Post-translational modifications of the nucleoid protein H-NS: sites, mechanisms, and regulatory cues

Abstract

Histone-like nucleoid structuring protein H-NS plays a pivotal role in orchestrating bacterial chromatin and regulating horizontal gene transfer (HGT) elements. In response to environmental signals, H-NS undergoes dynamic post-translational modifications (PTMs) that resemble the epigenetic codes of eukaryotic histones. This review explores how environmental cues regulate PTMs at specific sites within distinct domains of H-NS, thereby modulating its oligomerization and DNA-binding capabilities to reprogram bacterial responses. Notably, HGT elements commonly encode counter-silencing factors, including PTM-modifying enzymes, that counteract H-NS repression. We propose that combinatorial PTM patterns on H-NS form the bacterial histone-like epigenetic code, regulating the expression of HGT elements. Collectively, these interactions establish a sophisticated network of silencing and counter-silencing mechanisms that drive bacterial genome evolution.

This review underscores the key role of H-NS post-translational modifications in selectively silencing horizontal gene transfer elements, such as prophages, T3SS, and T6SS, while also considering the potential of these elements to carry PTM enzymes that regulate H-NS modification levels.

Introduction

Genome compaction and dynamic regulation are crucial challenges for organisms, ranging from unicellular bacteria to multicellular eukaryotes (Hulton et al. 1990, Ishihama and Shimada 2021, Millan-Zambrano et al. 2022). Eukaryotes address these challenges through nucleosome assembly mediated by histones, which utilize post-translational modifications (PTMs) to create “histone codes” that regulate genome-wide activity (Millan-Zambrano et al. 2022, Lopez-Hernandez et al. 2025). Correspondingly, bacterial genomes achieve approximately 1000-fold compaction within the nucleoid without nucleosome-like structures (Ishihama and Shimada 2021). This is accomplished by nucleoid-associated proteins (NAPs), such as H-NS and HU, which collaborate with sigma factors and transcriptional regulators to shape genome architecture and regulatory networks (Dorman 2004, 2007, Gavrilov et al. 2025). A key feature of bacterial genomes is the integration of horizontal gene transfer (HGT) elements, such as plasmids, phages, and pathogenicity islands, which are generally silenced by H-NS proteins through recognition of AT-rich DNA regions (Dame et al. 2001, Rimsky et al. 2001, Wang et al. 2010, Wang et al. 2011, Lamberte et al. 2017, Arnold et al. 2022). HGT element silencing is essential because its untimely expression can disrupt cellular processes, trigger immune responses, or impose metabolic burdens (Zhao et al. 2011, Chen et al. 2017, Xu et al. 2019, Li et al. 2021). Moreover, AT-rich sequences of HGT elements with nonspecific RNA polymerase binding ability may cause transcriptional noise that harms bacterial fitness (Lamberte et al. 2017, Forrest et al. 2022). Thus, H-NS has evolved into a dedicated silencer for AT-rich regions, optimizing energy allocation for growth and stress adaptation (Lucchini et al. 2006, Navarre et al. 2006, Navarre et al. 2007).

The genes encoding H-NS family proteins are widespread across the chromosomes and plasmids of various bacteria (Varshavsky et al. 1977, Hulton et al. 1990, Fitzgerald et al. 2020, Wang et al. 2025). These proteins include homologs such as those of Escherichia coli H-NS (Dorman 2004), Mycobacterium tuberculosis Lsr2 (Alqaseer et al. 2019, Ling et al. 2024), Pseudomonas aeruginosa MvaT/U (Castang et al. 2008, Lippa et al. 2021), and Bacillus subtilis Rok (Duan et al. 2018, Erkelens et al. 2024). Genomically, H-NS is abundant (~20 000 copies/cell) and preferentially binds AT-rich regions ​​through DNA bending, stiffening, bridging, and supercoiling mechanisms to stabilize chromosome organization (Dame et al. 2001, Rimsky et al. 2001, Dorman 2007, Wang et al. 2011, Dame et al. 2020). Transcriptionally, H-NS silences horizontally acquired genes (Navarre et al. 2006, Ma et al. 2022). Mass spectrometry analyses have identified multiple PTMs on H-NS (Dilweg and Dame 2018), including acetylation (Liu et al. 2024, Liu et al. 2024), phosphorylation (Alqaseer et al. 2019, Hu et al. 2019, Liu et al. 2021, Lukose et al. 2024, Guo et al. 2024b, Wang et al. 2025), 2-hydroxyisobutyrylation (Dong et al. 2022a), and lactylation (Dong et al. 2022b) (Table 1). These modifications dynamically regulate bacterial virulence, biofilm formation, thermotolerance, and acid resistance. However, the spatiotemporal dynamics, enzymatic regulation, and functional consequences of H-NS PTMs remain poorly understood. This review examines the structural features of H-NS family proteins and discusses how environmental signals alter PTM-specific modifications across each domain of H-NS. Furthermore, we explore the manner in which HGT element-encoding factors subvert H-NS repression, especially the PTM mechanisms involved. Collectively, we propose that PTMs act as counter-silencing mechanisms, temporarily overriding H-NS-mediated repression during environmental adaptation.

Table 1.

H-NS functional PTM sites.

Domain	Site	Modification	Species	Environmental cue	Function	Reference
N-terminal	T13	Phosphorylation	S. Typhimurium	-	Reduce protein dimerization, activate virulence	(Hu et al. 2019)
	K19	Acetylation	S. oneidensis	Nitrogen	Disrupt the biofilm formation	(Liu et al. 2024)
	S42	Phosphorylation	S. oneidensis	Temperature	Affect the H-NS-DNA oligomerization, Inhibit the excision of CP4So prophage	(Liu et al. 2021)
	Y61	Phosphorylation	E. coli	-	Disrupt oligomerization but retain dimerization, reduce condensates	(Lukose et al. 2024, Lukose et al. 2025)
	S78	Phosphorylation	S. Typhimurium	-	Reduce protein dimerization, activate virulence	(Wang et al. 2025)
Linker	T65	Phosphorylation	P. aeruginosa	Biofilm	Control of Pf phage lysogeny	(Guo et al. 2024b)
	S67	Phosphorylation	P. aeruginosa	Biofilm	Control of Pf phage lysogeny	(Guo et al. 2024b)
C-terminal	K120	Acetylation	E. piscicida	Amino acid	Reduce DNA binding ability, prone to be displaced by counter-silence proteins	(Liu et al. 2024)
	K121	2-hydroxyisobutyrylation	E. coli	Acid stress	Reduce DNA binding ability, express acid resistance genes	(Dong et al. 2022a)

The structure of H-NS

At the primary structural level, H-NS amino acid sequences are highly conserved within Enterobacteriaceae species (e.g. E. coli, Salmonella Typhimurium, Edwardsiella piscicida), with differences observed in the Shewanellaceae strain Shewanella oneidensis compared with Enterobacteriaceae (Fig. 1a). Compared with those in Enterobacteriaceae H-NS, functional homologs in other bacteria, such as P. aeruginosa MvaT/U, exhibit significant sequence differences (Fig. 1a). Despite these variations, all H-NS homologs share two key functional domains: the N-terminal oligomerization domain and the C-terminal DNA-binding domain (Fig. 1b, c). A flexible linker region connects these two domains, enabling conformational flexibility. Surface charge analysis shows that the oligomerization domain is predominantly negatively charged, while the flexible linker and adjacent DNA-binding domain are mainly positively charged (Fig. 1d, e). The distinct distribution of surface charge across the different domains of H-NS may underlie their functional differences.

Figure 1.

Structural features of H-NS. (a) Sequence alignment of H-NS family proteins. Representative H-NS homologs from E. coli (MG1655), S. Typhimurium (SL1344), E. piscicida (EIB202), S. oneidensis (MR1), and MvaT/U from P. aeruginosa (MPAO1) are shown, highlighting conserved domains and residues. The “QGR” and “R-GN” motifs are marked with red boxes. (b) Predicted structure of H-NS (E. coli) generated by AlphaFold3. (c) Predicted structure of MvaU (P. aeruginosa) generated by AlphaFold3. (d) Electrostatic surface potential of H-NS (E. coli). (e) Electrostatic surface potential of MvaU (P. aeruginosa). (f) H-NS mediates multiple DNA-binding modes through its distinct regions. The H-NS monomer, displayed from top to bottom in sequence. Long rounded rectangle: Dimerization site. Circle: Oligomerization site. Curved line: Linker. Short rounded rectangle: DNA binding domain.

The N-terminal domain facilitates protein‒protein interactions, allowing H-NS to form dimers or higher-order oligomers. In E. coli H-NS, the N-terminus comprises α1 (residues 3–9), α2 (residues 11–18), and a long α3 (residues 23–67) (Arold et al. 2010). α1 and α2, along with the head of α3, form dimerization site, while α4 (residues 72–83) and the tail of α3 contribute to oligomerization site (Yamanaka et al. 2018). Consequently, dimerization occurs through “head-to-head” interactions and oligomerization through “tail-to-tail” interactions between these respective interfaces (Grainger 2016). These interactions provide the basis for the multiple modes of H-NS-DNA binding (Fig. 1f). While environmental factors influencing the alteration of oligomerization states have long been documented (Stella et al. 2006, van der Valk et al. 2017), the underlying genetic mechanisms have only recently been identified (Liu et al. 2024, Lukose et al. 2024, Guo et al. 2024b, Lukose et al. 2025).

The flexible linker plays a crucial role in coordinating oligomerization and DNA binding (Ding et al. 2015, Gao et al. 2017). The amino acid sequence in this region is highly variable, but the positively charged residues are relatively conserved (Fig. 1c). In E. coli and S. Typhimurium, the sequence is “KAKR”; in E. piscicida, it is “KTKR”; and in S. oneidensis, it is “SKKR.” The MvaU and MvaT proteins exhibit “RRAR” and “KRAR,” respectively. These positively charged amino acids play crucial roles in the silencing of gene expression by H-NS family proteins (Gao et al. 2017, Riccardi et al. 2019).

The C-terminal domain preferentially binds to AT-rich DNA sequences, which are typically found in horizontally acquired genes, such as pathogenicity islands or phage DNA (Gordon et al. 2011, Liu et al. 2024). The C-terminal domain of H-NS and MvaU begins with two antiparallel β-strands, followed by two α-helices (Gordon et al. 2011, Ding et al. 2015). Both H-NS and MvaU utilize a small loop structure following β2 to insert into the minor groove of DNA (Ding et al. 2015, Riccardi et al. 2019). In addition to the core loop, the arginine and lysine residues in the linker and DNA-binding domain are crucial for the initial recognition and stable binding of H-NS to DNA (Ding et al. 2015, Riccardi et al. 2019).

PTMs of the H-NS N-terminal oligomerization domain

The function of PTMs at the N-terminus of H-NS is illustrated by the phosphorylation of H-NS in S. oneidensis (Fig. 2) (Zeng et al. 2016, Liu et al. 2021). Shewanella oneidensis MR-1, which is isolated from Lake Oneida sediments, experiences seasonal temperature fluctuations ranging from freezing to 25°C (Myers and Nealson 1988, Zeng et al. 2016). In warm environments, H-NS suppresses CP4So excision by inhibiting the excisionase gene alpA (Zeng et al. 2016). Specifically, H-NS phosphorylation by the CP4So-encoded PknB-family serine/threonine kinase So_1461 enhances H-NS binding to the alpA promoter, thereby blocking CP4So excision. Upon cold exposure, H-NS phosphorylation (especially at S42) decreases, lowering the affinity for the alpA promoter and increasing CP4So excision. The excision leads to the deletion of the U349 nucleotide in SsrA tmRNA and then disrupts the G·U wobble base pair. The impaired SsrA tmRNA promotes the formation of attached biofilms, protecting bacteria from tolerating cold conditions. Concurrently, reduced phosphorylation of H-NS increases the expression of the cold shock family protein (CSP) SO_1648 and a stress-related chemosensory system (SO_2119-SO_2126), further supporting bacterial adaptation to cold environments (Liu et al. 2021). This dynamic regulation of H-NS phosphorylation highlights how PTMs enable rapid environmental adaptation by modulating gene expression and physiological responses.

Figure 2.

Regulation of HGT-mediated adaptation by H-NS PTMs. (Left) Cold-induced H-NS dephosphorylation in S. oneidensis derepresses CP4So excision, modifying tmRNA to drive biofilm formation. (Middle) Biofilm activation in P. aeruginosa deploys prophage-encoded PfpC to inactivate MvaU, enhancing persistence. (Right) Host entry in E. piscicida promotes H-NS acetylation, derepressing T6SS effectors for immune evasion.

Specifically, S42 is located at the end of the α3 helix within dimerization site (Fig. 1a). Sedimentation velocity experiments via analytical ultracentrifugation revealed that H-NS^S42A-DNA formed an oversized complex with the sedimentation coefficient of 15 S (Liu et al. 2021). Moreover, electrophoretic mobility shift assay (EMSA) analysis revealed that the H-NS^S42A-DNA complex was trapped in the gel wells, and H-NS purified at a cold temperature also exhibited similar DNA binding patterns. These results suggest that S42 phosphorylation may influence H-NS oligomerization, potentially altering its head-to-head and tail-to-tail interaction patterns.

Other modifications at the N-terminus of H-NS include phosphorylation of the T13 residue at dimerization site in S. Typhimurium H-NS, which weakens dimerization, alleviating H-NS repression of the PhoP/PhoQ two-component system and activating downstream virulence factors (Hu et al. 2019). Additionally, phosphorylation of S78 at oligomerization site reduces H-NS dimerization and DNA binding, activating the type III secretion system (T3SS) and enabling S. Typhimurium to survive within host cells (Wang et al. 2025). In nitrogen-rich environments, S. oneidensis acetylates N-terminal oligomerization sites K19, K32, and K35, enhancing H-NS repression of the glnB promoter to limit unnecessary nitrogen transport (Liu et al. 2024). However, EMSA experiments simulating acetylation at these sites revealed no changes in H-NS-DNA affinity, suggesting that N-terminal acetylation modulates gene silencing through mechanisms independent of direct DNA binding.

PTM of key residues in the oligomerization site can also lead to a functional change of H-NS. The Y61E phosphomimetic mutation, which mimics phosphorylation at a site identified by mass spectrometry in E. coli H-NS (Dilweg and Dame 2018), abolishes higher-order oligomerization and weakens DNA binding, underscoring that robust DNA binding requires oligomerization beyond dimerization (Lukose et al. 2024, 2025). Structurally, while Y61E mutant retains dimerization capacity, it likely disrupts higher-order assembly by interfering with a critical salt bridge between K57 (in one chain) and D68 (in an adjacent chain) (Lukose et al. 2024). Moreover, a H-NS K57N mutation that eliminates the positive charge of K57 actives prophage Rac excision in E. coli, which is silenced by H-NS (Hong et al. 2010). Interestingly, K57 has been found to undergo acetylation, which can eliminate its positive charge (Dilweg and Dame 2018, Liu et al. 2024). Altogether, these studies indicate that acetylation of K57 may functionally mimic Y61 phosphorylation by disrupting the formation of a salt bridge, although its direct impact on oligomerization requires further investigation.

In summary, N-terminal PTMs primarily impact H-NS dimerization, oligomerization, and indirect DNA interactions. While these modifications may not directly affect DNA-binding affinity, they do influence phenotypic outcomes. For example, recent studies have shown that disrupting the N-terminus of H-NS does not affect its ability to bind DNA but impacts transposon insertion into the genome (Cooper et al. 2024). In addition to H-NS dimerization and DNA interactions, future research should investigate how N-terminal PTMs influence interactions between H-NS and its paralogs, such as StpA and Hha (Tendeng and Bertin 2003, Huttener et al. 2015).

PTMs of the H-NS middle flexible linker region

The role of PTMs at the flexible linker region is exemplified by the phosphorylation of MvaU in P. aeruginosa during biofilm formation (Fig. 2) (Li et al. 2019, Guo et al. 2024b). The opportunistic pathogen P. aeruginosa relies on biofilm formation and inovirus Pf4/6 activation for virulence (Guo et al. 2024b). Notably, Pf4/6 is silenced by the H-NS family proteins MvaT/U (Castang and Dove 2012, Li et al. 2019). MvaT/U are particularly interesting because they both recognize and silence AT-rich regions of the genome, suggesting that P. aeruginosa has acquired redundant H-NS family proteins (Winardhi et al. 2012, Winardhi et al. 2014). Previous studies focused mainly on MvaT, as mvaT deletion causes more severe phenotypic defects than does mvaU deletion (Castang et al. 2008). In the planktonic state, the kinases PfkA/PfkB in the Pf6-encoded toxin-antitoxin system (kinase-kinase-phosphatase; KKP) phosphorylate MvaU at S26, T50, T65, S67, and T91, not MvaT (Guo et al. 2024b). Upon transitioning to the biofilm state, dephosphorylation of MvaU S67 by the phosphatase PfpC in KKP reduces Pf4/6 inhibition, enabling superinfective phage release (Guo et al. 2024a, 2024b).

Mutations in the flexible linker region abolish H-NS-mediated gene silencing by impairing stiffened DNA filament formation, although genome structural regulation remains intact (Winardhi et al. 2012, Winardhi et al. 2014, Gao et al. 2017). Phosphorylation of S67 introduces the negative charge, replacing the hydrogen atom with a phosphate group. This not only alters the charge of serine but also may affect the overall charge distribution in the linker region, hindering DNA filament formation and Pf4/6 suppression.

The linker region of MvaT/U is particularly intriguing. Unlike typical H-NS family proteins, which rely on a “QGR” or “RGR” loop in the DNA-binding domain to insert into the minor groove (Gordon et al. 2011). MvaT/U lacks such motifs and only contains the “GN” sequence, which is insufficient for efficient DNA minor groove interactions (Ding et al. 2015). The positively charged motif “RRGR” (residues 72–75) in the linker region, with the last arginine (R75) and the “GN” sequence in the DNA-binding domain, is jointly inserted into the minor groove of DNA. Phosphorylation of S67 may interfere with the insertion of the nearby “RRGR” and “GN” into the minor groove of AT-rich DNA. This work also explains why P. aeruginosa has two similar H-NS family proteins. MvaT is expressed at relatively high levels and has dominant regulatory functions, while the KKP system solely modifies and alters the function of MvaU. MvaT likely serves as a housekeeping silencer, while KKP-modified MvaU fine-tunes specific pathways. Future studies should explore how the KKP system distinguishes structurally resembled MvaT/U.

PTMs of the H-NS C-terminal DNA-binding domain

The role of PTMs at the C-terminal DNA-binding domain is highlighted by H-NS acetylation in E. piscicida (Fig. 2) (Ma et al. 2022, Liu et al. 2024). Edwardsiella piscicida, a gram-negative pathogenic bacterium, infects aquatic animals primarily via the type III and type VI secretion systems (T3/T6SS) (Leung et al. 2022, Zhou et al. 2024). In nutrient-depleted environments, bacteria tend to shut down the energy-consuming T3/T6SS to survive (Liu et al. 2024). Upon entry into the host with sufficient amino acids, acetylation of H-NS lysine residues (K96, K107, K120, and K128) within the C-terminal DNA-binding domain releases T3/T6SS repression, allowing bacteria to disrupt the host's immune and metabolic pathways. Point mutations that mimic constitutive acetylation prevent H-NS from suppressing the T3/T6SS. Studies focusing on K120 have shown that acetylation weakens H-NS binding to T3/T6SS promoters, whereas non-acetylated modifications enhance this interaction (Liu et al. 2024).

Most transcriptional regulators and σ-factors typically promote transcription by binding to AT-rich DNA, while H-NS silences it. H-NS preferentially binds AT-rich DNA and excludes other DNA-binding proteins (Dame et al. 2001, Ishihama and Shimada 2021, Liu et al. 2024). In vitro assays revealed that K120 acetylation reduces H-NS-DNA binding and increases its susceptibility to be displaced by the counter-silencing protein EnrR at AT-rich promoters. In vivo ChIP-seq analysis revealed that acetylated H-NS still bound to the AT-rich DNA regions, but with reduced enrichment, indicating incomplete DNA occupancy. Thus, acetylation diminishes both H-NS binding and DNA occupancy, preventing the formation of filamentous H-NS-DNA complexes. This facilitates the displacement of H-NS by EnrR, and transcriptional regulators, driving T3/T6SS activation. Similar to lysine acetylation at the K120 site in E. piscicida, in E. coli, the K121 site of H-NS undergoes 2-hydroxyisobutyrylation by the acetyltransferase TmcA (Dong et al. 2022a). In acidic environments, this modification reduces the DNA-binding ability of H-NS, increasing the expression of stress response genes and promoting E. coli survival.

H-NS is a lysine-rich protein. In E. coli, H-NS contains 11 lysines within its 137 amino acid sequence, with 6 of which are located in the DNA-binding domain (Dilweg and Dame 2018). Lysine residues can undergo various modifications, including methylation, lactylation, acetylation, succinylation, and 2-hydroxyisobutyrylation, altering the charge, hydrophilicity, and spatial structure of lysines (Ren et al. 2017, Christensen et al. 2019, Dong et al. 2022a, 2022b, Li et al. 2025). Lysines in the DNA-binding domain are critical for H-NS recognition of AT-rich DNA (Ding et al. 2015, Riccardi et al. 2019). H-NS binding to DNA is not highly sequence-specific but primarily targets the minor groove of AT-rich DNA, where adenine (A) accumulation narrows the groove and increases its negative charge (Privalov et al. 2007, Haran and Mohanty 2009, Rohs et al. 2009). Consequently, H-NS family proteins bind DNA using positively charged arginine motifs that embed into the negatively charged minor groove, and adjacent lysine residues mediate initial interactions and stabilize this binding (Ding et al. 2015, Riccardi et al. 2019). Structural simulations suggest that four lysines in the H-NS DNA-binding domain interact with DNA via salt bridge interactions with phosphate groups (Riccardi et al. 2019). In the NMR structure of the MvaT-DNA complex, the “R-GN” motif interacts with the DNA minor groove. Additionally, six lysine residues (Lys81, Lys83, Lys97, Lys102, Lys105, and Lys108) in the DNA-binding domain form hydrophobic or electrostatic contacts with the DNA sugar‒phosphate backbone (Ding et al. 2015). This extensive network of lysine residues significantly increases the DNA contact surface area, serving as a distinguishing feature of the H-NS DNA-binding domain.

The core “QGR” motif lacks reported PTMs, but adjacent lysines undergo diverse modifications that modulate H-NS-DNA affinity and function (Dilweg and Dame 2018, Dong et al. 2022a, Liu et al. 2024). This regulatory mechanism aligns with the essential role of H-NS in bacterial physiology, as deletion of H-NS is lethal in many bacterial species (Navarre et al. 2006, Castang and Dove 2012). Modifications targeting critical residues, such as the arginine in the “QGR” motif, may compromise bacterial fitness. In contrast, fine-tuning PTMs at peripheral lysines within the H-NS DNA-binding domain enables bacteria to adjust specific functions, reflecting an evolutionarily optimized strategy for environmental adaptation.

HGT elements encode factors to subvert H-NS-mediated silencing

HGT is a major driver of bacterial evolution and diversity (Will et al. 2015, Singh et al. 2016, Arnold et al. 2022). While H-NS silences HGT-acquired genes, HGT elements frequently encode factors that counteract H-NS-mediated repression (Pfeifer et al. 2019), including PTM enzymes (Fig. 3). This mirrors the evolutionary arms race between phages and bacteria, where bacteria deploy antiphage defenses and phages evolve counter-defenses to bypass these barriers (Murtazalieva et al. 2024).

Figure 3.

HGT elements encode factors to subvert H-NS-mediated silencing. Phages, genomic islands, and plasmids, as HGT elements, face the H-NS-mediated silencing mechanism. Therefore, HGT elements frequently encode factors against H-NS-mediated silencing.

HGT element-encoded factors could modulate the PTM state of H-NS. In S. oneidensis, phosphorylation of the N-terminus of H-NS inhibits excision of the prophage CP4So. CP4So carries a serine/threonine protein kinase domain in SO_1641, which enhances H-NS phosphorylation (Liu et al. 2021). Similarly, in P. aeruginosa, Pf6 encodes a kinase-kinase-phosphatase system that modulates MvaU phosphorylation to regulate Pf4/6 lysogeny (Guo et al. 2024b). As the acetylation level of H-NS increases in E. piscicida, the EnrR protein encoded by EPI-1 (Edwardsiella pathogenicity island 1) displaces H-NS from the T3/T6SS clusters to enable virulence activation (Ma et al. 2022, Liu et al. 2024). However, the mechanisms underlying the occurrence and removal of H-NS acetylation remain to be further explored.

In addition to the PTM mechanism, phages such as P. aeruginosa LUZ24 encode the positively charged protein gp4, which binds to MvaT dimerization site to block DNA bridging (Wagemans et al. 2015, Bdira et al. 2021). Phage T7 produces gp5.5, which interacts with both tRNA and H-NS oligomerization site, derepressing phage T7 promoters to increase replication (Liu and Richardson 1993, Ali et al. 2011, Zhu et al. 2012). The early expression protein MotB of phage T4 disrupts H-NS silencing by wrapping around both phage and bacterial genomes, creating space for phage gene replication and assembly (Patterson-West et al. 2021, Son et al. 2021). Additionally, the DNA-mimic protein Arn competitively inhibits H-NS-DNA interactions (Ho et al. 2014). Other phages, such as T7, carry the DNA-mimic protein Ocr, which likely interacts with bacterial DNA recognition systems, such as H-NS-mediated silencing and restriction-modification systems (Melkina et al. 2016, Isaev et al. 2020).

Pathogenicity islands and plasmids also harbor counter-silencers. The S. Typhimurium SPI-2 virulence island encodes the SsrA/SsrB two-component system, which displaces H-NS from T3SS promoters upon activation in host vacuoles (Walthers et al. 2011, Choi and Groisman 2020). The large virulence plasmid pINV of S. flexneri carries VirB, which modulates DNA supercoiling and relieves H-NS-mediated pINV silencing after binding to CTP (Antar and Gruber 2023, Picker et al. 2023, Jakob et al. 2024). The virulence island LEE in E. coli contains Ler, which binds the DNA minor groove occupied by H-NS using the “VGR” motif, alleviating H-NS-mediated silencing of LEE (Cordeiro et al. 2011, Garcia et al. 2012, Bhat et al. 2014, Leh et al. 2017). Additionally, the E. coli EPEC AsnW genomic island produces H-NST, a truncated H-NS variant lacking the DNA-binding domain, disrupting H-NS oligomerization and alleviating silencing (Williamson and Free 2005, Levine et al. 2014). Collectively, these examples illustrate that HGT elements have evolved sophisticated mechanisms to subvert H-NS-mediated repression, ensuring their functional integration into the host physiological network.

PTMs as a counter-silencing mechanism

Environmental cues, such as temperature, metal ions, and pH, can alter the H-NS conformation to achieve counter-silencing effects, and underlying mechanisms have been preliminarily established (van der Valk et al. 2017, Will et al. 2018, Qin et al. 2020, Rashid et al. 2023, Lukose et al. 2024). Among these, the most extensively studied counter-silencing mechanisms involve proteins that displace H-NS from DNA, including global regulators PhoP, LuxR, and EnrR (Will et al. 2014, Chaparian et al. 2020). In addition to counter-silencing proteins, recent discoveries have shown that transcripts from nearby genes can dynamically alter chromosome structure to interfere with H-NS silencing (Figueroa-Bossi et al. 2022, 2024). Furthermore, bacterial metabolites such as c-di-GMP and methylerythritol cyclodiphosphate (MEcPP) also interact with H-NS, disrupting its silencing effect (Guo et al. 2024, Ling et al. 2024). In addition, the most direct mechanism involves Lon protease-mediated degradation of H-NS (Choi and Groisman 2020, Choi et al. 2022, Groisman and Choi 2023).

However, changes in H-NS itself are often overlooked. Here, we propose that PTMs serve as a counter-silencing mechanism (Fig. 4). If H-NS is compared to the “regulatory hand” of bacteria, its amino acid side chains are the “fingers” dynamically adjusted by PTMs. As a dual-function protein, H-NS maintains genome architecture and transcriptional fidelity. In response to specific environments, H-NS must preserve most functions while adjusting others. Environmental fluctuations in metabolites such as ATP, NAD⁺, and acetyl-CoA activate PTM enzymes that modify H-NS (Wang et al. 2010, Weinert et al. 2013). Site-specific PTMs modulate H-NS interactions with DNA, proteins, and metabolites, fine-tuning HGT gene silencing for rapid adaptation.

Figure 4.

PTMs counteract H-NS-mediated xenogeneic silencing. Foreign genetic elements (e.g. prophages and genomic islands) acquired via HGT integrate into bacterial genomes. The nucleoid-associated protein H-NS silences these elements by binding AT-rich DNA. PTMs of H-NS domains disrupt its silencing activity, enabling context-dependent expression of foreign genes.

PTMs serve as a counter-silencing mechanism, often acting in concert with other counter-silencing systems. A key example is that acetylation at the K120 site facilitates H-NS expulsion from AT-rich promoter regions through counter-silencing protein EnrR (Ma et al. 2022, Liu et al. 2024). Moreover, the relationship between H-NS degradation and PTMs is also noteworthy. Initially, we hypothesized that acetylation at K120 would lead to H-NS expulsion by proteins such as EnrR, followed by Lon protease-mediated degradation (Choi et al. 2022, Ma et al. 2022, Liu et al. 2024). However, we found that H-NS protein levels remained stable. This seemingly paradoxical result arises from acetylation at K120, which relieves the repression of its own promoter, leading to increased H-NS protein expression. Acetylated H-NS continues to bind DNA, albeit with reduced affinity. Lon selectively degrades unbound H-NS, meaning it cannot degrade acetylated H-NS. Thus, when considering the PTM functions of H-NS, the interplay network among H-NS self-regulation, counter-silencing proteins, metabolites, and enzymatic degradation should be considered in synergy.

Conclusion

Bacteria dynamically fine-tune H-NS activity through modifying its distinct functional domains, including the dimerization/oligomerization interface, the flexible linker, and the DNA-binding domain. This sophisticated regulatory mechanism allows for adaptive control of critical cellular processes such as biofilm formation, stress response, and intricate phage–host interactions. Notably, these reversible PTMs, such as acetylation and phosphorylation, redefine H-NS not merely as a silencer but as a bidirectional molecular switch. It can balance the essential tasks of xenogeneic silencing and targeted counter-silencing, thereby facilitating the integration of foreign genetic material and fine-tuning global transcription in response to environmental cues. The discovery of this complex PTM landscape on H-NS suggests possible evolutionary convergence between prokaryotes and eukaryotes in the realm of epigenetic regulation, where histone-like proteins in bacteria are modulated by mechanisms analogous to those governing chromatin in higher organisms. However, our current understanding of how specific modifications alter the biophysical properties of H-NS, particularly its capacity for higher-order structure formation and its subsequent impact on DNA binding and condensation, remains incomplete. Elucidating the precise molecular mechanisms will require further multidisciplinary research, as illustrated by recent work that used Micro-C to reveal H-NS-mediated chromosomal hairpin formation (Gavrilov et al. 2025).

Future research on bacterial H-NS PTMs should address population heterogeneity, as cells within a population exhibit different PTM patterns. For example, temperature shifts from warm to cold environments lead to reduced H-NS phosphorylation in S. oneidensis, which is correlated with increased CP4So excision frequency (Liu et al. 2021). Even a 0.1% excision rate in a subpopulation is enough to promote biofilm formation and cold adaptation (Zeng et al. 2016, Liu et al. 2021). Correspondingly, only a subset of the ~20 000 H-NS copies per cell undergo site-specific PTMs. In P. aeruginosa, phosphorylation of a small fraction of MvaU at T65 (20.73% of total MvaU) and S67 (0.47% of total MvaU) is sufficient to trigger the activation of Pf4/6 (Guo et al. 2024b). Future research should focus on the spatiotemporal dynamics and enzymatic regulation of H-NS PTMs. By integrating high-resolution structural studies with multi-omics approaches, we can gain a more comprehensive understanding of the PTM landscape under different environmental conditions. These advances will not only elucidate the epigenetic principles of bacteria but also inform the development of antimicrobial strategies targeting H-NS.

Author contributions

Xiaoxue Wang (Conceptualization, Project administration, Funding acquisition, Writing – review & editing), and Yabo Liu (Project administration, Funding acquisition, Writing – original draft)

Conflict of interest

The authors declare no conflict of interest.

Funding

This work was supported by the National Natural Science Foundation of China (NSFC) Program [42188102, 32400067], the Science & Technology Fundamental Resources Investigation Program [2022FY100600], the special fund of South China Sea Institute of Oceanology, Chinese Academy of Sciences [SCSIO2023QY03], the Ocean Negative Carbon Emissions (ONCE) Program, and the China Postdoctoral Science Foundation [2025T180856].