Divalent cations play a crucial role in organisms, serving as essential cofactors for numerous enzymes. They are vital for maintaining the integrity of biological membranes and facilitating various physiological functions (Lippa and Goulian, 2012). Mg²⁺ was initially identified as the environmental stimulus factor for the PhoP/PhoQ system, which plays a crucial role in maintaining Mg²⁺ homeostasis (Véscovi et al., 1996). When the cytoplasmic Mg²⁺ concentration falls below a certain threshold (e.g., when Salmonella typhimurium concentration below 0.5 mM Mg²⁺) (Véscovi et al., 1996), bacteria generally reduce the assembly of functional ribosomes and undergo auto-phosphorylation of the periplasmic PhoQ. PhoP is phosphorylated to PhoP-P, and PhoP-P specifically binds to the promoter region of Mg²⁺ transport-related genes (such as mgtA, mgtB, and mgtC), and thus activating gene transcription (Cromie and Groisman, 2010; Yeom et al., 2020; Yeom and Groisman, 2021). In the case of E. coli, when Mg²⁺ levels decrease to levels impairing protein production (below 10 μM Mg²⁺), PhoP-P promotes the expression of the iraP gene. This increases the intracellular content of RpoS, reducing the rate of protein synthesis to maintain essential cellular functions (Yin et al., 2019). Meanwhile, the expression level of the Mg²⁺ transporter protein MgtA is significantly upregulated, facilitating the transport of Mg²⁺ from the periplasm to the cytoplasm, thereby maintaining stable cellular Mg²⁺ concentrations (Park et al., 2018). When PhoQ is activated by cationic antimicrobial peptides or acidic environmental conditions, MgtA remains unaffected (Subramani et al., 2016).

Due to environmental stress, the PhoP/PhoQ cascade activates the transcription of downstream genes, which requires significant ATP consumption. The availability of ATP directly correlates with changes in the abundance of ClpXP (Groisman, 2016). Under normal conditions, upon binding with adaptor proteins, RpoS is transported to ClpXP for degradation, rapidly reducing RpoS levels (Groisman, 2016). The transcription factor RpoS regulates the expression of numerous bacterial genes, with its synthesis and degradation tightly controlled, varying in response to cellular growth stresses (Battesti and Gottesman, 2013; Schellhorn, 2020). In Salmonella enterica serovar Typhimurium (S. Typhimurium) under low Mg²⁺ conditions (≤20 μM Mg²⁺), the stability of the sigma factor RpoS plays a crucial role in the PhoP/PhoQ system cascade (Bougdour et al., 2008). PhoP-P promotes the upregulation of RssB anti-adaptors (IraM/IraP/IraD) expression (Bougdour et al., 2008). Acting as an intermediary in regulating RpoS stability, it interferes with RssB-mediated degradation of RpoS by interacting with RssB. Moreover, the PhoP/PhoQ cascade promotes the regulation of RpoS stability by iraP, and high levels of RpoS mediate transcriptional expression of its dependent genes (such as katE and esrB genes) (Bougdour et al., 2008).

SlyB, located in the outer membrane, is regulated by PhoP-P under decreased Mg²⁺ concentration (such as in Yersinia pestis when Mg²⁺ is below 50 μM) or increased osmotic pressure (such as in E. coli when stimulated with 300 mM NaCl) (Tu et al., 2006; Perez et al., 2009; Yuan et al., 2017). In addition, SlyB plays a negative regulatory role in some bacteria on PhoP/PhoQ (Tu et al., 2006; Perez et al., 2009). For instance, in Salmonella typhimurium, deletion of the slyB gene leads to decreased transcription levels of genes activated by PhoP-P. In contrast, such a negative regulatory mechanism is not observed in E. coli (Lippa et al., 2009). Additionally, SlyB can respond to outer membrane (OM) biogenesis defects by sensing the accumulation of lipopolysaccharide (LPS) and periplasmic unfolded outer membrane proteins (OMPs). The modification of LPS plays a crucial role in the PhoP/PhoQ cascade (Janssens et al., 2024). LPS modifications help bacteria reduce the electrostatic repulsion of phosphorylated residues and releases a certain amount of Mg²⁺ for MgtA-related proteins to transfer Mg²⁺ from the periplasmic space into the cytoplasm (Janssens et al., 2024). Studies on S. Typhimurium demonstrate that under low Mg²⁺ conditions (less than 50 μM Mg²⁺), the mgtA gene is activated in a PhoP-P-dependent manner, independent of other environmental stimuli (Yeom et al., 2020). When PhoQ detects signals like low pH or antimicrobial peptides, the expression level of the Mg²⁺ transporter gene mgtA remains unaffected (Yeom et al., 2020; Groisman et al., 2021). Additionally, Ca²⁺ and Mn²⁺ can serve as ligands for PhoQ with similar mechanisms of action, neutralizing electrostatic repulsion between negatively charged residues at the divalent cation binding sites (Regelmann et al., 2002; Barchiesi et al., 2008). Conversely, as the concentration of divalent cations increases, the expression levels of regulatory proteins produced by the PhoP/PhoQ cascade (such as PgtE, PhoN, MgtA, MgtB, and IraP) gradually decrease (Cho et al., 2006; Bougdour et al., 2008). When the Mg²⁺ concentration exceeds 50 μM, a stable bridge forms between the negatively charged outer and inner membranes, thereby inhibiting the PhoP/PhoQ cascade reaction (Regelmann et al., 2002).

Under conditions of low Mg²⁺ concentration (such as S. Typhimurium in a minimal medium containing 10 μM Mg²⁺), the PhoP/PhoQ system interacts with PmrA/PmrB (Hu et al., 2016). PmrA serves as the sensor responding to external stimulus signals, while PmrB acts as the downstream responder to PmrA (Kato et al., 2003; Paredes et al., 2023). PhoP-P stimulates the transcription of PmrD, which mediates the phosphorylation of another response regulator, PmrA. Sufficient PmrA-P is produced to promote the expression levels of genes such as pmrC, pmrE, pmrHFIJKLM, collectively modifying the outer membrane LPS (Chen and Groisman, 2013; Shprung et al., 2021; Paredes et al., 2023; Janssens et al., 2024). In pmrD deletion strains, it was found that the expression level of PmrA was significantly reduced compared to wild-type strains (Cho et al., 2006). Additionally, under high Fe³⁺ conditions, (such as S. enterica in a minimal medium containing 100 μM Fe³⁺) activate the PmrA/PmrB system (Bolard et al., 2019). PmrD also plays a role in promoting the activation of PmrA (Cho et al., 2006), serving as a crucial bridge between the PhoP/PhoQ and PmrA/PmrB systems, directly influencing the regulatory mechanism and abundance of PmrA (Cho et al., 2006). The above content is briefly described in Figure 2 .

Antimicrobial peptides are widely sourced from diverse origins, including animals, plants, microorganisms, and synthetic production, serving as integral components of the innate immune systems in most multicellular organisms (Kato et al., 2012). AMPs are predominantly concentrated within phagosomes, where they exert antimicrobial effects in macrophages (Li et al., 2022). AMPs are rich in positive charges, enabling them to bind with negatively charged molecules on bacterial surfaces (Yan et al., 2021; Zhu et al., 2022). They swiftly penetrate lipid membranes, forming pores in bacterial cell membranes and disrupting membrane permeability, ultimately causing bacterial cell lysis (Yan et al., 2021; Zhu et al., 2022). For pathogens, resistance to antimicrobial peptides is crucial for exerting their toxicity. It has been established that antimicrobial peptides serve as direct signals for activating the PhoQ histidine kinase (Ramezanifard et al., 2023). Cationic antimicrobial peptides competitively bind to the periplasmic domain of PhoQ with divalent cations, inducing a conformational change in the cytoplasmic dimer (Ramezanifard et al., 2023). This promotes the phosphorylation of PhoP and alters the total charge of the lipid A portion of bacterial lipopolysaccharide (LPS), modifying LPS to increase bacterial resistance (Bader et al., 2005; Yu and Guo, 2011; Ramezanifard et al., 2023). The inner membrane protein Mig-14 in extraintestinal pathogenic E. coli (ExPEC) and S. typhimurium) play a crucial role within macrophages, significantly enhancing bacterial resistance against AMPs (Zhuge et al., 2018; Martynowycz et al., 2019).

In recent years, polymyxins have garnered significant attention from researchers due to the rapid increase in bacterial antibiotic resistance (Brodsky et al., 2005; Wang et al., 2020). Polymyxins are important cyclic peptide antibiotics isolated from Bacillus species (Gahlot et al., 2024). They disrupt membrane integrity and induce bacterial outer membrane damage by interacting with negatively charged surface structures such as LPSs in Gram-negative bacteria and lipoteichoic acids in Gram-positive bacteria, thereby exhibiting bactericidal activity (Storm et al., 1977; Brodsky et al., 2005). The cascade of PhoP/PhoQ system modifying bacterial outer membrane LPS can lead to increased resistance of bacteria to polymyxins (Guo et al., 2022). Meanwhile, PhoP-P regulates the expression of PmrD, which effectively inhibits the dephosphorylation of PmrA-P, thereby mediating the involvement of the PmrA/PmrB system in the modification process of LPS (Brodsky et al., 2005; Zhang et al., 2022). PhoQ promotes the binding of PhoP-P to its downstream pmrHFIJKLM promoter (also known as arnBCADTEF or pbgPE operator) through the recognition of polymyxin (Chandler et al., 2020). Concurrently, the upregulation of PmrD expression indirectly enhances the cascade reaction of the PmrA/PmrB system. PmrA also binds to downstream pmrC, pmrE, and pmrHFIJKLM promoters (Shprung et al., 2012; Poirel et al., 2017; Paredes et al., 2023). The overexpression products of pmrC, pmrE, and pmrHFIJKLM are utilized for the synthesis of 4-amino-4-deoxy-L-arabinose (L-Ara4N) and phosphoethanolamine (PEA) ( Figure 3 ). These two components modify LPS by increasing the negative charge on the outer membrane (Hiroshi, 2003), reducing membrane permeability, thereby limiting the entry of antimicrobial peptides and playing a crucial role in promoting polymyxin resistance (Phan et al., 2017; Liu et al., 2021; Shahzad et al., 2023).

Typically, the regulation of its own and downstream target genes by the PhoP/PhoQ system to resist external stimuli is called positive feedback regulation. Negative feedback regulatory mechanisms collectively contribute in maintaining cellular homeostasis, and adverse feedback effects also play a crucial role in reducing intra-population cellular variability (Lippa et al., 2009). The membrane protein MgrB activation occurs through PhoP phosphorylation (Lippa et al., 2009). Subsequently, it binds to the periplasmic domain of PhoQ in order to attenuate its interaction with other stimulus signals, thereby inhibiting the phosphorylation of PhoP and establishing a negative feedback mechanism (Lippa et al., 2009; Poirel et al., 2017). The combination of positive and negative feedback in the PhoP/PhoQ system enhances bacterial sensitivity to signals and plays a crucial role in maintaining intracellular homeostasis (Lippa et al., 2009). When MgrB undergoes functional changes or is lost, the negative feedback regulation of PhoPQ is disrupted (Zafer et al., 2019; Kong et al., 2021). Although the absence of MgrB indirectly affects the activation of PmrD, MgrB appears to specifically target the PhoQ domain (Zafer et al., 2019). In PhoQ-deficient strains, MgrB does not exert its inhibitory effect and does not influence PmrD-mediated resistance to polymyxin B (Zafer et al., 2019). Recent studies have suggested that the CpxR/CpxA system may indirectly influence the antibiotic sensitivity of the PhoP/PhoQ and PmrA/PmrB systems by regulating the activity levels of MgrB (Wang et al., 2020).

pH regulates crucial biological processes such as genes expression, energy generation, and various enzyme functions. Many bacteria, including E. coli, S. enterica, P. aeruginosa, and Edwardsiella, have evolved distinct acid resistance mechanisms (Du et al., 2021; Mallick and Das, 2023). In addition to combating AMPs pressure within phagosomes, bacteria also face the challenge of phagosomal acidification that needs to be overcome (Di et al., 2017). The regulatory response to acid stress is achieved through the coordinated action of various regulators and regulatory systems (Krin et al., 2010). Two-component systems (TCS), such as PhoP/PhoQ, PmrA/PmrB, EvgS/EvgA, SsrA/SsrB, RstA/RstB, and CpxA/CpxR system consist of multiple components that enable bacteria to sense acidic environments and respond to acid stress (Perez and Groisman, 2007; Lin et al., 2017; Sen et al., 2017; Xu et al., 2020; Li and Yao, 2022; Wan et al., 2024). Deletion of phoPQ in E. coli leads to reduced expression levels of various acid-regulated proteins, highlighting the importance of PhoPQ under mildly acidic conditions. Multiple studies indicate that PhoPQ is an effective bacterial defense mechanism against phagosomal killing. PhoPQ directly regulates the lipid A deacylase PagL and the putative dehydrogenase/reductase (SDR) HlyF (Elhenawy et al., 2016; Martynowycz et al., 2019). The upregulation of their transcription levels mediates LPS modification and reshaping of lipid structures (formation of OMV) (Martynowycz et al., 2019). The periplasmic sensor PhoQ detects acidic stimuli and initiates a positive phosphorylation response, thereby activating the transcription of PhoP and its downstream acid resistance-related genes (Tu et al., 2006). Moreover, under acidic environmental conditions, PhoQ does not affect its response to other environmental stimuli (Prost et al., 2007; Choi and Groisman, 2017; Roggiani et al., 2017). For example, within macrophage phagosomes, the PhoP/PhoQ system in bacteria can simultaneously sense stimuli from cationic AMPs and mildly acidic environmental conditions. This capability reduces the damage caused by these stimuli to the outer membrane and maintains normal physiological functions of the bacteria (Han et al., 2023). PhoQ simultaneously sensing both signals rather than individually responding to one of them results in a significant increase in the abundance of PhoP and its downstream target genes (Prost et al., 2007). Under conditions of high or low concentrations of Mg²⁺, low pH can still be sensed by PhoQ (Prost et al., 2007). Be more specific about the concentration of Mg²⁺ and low pH. Research has reported that in S. enterica, low pH conditions lead to an increase in PhoP-P levels, which indirectly promotes transcription of pmrD (Perez and Groisman, 2007). This mediation enhances LPS modification effectiveness under acidic conditions, thereby strengthening bacterial resistance (Perez and Groisman, 2007). It can be seen that low pH can cooperate with other stimulating conditions to activate PhoQ, but currently, there is limited research on this aspect.

In S. enterica under weakly acidic conditions (pH 4.9), the UgtL protein is essential for activation of the PhoP/PhoQ system (Choi and Groisman, 2017). UgtL interacts with PhoQ, enhancing its autophosphorylation and increasing the intracellular abundance of phosphorylated PhoP (Choi and Groisman, 2017). This leads to the activation of downstream gene transcription by PhoP. However, under other stimulating conditions, the effect of UgtL on PhoQ is not significant (Choi and Groisman, 2017). Recent studies have shown that UgtS, a novel inner membrane protein homologous to UgtL, is upregulated at the transcriptional level by PhoP phosphorylation (Salvail et al., 2022). It acts as an antagonist to UgtL within macrophages of S. Typhimurium (Salvail et al., 2022). Following activation of the SsrB/SsrA system in response to weak acid conditions, further enhancement of ugtL gene expression can increase PhoP phosphorylation (Choi and Groisman, 2020a). Conversely, PhoP phosphorylation can also increase transcription of the ssrB gene (Choi and Groisman, 2020a). The PhoP/PhoQ and SsrB/SsrA systems play crucial regulatory roles in controlling genes within the (S. Typhimurium) pathogenicity island.

Additionally, the TCS EvgS/EvgA system is also a major player in acid resistance, activating the expression of numerous acid-resistant genes (Dan et al., 2021; Zeng et al., 2021). It primarily branches into two pathways: EvgSA-YdeO and EvgSA-SafA (Zeng et al., 2021). YdeO is a critical component of glutamate-dependent acid resistance AR2, whose transcriptional upregulation activates the expression of the gadE gene, mediating the upregulation of AR2 effector genes (gadABC) (Roggiani et al., 2017). The membrane protein SafA acts as a connector between the EvgS/EvgA system and the PhoP/PhoQ system (Schellhorn, 2020). SafA and UgtL are both short membrane proteins (65 and 132 residues, respectively) that interact with PhoQ to facilitate network regulation of PhoP/PhoQ (Choi and Groisman, 2017; Yoshitani et al., 2019). However, they lack sequence similarity between each other and independently exert their functions (Choi and Groisman, 2017; Yoshitani et al., 2019). Through binding with anti-adaptor proteins, RssB reduces its interaction with RpoS, thereby mediating the upregulation of RpoS expression levels and ultimately promoting the transcriptional upregulation of gadE (Xu et al., 2019). As mentioned above, the MgrB protein acts as a feedback inhibitor in the PhoP/PhoQ system. The expression of the mgrB gene may be associated with acid resistance, as its deletion can increase the transcription levels of iraM, thereby promoting the activation of the acid resistance gene gadE (Yuan et al., 2017; Xu et al., 2019). Figure 4 depicts a brief description of the PhoP/PhoQ system responding to acidic pH stimulation.

Bacteria encounter oxidative stress responses induced by reactive oxygen species (ROS) during both natural environments and host infection (Hillion and Antelmann, 2015). Bacteria have evolved complex oxidative stress regulatory networks (Hillion and Antelmann, 2015). Before oxidative repair can occur, there is a need for oxidative redox sensors to transmit oxidative information directly or indirectly between them, thereby further regulating the expression of relevant proteins (Hillion and Antelmann, 2015). DsbA, a member of the Dsb family of oxidoreductases involved in disulfide bond formation, is commonly found in the bacterial inner membrane and periplasmic space (Eckels et al., 2021). Its transcriptional regulation depends on the PhoP/PhoQ system cascade, and it plays a crucial role in disulfide bond synthesis (Cardenal-Muñoz and Ramos-Morales, 2013; Choi and Groisman, 2016; Eckels et al., 2021). DsbA acts as a potent oxidase involved in disulfide bond formation, while DsbB re-oxidizes DsbA to form new disulfide bonds (Kadokura and Beckwith, 2014; Santos-Martin et al., 2021). The DsbA-DsbB pathway functions as a redox cycle, continuously driving proper folding and function of substrate proteins in the bacterial envelope and periplasmic space (Kadokura and Beckwith, 2014; Santos-Martin et al., 2021). Studies have shown that the transcription of dsbA and mgrB is regulated by PhoP-P (Cardenal-Muñoz and Ramos-Morales, 2013). In E. coli, the absence of dsbA directly leads to the activation of the PhoP/PhoQ system (Cardenal-Muñoz and Ramos-Morales, 2013). Meanwhile, MgrB, a membrane protein, has been shown to interact with PhoQ, exerting a dephosphorylating effect on PhoP and thereby inhibiting the activation of the PhoP/PhoQ system (Cardenal-Muñoz and Ramos-Morales, 2013). Furthermore, deletion of mgrB in dsbA-deficient strains has been found to reduce the activation of DsbA by the PhoP/PhoQ system (Cardenal-Muñoz and Ramos-Morales, 2013). However, there is currently limited research on the regulatory role of the PhoP/PhoQ system on the Dsb family of proteins.

Osmotic pressure is also among the environmental factors encountered during microbial growth (Brauer et al., 2023). High osmotic pressure caused by excessive or insufficient extracellular solutes that may have detrimental effects on bacteria (Sun et al., 2021; Brauer et al., 2023). The TCSs are important regulatory mechanisms in prokaryotic microbes for coping with osmotic stress. Components involved in osmotic regulation include OmpR/EnvZ, CpxA/CpxR, and the PhoP/PhoQ system (Yuan et al., 2017). The OmpR/EnvZ system can perceive stimuli of both low and high osmotic pressure across the outer membrane (Yuan et al., 2017). Meanwhile, the CpxAR system, responds to signals of outer membrane stress, whereas the PhoP/PhoQ system is specifically associated with high osmotic pressure (Yuan et al., 2017). During hyperosmotic stress (300 mM NaCl), cells experience water loss, growth stagnation, and an increase in the thickness of the lipid bilayer (Poolman et al., 2002). When PhoQ senses high osmolarity, there is a reorganization of lipid bilayers and transmembrane domain conformations, promoting the accumulation of osmoregulatory proteins through PhoP-P (Yuan et al., 2017). In E. coli, mutants lacking PhoPQ show decreased sensitivity to high osmolarity (Xu et al., 2019). PhoP-P mediates the activation of the iraM gene, whose increased expression prevents the binding of RssB to RpoS, thereby further enhancing PhoP/PhoQ activation (Xu et al., 2019). This process regulates the balance of intracellular osmotic pressure in bacteria (Xu et al., 2019). In E. coli, through individual knockout studies of phoQ, phoP, envZ, and ompR, it was found that the PhoP/PhoQ and OmpR/EnvZ systems independently perceive and respond to osmotic pressure stimuli (Xu et al., 2019).

Exogenous long-chain unsaturated fatty acids (LCUFAs) are transported across the bacterial outer membrane and converted into acyl-CoA derivatives, which serve as substrates for β-oxidation or membrane phospholipid synthesis (Viarengo et al., 2013; Xia et al., 2024). LCUFAs inhibit the activity of the PhoP/PhoQ system by interacting with the PhoQ periplasmic sensor, disrupting its autophosphorylation activity, and subsequently downregulating the expression of PhoP-P and its downstream target genes (Viarengo et al., 2013). However, previous studies have shown that LCUFAs do not compete for binding sites with other stimuli (Viarengo et al., 2013; Carabajal et al., 2020). In S. Typhimurium, the PhoP/PhoQ system is inhibited in response to LCUFAs stimulation. LCUFAs may bind to Ca²⁺, aiding in the distinction between intracellular and extracellular environmental conditions (Viarengo et al., 2013). Furthermore, as signaling molecules, LCUFAs play a regulatory role in coordinating bacterial virulence expression (Xia et al., 2024). For instance, in S. enterica, their presence can interact with the transcription regulators HilC/HilD, leading to the expression of the type III secretion system (Xia et al., 2024).

Lysine acetylation is a typical post-translational modification in bacteria that can regulate various cellular functions (Weinert et al., 2013). Acetylation utilizes acetyl coenzyme A as a cofactor, transferring acetyl groups via acetyltransferases (Weinert et al., 2013). During aerobic microbial growth, acetate is secreted as part of metabolic processes. Acetate can be converted into acetyl coenzyme A, mediating the occurrence of PhoP acetylation During aerobic microbial growth, acetate is secreted as part of metabolic processes. Acetate can be converted into acetyl coenzyme A, mediating the occurrence of PhoP acetylation (Ren et al., 2019). Research indicates that acetylation plays a crucial role in modulating PhoP activity, regulating changes in bacterial virulence (Ren et al., 2016). In S. Typhimurium, PhoP undergoes acetylation at three lysine residues (K201, K88, and K102), which inhibits the binding of PhoP-P to downstream gene promoters (Ren et al., 2016). PhoP K201 undergoes acetylation and deacetylation mediated by Pat and CobB, while PhoP K88 and PhoP K102 are acetylated by non-enzymatic acetyl phosphate (AcP) modification (Ren et al., 2019; Li et al., 2021). Acetylation of PhoP inhibits its phosphorylation (Ren et al., 2019), resulting in a 2- to 5-fold reduction in transcriptional activation of PhoP-regulated genes (Ren et al., 2019).