## 4 Role of sRNA in Antibiotic Resistance
### Inhibiting Drug Uptake Systems
#### 4.1.1 GcvB  
One of the key players of antibiotic resistance in _E. coli_ and _Vibrio_ is the small RNA GcvB, a 205-nt sRNA, transcribed divergently from the _gcvA_; the latter encodes for the transcriptional activator of the glycine cleavage (GCV) pathway. In the presence of glycine, transcription of _gcvB_ is activated by GcvA and GcvR. Although GcvB was initially associated with the acid stress response [173], recent studies have elucidated its role in antibiotic resistance. Deleting _gcvB_ in _E. coli_ leads to heightened sensitivity to various aminoglycosotide antibiotics, including neomycin, streptomycin, kanamycin, and kasugamycin, even at low concentrations [174]. GcvB consists of five stem-loops (SL1-5) with three conserved seed sequences, R1, R2, and R3, to regulate multiple target genes [175]. The G/U-rich R1 seed sequence located between SL1 and SL2 can base pair with the majority of target mRNAs. Although the seed sequence R2 primarily repress CycA mRNA in _E. coli_ and _Salmonella_, whereas the R3 seed sequence, located in SL4, regulates several mRNAs, including global transcriptional regulators PhoP, Lrp, and SroC [176; 177] (Figure 3a). GcvB contributes to antibiotic resistance in _E. coli_ by posttranscriptionally downregulating the expression of the inner membrane protein CycA. In addition to its role in amino acid (D-glycine, D-serine, D-alanine) import, CycA facilitates the uptake of aminoglycosotide antibiotics such as streptomycin and gentamicin. GcvB mediates this regulation by base pairing with _cycA_ mRNA, promoting its degradation and thereby reducing CycA protein levels, ultimately limiting the entry of aminoglycosotide antibiotics into the cell [178]. Two regions of GcvB, spanning nucleotides +124 to +161 and +73 to +82, exhibit complementarity to the CycA mRNA. Transcriptional fusions of _cycA_ to _lacZ_ revealed that this CycA mRNA region is essential for GcvB-mediated regulation [179]. [PERSON] et al. deciphered that either of the abovementioned complementary regions of GcvB can independently base pair with and repress _cycA-lacZ_, with significant repression loss occurring only when both regions are mutated [174]. Additionally, mutations in _cycA_ can lead to increased resistance to aminoglycoside by reducing its uptake into the bacterial cell. Moreover, GcvB plays a role in promoting mutagenic break repair (MBR) by enabling a robust RpoS response while suppressing the competing SigE (membrane stress) response. _gcvB_ deletion impairs MBR and RpoS-dependent transcription without reducing RpoS protein levels. Restoring RpoS levels or inhibiting SigE response induction rescues these defects, suggesting that GcvB maintains membrane integrity, resulting in decreased SigE activation. This balance prevents SigE from outcompeting RpoS for RNA polymerase, thereby supporting mutagenesis [180]. Furthermore, using transcriptomics- and proteomics-based approaches, [PERSON] et al. identified the role of GcvB in regulating the type III secretion system in the marine bacterium _Vibrio alginolyticus_, thereby expanding its role in bacterial virulence [175]. Figure 3: **The mechanisms of bacterial antibiotic resistance. ****(a) Small RNA-mediated antibiotic resistance mechanisms:** Upon exposure to environmental stimuli, bacterial pathogens activate transcriptional regulators/factors (TF), which in turn induce the expression of cognate sRNAs. These sRNAs contribute to antibiotic resistance by either upregulating efflux pumps that expel antibiotics from the cell or downregulating outer membrane proteins involved in antibiotic uptake, thereby reducing intracellular antibiotic accumulation. A few components of this figure were created using Biorender. (**b.c**) **The role of small RNAs in antibiotic resistance through biofilm formation. ****(b) The role of AS1974 in biofilm formation:** The sRNA AS1974 is activated through epigenetic regulation and functions to inhibit antibiotic resistance, biofilm, and bacterial adherence to the host.