In contrast to eukaryotes, bacteria lack a complete nucleus, which initially led to the theory that DNA methylation was the only type of bacterial epigenetic mechanism (Ghosh et al., 2020). Bacterial DNA methylation has been extensively studied over the past half century, revealing its involvement in chromosome replication, DNA degradation, mismatch repair, gene expression regulation, and other important physiological activities ( Table 1 ) (Heusipp et al., 2007; Muhammad et al., 2022). Bacteria have three major forms of DNA methylation: 5-methylcytosine (m⁵C), N6-methyladenosine (m⁶A), and N4-methylcytosine (m⁴C). DNA methyltransferase (MTase) add methyl groups to specific DNA locations, such as the C5 or N4 position of cytosine and the N6 position of adenine ( Figure 2 ) (Dunn and Smith, 1955; Holliday and Pugh, 1975). The most commonly known DNA MTases are associated with the restriction-modification (R-M) system, which is a widely known defense mechanism in bacteria. While m⁵C and m⁶A are found in most bacteria, m⁴C is specific to bacteria and archaea (Sánchez and Casadesús, 2020).

In addition to DNA methylation, DNA modifications also include DNA phosphorothioation (PT) modification, which is a lesser known defence system that works in a way similar to that of the R-M system ( Table 1 ) (Wang et al., 2019; Muhammad et al., 2022). PT modification, in which the nonbridging oxygen in the phosphate moiety of the DNA sugar-phosphate backbone is replaced by sulfur, was originally developed via chemically synthesized for decades (Tong et al., 2018). However, some research have discovered that PT modification can occur naturally in bacteria (Zou et al., 2018). Previously, it has been reported that DNA PT system consists of two parts: a five-gene dndABCDE cluster function as the M component to control DNA modification in a stereo- and sequence-selective manner, whereas products of the dndFGH cluster function as the R component to distinguish and restrict non-PT-protected foreign DNA (Tong et al., 2018). Among them, dndA possesses cysteine desulfurase activity and assembles DndC in bacteria (An et al., 2012). The IscS (a DndA homolog) can perform the same function as DndA to collaborate with DndBCDE in generating DNA PT modification (An et al., 2012). DndB can bind to the promoter region of the dnd operon to regulate the transcription of dnd genes. dndCDE function as modification genes: DndC is an iron-sulfur cluster protein that has ATP pyrophosphatase activity; DndD has ATPase activity and possibly provide energy for PT modification, and DndE is involved in binding nicked dsDNA (Hu et al., 2012; Wang et al., 2019). According to some research, the defence mechanism of PT modification has been revealed roughly ( Figure 3 ). At first, the DndB function as a regulator to make response of environmental or cellular cues, and binds to the promoter region of the dnd operon. The DndA/IscS, DndC, DndD and DndE form a protein complex. Under the action of DndA/IscS, L-cysteine is used as a substrate to generate a persulphide group. Then, the sulphur is transferred to the DndACDE complex to complete the DNA PT modification (Wang et al., 2019). DNA PT modification has been reported in many bacteria. Except for function the similar way as the R-M system, DNA PT modification also plays important roles in antioxidant defenses, cellular redox homeostasis maintenance, environmental stress resistance, antibiotic resistance and cross talk with DNA methylation modification (Xie et al., 2012; Gan et al., 2014; Wu et al., 2017; Wu et al., 2020; Xu et al., 2023).

rRNA, a conserved macromolecule, is a structural component of the most abundant cellular molecule, the ribosome. In bacteria, ribosomes are composed of 16S, 23S, 5S rRNA and proteins. In eukaryotic cells, ribosomes are composed of 28S, 5S, 5.8S, 18S rRNA and proteins. In ribosomes, the rRNA is the main structural component and the core of structure and function, including (1) Synthesizing amino acids into peptide chains under the guidance of mRNA; (2) Providing binding sites for a variety of protein factors; (3) Having the activity of peptidyl transferase; (4) Providing binding sites for tRNA; (5) Targets of some antibiotics (Korobeinikova et al., 2012; Tafforeau, 2015; Srinivas et al., 2023). These functions are under tight transcriptional control to serve to meet cellular needs. Therefore, rRNA from all organisms undergoes post-transcriptional modifications that increase the diversity of its composition and activity.

Methylation of rRNA is a ubiquitous feature, and takes place during ribosomal biogenesis either by enzymes guided by an antisense small nucleolar RNA (snoRNA) or conventional protein enzymes (Lopez et al., 2020). Generally, rRNA methylation may promote the conformational rearrangement of rRNA, and regulate ribosome biogenesis and post-transcriptional modification (Wang et al., 2020). There are 25 rRNA modifications have been found in the 23S rRNA, including 13 methylations in Escherichia coli (E. coli) ( Sergeeva et al., 2015). Wang et al. found that the absence of a single methylation in 23S rRNA affected 50S assembly and impaired translation initiation and elongation (Wang et al., 2020). In addition, rRNA methylation has emerged as a significant mechanism of AMR in pathogenic bacterial infections, such as aminoglycoside and macrolide resistance (Bhujbalrao et al., 2022; Srinivas et al., 2023).

Post-transcriptional gene regulation, which includes ncRNAs, is another important epigenetic modification. There are various types of ncRNAs: including housekeeping ncRNAs such as tRNA, rRNA, and regulatory ncRNAs such as micro RNA (miRNA) and long non-coding RNA (lncRNA) (Gusic and Prokisch, 2020). These RNAs play significant roles in transcription and translation, and in eukaryotes, they are involved in regulatory processes such as development, cell death, and chromosomal silencing. Although three regulatory RNAs contained E. coli 6S RNA, Spot 42 and the eukaryotic 7SK RNA were first discovered by sequencing in the 1970s, but were uncharacterized until decades later (Griffin, 1971; Delihas, 2015). Until the 1980s, the E. coli micF RNA gene was the first regulatory RNA discovered and characterized. Recent research has shown that ncRNAs regulate various cellular processes in bacteria, including multidrug resistance, glucose metabolism, and biofilm formation (Hirakawa et al., 2003; Vanderpool and Gottesman, 2004; Zhao et al., 2022). As a result, the regulatory mode of ncRNAs has become a major focus in the bacterial regulatory network.

Bacterial DNA methylation plays a vital role in epigenetic regulation by controlling gene expression, genome modification, virulence, mismatch repair, transcriptional regulation, cell cycle control, and AMR (Marinus and Casadesus, 2009). The most well-known DNA MTases are associated with the defense mechanisms in bacteria known as restriction-modification systems (R-M systems). R-M systems prevent lethal cleavage of intracellular DNA by identifying their own DNA and methylating the same sequence as the restriction endonuclease cleavage site (Ghosh et al., 2020). However, foreign DNA such as plasmids carrying AMR genes, transposons, and insertable sequences cannot be methylated and will be recognized and degraded by endonucleases of the R-M systems. This defense mechanism can be circumvented if the foreign DNA carries a homolog methylase with the same specificity, and the sequence will be inserted into the genomic locus rather than degraded (Casadesús and Low, 2006; Ishikawa et al., 2010). This mechanism could explain why plasmids, phages, transposons, integrons, and gene islands can insert into bacterial genomes and contribute to the widespread dissemination of AMR genes.

The R-M systems are classified into four types (I, II, III and IV) based on their functional localization of restriction endonuclease (Rease), activity of MTases, and requirement for specific subunits or cofactors (Roberts et al., 2003). The R-M systems have reported to function as a barrier to horizontal gene transfer in many bacteria ( Figure 5 ) (Vasu and Nagaraja, 2013; Kumar et al., 2018). Li et al. found a carbapenem-resistant hypervirulent Klebsiella pneumoniae (K. pneumoniae) strain with a bla _kpc harboured conjugative plasmid and a pLVPK-like plasmid from the patient, and the type I R-M system on plasmids protected the plasmids from cleavage (Li et al., 2020). Bubendorfer et al. concluded that R-M systems inhibited genomic integration of exogenous sequencs, while they pose no effects to homeologous recombination in H. pylori (Bubendorfer et al., 2016).

The type I and III includes genes encoding the DNA MTase mod. Many studies have described that mod gene-mediated DNA methylation can regulate phase-variable expression associated with various resistant clinical strains ( Table 3 ) (Phillips et al., 2019). For instance, the ability of N. gonorrhoeae to form biofilms is affected by allele modA13 ON/OFF switching (Srikhanta et al., 2009); Neisseria meningitidis susceptibility to ceftazidime and ciprofloxacin result from ON/OFF of modA11 and modA12 OFF switching (Jen et al., 2014). A typical H. influenzae expressing modA2 MTase produces more biofilms in an alkaline environment than modA2-deficient populations, and these biofilms have a larger biomass and less apparent structure (Brockman et al., 2018). Bacterial biofilms and AMR are closely connected. Biofilms are organized multicellular communities surrounded by an extracellular polymeric substances and can decrease bacterial metabolism, growth rate, and resistance to antibiotic penetration, all of which contribute to biofilm resistance (davies, 2003). Even in Streptococcus suis, Tram et al. found biaphasic switching of phase-variable DNA MTase ModS2 results in the expression of distinct phase varions. Proteins involved in general metabolism increased expression in ModS2 ON. Adversely, a glyoxalase/bleomycin resistance/extradiol dioxygenase family protein which has been described as involved in resistance to beta-lactam and glycopeptide antibiotics was upregulated in strains that did not express ModS2 OFF (Tram et al., 2021).

In addition to the well-known R-M systems, there exists a group of bacterial DNA MTases called orphan MTases, which function independently without association with any R-M system (Ishikawa et al., 2010). Orphan MTases are unique, as they do not have functional counterparts in the restriction enzyme (Reases) family. The common categories of orphan MTases include DNA adenine methyltransferase (Dam), cell cycle regulated methyltransferase (CcrM) and DNA cytosine methyltransferase (Dcm). Bacteria exhibit complex stress responses when exposed to antibiotics, leading to the phenomenon of adaptive resistance. Recent research has revealed that these three orphans MTases play a crucial role in regulating adaptive resistance and the genetic pathways involved in drug sensitivity.

The DNA PT modification, a novel R-M system, has been discovered widely in bacteria and archaea. As a defense barriers, DNA PT modification plays a vital part in bacterial AMR. Nonetheless, the potential role of the DNA PT modification in AMR is still unclear. By analyzing the functions of DNA PT modification in AMR with a serious of clinical pathogenic bacteria, Xu et al. demonstrated DNA PT modification reduced the distribution of horizontal gene transfer (HGT)-derived AMR genes in the genome, meanwhile the modification could suppress HGT frequence (Xu et al., 2023). To understand the mechanism of antibiotic resistance genes (ARGs) in drinking water supply systems, Khan et al. found the relative abundance of dndB and ARGs increased in the effluent, as well as, considered that DNA PT modification protected mcr-1 and bla _NDM-1 carrying bacteria from chloramine disinfection during the water treatment process (Khan et al., 2021). DNA PT modification can recognize and cleave unmodified exogenous DNA, such as HGT, ARGs and phage. Therefore, the modification is significant for bacteria to resist foreign invasion and maintain own genetic stability. Up to now, there is few systematic studies on AMR base on DNA PT modification, while we need to study the impact on AMR further.

RNA modifications, such as rRNA methylation, have emerged as important mechanisms associated with AMR. Ribosomes are a common target for antibiotics. Methylation of specific sites in rRNA can prevent antibiotics from binding to their target sites, thereby leading to antibiotic resistance. Thus AMR via rRNA methylation is one of the most common strategies adopted by multidrug resistant pathogens. One such example is 16S rRNA methylation, which is a major mechanism of aminoglycoside resistance in clinical pathogens (Tada et al., 2013; Liu et al., 2015). Two different methylation sites in 16S rRNA lead to different aminoglycoside-resistant phenotypes. Methylation of residue A1408 confers resistance to kanamycin and apramycin in E. coli, but sensitivity to gentamicin, while methylation of residue G1405 confers resistance to kanamycin and gentamicin, but sensitivity to apramycin (Liu et al., 2015). The multidrug resistance gene cfr, found in Staphylococcus, encodes an MTase that modifies the A2503 site in 23S rRNA, leading to resistance to antibiotics such as amide alcohols, lincomycins, oxazolidinones, pleuromutilin, and streptogramin A (Long et al., 2006). In S. pneumoniae, U747 methylation mediated by RlmCD promotes efficient G748 methylation by the MTase RlmA^II in 23S rRNA, affecting the susceptibility to telithromycin (Shoji et al., 2015). Another research indicated the erythromycin-resistance MTase methylates rRNA at the conserved A2058 position, and imparts resistance to macrolides, such as erythromycin (Bhujbalrao et al., 2022). Up to now, the number of rRNA MTases related to AMR mechanisms have increased, but the source of MTases and the exact mechanisms of AMR are still unclear.

Advancements in high-throughput sequencing technology and bioinformatics have facilitated the discovery of various ncRNAs and their functions in bacteria. Recent studies have found that exposure to environmental stress, especially antibiotics, bacteria produce specific ncRNAs profiles, which may regulate the expression of downstream genes. When bacteria sense antibacterial stress, a large number of ncRNA regulators are upregulated, and one of their roles is to improve bacterial adaptation in a dynamic environment (Morita and Aiba, 2007). Thus, ncRNAs play an essential role in the bacterial regulatory network that controls the expression of bacterial genes through regulating proteins and target mRNAs. In comparison to regulatory proteins, ncRNAs are considered a better class of regulatory molecules for controlling gene expression (Toledo et al., 2007).

ncRNAs play an essential role in the regulation of bacterial gene expression and can affect AMR mechanisms. Although ncRNAs are a major form of post-transcriptional gene control in bacteria, some research indicate ncRNAs also influence transcription (Rodgers et al., 2023). For instance, Majdalani et al. found that RprA ncRNA reduced type IV secretion-mediated transfer of pSLT (Salmonella virulence plasmid) (Papenfort and Melamed, 2023). In particular, RrpA controls the transcription and translation of ricI, which encodes a membrane protein that interacts with and suppresses the anchor protein Trav of the type IV secretion apparatus (Majdalani et al., 2001). It is reported that antisense vicR (a kind of ncRNAs) is transcribed from the opposite strand of vicR mRNA and regulates the biofilm formation of Streptococcus mutans via affecting the production and function of VicR protein (Lei et al., 2018).

The incomplete complementary pairing of most ncRNAs with the target mRNA sequence can lead to two results: (1) Blocking the ribosome binding sites and suppressing translation; (2) Secondary structure melting, exposing the nucleose binding site and translation start site, leading to translation activation (Vogel and Sharma, 2005; Fröhlich and Vogel, 2009). Moreover, since the instabilized base pairing between the ncRNAs and their target mRNAs, the RNA chaperone protein Hfq, binding protein Fino/ProQ family, CsrA/RsmA family and other regulators usually facilitate imperfect base pairing between ncRNAs and mRNAs, leading to regulate the translation initiation frequency or the stability of target mRNAs (Liao and Smirnov, 2023; Wang et al., 2023; Yu and Zhao, 2023). In this chapter, we will explore some research on ncRNAs that regulate the mechanisms of AMR from two perspectives.