit

environmental signals to the HK55. The transcriptome of the BceSV124A mutant reveals a 9-gene regulon, including the _bceRS_ operon and adjacent genes (Fig. 5A and Supplementary Figs. 2 and

3). Further validation by RT-qPCR confirmed the 10- to 1000-fold activation of the regulon in the absence of drugs in the BceSV124A mutant, as well as the absence of significant

transcriptional changes of the CovR-regulated genes (Fig. 5B). As expected, mutation of the BceR regulator to a non-phosphorylated form (BceRD55A) abolishes the activation of the signalling

pathway in the BceSV124A mutant (Fig. 5B). Interestingly, deletion of the transporter/sensor (∆_bceAB_) in the BceSV124A mutant also switches off the signalling pathway (Fig. 5B), showing

the essential role of the BceAB transporter in activating BceRS signalling in the absence of inducing signals. Typically, this TCS family confers resistance to antimicrobials targeting lipid

II cell wall metabolites such as nisin or bacitracin. Genetic activation of the pathway renders the BceSV124A mutant insensitive to nisin, which has a marked effect on the lag phase but not

on the growth rate of the WT strain, and increases resistance to gramicidin and, to a lesser extent, bacitracin (Fig. 5C and Supplementary Fig. 5). Interestingly, the BceSV124A mutant is

also more susceptible to antimicrobial peptides (colistin and polymyxin D) compared to the WT strain, while equally susceptible as the WT to vancomycin (Fig. 5C and Supplementary Fig. 5).

These results show that BceRS activation confers protection against structurally unrelated drugs targeting lipid II intermediates at the cost of increased susceptibility to antimicrobial

peptides. We then tested the phenotypes associated with loss-of-function of BceRS. Inactivation of the pathway in the BceSV124A background by additional BceRD55A or ∆_bceAB_ mutations leads

to nisin hyper-susceptibility while restoring WT level of colistin susceptibility (Fig. 5D). Nisin hyper-susceptibility is also observed for the single BceRD55A mutant (Fig. 5D), a phenotype

not linked to down expression of BceRS regulated genes (Fig. 5B). These results show that the BceRS system is constitutively active in the absence of drugs and that the basal activity is

necessary and sufficient to counteract the effects of sub-inhibitory concentrations of nisin. To test the current model of nisin resistance based on drug efflux and cleavage, we inactivated

the BceCDE transporter, the BceO substrate-binding protein, and the BceP extracellular protease (Fig. 5A). Deletion of ∆_bceD_ and ∆_bceO_ in the WT or BceSV124A backgrounds has no impact on

the nisin phenotypes of the respective parental strains (Fig. 5E), excluding a major function in drug export or binding. In contrast, the ∆_bceP_ mutant is slightly more susceptible to

nisin compared to the WT parental strain (Fig. 5E). However, deletion of _bceP_ in the BceSV124A background was always associated with secondary mutations inactivating the whole signalling

pathway in five independent mutants (Supplementary Data 2). Altogether, these results show that individual genes do not provide drug resistance and suggest that the BceP extracellular S41

protease56 has a buffering role when the pathway is activated, rather than directly cleaving drugs through an atypical mechanism, as previously suggested57. To test if BceRS regulates an

adaptive response rather than a resistance mechanism per se, we pre-incubated the WT strain with nisin for four hours. Prior exposure to the drug decreases the lag phase in a dose-dependent

manner upon subsequent exposure to higher nisin concentrations (Fig. 5F). For instance, adaptation with 8 µg/ml nisin, a WT sub-inhibitory concentration, confers a BceSV124A-like resistance

against a subsequent 64 µg/ml nisin challenge (Fig. 5F). More generally, prior adaptation with a given nisin concentration increases by a 4-fold factor the inhibiting concentration. These

results show that the BceRS response is adaptative and suggests that the BceRS system actively monitors and adjusts surface-exposed lipid II metabolites, rather than directly detoxifying

drugs or drug-lipid II complexes. DISCUSSION Our systematic analysis highlights the benefits of the HK+ gain-of-function approach to characterise TCS signalling, both for mapping regulatory

networks and for characterising individual systems. This study was made possible by the conserved mechanism of HK phosphatase activity originally proposed31,32, which allowed a single

residue to be targeted to activate the corresponding signalling pathway. By systematically testing all HisKA and HisKA_3 systems in a bacterium, we show the broad potential of this approach

to reveal specialised, connected, and global regulatory systems covering the functional diversity that has evolved from a simple two-component architecture. Targeting the HK phosphatase

catalytic residue has the advantage of leaving a quasi-native system. The gain-of-function is solely dependent on the HK mutation, with no change to the RR and preservation of the

physiological feedback loops. A second major advantage is that it bypasses the need for environmental signals, which are often unknown or confounding when having a wide effect on bacterial

physiology. In this respect, the SaeRS system is a remarkable example. Previous studies demonstrated SaeR regulation of _pbsP_ and _bvaP_ during vaginal colonisation, among transcriptomic

perturbations affecting nearly 40% of the genome49. However, the regulon remained elusive due to a lack of activation in vitro49. The HK+ approach resolves the signalling pathway by

revealing a specialised and CovR-connected, pathway. Comparison with the well-characterised _Staphylococcus aureus_ homologous system58 highlights the evolutionary divergence between

regulatory circuits, particularly for those regulating host-pathogen interactions, which need to be studied in each species. Originality is a mechanism linking the SaeRS and CovRS systems.

Complex regulatory wiring can be selected to mount co-ordinated responses, primary trough transcriptional cascades (a TCS regulating transcription of a second TCS) or connectors (usually a

TCS-regulated transmembrane protein modulating the activity of a second TCS)59. The C-peptide of adhesins can act as connectors when the transmembrane end remaining after cleavage of the

LPxTG motif by sortase A interacts with a histidine kinase53. The mechanism differs in GBS in which the PbsP adhesin domain acts as an extracellular signalling molecule to activate CovR

signalling, independently of cell wall anchoring. We hypothesise that the lysin-rich and positively charged PbsP adhesin interacts with CovS, with the co-regulatory proteins Abx1 and

Stk154,60, or with the negatively charged membrane, recalling the activation of the homologous CovRS system in _Streptococcus pyogenes_ by cationic peptides61. To complete the regulatory

circuit, CovR has previously been shown to repress _pbsP_ in a strain-specific manner45,50,51. The intertwining of SaeR and CovR signalling through PbsP constitutes an adaptive mechanism for

balancing adhesion and invasion and could contribute to the phenotypic and pathogenicity variabilities observed within the species. HK+ mutations resolve TCS regulatory networks but reveal

discrepancies in the activation of signalling pathways. While primary sequence analysis of TCSs did not uncover specific motifs correlating with high, intermediate, or low pathway

activation, two underlying factors may dampen the effect of HK+ mutations. First, HK kinase activity can be inhibited by interacting proteins, such as the small LiaF protein inhibiting

LiaS62,63 and the Pst/PhoU proteins inhibiting PhoRS64,65. Genes encoding co-regulatory proteins are often themselves regulated by the TCS, creating feedback loops that lock HKs in

kinase-deficient conformation and thus obliterate the effect of HK+ mutations. However, the presence of auto-inhibitory proteins is not a sufficient condition for preventing activation, as

demonstrated by VicK, which is inhibited by YycH/YycI66,67 but still activated by an HK+ mutation. Second, intermediary activation of the RelRS and CiaRH pathways suggests buffering

mechanisms for TCSs regulating multiple independent loci and integrated cellular response. However, detailed analysis is required to decipher phosphorylation dynamics in each

phosphatase-deficient HK+ and correlate in vivo RR phosphorylation with regulatory network activation, considering variable factors like the source of RR phosphorylation (kinase activity of

the HK+ variant, cross-talk by other HK, small metabolites) and specific spontaneous dephosphorylation rates of the labile aspartate residues27,68,69. The systematic approach validates the

conservation of the dephosphorylation mechanism. It also uncovers an unanticipated activation of the BceRS system with a degenerate QMKV motif. Recent structural insights from _Bacillus

subtilis_ complexes into the membrane environment support a highly dynamic model of interactions between the BceAB transporter and the BceS kinase- and phosphatase-competent

conformations70,71. Our results with the HK+ BceS indeed suggest that BceAB is necessary to stabilise the kinase-competent conformation of BceS. Alternatively, BceAB could also provide the

catalytic residue on the models suggested for the auxiliary phosphatases RapH and Spo0E31,72. At the phenotypic level, our results point towards a need-based mechanism of target protection,

as recently suggested for the Bce-like system73,74,75, and not towards a drug cleavage-exclusion mechanism as initially suggested76,77. The target protection mechanism relies on the binding

of lipid II intermediates on a binding pocket of BceAB70. However, it is still unclear how the system releases lipid II when it is complexed with drugs. Our results suggest an alternative

scenario in which BceAB constantly monitor free lipid II intermediate to minimise target exposure78,79. This alternative is supported by the steady-state activity of the BceRS pathway in the

absence of drugs and is compatible with a need-based mechanism. Further studies should test the entire BceRS pathway without relying on a lipid II-drug detoxification mechanism but rather

on a mechanism that maintains the steady-state level of free lipid II in the presence of drugs. It is also interesting to note that the activation of the BceRS-BceAB system impacts the

retention or secretion of the ß-h/c toxin, potentially as a result of the interaction between the polyene backbone of the toxin and the membranes46,47,48, suggesting functional links between

cell envelope homoeostasis, drug resistance, and virulence. To conclude, genetic activation by HK+ is a powerful approach to characterise positive regulation by TCS. It circumvents the

major drawback of studying systems that are usually non-activated in standard conditions. Previous studies on individual TCSs have demonstrated the potential of the approach, but it has

unfortunately not been widely adopted to date. Our systematic analysis based on the conserved mechanism of phosphatase activity provides a blueprint to decipher signalling, response

dynamics, evolution of gene regulation, and regulatory networks. The HK+ approach is recommended for the study of TCS in any species, either as a complement or as a first choice alongside a

classical deletion mutant. METHODS STRAIN, BACTERIAL GENETICS, AND GENOME SEQUENCING The BM110 strain is a clinical isolate representative of the hypervirulent CC-17 clonal complex

responsible for most neonatal meningitis80. The 2.2 Mb annotated genome is available under the NCBI RefSeq reference NZ_LT714196. The standard growth condition is in Todd-Hewitt medium

supplemented with 1% yeast extract and 50 mM Hepes pH 7.4 (THY) incubated in static condition at 37 °C. Oligonucleotides and construction of vectors for site-directed mutagenesis and

deletion are detailed in Supplementary Datas 5 and 6, respectively. Splicing-by-overlap PCR with high-fidelity polymerase (Thermo Scientific Phusion Plus) were done with complementary

primers containing the desired mutations. The final PCR products contain mutations (SNP or deletion) flanked on either side by 500 bp of sequence homologous to the targeted loci. Cloning is

done by Gibson assembly in pG1, a thermosensitive shuttle vector similar to the pG + host5 vector81. Constructs were introduced and maintained in _E. coli_ XL1-blue (Stratagene) with

erythromycin selection (150 µg/ml) at 37 °C. Inserts were validated by Sanger sequencing (Eurofins Genomics). Mutant construction in GBS was performed through a three-step process involving

episomal replication, chromosomal integration, and vector loss. Initially, pG vectors were introduced into GBS via electroporation, and transformants were selected on THY agar supplemented

with 5 µg/ml erythromycin at 30 °C, the permissive temperature for episomal replication. After 24–36 h of growth, two single transformant colonies were isolated on THY agar containing

erythromycin and incubated another 24–36 h at 37 °C, the non-permissive temperature for vector replication, thereby promoting chromosomal integration of the vector at the targeted locus

through homologous recombination. Subsequently, rare colonies that had integrated the vector into the chromosome were isolated on THY agar with erythromycin at 37 °C for 16 h. The isolated

integrants were inoculated into 10 ml of THY medium without antibiotic, incubated at 30 °C to activate the rolling circle origin of replication, and subjected to serial subculture twice

daily. By day 3, cultures were diluted (typically 10−5), spread onto THY and Columbia agar supplemented with 10% horse blood (BioMerieux), and incubated at 37 °C. Isolated colonies (_n_ = 

24–48) were picked into 150 µl of THY in 96-well plates, incubated for 4–6 h at 37 °C, and replica-plated using a 96-pin replicator (Boekel Scientific) onto THY agar plates with and without

erythromycin. After 16 h at 37 °C, erythromycin-susceptible colonies that had lost the vector were identified and confirmed by discriminatory PCR (MyTaq HS - Bioline) using specific

oligonucleotides with the expected mutation at their 3’ extremity (Supplementary Data 5) to distinguish mutant from wild-type genotypes. Mutants were then isolated on THY at 37 °C, single

colonies inoculated in THY incubated at 37 °C for 16 h, centrifuged and resuspended in 20% glycerol for long-term storage at − 80 °C. Genomic DNA of at least two independent mutants for each

construction were purified from 1 ml of culture following manufacturer instruction for Gram-positive bacteria (DNeasy Blood and Tissue – Qiagen) and sequenced (Illumina sequencing at Core

facility or Eurofins Genomics). High-quality reads in Fastq were mapped against the BM110 genomes and analysed with Geneious Prime (2019.2.3 – Biomatters Ltd) using default parameters

(mapping: up to 5 iterations, minimum mapping quality Phred score 30, medium/low sensitivity; SNP calling: minimum variant frequency 0.25, maximum variant _P_-value 10−6, minimum strand-bias

_P_-value 10−5, with visual inspection for coverage and validation of SNPs). Results of genome sequencing for all mutants used in this study (55–419 x coverage, mean 181 x) are summarised

in Supplementary Data 2. RNA SEQUENCING RNA purification, sequencing and analysis were conducted essentially as described for the characterisation of the virulence regulator CovR45. The 14

HK+ mutants were split into two series of 8 strains (7 mutants and one WT strain), and RNA was purified using three independent replicates that were grown on different days. Overnight

cultures were used to inoculate THY (1/50), and 10 ml of culture were harvested in the exponential growth phase (OD600 = 0.5) after incubation at 37 °C. Bacterial pellets are washed with

cold PBS containing RNA stabilisation reagents (RNAprotect, Qiagen) before flash freezing and storage at − 80 °C. Total RNA are extracted after cell wall mechanical lysis with 0.1 µm beads

(Precellys Evolution, Bertin Technologies) in RNApro reagent (MP Biomedicals), and purified by chloroform extraction and ethanol precipitation. Samples were treated to remove residual DNA

(TURBO DNase, Ambion) before fluorescent-based quantification (Qubit RNA HS, Invitrogen) and quality validation (Agilent Bioanalyzer 2100). Depletion of rRNA (FastSelect Bacterial, Qiagen),

library construction and sequencing were done following manufacturer instructions (TruSeq Stranded mRNA, NextSeq 500, Illumina). Single-end strand-specific 75 bp reads were cleaned (cutadapt

v2.10) and mapped on the BM110 genome (Bowtie v2.5.1, with default parameters). Gene counts (featureCounts, v2.0.0, parameters: -t gene -g locus_tag -s 1) were analysed with R (v4.0.5) and

the Bioconductor package DESeq2 (v1.30.1)82. Normalisation, dispersion, and statistical tests for differential expression were performed with independent filtering. For each comparison, raw

_p_-values were adjusted using Benjamini and Hochberg multiple tests83 and adjusted _p_-values lower than 0.05 were considered significant. Raw sequencing reads and statistical analysis are

publicly available (GEO accession number GSE261394). In addition to HK+ RNA-sequencing, we have included an independent CovST282A transcriptome that was done simultaneously with the CovRD53A

transcriptome45, the latter being already reported altogether with CovR ChIP-seq experiment (GEO accession number GSE158049). Gene networks are represented with the open-source software

Cytoscape (v3.9.1)84. RT-QPCR AND PROMOTER ACTIVITY For validation, independent RNA purifications from biological triplicates were done using the same protocol, except that the cultures were

grown on the same day and only 1 ml was harvested. Reverse transcription and quantitative PCR (iScript Reverse Transcription and SsoAdvanced Universal SYBR Green, BioRad) were done using

specific primers (Supplementary Data 5). Fold changes are calculated for each target relative to the WT strain whose RNA was purified in parallel. For promoter activities, promoters were

amplified and cloned in the pTCV-lac vector containing a ß-galactosidase reporter (Supplementary Datas 5 and 6) and introduced in GBS. Reporter activity was quantified in microplate format

by colourimetric assay with ONPG as substrate and permeabilized overnight cultures54. Reaction kinetics at 28 °C were followed by OD at 420 nm every 5 min (Tecan Infinite). Linear slopes

(OD/min) were used to infer enzymatic activities and were normalised for the initial cell density (OD 600 nm) of each replicate. GROWTH CURVES AND ANTIBIOTIC SUSCEPTIBILITIES Growth curves

are done in a volume of 150 µl of THY inoculated with diluted overnight cultures (1/500) in 96-wells microplates and incubated at 37 °C with automatic recording of OD 600 nm every 10 minutes

and 1 min agitation by cycle (TECAN Infinite). Doubling times are determined by fitting non-linear regression with a Malthusian growth model (GraphPad Prism 10) in the exponential phase

(_R_2 > 0.99) for each replicate. Fitness is calculated by dividing the mean doubling time of the WT by the doubling time of the mutant. For antibiotic susceptibilities, concentrated

drugs (10 x) were added to an aliquot of the starting cultures and serial two-fold dilutions were done in the starting culture without drugs before incubation in the microplate reader.

Minimal Inhibitory Concentration (MIC) is done following EUCAST guidelines in Muller-Hinton Fastidious culture media (MH-F, Becton Dickinson) media using custom AST Sensititre 96 wells

plates (ThermoScientific) and 18 h of incubation at 37 °C. SS-HAEMOLYTIC ACTIVITY Columbia agar supplemented with 5% horse blood and Granada medium (BioMerieux) were used to visualise

ß-haemolytic activity and pigmentation, respectively. Serial ten-fold dilutions of cultures were spotted onto media plates and then incubated under anaerobic conditions (AnaeroGen, Oxoid) at

37 °C. To highlight the halo of lysis around colonies, images are converted to greyscale and uniformly processed (Photoshop, Adobe) to adjust contrast and brightness. Haemolytic titres were

determined by a semi-quantitative method54. Serial 2-fold dilution of cultures initially adjusted to 109 CFU/ml in PBS were added (V/V) to 1% defibrinated horse blood (Oxoid) in PBS

supplemented with 0.2% glucose. After 1 hour of incubation at 37 °C, cells were gently pelleted, and haemoglobin in supernatants was quantified by optical absorbance at 420 nm. The

haemolytic activity of each strain was defined as the minimum dilution that lysed at least 50% of red blood cells. Haemolytic titres are the ratio between the haemolytic activity of each

replicate against the haemolytic activity of the WT strain. Haemolytic titres are then normalised against the WT strain (normalised WT titre = 1). RR PHOSPHORYLATION LEVEL Genes encoding RR

were amplified and cloned by Gibson assembly (Supplementary Data 5 and 6) in a custom-made pEX-CterFLAG vector containing a synthetic cassette with a translational initiation site, a

flexible Gly-Ala linker, a 3xFLAG epitope, and a transcriptional terminator. Cassettes with genes of interest cloned in frame with the linker were excised with restriction enzymes and cloned

into the anhydro-tetracycline (aTc) inducible expression vector pTCV_PtetO45. Expression vectors were introduced in the corresponding HK+ mutants by electroporation with kanamycin

selection. Total protein extracts were prepared from 45 ml of cultures in exponential phase in the presence of 100 ng/ml aTc (Sigma) by mechanical lysis of bacterial pellet (Precellys

Evolution) resuspend in cold TBS buffer with EDTA-free protease inhibitors (cOmplete, Roche). Following clearance by centrifugation, 15 µg of proteins were loaded in 12.5% Phos-Tag SDS

polyacrylamide gels (SuperSep Phos-Tag, Wako Pure Chemical Industries Ltd) in loading dye buffer without EDTA and without sample heating to avoid dephosphorylation of the labile aspartate85.

Electrophoresis (2 h, 100 V, 30 mA) in Tris-glycine buffer was performed on an ice bath. Semi-dry transfer on nitrocellulose membrane (15 min, 15 V, Mini-Protean, BioRad) was followed by

blocking (TBS buffer with 0.05% Tween20 and 5% BSA), and hybridisation with rabbit polyclonal anti-FLAG antibodies (1:1500, Millipore F7425) and finally with secondary antibodies coupled to

infra-red dyes (1:15000, Li-Cor 926-32211 IRDye 800CW). After final washing in TBS buffer without Tween20, fluorescent signals were acquired (Odyssey Imager, Li-Cor). The ratio of

phosphorylated and non-phosphorylated proteins was analysed with ImageJ from three independent protein extracts. REPORTING SUMMARY Further information on research design is available in the

Nature Portfolio Reporting Summary linked to this article. DATA AVAILABILITY The RNA-seq data generated in this study have been deposited in the Gene Expression Omnibus database under

accession code GSE261394 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE261394]. Additional RNA-seq and ChIP-seq data already reported are available under accession code GSE261394

[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE158049]. Source data are provided in this paper. REFERENCES * Stock, A. M., Robinson, V. L. & Goudreau, P. N. Two-component signal

transduction. _Annu. Rev. Biochem._ 69, 183–215 (2000). Article  CAS  PubMed  Google Scholar  * Groisman, E. A. Feedback control of two-component regulatory systems. _Annu. Rev. Microbiol._

70, 103–124 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Groisman, E. A., Duprey, A. & Choi, J. How the PhoP/PhoQ system controls virulence and Mg(2+) homeostasis:

Lessons in signal transduction, pathogenesis, physiology, and evolution. _Microbiol. Mol. Biol. Rev._ 85, e0017620 (2021). Article  PubMed  Google Scholar  * Francis, V. I. & Porter, S.

L. Multikinase networks: Two-component signaling networks integrating multiple stimuli. _Annu. Rev. Microbiol._ 73, 199–223 (2019). Article  CAS  PubMed  Google Scholar  * Sastry, A. V. et

al. The _Escherichia coli_ transcriptome mostly consists of independently regulated modules. _Nat. Commun._ 10, 5536 (2019). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Xie,

Y. et al. An atlas of bacterial two-component systems reveals function and plasticity in signal transduction. _Cell Rep._ 41, 111502 (2022). Article  CAS  PubMed  Google Scholar  * Capra,

E. J. & Laub, M. T. Evolution of two-component signal transduction systems. _Annu. Rev. Microbiol._ 66, 325–347 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Salazar,

M. E. & Laub, M. T. Temporal and evolutionary dynamics of two-component signaling pathways. _Curr. Opin. Microbiol._ 24, 7–14 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar

  * Perez, J. C. & Groisman, E. A. Evolution of transcriptional regulatory circuits in bacteria. _Cell_ 138, 233–244 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Rapun-Araiz, B., Haag, A. F., Solano, C. & Lasa, I. The impact of two-component sensorial network in staphylococcal speciation. _Curr. Opin. Microbiol._ 55, 40–47 (2020). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Perez, J. C. & Groisman, E. A. Transcription factor function and promoter architecture govern the evolution of bacterial regulons. _Proc. Natl.

Acad. Sci. USA_ 106, 4319–4324 (2009). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Choi, J. & Groisman, E. A. Horizontally acquired regulatory gene activates ancestral

regulatory system to promote _Salmonella_ virulence. _Nucleic Acids Res._ 48, 10832–10847 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ghose, D. A., Przydzial, K. E.,

Mahoney, E. M., Keating, A. E. & Laub, M. T. Marginal specificity in protein interactions constrains evolution of a paralogous family. _Proc. Natl. Acad. Sci. USA_ 120, e2221163120

(2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Rapun-Araiz, B. et al. Systematic reconstruction of the complete two-component sensorial network in _Staphylococcus aureus_.

_mSystems_ 5, e00511–e00520 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Villanueva, M. et al. Sensory deprivation in _Staphylococcus aureus_. _Nat. Commun._ 9, 523

(2018). Article  ADS  PubMed  PubMed Central  Google Scholar  * Galagan, J. E. et al. The _Mycobacterium tuberculosis_ regulatory network and hypoxia. _Nature_ 499, 178–183 (2013). Article 

ADS  CAS  PubMed  PubMed Central  Google Scholar  * Minch, K. J. et al. The DNA-binding network of _Mycobacterium tuberculosis_. _Nat. Commun._ 6, 5829 (2015). Article  ADS  CAS  PubMed 

Google Scholar  * Trouillon, J. et al. Determination of the two-component systems regulatory network reveals core and accessory regulations across _Pseudomonas aeruginosa_ lineages. _Nucleic

Acids Res._ 49, 11476–11490 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gao, R. & Stock, A. M. Biological insights from structures of two-component proteins. _Annu.

Rev. Microbiol._ 63, 133–154 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Jacob-Dubuisson, F., Mechaly, A., Betton, J. M. & Antoine, R. Structural insights into the

signalling mechanisms of two-component systems. _Nat. Rev. Microbiol._ 16, 585–593 (2018). Article  CAS  PubMed  Google Scholar  * Buschiazzo, A. & Trajtenberg, F. Two-component sensing

and regulation: How do histidine kinases talk with response regulators at the molecular level? _Annu. Rev. Microbiol._ 73, 507–528 (2019). Article  CAS  PubMed  Google Scholar  * Pioszak, A.

A. & Ninfa, A. J. Genetic and biochemical analysis of phosphatase activity of _Escherichia coli_ NRII (NtrB) and its regulation by the PII signal transduction protein. _J. Bacteriol._

185, 1299–1315 (2003). Article  CAS  PubMed  PubMed Central  Google Scholar  * Dutta, R., Yoshida, T. & Inouye, M. The critical role of the conserved Thr247 residue in the functioning of

the osmosensor EnvZ, a histidine Kinase/Phosphatase, in _Escherichia coli_. _J. Biol. Chem._ 275, 38645–38653 (2000). Article  CAS  PubMed  Google Scholar  * Hsing, W., Russo, F. D., Bernd,

K. K. & Silhavy, T. J. Mutations that alter the kinase and phosphatase activities of the two-component sensor EnvZ. _J. Bacteriol._ 180, 4538–4546 (1998). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Kenney, L. J. How important is the phosphatase activity of sensor kinases? _Curr. Opin. Microbiol._ 13, 168–176 (2010). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Laub, M. T. & Goulian, M. Specificity in two-component signal transduction pathways. _Annu. Rev. Genet._ 41, 121–145 (2007). Article  CAS  PubMed  Google Scholar  *

Gao, R. & Stock, A. M. Quantitative kinetic analyses of shutting off a two-component system. _MBio_ 8, e00412–e00417 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Trajtenberg, F. et al. Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action. _Elife_ 5, e21422 (2016). Article  PubMed  PubMed Central 

Google Scholar  * Casino, P., Rubio, V. & Marina, A. Structural insight into partner specificity and phosphoryl transfer in two-component signal transduction. _Cell_ 139, 325–336 (2009).

Article  CAS  PubMed  Google Scholar  * Skerker, J. M. et al. Rewiring the specificity of two-component signal transduction systems. _Cell_ 133, 1043–1054 (2008). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Huynh, T. N. & Stewart, V. Negative control in two-component signal transduction by transmitter phosphatase activity. _Mol. Microbiol._ 82, 275–286

(2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Huynh, T. N., Noriega, C. E. & Stewart, V. Conserved mechanism for sensor phosphatase control of two-component signaling

revealed in the nitrate sensor NarX. _Proc. Natl. Acad. Sci. USA_ 107, 21140–21145 (2010). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Willett, J. W. & Kirby, J. R.

Genetic and biochemical dissection of a HisKA domain identifies residues required exclusively for kinase and phosphatase activities. _PLoS Genet._ 8, e1003084 (2012). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Mikkelsen, H., Hui, K., Barraud, N. & Filloux, A. The pathogenicity island encoded PvrSR/RcsCB regulatory network controls biofilm formation and

dispersal in _Pseudomonas aeruginosa_ PA14. _Mol. Microbiol._ 89, 450–463 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Horstmann, N. et al. Phosphatase activity of the

control of virulence sensor kinase CovS is critical for the pathogenesis of group A _Streptococcus_. _PLoS Pathog._ 14, e1007354 (2018). Article  PubMed  PubMed Central  Google Scholar  *

Wayne, K. J., Li, S., Kazmierczak, K. M., Tsui, H. C. & Winkler, M. E. Involvement of WalK (VicK) phosphatase activity in setting WalR (VicR) response regulator phosphorylation level and

limiting cross-talk in _Streptococcus pneumoniae_ D39 cells. _Mol. Microbiol._ 86, 645–660 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sharkey, L. K. R. et al. The

two-component system WalKR provides an essential link between cell wall homeostasis and DNA replication in _Staphylococcus aureus_. _mBio_ 14, e0226223 (2023). Article  PubMed  Google

Scholar  * Armistead, B., Oler, E., Adams Waldorf, K. & Rajagopal, L. The double life of group B _Streptococcus_: asymptomatic colonizer and potent pathogen. _J. Mol. Biol._ 431,

2914–2931 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Vornhagen, J., Adams Waldorf, K. M. & Rajagopal, L. Perinatal group B streptococcal infections: virulence

factors, immunity, and preventionstrategies. _Trends Microbiol._ 25, 919–931 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Thomas, L. & Cook, L. Two-component signal

transduction systems in the human pathogen _Streptococcus agalactiae_. _Infect. Immun._ 88, e00931–19 (2020). Article  PubMed  PubMed Central  Google Scholar  * Dobihal, G. S., Brunet, Y.

R., Flores-Kim, J. & Rudner, D. Z. Homeostatic control of cell wall hydrolysis by the WalRK two-component signaling pathway in _Bacillus subtilis_. _Elife_ 8, e52088 (2019). Article  CAS

  PubMed  PubMed Central  Google Scholar  * Monk, I. R., Howden, B. P., Seemann, T. & Stinear, T. P. Spontaneous secondary mutations confound analysis of the essential two-component

system WalKR in _Staphylococcus aureus_. _Nat. Commun._ 8, 14403 (2017). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Winkler, M. E. & Hoch, J. A. Essentiality, bypass,

and targeting of the YycFG (VicRK) two-component regulatory system in gram-positive bacteria. _J. Bacteriol._ 190, 2645–2648 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Joubert, L. et al. Visualization of the role of host heme on the virulence of the heme auxotroph _Streptococcus agalactiae_. _Sci. Rep._ 7, 40435 (2017). Article  ADS  CAS  PubMed  PubMed

Central  Google Scholar  * Mazzuoli, M. V. et al. The CovR regulatory network drives the evolution of Group B _Streptococcus_ virulence. _PLoS Genet._ 17, e1009761 (2021). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Whidbey, C. et al. A hemolytic pigment of Group B _Streptococcus_ allows bacterial penetration of human placenta. _J. Exp. Med._ 210, 1265–1281

(2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Armistead, B. et al. Lipid analogs reveal features critical for hemolysis and diminish granadaene mediated Group B

_Streptococcus_ infection. _Nat. Commun._ 11, 1502 (2020). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Armistead, B. et al. Hemolytic membrane vesicles ofgroup B

_Streptococcus_ promote infection. _J. Infect. Dis._ 223, 1488–1496 (2021). Article  CAS  PubMed  Google Scholar  * Cook, L. C., Hu, H., Maienschein-Cline, M. & Federle, M. J. A vaginal

tract signal detected by the GBS SaeRS system elicits transcriptomic changes and enhances murine colonization. _Infect. Immun._ 86, e00762–17 (2018). Article  PubMed  PubMed Central  Google

Scholar  * Buscetta, M. et al. PbsP, a cell wall-anchored protein that binds plasminogen to promote hematogenous dissemination of group B _Streptococcus_. _Mol. Microbiol._ 101, 27–41

(2016). Article  CAS  PubMed  Google Scholar  * Lentini, G. et al. The plasminogen binding protein PbsP is required for brain invasion by hypervirulent CC17 Group B streptococci. _Sci. Rep._

8, 14322 (2018). Article  ADS  PubMed  PubMed Central  Google Scholar  * Thomas, L. S. & Cook, L. C. A novel conserved protein in _Streptococcus agalactiae_, BvaP, is important for

vaginal Colonization and Biofilm Formation. _mSphere_ 7, e0042122 (2022). Article  PubMed  Google Scholar  * Hall, J. W. et al. An intramembrane sensory circuit monitors sortase A-mediated

processing of streptococcal adhesins. _Sci. Signal._ 12, eaas9941 (2019). Article  PubMed  PubMed Central  Google Scholar  * Firon, A. et al. The Abi-domain protein Abx1 interacts with the

CovS histidine kinase to control virulence gene expression in group B _Streptococcus_. _PLoS Pathog._ 9, e1003179 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mascher, T.

Bacterial (intramembrane-sensing) histidine kinases: signal transfer rather than stimulus perception. _Trends Microbiol._ 22, 559–565 (2014). Article  CAS  PubMed  Google Scholar  *

Sommerfield, A. G. & Darwin, A. J. Bacterial carboxyl-terminal processing proteases play critical roles in the cell envelope and beyond. _J. Bacteriol._ 204, e0062821 (2022). Article 

PubMed  Google Scholar  * Khosa, S. et al. Structural basis of lantibiotic recognition by the nisin resistance protein from _Streptococcus agalactiae_. _Sci. Rep._ 6, 18679 (2016). Article 

ADS  CAS  PubMed  PubMed Central  Google Scholar  * Jenul, C. & Horswill, A. R. Regulation of _staphylococcus aureus_ virulence. _Microbiol. Spectr._ 7,

https://doi.org/10.1128/microbiolspec.GPP3-0031-2018 (2019). * Mitrophanov, A. Y. & Groisman, E. A. Signal integration in bacterial two-component regulatory systems. _Genes Dev._ 22,

2601–2611 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Lin, W. J. et al. Threonine phosphorylation prevents promoter DNA binding of the Group B _Streptococcus_ response

regulator CovR. _Mol. Microbiol._ 71, 1477–1495 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gryllos, I. et al. Induction of group A _Streptococcus_ virulence by a human

antimicrobial peptide. _Proc. Natl. Acad. Sci. USA_ 105, 16755–16760 (2008). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Jani, S. et al. Low phosphatase activity of LiaS and

strong LiaR-DNA affinity explain the unusual LiaS to LiaR in vivo stoichiometry. _BMC Microbiol._ 20, 104 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sanson, M. A. et

al. The LiaFSR transcriptome reveals an interconnected regulatory network in group A _streptococcus_. _Infect. Immun._ 89, e0021521 (2021). Article  PubMed  Google Scholar  * Lamarche, M.

G., Wanner, B. L., Crepin, S. & Harel, J. The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis. _FEMS Microbiol. Rev._

32, 461–473 (2008). Article  CAS  PubMed  Google Scholar  * Baek, S. & Lee, E. J. PhoU: a multifaceted regulator in microbial signaling and homeostasis. _Curr. Opin. Microbiol._ 77,

102401 (2024). Article  CAS  PubMed  Google Scholar  * Szurmant, H., Bu, L., Brooks III, C. L. & Hoch, J. A. An essential sensor histidine kinase controlled by transmembrane helix

interactions with its auxiliary proteins. _Proc. Natl. Acad. Sci. USA_ 105, 5891–5896 (2008). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Szurmant, H., Mohan, M. A., Imus,

P. M. & Hoch, J. A. YycH and YycI interact to regulate the essential YycFG two-component system in _Bacillus subtilis_. _J. Bacteriol._ 189, 3280–3289 (2007). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Gao, R. & Stock, A. M. Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation

profiling. _Proc. Natl. Acad. Sci. USA_ 110, 672–677 (2013). Article  ADS  CAS  PubMed  Google Scholar  * Butcher, R. J. & Tabor, J. J. Real-time detection of response regulator

phosphorylation dynamics in live bacteria. _Proc. Natl. Acad. Sci. USA_ 119, e2201204119 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * George, N. L., Schilmiller, A. L.

& Orlando, B. J. Conformational snapshots of the bacitracin sensing and resistance transporter BceAB. _Proc. Natl. Acad. Sci. USA_ 119, e2123268119 (2022). Article  CAS  PubMed  PubMed

Central  Google Scholar  * George, N. L. & Orlando, B. J. Architecture of a complete Bce-type antimicrobial peptide resistance module. _Nat. Commun._ 14, 3896 (2023). Article  ADS  CAS 

PubMed  PubMed Central  Google Scholar  * Parashar, V., Mirouze, N., Dubnau, D. A. & Neiditch, M. B. Structural basis of response regulator dephosphorylation by Rap phosphatases. _PLoS

Biol._ 9, e1000589 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fritz, G. et al. A new way of sensing: Need-based activation of antibiotic resistance by a flux-sensing

mechanism. _mBio_ 6, e00975 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kobras, C. M. et al. BceAB-Type antibiotic resistance transporters appear to act by target

protection of cell wall synthesis. _Antimicrob. Agents Chemother._ 64, e02241–19 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Koh, A., Gibbon, M. J., Van der Kamp, M. W.,

Pudney, C. R. & Gebhard, S. Conformation control of the histidine kinase BceS of _Bacillus subtilis_ by its cognate ABC-transporter facilitates need-based activation of antibiotic

resistance. _Mol. Microbiol._ 115, 157–174 (2021). Article  CAS  PubMed  Google Scholar  * Reiners, J. et al. The N-terminal region of Nisin is important for the BceAB-type ABC transporter

NsrFP from _streptococcus agalactiae_ COH1. _Front. Microbiol._ 8, 1643 (2017). Article  PubMed  PubMed Central  Google Scholar  * Yang, Y. et al. Role of two-component system response

regulator bceR in the antimicrobial resistance, virulence, biofilm formation, and stress response of group B _streptococcus_. _Front. Microbiol._ 10, 10 (2019). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Piepenbreier, H., Diehl, A. & Fritz, G. Minimal exposure of lipid II cycle intermediates triggers cell wall antibiotic resistance. _Nat. Commun._ 10, 2733

(2019). Article  ADS  PubMed  PubMed Central  Google Scholar  * Kingston, A. W., Zhao, H., Cook, G. M. & Helmann, J. D. Accumulation of heptaprenyl diphosphate sensitizes _Bacillus

subtilis_ to bacitracin: implications for the mechanism of resistance mediated by the BceAB transporter. _Mol. Microbiol._ 93, 37–49 (2014). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Da Cunha, V. et al. _Streptococcus agalactiae_ clones infecting humans were selected and fixed through the extensive use of tetracycline. _Nat. Commun._ 5, 4544 (2014). Article 

PubMed  Google Scholar  * Biswas, I., Gruss, A., Ehrlich, S. D. & Maguin, E. High-efficiency gene inactivation and replacement system for gram-positive bacteria. _J. Bacteriol._ 175,

3628–3635 (1993). Article  CAS  PubMed  PubMed Central  Google Scholar  * Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with

DESeq2. _Genome Biol._ 15, 550 (2014). Article  PubMed  PubMed Central  Google Scholar  * Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: A practical and powerful

approach to multiple testing. _J. R. Stat. Soc. Ser. B_ 57, 289–300 (1995). Article  MathSciNet  Google Scholar  * Shannon, P. et al. Cytoscape: a software environment for integrated models

of biomolecular interaction networks. _Genome Res._ 13, 2498–2504 (2003). Article  CAS  PubMed  PubMed Central  Google Scholar  * Barbieri, C. M. & Stock, A. M. Universally applicable

methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag-based reagents. _Anal. Biochem._ 376, 73–82 (2008). Article  CAS  PubMed  PubMed

Central  Google Scholar  Download references ACKNOWLEDGEMENTS This study was supported by Agence Nationale de la Recherche (VirEvol - ANR-22-CE15-0024) to A.F., and the National Laboratory

of Excellence programme - Integrative Biology of Emerging Infectious Diseases (LabEx IBEID, ANR-10-LABX-62-IBEID) to P.T.C. C.C. and M.V.M. are recipients of National PhD grants from Ecole

Doctorale BioSpc (ED562) - Université Paris Cité. AUTHOR INFORMATION Author notes * These authors contributed equally: Cosme Claverie, Francesco Coppolino. AUTHORS AND AFFILIATIONS *

Institut Pasteur, Université Paris Cité, Department of Microbiology, Biology of Gram-Positive Pathogens, Paris, France Cosme Claverie, Francesco Coppolino, Maria-Vittoria Mazzuoli, Odile

Sismeiro, Patrick Trieu-Cuot & Arnaud Firon * University of Messina, Department of Human Pathology, Messina, Italy Francesco Coppolino, Giuseppe Valerio De Gaetano & Concetta

Beninati * Assistance Publique-Hôpitaux de Paris, Hôpital Cochin, Department of Bacteriology, French National Reference Center for Streptococci, Paris, France Cécile Guyonnet & Asmaa

Tazi * Université Paris Cité, Institut Cochin, Institut National de la Santé et de la Recherche Médicale U1016, Centre National de la Recherche Scientifique UMR8104, Team Bacteria and

Perinatality, Paris, France Cécile Guyonnet & Asmaa Tazi * Fédération Hospitalo-Universitaire Fighting Prematurity, Paris, France Cécile Guyonnet & Asmaa Tazi * Institut Pasteur,

Université Paris Cité, Bioinformatics and Biostatistics Hub, Paris, France Elise Jacquemet & Rachel Legendre * Scylla Biotech Srl, Messina, Italy Giuseppe Teti Authors * Cosme Claverie

View author publications You can also search for this author inPubMed Google Scholar * Francesco Coppolino View author publications You can also search for this author inPubMed Google

Scholar * Maria-Vittoria Mazzuoli View author publications You can also search for this author inPubMed Google Scholar * Cécile Guyonnet View author publications You can also search for this

author inPubMed Google Scholar * Elise Jacquemet View author publications You can also search for this author inPubMed Google Scholar * Rachel Legendre View author publications You can also

search for this author inPubMed Google Scholar * Odile Sismeiro View author publications You can also search for this author inPubMed Google Scholar * Giuseppe Valerio De Gaetano View

author publications You can also search for this author inPubMed Google Scholar * Giuseppe Teti View author publications You can also search for this author inPubMed Google Scholar * Patrick

Trieu-Cuot View author publications You can also search for this author inPubMed Google Scholar * Asmaa Tazi View author publications You can also search for this author inPubMed Google

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inPubMed Google Scholar CONTRIBUTIONS C.C., F.C., M.V.M., C.G., O.S., and G.V.D.G. performed experiments and analysed data. E.J. and R.L. analysed RNA-seq data. G.T., P.T.C., A.T., C.B., and

A.F. designed and supervised experiments. A.F. conceived the study and wrote the manuscript with input from all the authors. CORRESPONDING AUTHOR Correspondence to Arnaud Firon. ETHICS

DECLARATIONS COMPETING INTERESTS GT is an employee and CB is the founder and owner of Scylla Biotech Srl. The company did not provide funding and had no role in the design, conduct, or

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_et al._ Constitutive activation of two-component systems reveals regulatory network interactions in _Streptococcus agalactiae_. _Nat Commun_ 15, 9175 (2024).

https://doi.org/10.1038/s41467-024-53439-3 Download citation * Received: 04 April 2024 * Accepted: 07 October 2024 * Published: 24 October 2024 * DOI:

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